BLOCK II STUDY GUIDE: INCLUDES TERMS AND LECTURE OBJECTIVES

Lecture XVII
Introduction to Part II - Presentation of a Case Study

Terminology
Term Hereditary Hemorrhagic Telangiectasia (HHT) Anemia Focal Vascular Lesion Epistaxis Telangiectasias Arteriovenous Malformation Nidus Autosomal Dominant Haploinsufficiency Incomplete Penetrance Variable Expressivity Definition Genetic (autosomal dominant) disorder that leads to abnormal blood vessel formation in the skin, mucous membranes, and often in organs such as the lungs and brain. This can cause nosebleeds, GI bleeding. Common blood disorder attributed to a general decrease in number of erythrocytes or decrease in hemoglobin. Abnormality near the surface of epidermal tissue attributed from vascular origins, particularly through dilation of blood vessels. Common occurrence of nosebleeds, where blood drains from the nostril, attributed to rupture of the blood vessels in the nose. Small, dilated blood vessels near the surface of the skin or mucous membranes. Abnormal connection between veins and arteries, typically from congenital origins. Large, fragile tangle/accumulation of arteries and veins. This refers to the inheritance pattern of a disease. If it is autosomal dominant, then only one abnormal gene from a parent is necessary to attain the disorder. When an organism only has one functional copy of the gene, with the other inactivated, which can cause abnormal presentations such as in disease-states. Proportion of patients carrying the genotype that do not express the phenotype. Variations in phenotype carrying a particular genotype. It is analogous to the severity of a condition in clinical medicine.

Lecture Objectives
Introduction to Block II and the case study. Block II surrounds the elements of signal transduction pathways that alter gene expression. This strategy is mainly the way to target genes. This can occur in elements of development and can be utilized to our advantage in stem cell research. In short, signal affects expression. The case study involved in this block is the medically unique tale of a patient named Sarah. She is a 32-year old martial arts instructor in South Arizona, married with kids. She has experienced elements of fatigue (tired and sluggish). She initially attributed it to stress until she observes blood in her stool. Sarahs doctor does a history and physical, with no indication of colon cancer in the family. A fecal occult blood test confirms blood in her stool, and Sarah is then scheduled for a colonoscopy. Her doctor finds numerous small red spots on her skin, distributed mostly in her mouth and on the top of her hands. They increased in number, as she got older. She also gets small lesions on the inside of her mouth or tongue from time to time. Sarah also tells her doctor she had a history of nosebleeds, even though her sister does not get nosebleeds. Her mom also had a history of nose bleeds, and that one of her sons had a lot of nosebleeds. The colonoscopy revealed vascular lesions (known as telangiectasias) in her colon, and laboratory test revealed anemia in her blood. This led to the diagnosis of hereditary hemorrhagic telangiectasia, and DNA testing later revealed that she has specifically HHT Type II. Learn about the presentation, diagnosis, complications, and inheritance pattern of HHT. Hereditary hemorrhagic telangiectasia (HHT) is a vascular disorder found in ~1 in 5-8000 people, with 1.2 million people affected. It is a multisystem vascular disorder consisting of focal vascular lesions (telangiectasias or arteriovenous malformations). It is an autosomal dominant disease, with the bad gene inhibits the good gene, and so the bad protein is created so that it inhibits the desired effects from normal proteins. It exhibits haploinsufficiency in which the on good copy is not enough for normal effects. The disease has marked intrafamilial variation in terms of both penetrance and variable expressivity. The presentation is typically observed in childhood but the symptoms can be aggravated with age, with patients exhibiting only nosebleeds and telangiectasia. Of the patient population, approximately a third of the patients exhibit chronic anemia and GI bleeding. However, a majority can have typically silent arteriovenous malformations in the pulmonary, hepatic, cerebral, and spinal circulations. Complications can involve not only anemia due to chronic GI and epistaxis, but also in the larger arteriovenous malformations in lungs, brain, liver, and GI tract (such as in stroke, hemorrhage, and brain infection). More rare complications tend to include congestive high output heart failure from the decrease in capillary bed size.

Diagnosis involves three of the following criteria: (1) spontaneous and recurrent epistaxis, (2) multiple telangiectasias at characteristic sites, (3) visceral vascular lesions (gastrointestinal telangiectasias and proven arterioveous malformations on lung, liver, brain, or spine), (4) family history of HHT (first-degree relative) Treatment and management involves strategies to: (1) stop or prevent excessive bleeding, (2) increase clotting ability, (3) replenish lost blood, (4) monitor for more serious complications, and (5) procedures to block off local sites where there is a blood vessel defect. Know the underlying structural disorder in HHT. Distinguish between Telangiectasias and Arteriovenous Malformations. The underlying disorder in HHT is attributed to the abnormal vascular architecture. There are two types of vascular abnormalities: telangiectasis and arteriovenous malformations (AVMs). Telangiectasis are focal dilatations of microvessels. There is a decrease in the number of capillaries, and are convoluted in larger lesions in later stages. This implies that there is really a direct connect between arteries and veins, and thus does not allow appropriate capillary exchange. They are common on skin and mucous membranes. Arteriovenous malformations are essentially direct connections between larger arteries and veins. These are typically larger than telangiectasis, up to several centimeters. At this point , there is a decrease (if not a lack) of capillaries, and can be present as a nidus (essentially a big, fragile tangle of arteries and veins). AVMs can particular develop in distinct parts of the vasculature to limit bypasses in normal circulation. Think about what we might learn by examining this case. From observing the pathogenic mechanisms underlying the vascular malformations in HHT, we can learn about how the genetic mutations can overall affect signaling patterns in the body and disrupt normal physiological processes. In HHT, one of the physiological consequences is essentially excessive bleeding and poor capillary exchange.

Lecture XVIII
Cardiovascular System : Organization, Functions, Properties

Terminology
Term Transport and Exchange Closed versus Open Circulation Definition Major function of the cardiovascular system, involving movement of a media (particularly blood) through a circulatory system to allow for tissue exchange of nutrients and metabolic waste. Closed Circulation: Blood never leaves the network of arteries, veins and capillaries, with oxygen and nutrients diffusing into interstitial fluid. Allows for diffusion of nutrients at low pressure states. Open Circulation: Blood leaves network of arteries, veins, and capillaries in the form of hemolymph, which provides no distinction between blood and interstial fluid. Allows for movement at higher pressure. English physician who first described the systemic circulation and properties of blood in a closed circulation. Portion of the cardiovascular system carrying oxygen-depleted blood away from the heart to the lungs, to bring oxygenated blood to the heart. Portion of the cardiovascular system where oxygenated blood is carried to the various organs (excluding lungs) and brings oxygen-depleted blood to the heart. Type of organization consisting of consecutive elements, such as A B. Type of organization consisting of one attending to multiple events, such as A B + C + D (simultaneously). Blood vessels that carry blood away from the heart. Blood vessels that carry blood towards the heart. Small vessels in the vasculature that are embedded in organs and are the main element in the distribution of nutrients from blood to tissue. Blood vessel in microcirculation that moves out of the artery and into the capillaries. They are also the primary sites of vascular resistance, where the greatest change in blood pressure and velocity of blood flow occurs. Blood vessel in microcirculation that return blood from the capillary to the larger blood vessels. Smallest blood vessels and are involved in exchange of nutrients and materials, and connect arterioles to venules. Chamber of the heart involved in receiving blood. The right atrium receives blood

William Harvey Pulmonary Circulation Systemic Circulation

A' B' In Series
Components'in'series'
Components'in'series' In Parallel

A'

B'

A' B' A' B' C' C' Components'in'parallel' Artery

Components'in'parallel'

Vein Microcirculation Arteriole Venule Capillary Atrium

Ventricle Myocardium Atrioventricular Valves Cardiac Output Total Cross-Sectional Area Velocity of Blood Flow Volumetric Flow Rate Blood Pressure Arteriovenous Malformation Telangiectasia

rom the superior or inferior vena cava, while the left atrium receives blood from the pulmonary veins. Large chamber that collect and expel blood from the atrium towards either lungs (right ventricle) or to the body (left ventricle). The left ventricle is typically bigger than the right ventricle. Muscular layer of the heart involved in contraction and pumping of blood, consisting of cardiac muscle. Valves of the heart between the atria and ventricle, known as the mitral valve (left) and the tricuspid (right). Volume of blood being pumped by the heart, by a left or right ventricle within one minute. It is the multiplication product of stroke volume and heart rate. = . Total cross sectional area of all blood vessels of a particular type. Capillaries (through their numbers) have the largest cross sectional area despite their small size. A value equal to the total volume flow divided by the cross-sectional area: !"#$% !"#$%& !"#$ = !"#$$!!"#$%&'() !"#$ Volume of fluid that passes through a given surface per unit time. Pressure exerted by circulating blood upon the walls of blood vessels. Abnormal connection between veins and arteries, typically from congenital origins. Small, dilated blood vessels near the surface of the skin or mucous membranes.

Lecture Objectives
Know the functions of the CV system. The cardiovascular system has the following major functions: (1) transport and exchange of nutrients, (2) fluid balance to regulate intracellular and extracellular volume of body cells, (3) dissipation of excess heat, (4) as a buffer system to stabilize pH. The ultimate goal of this is to maintain homeostasis, or a relatively constant Func:onal'Organiza:on' environment around cells. Learn how the CV system is organized. The cardiovascular system is organized in a closed circulation, in which blood is contained within vessels (of varying size and thickness) at all times. The components of the human cardiovascular system are: (1) the heart, (2) blood vessels, and (3) blood. The heart is the muscular pump that provides the driving force of the movement of blood. The blood vessels are ultimately the plumbing of the heart, directing movement and keeping the blood confined. Finally, the blood is the tissue involved in carrying materials.
Site$for$gas$ exchange$ Goal:''Transport'and'exchange' Distribu=ng$ tubes$ Large$network$of$ very$thin$tubes$ where$exchange$ occurs$at$=ssues$ Collec=ng$ tubes$

Pump$

'

On a larger scale, it is organized into two circulations: (1) the pulmonary circulation (involving the right side of the heart) and (2) the system circulation (involving the left side of the heart). The systemic circulation is typically in series with the pulmonary circulation, while the other organ systems are supplied with blood in parallel by the systemic circulation. It can also be organized by function. Lungs represent a site of gas exchange. The heart is the pump. The arteries are the distributing tubes. The capillaries are the large networks of very thin tubes where exchange occurs at tissues. Veins are the collecting tubes. Trace the flow of blood through the CV system. The flow of blood through the cardiovascular system is typically in the following manner. Blood flows into the right atrium from the superior and inferior vena cava and consequently flow into the right ventricle. From the right ventricle, blood will then flow (via the pulmonary arteries) to the lungs, where they will be replenished with oxygen while simultaneously releasing carbon dioxide. The newly oxygenated blood will then flow into the left atrium via the pulmonary veins. It will then flow into the left ventricle, and then be pumped into the aorta and thus into the systemic circulation. Know about cardiac output and its distribution and how it can change. Cardiac output is the volume of blood pumped by each ventricle per minute (approximately 5 liters/minute). The cardiac output from right to left ventricles is

Superior/ Inferior Vena Cava Systemic Circulation Right Atrium

Aorta

Right Ventricle

Left Ventricle

Pulmonary Arteries

Left Atrium Pulmonary Veins

Lungs

typically the same (at 5 liters/minute as it is a closed circulatory system). If not the same, it may imply backup of flow. The distribution of the cardiac output to the different organ systems Distribu:on'of'the' is rather unequal, determined by function, size, and metabolism. Some changes in cardiac output are rather normal, especially in states of exercise, where the cardiac'output'to'the' metabolic demands (by neurological excitation) require greater flow of blood. dierent'organ'systems'
What$determines$the$ Learn how CV parameters change at different levels of the circulation. Think about distribu=on$of$the$ how these might change in HHT. cardiac$output?$ Blood'pressures'in'the'systemic'circula:on' The cardiovascular parameters can change at different levels of the circulation. In terms of total cross-sectional Sherwood'Fig.'10T1' area, the capillaries (collectively) have the greatest crosssecitonal area, because of their numbers. The total crosssectional area is the key determinant of the velocity of blood flow. Typically, as the cross sectional area increases the velocity of flow decreases (at constant blood flow rate). This is important because we can also tell that the pressure is the lowest at the capillaries, while highest at the aorta. The mean arterial pressure remains constant through larger arteries and then drops at the arterioles and troughs at the venules and veins. This can change in hereditary hemorrhagic telangiectasia (where direct connections allow paths of lower resistance) by making venous pressure increase. People with hereditary hemorrhagic telangiectasia can have arteriovenous malformations that spur a shunt (a low resistance shortcut) between the arteries and veins. The problem is that the veins are not as structurally stable in comparison to the muscular arteries, and can easily hemorrhage because of such relatively extreme pressures by such bypasses. Patients with this same disorder can develop a shunt in the hepatic circulation, with the chronic high cardiac output that can spur cardiac failure.
Systolic'pressure' Mean'pressure' Diastolic'' pressure'
Sherwood'Fig'10T9'

Lei' ventricle'

Large' arteries'

Arterioles'

Capillaries'

Venules'and'veins'

Lecture XIX
Blood Vessel Structure and Function

Terminology
Term Tunica Interna Tunica Media Tunica Externa Vascular Endothelium Vascular Smooth Muscle Basement Membrane Elastin Collagen fibers Arteries Pressure Reservoirs Pulsatile Blood Pressure Systolic Pressure Diastolic Pressure Mean Arterial Blood Pressure Veins Volume Reservoirs Distribution of Blood Definition Most interior layer of the blood vessel, which includes endothelial cells and connective tissue. Medial layer of the cell, containing vascular smooth muscle cells in loose connective tissue and elastic fibers. Most external layer of the blood vessel, which is essentially a connective tissue sheath, also known as an adventitia. Thin layer of cells that line the interior surface of blood vessels, with the individual cells called endothelial cells. Smooth muscle found within, and composing the majority of the wall of blood vessels, particularly the arteries and arterioles. Sheet of fibers that underlie the epithelium, which line cavities and surfaces such the endothelium. Protein in elastic connective tissue to allow consistency in shape after expansion or contraction. Major component of connective tissue that allows maintenance in shape. This is seen in greater amounts in venules and veins. Blood vessels that carry blood away from the heart, exhibiting more smooth muscle in comparison to veins. Type of reservoir in which pressure, not volume, is the main element regulated. This is seen in arteries, where the mean arterial pressure is kept constant. Quantity of pressure required creating the feeling of a pulse. Maximal blood pressure, or the pressure exhibited in contraction. Minimal blood pressure, or the pressure exhibited in relaxation. Average arterial pressure during a single cardiac cycle, or the average blood pressure in an individual. Calculated as the product as the sum of the product of cardiac output and stroke volume and central venous pressure [() + = ]. Blood vessels that carry blood towards the heart, exhibiting more collagen in comparison to arteries. Type of reservoir where volume, not pressure, is under regulation. Veins are considered volume reservoirs, retaining the majority of blood volume. Distribution of blood is where volume changes to different parts of the body

Volume Venous Valves Microcirculation Arterioles Venules Volumetric Flow Rate

despite the constant pressure gradient. Main force involved in allowing blood to flow towards the heart unidirectionally. Smaller vessels in vasculature involved in distribution of blood within tissues. Blood vessel in microcirculation that moves out of the artery and into the capillaries. They are also the primary sites of vascular resistance, where the greatest change in blood pressure and velocity of blood flow occurs. Blood vessel in microcirculation that return blood from the capillary to the larger blood vessels. Volume of fluid that passes through a given surface per unit time. It is proportional to the pressure gradient across the vessel and is inversely related to !! the resistance to blood flow: = . Difference in pressure along a blood vessel. Despite a constant pressure, the major variable involved in altering volume and distribution of blood. It determines how much blood flow goes through a particular blood vessel, and also determines how much flow goes into a particular organ. Physical law that provides a pressure drops along a blood vessel. Calculated by: =
!!! ! !!" !

Pressure Gradient Resistance to Flow

Poisseuilles Law Smooth Muscle Cell Contractility Primary Resistance Vessels Vasoconstriction Vasodilation Capillaries

, where r is radius, P is pressure, is viscosity, and L is length.

Ability of the smooth muscle to relax or contract independent of volume. Vessels involved in altering blood volume despite constant pressure. Arterioles are the primary resistance vessels. Narrowing of blood vessels from contraction of smooth muscles, found particularly in larger arteries and arterioles. The widening of blood vessels from relaxation of smooth muscles. Smallest blood vessels and are involved in exchange of nutrients and materials, and connect arterioles to venules.

Lecture Objectives
Learn about the anatomy of blood vessels. The anatomy of blood vessels is distinctly different among the arteries and veins. Remember that their structure is reflective upon function, but as blood vessels (excluding the capillaries), they have similar parts. The blood vessel contains three parts: the tunica interna, the tunica media, and the tunica externa. The tunica interna is the innermost layer of the blood vessel, containing the endothelium and connective tissue. All blood vessels are lined with the endothelial cells, which function in clot prevention, cell signaling, and capillary exchange. The tunica media is the smooth muscle layer, containing the smooth muscle in loose connective tissue with elastin. Elastin is important in allow the recoil from the larger volume of blood flow. The tunica externa (also known as the adventitia) represents a connective tissue sheath that encapsulated the blood vessel. Though arteries and veins are classified under blood vessels, they have observable differences. Arteries have a larger amount of smooth muscle and elastin, while veins tend to have greater amounts of collagen fibers. Thus, compensate in order to establish unidirectional flow, the veins are accommodated with valves made from endothelial tissue. The exclusion of capillaries is simply because they are one cell thick and are the main exchange points between the blood and tissue. See how the anatomy of different types of blood vessels reflects function. The anatomy is reflective upon function. Arteries typically move blood away from the heart, while veins move blood towards the heart. Their function can be represented structurally in the fact that arteries have larger amounts of elastin and vascular smooth muscle lining this type of blood vessel. This allows the artery to propagate pressure waves from the heart (allowing transient increases in blood), and allow it to recoil back from expansion. Thus, the arteries are treated as the pressure reservoirs of the circulatory system, keeping the mean arterial pressure constant and allow even pressure distribution to the system.

Meanwhile, veins display a different structure for their function. They are involved in carrying tissues back to the heart. They have thinner walls compared to arteries and less elastic tissue and smooth muscle, and are typically adapted for lower pressures. To facilitate venous return to the heart in a unidirectional fashion, they have a series of valves. Thus, they are typically considered to be volume reservoirs, as they are the major storage of blood volume (containing approximately 64% of volume distributed). Know the functions of endothelial cells and smooth muscle cells. The endothelial cells of the body are involved in (1) provision of a smooth interface, (2) secretion of cellular signals, and (3) in the capillaries, provide a penetrable barrier for exchange. Smooth muscle cells in the vasculature can contract or relax depending on what kinds of signals they receive. Arterioles are the major resistance vessels. A change in arterioles will cause a change in flow. The largest drop in pressure occurs at the arterioles, and arterioles control the flow of blood amid the constant pressure. They typically act by vasoconstriction and vasodilation. Vasoconstriction occurs by increased contraction of smooth muscle, where there is increases in resistance and eventual decrease in flow. Vasodilation occurs when there I a decreased contraction of smooth muscle, where there is a decrease in resistance and an increase in flow. Resistance is what determines how much flow goes into each organ. Local and extrinsic factors can affect the activity of the vascular smooth muscle. It can be affected by (1) autonomic nervous system activity, such as in state of physical activity, (2) local factors, or metabolites, released by the nearby tissue, giving a metabolic control of blood flow, (3) local changes in blood flow, showing myogenic control, and (4) signals released by the vascular endothelium, such as nitric oxide and endothelin. Know the different parameters that affect blood flow through a blood vessel. There are several general parameters that can affect blood flow through a blood pressure, as Poiseuilles Law can describe. We do know that pressure and resistance are the two major variables that can affect flow, but there are others. The radius is inversely proportional to resistance, such that an increase in the radius can cause a decrease in resistance. However resistance is directly proportional to the viscosity and length, so that increasing either viscosity or length can cause an increase in resistance. However, we should also examine the role of blood pressure. It is not dependent on the absolute values of pressure along the blood vessel, but more of the difference between the pressures along the blood vessel, which is known as a pressure gradient. In physiological states, however, we should remember that there is going to be change in blood vessel structure as the absolute pressures increase. Think about how the above relate to the case study. What changes occur with HHT? In hereditary, hemorrhagic telangiectasia, we should remember that the arteriovenous malformations allow a low resistance bypass into the vein. Higher pressures in the draining veins at focal vascular lesions in HHT can lead to consequential structural changes in the veins. However, veins are particularly thinner relative to arteries, and the compensatory measures are inadequate. If the pressure is too high and the structural compensations are insufficient, it can lead to strokes, aneurysm, and hemorrhage due to such structural insufficiency. The laminar flow is disrupted in the arteriovenous malformation and spur turbulent flow. Such turbulent flow can cause structural changes and greater proliferation to accommodate such changes in flow.

Lecture XX
Transport and Exchange in the M icrocirculation

Terminology
Term Microcirculation Arterioles Metarterioles Precapillary sphincters Definition Smaller vessels in vasculature involved in distribution of blood within tissues. Blood vessel in microcirculation that moves out of the artery and into the capillaries. They are also the primary sites of vascular resistance, where the greatest change in blood pressure and velocity of blood flow occurs. Short vessel that links arterioles to capillaries, which contain smooth muscle cells a short distance apart that forms a precapillary sphincter that surrounds entrance to capillary bed. Band of smooth muscle that adjusts flow into capillary.

Venules Capillaries

Capillary Recruitment Continuous Capillaries

Blood vessel in microcirculation that return blood from the capillary to the larger blood vessels. Smallest blood vessels and are involved in exchange of nutrients and materials, and connect arterioles to venules. Capillaries are suited as a site of exchange because of the minimal diffusion distance, maximal surface area, and the maximal time for exchange. Increase in the number of perfused capillaries in response to stimuli. Type of capillary that has an uninterrupted lining in the endothelial cells, to allow water and ions to diffuse with two types of tight junctions: (1) one with many transport vesicles such as those in skeletal muscle, and (2) those with few vesicles, such as in the central nervous system. Type of capillary with pores in the endothelial cells that allow small molecules and small proteins to diffuse, found in kidneys and exocrine glands.

Fenestrated Capillaries

Sinusoidal Capillaries

Type of capillary with larger openings in the endothelium due to a discontinuous basal lamina, which allow cells and larger proteins to enter. These are found in bone marrow, lymph nodes, liver, spleen and adrenal gland.

Diffusion Ficks Law

Concentration Gradient Lipid-Soluble Substances Small, Water-Soluble Substances Exchangeable Proteins Plasma Proteins Endocytosis Exocytosis Secondary Polycythemia Interstitial Fluid Plasma Fluid Intracellular Fluid Bulk Flow Ultrafiltration Reabsorption Lymphatics Net Fluid Exchange Pressure

Capillary Pressure Interstitial Fluid Exchange Pressure Plasma Colloid Osmotic Pressure Interstitial Fluid Colloid Osmotic Pressure Starlings Law

Movement of particles from areas of higher concentration to areas of low concentration. !!! Mathematical relationship in diffusion: = , where C !"! is concentration, P is permeability of membrane to substance, A is surface area of membrane, MW is the molecular weight of substance, and X is distance. Gradual difference in the concentration of solutes in a solution between two regions. Rate of diffusion depends on the concentration gradient. Nonpolar substances that can dissolve in fats thus can pass through the phospholipid bilayer. Polar or charged solutes that need to be dissolved in water thus will diffuse across certain pores or gaps in cells. Proteins that are shuttled across the membrane by a general vesicular mechanism of transport, such as endocytosis and exocytosis. Proteins that are generally too large to pass through the water-filled pores thus remain in the plasma. Cells absorb molecules through engulfment. Cells direct contents of solutes out of the cell membrane via secretory vesicles. Type of increase in erythrocytes either by natural or artificial causes, with a known underlying cause. Fluid that bathes and surround cells of tissue, and is the major component of extracellular fluid. Liquid component of blood where blood cells in whole blood are suspended. Fluid found inside cells, which is also known as cytosol or cytoplasm. Movement of a fluid driven by pressure. Type of membrane filtration involving hydrostatic pressure pushing against a semipermeable membrane. In this case, there is a push for fluid out of the capillary. Typically, ultrafiltration is larger than reabsorption by 3 L. Type of membran filtration in which there is a uptake of fluid into the capillary. Organ system involved in filtration of lymph and reuptake of fluid that is not reabsorbed into the capillary. Consist of blind-ended capillaries that overlap the capillary system to transport fluid back into the venous system. Contributor of bulk flow which is the difference in the outward and inward pressures, such is also influenced by derivatives of the outward and inward pressure, such as capillary pressure (PC), interstitial fluid pressure (PIF), plasma colloid osmotic pressure (P), and the interstial fluid colloid osmotic pressure (IF). Thus: = !"#$%&' !"#$%& = ! + !" (!" + ! ). Outward force for fluid exchange. Inward force for fluid exchange. Inward Force for fluid exchange. Outward force for fluid exchange. Mathematical illustration of hydrostatic and oncotic forces to determine amount of fluid movement from blood to tissue. Calculated as: ! = ! = ! [ ! +

!" (!" + ! )]. Exchange pressure determines direction of movement, while both exchange pressure, and permeability will determine magnitude of the movement.

Lecture Objectives
Know the organization, anatomy, and function of the microcirculation. We need to remember that microcirculation is the site of exchange of solutes and fluid. The microcirculation consists of three general anatomical parts: the arteriole, the capillaries, and the venule. On the side of the arterioles, there are smooth muscle cells in areas such as the metarteriole and the precapillary sphincter that allow regulation of flow through the capillaries. It should also be remembered that the capillary is onecell thick to allow a site of exchange. Learn how the exchange of solutes occurs in capillaries. The exchange of solutes between the plasma and tissue cells occurs via a mechanism known as diffusion in the capillaries. Diffusion is the movement of solutes from areas of high concentration to areas of low concentration. One can calculate the net rate of diffusion Q by Ficks Law: = , where C is !"! Tissue'metabolic'ac8vity' Eect'of'capillary' concentration gradient, P is permeability of membrane to substance, A is recruitment'and' surface area of membrane, MW is the molecular weight of substance, and O ,''CO 'other'metabolites' arteriolar'dila8on' X is distance. Capillries are well suited as a site of exchange because of Relaxa8on'of' the minimal diffusion distances (low X), maximal surface area (large Arteriolar'vasodila8on' precapillary'sphincters' A), and a low flow rate (giving it a lot of time for the necessary exchange Capillary'blood'ow' to occur, because flow is inversely proportional to cross-sectional area). Number'of'open'' However, at any particular moment, not all the capillaries are open. In capillaries' Delivery'of'O ,'more' rapid'removal'of'CO ' skeletal muscle, only 10% of the capillaries are open. In more physical and'other'metabolites' states, such as in exercise, metabolites diffuse in the precapillary Diusion'distance'' Concentra8on'gradient'' Capillary'surface' from'cell'to'open'' between' area'available'for' sphincters and allow blood to flow into the capillaries due to dilation capillary' blood'and'8ssue'cells' exchange' (relaxing) of the sphincters and arterioles. The major strategy of this is to maintain the concentration gradient. Capillary recruitment is also Sherwood'Fig.'10X20' Exchange'between'blood' and'8ssue' self-regulatory, because decreases in physical activity will cause a decrease in flow and removal of the vasodilating metabolites and cause the precapillary sphincter and arterioles to contract.
2 2', 2 2

!!!

In the capillary, the exchange of solutes is known as transcapillary exchange, in which the solutes will move based on the intrinsic diffusion characteristics. Lipid-soluble substances, such as O2 and CO2, will diffuse across the membrane because they are chemically nonpolar and are able to dissolve in lipids. Other substances, particularly those that are water-soluble and small, need to be dissolved in water first, but can diffuse across gaps in cells. Remember, the rate of diffusion is dependent on the concentration gradient, size of the solute, and the polarity of the solute. With this, there are also three different types of capillaries, the (1) continuous (which is the most common) and found in skeletal muscle, skin, the (2) fenetrated, which is found in kidneys and exocrine glands for filtration purposes, and (3) sinusoidal, found in the liver, spleen, where there is necessary movement of cells and larger proteins. The capillary pore sizes vary in different tissues. In general, permeability to small solutes is greater than permeability to large molecules. However, the capillary permeability can be altered under certain conditions, such as histamine (an inflammatory response chemical released by mast cells that increases the size of the capillary pores). For larger molecules, such as proteins, diffusion is not the optimal mechanism, so vesicular mechanisms such as endocytosis and exocytosis are utilized to shuttle proteins to the interior or the exterior of the cell. So, we can summarize the factors of transcapillary exchange of small solutes in the following diagram:
Factors affecting transcapillary exchange of small solutes

Concentration gradients: Determines direction of movement and magnitude of movement of solutes

Volumetric Flow Rate: Maintains fresh diffusion gradients

Velocity of blood ow: Determines time available for exchange by diffusion.

Capillary surface area: The greater the number of capillaries open, the greater the surface area for diffusion.

Permeability of Capillaries to Solutes: The size of the waterlled pores between endothelial cells is important.

Know how exchange of fluid occurs in capillaries and learn about Starlings Law. The movement of solutes occurs through the interstitial fluid, which serves as a passive intermediary. Interstitial fluid is the majority of extracellular fluid (approximately 80%) and serves a homeostatic role of the cell. Intracellular fluid constitutes the majority of fluid, but plasma fluid and interstitial fluid play a role in providing the optimal environment for cells. The exchange of fluid between capillaries and interstitial fluid typically occurs via bulk flow. In bulk flow, there are two movements: (1) ultrafiltration, in which the fluid

Determinants'of'net'uid'exchange'pressure''

moves out of the capillaries, and (2) reabsorption, in which the capillaries take up the fluid. Ultrafiltration is typically greater than reabsorption by approximately 3 liters a day, but the fluid is returned to the plasma by the lymphatic system, which transports the excess fluid (known as lymph) back into the venous system. Bulk flow is influenced by net fluid exchange pressure, which is an outward and inward pressure difference. Four forces determine them: capillary pressure (outward), interstitial fluid pressure (inward), plasma colloid osmotic pressure (inward), and interstitial fluid colloid osmotic pressure (outward). Thus, we can calculate the net fluid exchange pressure: = !"#$%&' !"#$%& = ! + !" (!" + ! ). From there, we can determine the fluid movement between blood and tissue, which is known as Starlings Law/Equation, which is: ! = ! = ! [ ! + !" (!" + ! )]. From there, we can draw two conclusions: (1) the pressure difference determines the direction of fluid movement, and (2) either fluid permeability coefficient, capillary surface area, or pressure difference will determine the magnitude of the fluid movement.

Capillary' pressure'(PC)'

Plasma'colloid' osmo8c'pressure' (P)'

Inters88al'uid' pressure'(PIF)'

Typical'values..'

Inters88al'uid' colloid'osmo8c' pressure'(IF)'

Inters88al'uid'colloid'osmo8c'pressure'is'an' Ini8al'lympha8c' outward'force'for'uid'exchange'

vessel' Inters88al' uid'

11'mm'Hg' (ultraltra8on)'

9'mm'Hg' (reabsorp8on)'

From' arteriole'
Sherwood'Fig.'10X22'

Blood' capillary'

To' venule'

Discuss how the above relate to the case study. In hereditary hemorrhagic telangiectasia, the transfer of small solutes can be locally affected at the site of the vascular lesions. Thus, the decrease in number of capillaries can spur less exchange occurring at the local areas. If there is a pulmonary arteriovenous malformation in a patient, individuals can develop secondary polycythemia, which is essentially an elevated hematocrit. This response is mainly to compensate for the low oxygen concentration in the blood. Thus, the adrenal glands secrete erythropoietin to increase carrying of oxygen to the tissues in response to the presence of the pulmonary arteriovenous malformation. In terms of pressure, one can relate the low-resistance bypass as not beneficial because they do not retain the properties that allow for optimal exchange (which is the complete opposite of a capillary). Thus there is little diffusion across the membrane in the between the capillary and lung or between the capillary and tissues. This is why patients with HHT often will present with fatigue and secondary polycythemia.

Lecture XXI
Vascular Developm ent

Terminology
Term Cell Differentiation Cell Proliferation Cell Movement Cell Interaction Inductive Interaction Gene Expression Vasculogenesis Angiogenesis Angioblasts Mesoderm Yolk Sac Blood Islands Primary Capillary Plexus Embryonic versus ExtraEmbryonic Vasculature Definition Process by which a less specialized cell becomes more specialized in a multicellular organism. The growth or production of cell by multiplication of parts Movement of the cell in response to Direct interactions between cells that play a role in development and function of multicellular organisms. Interaction between two groups of cells in which a signal passed from one group of cells causes the other group of cells to change their developmental state (or fate). Process by which information from a gene is used to synthesize a protein or gene product. Differentiation of angioblasts into endothelial cells and their assembly into a primary vascular plexus. Formation of the vascular network in some organs occurs mainly by vasculogenesis. The process by which new vessels form by sprouting or splitting of pre-existing vessels. In other organ systems, angiogenesis may be more prominent. Differentiated cell from the mesoderm formed from hemangioblasts. Primary germ cell layer that yields the cardiovascular system. In response to BMP, they form hemangioblast, which eventually forms parts of the vasculature. Membranous sac attached to the embryo, providing nourishment to the embryo. Around the 4th week of embryonic development, the functioning vasculature is present in the yolk sac, and angioblasts develop in the blood islands. Structures in the developing embryo that can yield different parts of the circulatory system, derived from plexuses formed from angioblasts. Initial site of vascular remodeling. It is formed by vasculogenesis. Embryonic: Involving angioblasts to form tubes that yield mature vessels. Extra-Embryonic: Involving angioblasts developing blood islands, which form a primary plexus to yield a mature vessel.

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Vascular Endothelial Cell Growth Factor (VEGF) Receptor Tyrosine Kinase Transphosphorylation Angiogenesis Sprouting Angiogenesis Non-Sprouting (Intussusceptive) Angiogenesis Activation Phase Resolution Phase Basement Membrane Mesenchymal Cells Tip Cells Platelet-Derived Growth Factor (PDGF) Mural Cells Transforming Growth Factor (TGF-) Postnatal Angiogenesis

Signaling protein produced to stimulate vasculogenesis and angiogenesis. High-affinity cell surface receptors for many polypeptide growth factors, cytokines, and hormones. Play a critical role in binding of VEGF for angiogenesis and vasculogenesis. Movement of one phosphate group of one molecule to another molecule. VEGF causes receptor dimerization and transphophorylation of tyrosine residues. Process by which new vessels form by sprouting or splitting of preexisting vessels. Form of angiogenesis in which tip cells (designated endothelial cells) extend into the extracellular matrix creating vessels. Form of angiogenesis in which the capillary wall extends into lumen to split the vessel into two. Phase in which vascular permeability and degradation of basement membrane allowing endothelial cells to migrate into the matrix to proliferate. Endothelial cells discontinue growth and migration and allow reformation of basement membrane. Sheet of fibers that underlie the endothelium of the vasculature. Multipotent stem cells that can differentiate into different cells. In the presence of activated TGF-, mesenchymal cells differentiate into mural cells, which consequently differentiate into vascular smooth muscle or pericytes. Designated endothelial cells that are the point men of the activation phase. Specific inductive signaling occurs to regulation formation and migration of tip cells, particularly VEGF-A and Notch signaling. One of many growth factos involved in regulation of cell growth and division. Vascular smooth mucle cells or pericytes that are involved in the formation of normal vasculature, which respond to VEGF. Protein that controls proliferation, differentiation in of endothelial cells. Important in vessel maturation and balances proliferation and differentiation. Angiogenesis that occurs after the birth and later development of the human, which can occur in states of hypoxia and trauma.

Lecture Objectives
Know some general principles of development. The general principles of development typically involve starting simple and simply refining. Vascular development involves a series of maturation and refinement, and recruitment of other cells for the associations, leading to stabilization and maturation. The remodeling and maturation then allows formation of vasculature, with tissuespecific accommodations. This process occurs in the embryo. The angioblasts, the precursor cells, become defined to move from vasculature without forming intermediate primary plexus. There are two major phases of vascular development: (1) vasculogenesis and (2) angiogenesis. Vasculogenesis is the formation of blood cells from undifferentiated cells to tube. Angiogenesis involves formation of vessels from pre-existing vessels, which can involve splitting of preexisting vessels. Vascular development involves the differentiation, proliferation, and migration of cells that will form the blood vessel tubes (endothelial cells). Within this process, there is a formation of a primitive network of tubes of endothelial cells known as the primary capillary plexus. Recruitment and differentiation of mural cells will yield smooth muscle cells or pericytes. From this, sprouting, branching, intussusception and remodeling occurs to form a more mature network, with eventual further maturation of blood vessels to reflect specific functions of vessel types. These processes are mediated by cellular signaling. Multicellular animals have proteins (that are typically conserved) that are involved in the mediation of cellular interactions and signal transduction. The inductive signals are

Vascular'Development'Overview'
Extraembryonic'
Tube'formaPon' primary'plexus' Recruitment'of' mural'cells,' remodeling,' maturaPon'' mature'vessel' mural'cell' DierenPaton' Angioblasts' ProliferaPon' MigraPon' Tube'formaPon'
L.'Coultas'et'al.,'Nature$438,#937-45#(2005).'

Angioblasts'

DierenPaton' ProliferaPon' MigraPon'

Intraembryonic'

11

important to spur orderly differences from initially identical cells. Signals are typically involved in gene expression, meaning that the genes are in control of the developmental process, sometimes repeatedly. Learn about the process of vasculogenesis during embryonic development. Vasculogenesis is considered the first phase in vascular development. Endothelial cells are derived from precursor cells, known as angioblasts, that originate from the mesoderm. Remember that the mesoderm gives rise to the cardiovascular system. By approximately the fourth week of embryonic development, there is a functioning vasculature, and nutrients are from the placenta as well as the yolk sac. Angioblasts typicaly develop from the blood islands of the yolk sac. From this, a primary capillary plexus in the yolk sac is formed by vasculogenesis and then goes through remodeling. The larger vessel is giving rise to a smaller vessel. On top of this, the angioblasts arise in the embryo proper as well as migrate out of the mesoderm in somites and form vessels in various parts of the body, including part of the aorta. They will eventually spur formation of blood vessels. However, there is such a thing as too much of a good thing in this case. Obviously, having not enough angioblasts can spur poor formation of the cardiovascular system, but having too much can compromise the embryo due to nutrient depletion and excessive remodeling. Thus, the embryo must regulate the formation of angioblasts. One factor that is important in vasculogenesis is known as vascular endothelial cell growth factor (VEGF). They promote angioblast formation and proliferation and allow formation of blood vessels by inductive interaction.
There'are'dierent'types'of'VEGFs'and'VEGFRs'

Signaling Pathways Activated by VEGF Vasculogenesis'

Angiogenesis'

SIGMA-ALDRICH

Lymphangiogenesis'

Inductive interaction is a stepwise interaction between two groups of cells in which a signal passed from one group of cells causes the other group of cells to change their evelopmental state (or fate). VEGF binds to a receptor tyrosine kinase, in which the binding of VEGF to the extracellular domain causes protein changes to spur a signaling cascade and desired effect. Though there are different types of VEGFs and

associated receptors, there are different effects present that are dependent on the receptor (not on the factor). The receptor dictates the effect of the binding, NOT the factor. VEGF can promote vasculogenesis and angiogenesis, but also lymphangiogenesis (formation of lymphatic vessels). VEGF causes the receptor dimerization and transphosphorylation of tyrosine residues, similar to growth factors such as EGF, and causes attraction of proteins to the signaling complex Cell'survival' Gene'expression' Cell'proliferaPon' as well as recruitment of different enzymes for the signaling pathway. Cell'proliferaPon' Vasopermeability' Focus'on'the' physiological' Different effects can occur depending on the signaling pathway by VEGF. eects'here' Vasculogenesis' VEGF can ensure (1) cell survival, (2) gene expression and cellular Angiogenesis' proliferation, (3) cellular proliferation and vasopermability, which ultimately leads to (4) vasculogenesis and angiogenesis. Learn about the process of angiogenesis during embryonic development and postnatal angiogenesis. Angiogenesis, as stated before, is the growth of blood vessels from preexisting vessels. The second phase of vascular development has two types: (1) non-sprouting (or intussusceptive) angiogenesis and (2) sprouting angiogenesis. They typically can either sprout from the primary plexus or be non-sprouting. Intussusceptive angiogenesis typically presents with a formation of a blood vessel pillar that splits off into two different vessels. The holes will eventually connect with each other to produce a much finer network. In some body regions (such as the limbs), the blood vessels develop during embryogenesis mainly by angiogenesis not by vasculogenesis. Sprouting angiogenesis involves two phases: (1) Activation and (2) Resolution. Activation involves the preexisting tube to sprout out and form tip cells. In the Activation Phase, cells loosen connections and degrade the extracellular matrix to allow for proliferation and migration from tube. In this phase, vascular permeability increases, degradation of the basement membrane is permitted to allow endothelial cell migration and proliferation. Tip cells are the endothelial point men that move in the Activation Phase. The specific inductive signaling occurs to regulate formation and migration of tip cells, involving VEGF-A signaling and the Notch signaling pathway. VEGF-A is the signal that designates the endothelial cell to be a tip cell and sprout. The Notch Signaling Pathway is simply a preventive tool involving lateral inhibition of neighboring endothelial cells to prevent unnecessary sprouting in proximity of the designated tip cell. The Resolution Phase involves reformation of the extracellular matrix as well as recruitment and differentiation of mural cells to become endothelial tubes. Endothelial cells halt migration and

12

proliferation, the cell-cell junctions are tightened, and reformation of the basement membrane occurs, allowing mesenchymal cells to be recruited to differentiate into mural cells. At this time, the pericytes (which are sparsely covered) involves the recruitment of mesenchymal cells to migrate and associate with the newly developed blood vessels. Recruitment and differentiation of mural cells to the endothelial tubes involves PDGF- secretion from the tip cells to recruit mesenchymal cells to migrate and contact endothelial cells. The cell-cell contact results in activation of latent transforming growth factor (TGF-) in the extracellular matrix. The activated TGF- causes mesenchymal cells ot differentiate into the mural cells (either vascular smooth muscle cells or pericytes). TGF- signaling is needed for formation of vascular smooth muscle cells, and also occurs in endothelial cells (in a paracrine fashion) to control balance between proliferation/migration and differentiation. Other signaling molecules are also involved in the development of new blood vessels, and their numbers can be altered depending on the body organ involved. Postnatally, angiogenesis can also occur, such as in states of wounding or hypoxia. In states of wounding or vascular trauma, the factors are present and can allow proliferation of these factors and spur a much finer network. In states of hypoxia, VEGF is also present. Tissue cells produce a factor known as hypoxia inducible factor that is sensitive to O2 levels in the blood. Normally, HIF is inhibited due to high oxygen levels, but can be turned on in low oxygen states and allow capillary sprouting through increased secretion of VEGF. This leads to this case study. Local hypoxia can affect the progression of arteriovenous malformation. The hypoxia is not from the decreased concentrations of oxygen in the blood, but the poor diffusion of oxygen into the tissues from a low-resistance bypass. Thus, there is increased production of VEGF and proliferation of vascular endothelial cells. However, they promote the formation of vessels called torturous vessels that worsen the arteriovenous malformation due to contortion and capillary bed destruction. Look at a few selected signaling systems (VEGF, PDGF, TGF-) that are important for vasculogenesis and angiogenesis. Growth Factor Acronym Role in Vasculogenesis and Angiogenesis Vascular VEGF Important in vasculogenesis. It binds to receptor tyrosine kinase, causing Endothelial Cell vasculogenesis, angiogenesis, or lymphangiogenesis depending on te Growth Factor receptor. Important in (1) cell survival, (2) cell proliferation and vasopermeability, (3) gene expression, and ultimately (4) vasculogenesis and angiogenesis. Platelet-Derived PDGF Secreted by the tip cells, allow recruitment of mesenchymal cells to migrate Growth Factor and contact endothelial cells. Like VEGF, it acts through receptor tyrosine kinase. Transforming Important in vessel maturation, and allow mesenchymal cells to TGF- Growth Factor, Type differentiated into mural cells. Hypoxia-Inducible HIF Allow postnatal angiogenesis in response to hypoxic states. Factor

Lecture XXII
TGF- Ligand Superfam ily: Properties, Cell Biology, Physiological Effects

Terminology
Term HHT Types Definition There are five major types of HHT depending on the problem area: HHT Type I: Involves a mutation in the endoglin (ENG) gene, which is a type III TGF receptor family co-receptor. HHT Type II: Involves a mutation in the activin receptor-like kinase (ALK-1) gene, which is a Type I TGF superfamily receptor. HHT Type III: Mutation on a locus on chromosome V. HHT Type IV: Mutation on a locus of chromosome VII. Juvenile Polyposis/HHT Syndrome: Mutations in the gene for SMAD4, a transcription factor. Receptor in the TGF Signaling Pathway that allows increases in the endothelial cell proliferation and migration. Glycoprotein on surface of cells that participates in the TGF signaling pathway. Group of structurally related proteins involved in the TGF signaling pathway. They are involved in embryogenesis, cell differentiation, cell cycle arrest, and apoptosis.

Activin Receptor-Like Kinase 1 (ALK-1) Endoglin TGF- Superfamily

13

Transforming Growth Factors Transformed Cells Soft Agar Colony Growth Assay Anchorage-Independent Growth Autocrine Signaling Paracrine Signaling Endocrine Signaling Apoptosis Proteinase Inhibitors Extracellular Matrix

Polypeptide growth factors that are involved in cellular proliferation and differentiation, but have structural and functional differences. Cells that have been altered to a cancer-like state due to the direct uptake, incorporation, and expression of exogenous genetic material. Type of assay in which cell colonies are grown independent of attachment to a surface an essentially suspended in agar. A type of growth in which cells are not suspended in a solid environment (to allow determination of growth in certain factors by not being attached). Type of signaling in which cell secretes a messenger that binds to receptors on the same cell Type of signaling in which cell secretes a messenger that binds to receptors on a neighboring cell. Type of signaling in which cell secretes a messenger into the bloodstream which binds to receptors on a distant cell. Process of programmed cell death by certain biochemical events. Type of inhibitor involved in the repression of extracellular matrix degradation. Part of tissue that provides structural support to cells, consisting of the interstitial matrix and the basement membrane. It provides support, segregation, and regulation of intercellular communication, maintaining stability amid the cells dynamic behavior. Group of proteinases that are classified by the most prominent functional group on their active site. They are proteolytic enzymes involving a metal in their catalytic enzyme. Culturing cells in flat plastic petri dishes (where the cells adhere to the surface), which allows for a 2-dimensional environment for the cells. Culturing cells in a suspension to provide a more physiologically relevant enevironment for the cells, without attachment. Dimer that is involved in cellular proliferation, differentiation, apoptosis, metabolism, homeostasis, immune response, wound repair, and endocrine function. Type of growth factors that are interact with receptors on the cell surfafe that are involved in growth and differentiation. Chemical that binds to receptor of a cell that triggers a response by the cell, typically mimicking a naturally occurring susbstance. Type of chemical that blocks a biological response when binding to a receptor, dampening cellular response. Type of patterning that involves compartmentalization the embryo to account for certain features at the dorsal-ventral locations. No symmetry at the left and right side of the body. The n-terminus is the first part of the protein that emerges from the ribosome in translation. It has signal peptide sequences that spurs delivery to the proper organelle. The peptide is removed at the destination by a signal peptidase. Protein derived rom N-terminal region of the TGF that interacts with the TGF homodimer to for a complex. Complex consisting of the N-terminal region of the TGF gene product and the TGF homodimer. Larger complex secreted in the extracellular matrix that consists of latent TGF- binding to the Small Latent Complex. Activation of latent TGF to an active state by signaling pathways or various other factors.

Metalloproteinases 2D Culture 3D Culture Activins BMPs Agonist Antagonist Dorsal-Ventral Patterning Left-Right Asymmetry N-terminal Signal Peptide Latency Associated Peptide Small Latent Complex Large Latent Complex TGF- Activation

Lecture Objectives
Learn about the molecular genetics of HHT. There are several types of HHT, which can be determined via laboratory testing involving DNA markers. The mutations in the Endoglin and ALK1 are the most common cause of hereditary hemorrhagic telangiectasia. These mutations can cause alterations in splicing at the donor and receptor sites and spur a loss of function due to their scattered distribution of the key sites. Most mutations are a loss of function, possibly haploinsufficiency from a having at least one bad-coding copy in the genotype. Type I II Mutation Endoglin (ENG) Gene Activin Receptor-Like Kinase 1 (ALK-1) Gene

14

III IV Juvenile Polyposis/HHT Syndrome

Candidate Locus on Chromosome 5 Candidate Locus on Chromosome 7 Mutations in gene for SMAD4 (transcription factor)

Learn about the discovery of TGF-. The genes that are mutated in HHT are part of the TGF- signaling pathway, in which binding to the receptor causes a signal transduction that can alter gene expression. And remember that latent TGF needs to be activated to spur the vascular development. This signaling pathway is one of many that can influence gene expression. TGF (known as Transforming Growth Factor-) was discovered about 30 years ago around the late 1970s. It was discovered when researchers began testing the hypothesis that some tumor viruses could transform cells into a cancer-like state by inducing cells to secrete molecules that acted on the same cells, causing transformation via autocrine control. These factors were partially purified which could cause normal fibroblasts to form progressively growing colonies in soft agar with reversible effects. After isolation and purification by column chromatography and various other testing, they found two proteins that are involved: TGF- and TGF-. TGF- is structurally related to epidermal growth factor and was the factor involved in the growth. However, TGF- was the protein that had the growth-inhibitory effect, among several other effects. When grown in a 2dimensional culture (meaning that the cells attached to the flat petri dish), there is actually an inhibition of proliferation and migration of cells. However, in the three-dimensional soft agar culture, there is the formation of tube-like structures. Learn about the diverse functions of the TGF- superfamily. The TGF- superfamily is involved in many functions: 1. Cell growth (inhibition) 2. Cell differentiation (promotion) 3. Apoptosis (induction) 4. Embryonic Development 5. Wound Healing 6. Vascular Remodeling 7. Roles in immunity, fibrosis, cancer, heart disease, diabetes, and Marfan Syndrome Scientists determining other functions eventually converged upon the finding of TGF-. It contributes to vascular development in the following ways: 1. Regulation in the proliferation, differentiation, and migration of endothelial cells and mural cells. 2. Vascular remodeling and vessel maturation 3. Stimulation in the production of extracellular matrix proteins and proteinase inhibitors (repressing matrix degradation) Know the general properties of TGF- superfamily ligands. This family of proteins is often context dependent, in which the response is dependent on: (1) ligand and receptors, (2) other signals, (3) cell type, (4) signal intensity, (5) signal duration. However, experimentally with endothelial cells, there is a negative feedback mechanism associated with the context-dependent response. Low levels of TGF are associated with an enhancement of proliferation and migration of cells, while high levels of the same protein can inhibit the effect. One way to regulate the levels is through binding affinity of the receptor. The TGF-s are often associated with embryogenesis, cell differentiation, cell cycle arrest, and apoptosis. The properties within the TGF- superfamily are similar. They are a secreted protein containing an N-terminal signal peptide (a zip code that ensures that the protein goes to the specific organelle). The C-terminal region consists of approximately 112 amino acids that become the mature TGF that is the active ligand (as a dimer). The middle portion of the TGF- encodes the latency-associated peptide. All three of the forms of TGF- are initially released from the cell in a latent/inactive form, and need to be activated to achieve an effect. There are several advantages of having the latent form: 1. Inactive complex provides an opportunity for intricate regulation. Activation pathways may be cell or tissue specific. 2. Differences in the LAPs provide different TGF complexes that are selective to specific stimuli. 3. Signaling can be locally activated by proteolysis or by mechanical strain. Know about the process of TGF- activation from a latent state. TGF- activation involves the latencyassociated protein combining with the active TGF- (in various catalysts) to

15

form the small latent complex. This complex can make further associations, by attaching the small latent complex with latent TGF- binding protein via a disulfide bond, forming the Large Latent Complex. This Large Latent Complex can then covalently link with the extracellular matrix. The mature ligand dimerizes to form the active molecule. From there, there are several mechanisms to fully activate TGF-: 1. Degradation of ECM proteins (fibrillin or LTBP) by proteases. 2. Cleavage, dissociation, or conformational change in LAP. 3. Integrin-dependent activation (by activating metalloproteases or by a mechanical strain-dependent form). Learn about the role of antagonists of TGF- superfamily ligands. The antagonists of the TGF- signaling can function to establish complex spatial patterns of functional activity because there is regulation. Antagonists are important to establish the dorsal-ventral patterning, such as in Noggin. Antagonists are also important for establishing left-right asymmetry. The inhibitors can be expressed in interesting patterns. The protein involved is Lefty, a TGF- superfamily member that inhibits Nodal signaling (another TGF- family member), helping to restrict Nodal expression to the left side of the embryo to push for molecular asymmetry.

Lecture XXIII
TGF- Signaling: Receptors and Coreceptors

Terminology
Term Receptor Serine/Threonine Kinase Catalytic Receptor Type I Receptors Type II Receptors Homodimer Heterodimer Constitutively Active Induced Proximity Model GS Region SMAD Protein Transcription Phosphorylation SMAD Signaling Non-SMAD Signaling Endoglin Co-Receptor (Accessory Receptor) Endocytosis Clathrin-coated Pit Lipid-Raft Transcription Factors Definition Type of kinase enzyme that phosphorylates the OH group of serine or threonine, playing a role in regulation of cell proliferation, apoptosis, differentiation, and embryonic development. TGF Transmembrane receptor where the binding of an extracellular ligand causes intracellular enzymatic activity. Protein receptor in heteromeric complex which is the main switch involved in the signal transduction. Protein receptor in heteromeric complex which can remain active, but needs a allosteric, conformational change in order to fully activate signal transduction. Macromolecular complex formed by two identical molecular subunits. Macromolecular complex formed by two non-identical molecular subunits. Type of activity in which the molecule is constant and active, but is not in appropriate conformation to phosphorylate another receptor. Ligand binding brings proteins together in the membrane and this close proximity allows for signaling to occur. Regulatory site for TGF- type I receptor. It needs to be phosphorylated by TGF- type II receptor for it to occur. Intracellular proteins that transduce extracellular signals from TGF- ligands to the nucleus to activate downstream gene transcription. Exist in three classes: (1) Receptor-regulated, (2) Common-mediator, and (3) Antagonistic Production of a complementary RNA copy (mRNA) from a sequence of DNA. Addition of a phosphate group to a protein or another organic molecule, activating or deactivating protein enzymes. Signaling involves transduction of extracellular signaling to the nucleus. They form a trimer or two receptor-regulated SMADs and one co-SMAD to create a transcription factor to regulate expression of certain genes. Conveyance of non-SMAD signaling proteins such as p38 or MAPK, with no use of SMAD proteins. Type I membrane glycoprotein on cell surfaces that is part of the TGF- receptor complex. Cell surface receptor that binds a signaling molecule in addition to a primary receptor to facilitate ligand recognition and initiate signaling. Process by which cells absorb molecules through engulfment. Involved in clathrin-mediated endocytosis, which is formed from the inward budding of plasma membrane vesicles to allow internalization of the molecules. Organization of glycosphingolipids and protein receptors to compartmentalize cellular processes by serving as centers for assembly of signaling molecules. Protein that binds to specific DNA sequences to regulate transcription.

16

Lecture Objectives
Learn about the different types of receptors and co-receptors in the TGF- superfamily. Type Examples I TRI (ALK5), ActRIB (ALK4), ALK 7, ALK1, ALK2, ALK3, ALK6 II TRII, ActRII, ActRIIB, BMPRII, MISRII Generally there are two types of receptors: Type I and II. In the signaling pathway, be sure to remember that Type II must be activated in order for activation of Type II to occur. They are quite structurally similar, and can only be differentiated by peptide mapping. They form homodimers and heterodimers. The number of ligands very much exceeds the number of receptors, so there can be convergence on signaling, particularly to a receptor. Consequently, there are combinatorial interactions, or diverse responses by small numbers of receptors and communication. The significance is simply because there is one way for cells to generate the capacity for diverse responses derived from a small number of ligands and receptors (with TGF- superfamily responding to a specific the TGF- receptor). It also allows for crosstalking between different signals. However, cells need to respond appropriate to the signal. Know how signal transduction occurs through the receptors. The general signaling pathway to understand is that it is activated by an extracellular ligand. The receptors involved in TGF- Signaling typically involved Receptor Serine/Threonine Kinases, which are consequently involved serine/threonine phosphorylation, which has intrinsic catalytic activity. The pathway generally involves several steps: 1. TGF- brings together two Type I Receptors with Type II Type'I'receptors'acCvate'dierent'SMADs' Receptors. 2. Type II Receptors phosphorylate and activate type I receptors. 3. The R-SMAD proteins bind to Type I Receptors to be in Ligand' Type'II'R' Type'I'R' SMAD' complex with SARA. (ALK5)' TGF.s' 4. Type I receptor phosphorylates R-SMAD, promoting SMAD'' (ALK4)' 2,'3' AcCvins' dissociation from the kinase and SARA. Nodal' 5. The product binds to a Co-SMAD. MyostaCn' 6. SMAD hetero-oligomer enters the nucleus. BMPs' SMAD'' 7. The SMADs associate with other DNA-binding proteins to 1,'5,'8' GDFs' activate or inhibit transcription of specific genes.
MIS'

The TGF- dimer binding results in a heterotetrameric complex of receptor subunits. The receptos essentially cluster to form heterotetrameric complex, consisting of 4 receptors and 2 ligand molecules [4+2]. A jigsaw arrangement can explain the specificity of the TGF- superfamily to the TGF- receptor. It is simply a stepwise, allosteric, cooperative mechanism. When the Type II receptor binds to TGF, it allows a conformation that allows the Type I receptor to fit, yielding the TGF- receptor complex. Essentially, the Type I receptor binds much more favorable in the presence of another factor (Type II), because the receptor affinity is higher for Type I after Type II enters. This is not necessary for other proteins such as BMP, which binding and activation of the complex can occur simultaneously. The system is to bring the Type I and II receptors for an intimate embrace. The complex puts the cytoplasmic kinase domains in a catalytically favorable orientation. This system is also associated with the induced proximity model, in which the ligand binding causes proteins to move closer together in the membrane and this close proximity causes the signaling to occur. The phosphorylation of the GS region of the TGF- 1 receptor activates the kinase. The GS region is the regulatory factor that interferes with the kinase. The GS region will move out of the way when the serines of the Type I receptor are phosphorylated. Type II does not have the GS, but Type I needs to move the GS region to cause the desired activation. Phosphorylation allow allows the type I receptor to phosphorylate SMAD proteins, and the phosphorylation of the SMAD proteins allow SMADs to accumulate in the nucleus. Phosphorylation is used both to activate Type I receptors and SMAD proteins, and thus one can conclude that phosphorylation is the mechanism to activate or deactive proteins or recruit proteins to a complex.
'K.'M.'Rik'Derynck,'The%TGF9[beta]%familyVolume%50%of%Cold%Spring% Harbor%monograph%series%(CSHL%Press,%2008),%pp.%1114.'

Endoglin has been noted as a co-receptor that is abundant in the vascular endothelial cells, mainly to potentiate signaling through the Type I and Type II receptors, but signaling can occur without the co-receptor. Endoglins function still remains a clinical mystery, but co-receptors play a role in endocytosis (especially in clathrin-mediated uptake) of TGF- receptors, and the signaling may be ongoing. Know the difference between SMAD and non-SMAD signaling via the TGF- pathway. TGF- signaling typically involves SMAD-mediated responses, but non-SMAD responses have also been noted. Some receptors act in other ways without utilizing SMAD, and was discovered through observation of rapid effects that were not involving SMAD proteins. This may allow for a bypass mechanism to allow for a desired effect in the event of faulty SMAD signaling.

17

Discuss the parameters that determine what kind of response is elicited in cells during signaling. Now this leads to another question: what determines what kind of response will be elicited by TGF- superfamily ligands in any particular cell? Experiments and studies have lead to these factors: 1. Ligands 2. Receptors and Co-Receptors 3. SMADs 4. Cell type-specific cooperating transcription factors

Lecture XXIV
Transcriptional Regulation in Physiology

Terminology
Term Central Dogma of Molecular Biology Differential Expression Messenger RNA (mRNA) Ribosomal RNA (rRNA) Transfer RNA (tRNA) Small, noncoding RNAs Transcription Unit Transcriptional Control Acute Regulation Long-Term Regulation RNA Polymerases (I, II, III) Definition Framework of comprehsnion in the transfer of information between DNA, RNA, and protein among living organisms, in which DNA can yield RNA, which then yields protein. However, this does not happen in the reverse, generating a oneway flow of information. Differences among cell expression even though the cells have the same DNA, mainly because they express different parts of the DNA. Molecule of RNA that encodes information for the protein product. RNA component of the ribosome, providing the mechanism for decoding mRNA into amino acids and interacts with tRNAs during translation. Adaptor molecule composed of RNA utilized in bridging genetic code in mRNA. Short ribonucleic acid molecule in eukaryotes that are regulatory involved in post-transcriptional regulation. Stretch of DNA transcribed into an RNA molecule to allow for eventual translation to protein. Regulation of gene expression by controlling number of RNA transcripts of a region of DNA. Major regulatory mechanism of protein synthesis. Processes that need to be regulated in the range of seconds to minutes involving changes in protein activity, brecause these changes can occur rapidly. Processes that are regulated over a longer time period typically involving transcriptional regulation. Enzyme that produces RNA. There are three types: RNA Polymerase I: Involved in the transcription of DNA to rRNA RNA Polymerase II: Involved in the transcription of DNA to mRNA or snRNA. RNA Polymerase III: Involved in the transcription of DNA to tRN, 5S rRNA, or 7S RNA of signal recognition particle. Process by which the DNA transcribes an RNA product. Contains three phases: (1) Initiation, (2) Elongation, and (3) Termination Phase of transcription where promoters designate area and allow binding of RNA polymerase to DNA promoter. Phase of transcription where DNA template strands allows for the synthesis of an RNA copy. Phase of transcription consisting of the halting of RNA synthesis and release of mRNA. Region of DNA that facilitates transcription of a gene. DNA sequence found in promoter region of genes. Part of the promoter sequence, it is the binding site of general transcription factors and involved in transcription. DNA sequence element that overlaps a transcription start site and allow determination of start site location in a promoter and enhancing strength of a promoter with a TATA box. Promoter elements that can direct low levels of transcription, but are insufficient for full expression of a gene. Large complex of proteins necessary for transcription of protein-coding genes. Transcription factors involved in the transcription of class II genes to mRNA templates. General transcription factor that binds to TATA box. Complex state of a protein where the aqueous environment is isolated with the

Transcription Cycle Initiation Phase Elongation Phase Termination Phase Gene Promoter TATA Box Initiator Element Basal Promoter Preinitiation Complex General Transcription Factors (TATA Box Binding Protein ) TBP Closed Complex

18

Open Complex

Conformational Change Promoter Proximal Elements Enhancer Elements Gene-Specific Transcription Factors Chromatin Structure

non-aqueous. Complex state of a protein where the protein opens, allowing for aqueous and nonaqueous environment to interact. In this case, the open complex allows the transcription start site to be unpaired and allows exposure to the template strand to allow nucleotides to join. Change in the proteins structure in response to the environment or other factors. Short regions of DNa involved for constitutive expression, or regulated expression. Short region of DNA that can be bound with proteins to enhance transcription levels of genes, which work by increasing the rate of initiation from a basal promoter. Diverse protein involved in gene regulation that are specific to a seuqnece of DNA. Combination of DNA nad proteins that make up the contents of the nucleus of a cell. Its function is to package DNA to a smaller volume to fit into the cell and to strengthen DNA to allow cell division and control gene expression and DNA replication. Chemical orientation of a strand of nucleic acid, with the start designated as 5 and the end as 3. Process in elongation phase consisting of halting transcription to allow replacement or removal of nucleic acid. With the help of enzymes, a process in which a cap is added to the 5 end of the nascent transcript, allowing protection against degradation and to promote translation. String of adenines added to the 3 end of the transcript to designate end of sequence. Release of mRNA from nucleus through nuclear pore complex. Pore in the nucleus in which the nascent strand of mRNA is release into to arrive at the cytoplasm.

5 to 3 Direction Pausing and Editing mRNA Capping Poly-A Tail mRNA Export Nuclear Pore Complex

Lecture Objectives
Know the difference between transcriptional and non-transcriptional regulation of gene expression. In order to understand transcriptional and non-transcriptional regulation of gene expression, we need to first understand the elements of transcription. Transcription is simply the generation of mRNA from DNA, which (in eukaryotes) occurs in the nucleus. It is one of the processes within the Central Dogma, in which DNA synthesizes RNA, and RNA consequently produces a protein. This one-way flow of information is key to the biological processes at a unicellular and multicellular level. From this, researchers eventually discovered mRNA, tRNA, rRNA, miRNA, and snRNA. All cells have the same genes (as ingrained in the form of DNA), but the cells are specialized due to the expression of different sets of genes. Expression of genes is different in each type of cell. Some are on all the time (constitutively), or only on at certain times. It can be also expressed in a cell-type specific manner. At times, some are expressed at high levels, while other are expressed at lower levels, and even some are expressed at higher levels only when specific cell surface receptors or cytoplasmic receptors are activated by ligand binding. Now, to discuss the genetic material, there are two major types of genetic material: DNA and RNA. DNA is a double helix with deoxyribose sugars consisting of one side with a strand of nucleotides while the other has a strand of the complementary sequence, according to Watson-Crick base pairing. RNA is a single-stranded molecule with a ribose sugar and contains uracil instead of the thymine in DNA. There are several types of RNA, but only one type of DNA. The types of RNA are listed at the table below: Type (Abbreviation) Description % Of RNA Messenger RNA (mRNA) Major encoding element for proteins. < 10 Ribosomal RNA (rRNA) Major component of ribosomes. 75 Small Stable RNAs Encompass RNAs that are mainly adaptor proteins. 15 Transfer RNAs (tRNAs) are involved in protein synthesis. Small Nuclear RNAs (snRNAs) are involved in RNA splicing, regulation of transcription Small noncoding (ncRNAs) Regulatory RNAs involved in controlling gene expression. Small. or microRNAs (miRNA) The strand of DNA that is being transcribed is typically referred to as the transcription unit. After transcription occurs, the mRNA is produced, but undergoes editing and splicing (to remove introns will keeping the exons at certain splice sites) to yield the mature mRNA. Transcriptional control is especially crucial to prevent expression of possible mutations. Transcriptional regulation is altering the rate of gene expression by altering the rate of transcription. Non-transcriptional regulation is associated with the rate of gene expression by

19

The'1me'scale'of'gene'expression'versus' changes'in'protein'ac1vity'

altering the protein activity, typically after mRNA has been synthesized. For example, non-SMAD mediated responses dont involve changes at the level of transcription, so it can be considered a non-transcription regulation. Learn about acute responses versus longer-term responses. Acute regulation typically encompass processes that need to be Seconds=minutes' regulated in the range of seconds to minutes that involve changes in Acute'regula1on' the protein activity, because these changes that can occur rapidly. Altering the environment of the protein to chemical phosphorylation of isomerization can affect the proteins activity. Long-term regulation involves processes that are regulated over a longer time period, typically involving transcriptional regulation. There are several advantages of this. One, it is energetically efficient, only producing a protein when it is needed. Second, it allows the cell to switch General'Eukaryo1c'Transcrip1on'Factors' expression of one type of gene to another, particularly during development or differentiation. Finally, it can also do large-scale expression of a whole network of genes involved in a particular pathway or process. Table'15=1.'Summary'of'RNA'Pol'II'General'Transcrip1on'Factors'
6=10'h' Longterm'regula1on'
Figure 7-5 Molecular Biology of the Cell ( Garland Science 2008)

Know the basics about the basal transcription machinery. Learn about TFIID, TBP, and TATA boxes. In order to understand the machinery, it is necessary to comprehend the various parts involved. Recall that RNA polymerase is involved in the synthesis of RNA from DNA. However, there are three types of RNA. RNA polymerase I is involved in the synthesis rRNA, and RNA polymerase III is involved in the production tRNA, 5S rRNA, and 7S RNA of signal recognition particle. RNA Polymerase II is the Modied'from'Pollard,'Table'15=1' enzyme of interest for transcription, which yields mRNAs and some snRNAs. RNA Polymerase II is a large multiprotein complex with a repeptive carboxyl-terminal domain. The conserved portions of the residues are located in the inner Preini1a1on'Complex' surfaces of the enzymes. The structure of the enzyme contains a cleft for the DNA and a site for nucleotides to enter, as well as a channel for newly synthesized RNA to exit. RNA'Pol'II'on'its'own'
from'promoters.''Addi1onal' Know the transcription cycle. factors,'called'General' The transcription cycle is a process creating RNA from DNA. It contains three steps: (1) Transcrip1on'Factors,'are' Initiation, (2) Elongation, and (3) Termination. Among these steps (which are all required.' regulated), initiation is the most regulated step. Prior to Initiation, a pre-initiation complex For'RNA'Pol'II,'there'are' must be formed because RNA polymerase II on its own cannot initiate transcription from more'than'20'proteins' promoters. Consequently, factors, known as general transcription factors, are required to form a complex. For RNA Pol II, there are more than 20 proteins. Though there are other factos involved in this complex, the first one to go in is transcription factor IID (TFIID). The binding of TBP to the TATA box leads to a pronounced bend in the DNA and recruitment of other factors. At'some'point,'the'RNA'
Pollard'Fig.'15=7'

cannot'ini1ate'transcrip1on'

Transcrip1on' Ini1a1on'

Polymerase/DNA'complex' shids'from'a'Closed' Initiation of transcription complex'to'an'Open' complex'conforma1on.'

(the Initiation Phase) involves a shift of the RNA Polymerase/DNA Complex from a closed complex to an open complex conformation. This conformation change T in his'conforma1onal' the polymerase causes a region of DNA around the transcription start site to become change'in'the'polymerase' unpaired, allowing exposure of the template strand. Consequently the first ribonucleotides are causes'a'region'of'DNA' around'the'transcrip1on' joined. At this point, several sequences, such as promoters, proximal promoter elements, and start'site'to'become' unpaired,'exposing'the' enhancers, are also add to spur effects on the rate of initiation from the basal promoter.
template'strand.'
Pollard'Fig.'15=6'

The'rst'few' You'are' ribonucleo1des'are'joined.'

The Elongation Phase involves the clearance of promoter that needs to occur before synthesis terminated!' begins. The transcript is extended in a 5 to 3 direction at a rate of approximately 30-100 Termina1on'Phase:'As' RNA'Pol'II'reaches'the' nucleotides per second by the following chemical reaction: ()! + ()!!! + ! . end'of'a'gene'(3'end),' During this process, pausing and editing may occur. As the new (or nascent) transcript the'RNA'gets'cleaved'at' the'polyadenyla1on'site' emerges, a special cap structure is added on to the 5 end. This cap help to protect against and'a'poly=A'tail'as'added' degradation and later will promote translation of the message to'the'transcript.''The' polymerase'may'con1nue' in the cytoplasm.
transcribing'for'a'while' but'soon'falls'o.'

Figure 6-38 (part 2 of 3) Molecular Biology of the Cell ( Garland Science 2008)

Termination of transcription (the Termination Phase) involves RNA Polymerase II reaching the end of the gene at the 3 end. Once this occurs, the RNA gets cleaved at the polyadenylation site and a poly-A tail is added to the transcript. The polymerase may continue transcribing for a while but soon falls off. After splicing occurs, the mature mRNAs are exported from the nucleus through the nuclear pore complex. Be aware of the role of promoters, proximal promoter elements, and enhancers. Promoters are the sum of DNA sequences necessary for transcription initiation. Other elements include a TATA box and initiator element, which is found near the

Transcrip1on' Regula1on' Mechanisms'

Eect'of'gene= specic'transcrip1on' factors' Eects'on'chroma1n' structure'

Pollard'Fig.'15=20'

20

start site of transcription, and consequently part of the basal promoter. Basal promoter elements can direct low levels of transcription, but are insufficient for full expression of a gene, so (as a result) promoters also contain promoter proximal elements that are needed for constitutive expression or regulated expression. Another group of sequences, known as enhancers, typically increase the rate of initiation from a basal promoter. They can be distant from the start site, and exhibit flexibility (allowing itself to work in different positions or orientations), and contain clusters of regulatory elements. Such regulation can have an effect on gene-specific transcription factors as well as chromatin structure. The following table summarizes promoters, proximal promoter elements, and enhancers. Sequence Promoter
Pol'II'Promoters'

Diagram

Promoter'proximal'elements'

Proximal Promoter Elements Enhancers

A'promoter'can'be'loosely'dened'as'the'sum'of'DNA' sequences'necessary'for'transcrip1on'ini1a1on.'

Pollard'Fig.'15=14'

Enhancers'are'elements'that'increase'the' TATA'box'and'Ini1ator'element'are'commonly'found' near'the'start'site'of'transcrip1on,'part'of'a'Basal' rate'of'ini1a1on'from'a'basal'promoter'

promoter'
Pollard'Fig.'15=5'

Description Loosely defined as the sum of DNA sequences necessary for transcription initiation. TATA box and initiator element are found near start of transcription as part of a basal promoter. Needed for constitutive/regulated expression.

Basal'promoter'elements'can'direct'low'levels'of' distances'away' transcrip1on,'but'they'are'insucient'for'full'expression' from'the'start'site' of'a'gene.''Most'promoters'contain'several'dierent' Promoter'proximal'elements'that'are'needed'for' Will'work'in' cons1tu1ve'expression'or'regulated'expression.' dierent'posi1ons'

Can'be'great'

Flexible part of the sequence that can be far from the start site that increase the rate of initiation from a basal promoter. They can contain clusters of regulatory elements.

or'orienta1ons'

Contain'clusters' of'regulatory' elements'

Pollard'Fig.'15=15'

Lecture XXV
Strategies for Transcriptional Regulation in Eukaryotes

Terminology
Term Promoter proximal elements Enhancer Elements Insulator Elements Locus Control Regions Gene Specific Transcription Factors Major Groove Definition Proximal sequence upstream of the gene that tends to contain primary regulatory elements. Elements that increase the rate of initiation from a basal promoter, which can be distant from start site and contain clusters of regulatory elements. Genetic boundary eleent that is an enhancer-blocking element or a barrier against condensed chromatin proteins spreading onto active chromatin. Regions defined by their ability to enhance the expression of linked genes. Binding sites for proteins allow differential control of gene transcription. Type of groove seen in DNA structure containing the nitrogen and oxygen atoms of the base pairs pointing inward toward the helical axis. It is more dependent on base composition and may be the site for protein recognition of specific DNA sequences or regions. Attractive interaction of a hydrogen atom with an electronegative atom. Complex regulatory regions (in enhancers and promoters) are constructed from different combinations of simple regulatory molecules. Two or more elements functioning together to produce a result not independent obtainable. Behavior observed in enzymes and receptors that have multiple binding sites where the affinity of the binding sites for a ligand is increased or decreased as a consequence of binding of the ligand to the receptor. Multiprotein complex that functions as a transcriptional coactivator. Switch in the lifecycle of a bacteriophage responsible for maintenance of lambda phage. It binds to operator associated with the RNA polymerase promoter to prevent RNA polymerase from initiating transcription, and cannot enter the lytic cycle. Independently folded protein domain that contains at least one motif that recognizes DNA. Protein domains involved in the formation and stability of the preinitiation complex, activating transcription. DNA-binding protein that regulates expression of one or more genes by binding to the operator and blocking attachment of RNA polymerase to the promoter, blocking transcription.

Hydrogen Bonding Combinatorial Control Synergy Cooperativity Mediator Complex Lambda Repressor

DNA Binding Domain Activation Domain Repressors

21

Competition Masking De Novo Synthesis Coactivator Histone Acetyltransferase Corepressor Gene Silencing DNA Methylation Histone Methylation Genetic Program Network Motifs

Method of utilizing transcription factors as repressors by competitive DNA binding. Method of utilizing transcription factors as repressors by masking the activation surface. Synthesis of complex molecules from simple molcules. Protein that increases gene expression by binding to an activator containing the DNA binding domain. Coactivators cannot bind DNA by itself. Enzymes that acetylate conserved lysine amino acids on histone proteins. Substance that inhibits the expression of genes, by indirect means (interaction with repressor proteins that in turn bind to the promoter) The deactivation of a gene by a mechanism other than genetic modification, meaning that it would be expressed under normal circumstances. Addition of a methyl group to the 5 position of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring on DNA. Modification of certain amino acids in a histone protein by addition of one, two, or three methyl groups. Physiological change brought about by a temporal pattern of activation of a particular subset of genes. Connectivity patterns that occur more often in comparison to random networks.

Lecture Objectives
Know the different types of DNA regulatory elements that control transcription of a gene. Regulatory Illustration Function Characteristics Promoter'proximal'elements' Element Promoter Proximal sequence Can be within 300 base pairs upstream Proximal upstream of the gene that or downstream of the basal promoters. tends to contain primary regulatory elements. Most'promoters'contain'several'dierent'Promoter' proximal'elements'that'are'needed'for'cons4tu4ve' Enhancer Elements that increase the Can be distances away from the start expression'or'regulated'expression.' rate of initiation from a site. They will work in different basal promoter, which can positions or orientations. They also be distant from start site contain clusters of regulatory elements. and contain clusters of regulatory elements.
Pollard'Fig.'15D14'

Insulator'Elements'

Insulator

Insulators'protect'regions'of'a'chromosome'from'the' eects'of'neighboring'regions.''

Locus'control'regions'
Locus Control Regions
Figure 7-62 Molecular Biology of the Cell ( Garland Science 2008)

Genetic boundary element that is an enhancer-blocking element or a barrier against condensed chromatin proteins spreading onto active chromatin. Regions defined by their ability to enhance the expression of linked genes.

Figure 7-61 Molecular Biology of the Cell ( Garland Science 2008)

Insulators are involved in the protections of regions of a chromosome from the effects of neighboring regions. They suppress activation of one gene, so that the enhancer only affects a specific gene. Locus control regions are short regions of DNA rich in binding sites for transcription regulators, which create open chromatin promoting the expression of nearby genes. These regions can be important in regulating the expression of a cluster of nearby genes, so that they are expressed in the correct order during development or in the current location in the embryo. All these regulatory elements contain binding sites for proteins called genespecific transcription factors.

It is important to remember that all cells have the same genes in the form of DNA, but cells are specialized because of the differences in expression of different sets of genes. It can also respond to its environment by turning on or off specific genes. Transcription initiation is key. A host of general transcription factors are required to form the preinitiation complex. Basal promoters only allow for basal levels of expression. Anything that promotes the formation of the preinitiation complex or stabilizes it will elevate transcription of the gene. It yields contact-mediated effects of gene-specific transcription factors and effets on chromatin structure, which alter DNA accessibility for other transcription factors. There are four types of regulatory elements: (1)

22

promoter proximal elements, (2) enhancer elements, (3) insulator elements, and (4) locus control regions. Promoter proximal elements are sequences that are upstream or downstream in proximity of the basal promoter (landmarked by TATA box). It has all the binding sites upstream of the promoter, within the first 300 base pairs, but must be near the transcription site. They are identified by a reporter gene assay, with the promoter gene being attached the reporter gene. Enhancers are elements that increase the rate of initiation from a basal promoter. They can be great distances away from the start site, maintain flexibility, and contain clusters of regulatory elements. Insulators are involved in the protections of regions of a chromosome from the effects of neighboring regions. They suppress activation of one gene, so that the enhancer only affects a specific gene. Locus control regions are short regions of DNA rich in binding sites for transcription regulators, which create open chromatin promoting the expression of nearby genes. These regions can be important in regulating the expression of a cluster of nearby genes, so that they are expressed in the correct order during Coopera4ve'binding' development or in the current location in the embryo. All these regulatory elements contain binding sites for proteins called gene-specific transcription factors. Learn about gene-specific transcription factors. classical' coopera4vity' Remember that all the regulatory elements contain binding sites for proteins called Combinatorial'control:''Complex'reguatory'regions' (in'enhancers'and'promoters)'are'constructed'from' gene-specific transcription factors. They make up approximately 6-8% of human dierent'combina4ons'of'simple'regulatory'modules.'''' genes. They are critical for a diverse array of cellular processes. There are about 1,400 transcription factors out of 20,000-25,000 genes. Transcription factors recognize and bind to specific DNA sequences, such as homeodomains, zinc fingers, glucocorticoid receptors, and leucine zippers. Most factors recognize Transcrip4on'factors'olen'work' exposed chemical groups in the major groove of the DNA double helix. The type of synergis4cally' bonding involved is hydrogen bonding. The specificity (to regulate only the right genes) comes binding sites. If the factor forms a dimer with itself or another protein, this will increase the specificity because it requires two adjacent sites to Allows'for'the'integra4on'of'mul4ple'regulatory'signals'by'cells'and' for'very'complex'spa4al'paeerns'of'gene'expression.' be present. Different dimer pairs can generate the novel binding sites.
Watson,'Molec.'Biol.'of'the'Gene,'Fig.'17D14'

Watson,'Molec.'Biol.'of'the'Gene,'Fig.'15D14'

Transcrip4on'factors'have'modular'ac4va4on' domains'and'DNA'binding'domain'

Be familiar with the concepts of combinatorial control and synergy with regard to transcription factors. Combinatorial control is when complex regulatory regions (in enhancers and promoters) are constructed from different combinations of simply regulatory modules. It allows for the integration of multiple regulatory signals by cells and for very complex spatial patterns of gene expression. In certain positions along a developing embryo, certain genes are enhanced and suppressed (and the concentrations of the regulatory proteins increase or decrease) along certain position of the embryo.

Pollard'Fig.'15D19'

Figure 7-48 Molecular Biology of the Cell ( Garland Science 2008)

Ac4va4on'domains'' come'in'dierent' avors:' Acidic'regions' Proline'rich'regions'

Transcription factors can work synergistically. Transcription with one protein can increase the number of regulatory proteins in transcription. Synergy is achieved in two ways: (1) classical cooperativity involves the binding of one factor makes it easier for the other to bind, or (2) two factors may be better able to recruit a coactivator by touching different parts of it. Binding of one factor may result in an alteration in chromatin structure so that another binding site becomes more accessible. The functions of synergy is to allow a sensitive switch to turn genes on or off. The DNA binding specificity increases. The integration of different signals is possible. Cooperative binding can affect the life of the various cells.

Glutamine'rich' regions' SFcky#surfaces#that# TranscripFon#factors#bind#to#the#DNA,#but#how# could#interact#with# do#they#acFvate#or#repress#transcripFon?# several#protein#surfaces#

Directly' Indirectly'(with' coac4vators'or' corepressors)'

By#aecFng#the#formaFon#or#stability#of#the# ways,'depending'on'the'factor'and'pathway' PreiniFaFon#Complex#

Cells'regulate'transcrip4on'factors'in'several'

Pollard'Fig.'15D20'

Know the basics about how transcription factors activate or repress transcription. Transcription factors bind to the DNA, but can directly or indirectly activate or suppress transcription by affecting the formation or stability of the preinitiation complex. They have Transcrip4on'factors'can'be'repressors,' modular activation domains and DNA binding domains. The activation domains have too' different regions: acidic, proline-rich, and glutamin-rich regions. They are, Alberts,'Fig.'7D50' simply put, stick surfaces that could interact with several protein surfaces. These transcription factors can be repressors also, through (1) competition (competitive DNA binding), (2) masking (masking the activation surface, or Compe44on' (3) direct interactions with general factors.
Pollard'Fig.'15D21'

How#does#this#relate# to#signal#transducFon# pathways#in#the#cell?#

Know the different ways that transcription factors can be regulated. Cells regulate transcription factors in several ways, depending on the factor and pathway. There are several ways to regulate transcription factors: (1) de novo synthesis, (2) ligand bindng, (3) phosphorylation, (4) heterodimer formation, (5) dimer dissociation, and (6) subcellular localization. Know that transcription factors can also work by affecting chromatin structure. Transcription factors can also exert their effects by altering chromatin structure rather than by direct contact-mediated effects on the pre-initiation

Masking'

Direct'interac4on' with'general'factors'

23

Transcrip4on'factors'can'also'exert'their'eects'by' altering'chroma4n'structure'rather'than'by'direct' contactDmediated'eects'on'the'preDini4a4on'complex'

complex. The activating factor may recruit a coactivator that is a histone acetyltransferase. The repressor may recruit a corepressor that is a histone acetylase. Acetylation of histone tails loosens up the chromatin structure.

Pollard'Fig.'15D20'

Lecture XXVI
Role of SM ADS in Transcriptional Regulation via the TGF- Pathw ay

Ac4va4ng'factor'may' recruit'a'coac4vator'that' is'a'histone' acetyltransferase.' Repressor'may'recruit'a' corepressor'that'is'a' histone'deacetylase' AcetylaFon#of#histone#tails#loosens#up#chromaFn#structure#

Terminology
Term Nuclear Pore Complexes MH1 Domain MH2 Domain Linker Region Receptor activated SMAD (R-SMAD) Common Mediator SMAD (Co-SMAD) Inhibitory SMAD (I-SMAD) SMAD Box -Hairpin Loop Definition Large protein complexes that cross the nuclear envelope. Domain in SMAD proteins that is involved in DNA binding and interaction with transcription factors. Domain in SMAD proteins that are involved in receptor interaction, Smad oligomerization, transcriptional activation, interaction with CBP/p300, and interaction with transcription factors. A short synthetic double-stranded DNA that is usually ligated to double-stranded DNA to introduce restriction sites or sequence tags. Transcription factors that transduce extracellular TGF- ligand signaling from cell membrane bound TGF- receptors into the nucleus where they activate transcription TGF- target genes. Includes SMAD1, SMAD2, SMAD3, SMAD5, and SMAD8/9. Transcription factor that interacts with R-SMADs to participate in signaling. Includes only SMAD4. Type of SMAd involved in the modulation of TGF- ligands, which includes SMAD6 and SMAD7. They inhibit transcription. Site where -hairpin loop makes H-bonds with unpaired groups, which allows binding in a sequence-specific manner. The sequence of the box is GTCT. Simplest structural motif involving two strands that look like a hairpin, which consists of two strands that are adjacent in primary structure oriented in an antiparallel arrangement (where the N-terminus of one sheet is adjacent to the Cterminus of the next). Region of MH2 domain that actually interacts with the Type I receptors. Small sequence differences along the L3 loop residues allow determination of the right structure. Common motif in the secondary structure of protiens containing a right-handed coil or spiral conformation. Wider region of the DNA helix Common affinity electrophoresis technique used to study protein-DNA or proteinRNA interactions. This can determine if a protein or mixture of proteins is capable of binding to a given DNA or RNA sequence, and ca indicate if more than one protein is involved in the binding complex. Protein domain that contains at least one motif that recognizes double or singlestraned DNA. It can recognize a specific DNA sequence or have a general affinity to DNA. Transcription factors that bind to specific sequences along DNA, which allow for a coding system for transcription. Protein that increases gene expression by binding to an activator, which contains a DNA binding domain. Substance that inhibits the expression of genes, in which is downregulates the expression of genes not by direct interaction with a gene promoter but indirectly through interaction of repressor proteins that bind to the promoter. Gene encoding the regulated product, including any linked regulatory elements and selectable markers. Methods of signaling in which the signaling may induce the expression of another transcription factor that can then cooperate with the signaling to regulate other targets. Pathway within a control system that passes a controlling signal from the source in the control systems external environment to a load elsewhere in its external environment. This allows the cell to be sensitive to the duration of a signal. Connectivity-patterns that occur much more often than they do in random networks. Collection of DNA segments in a cell that interact with each other indirectly and with other substances in the cell, governing the rates at which genes in the

L3 Loop -Helix Major Groove of DNA Gel Shift Assay

DNA Binding Motif Sequence-Specific Transcription Factors Coactivators Corepressors Target Genes Sequential and SelfModifying Signaling Feed-Forward Loop Network Motif Gene Regulatory Network

24

Post-Translational Modification Phosphorylation Mitogen Activated Protein (MAP) Kinases Dephosphorylation SMAD Phosphatases Ubiquitination Ubiquitin Ligases Crosstalk Subcellular Localization

network are transcribed into mRNA. Chemical modification of a protein after its translation. Addition of a phosphate group to a protein or other organic molecule. Serine/threonine-specific protein kinase that respond to extracellular stimuli and regulate various cellular activities. Removal of phosphate groups from an organic compound by hydrolysis. Enzyme that regulates the removal of phosphate groups from active SMAD proteins. Enzymatic, protein post-translational modification process in which ubiquitin is added to the protein. It targets SMAD for degradation by the proteasome. Protein that in combination with an E2 enzyme that causes the attachment of ubiquitin. It is involved in polyubiquitination, marking proteins for degradation by the proteasome. Instances in which one or more components of a signal transduction pathway affect a different pathway. Subdivisions of a eukaryotic cell that allow functionally distinct membrane bound The'distribu8on'of'SMADs'between'cytoplasm' compartments.
and'nucleus'is'altered'during'signaling'

Lecture Objectives
Know the role of SMADs in the TGF- family-signaling pathway. Remember that anything that promotes or stabilizes the preinitiation complex will activate transcription, and this is the same for the opposite. Transcription factors bind to specific DNA sites and act synergistically with other factors to achieve combinatorial control. Consquently, transcription factors can be regulated, and whether or not a gene is transcribed depends on what regulatory elements it has, what transcription factors are around, and how accessible is the DNA. Transcription factors work by contact-mediated effects or by altering chromatin structure. Thus transcription factors have modular activation domains and DNA binding domains. They have different types: (1) acidic regions, (2) proline-rich regions, and (3) glutamine-rich regions. These are essentially surfaces that could interact with several protein surfaces. It should also be remembered that transcription factors can also act as repressors also, either by competition, masking, or direct interactions. Cells regulate these transcription factors in several ways, depending on the factor and pathway. The ligand/receptor complex puts the cytoplasmic kinase domains in a catalytically favorable orientation. The Type II receptor phosphorylates the Type I receptor. The phosphorylation of the GS region of the TGF- type I receptor activates this kinase. This cannot happen in Type II receptors because the Type II receptors do not have this region. The phosphorylation of the Type I receptor activates it to phosphorylate SMAD proteins. The SMADs are the gene-specific transcription factors that are used to regulate target genes affected by TGF- family ligands. The SMADs will bind to specific DNA sites. Type I receptors activate different SMADs, and the distribution of SMADs between cytoplasm and nucleus is altered during signaling. When ligand binds, there is a steady state shift towards the accumulation in the nucleus, and difficulty to export out active SMADs. Consequently, there is an accumulation of the SMADs in the nucleus. SMADs enter and exit the nucleus through nuclear pore complexes. The R-SMADs are associated in the cytoplasm with SMAD Anchor for Receptor Activation (SARA). Thus, SMADs can either be recycled (by dephosphorylation) or degraded (by ubiquination).
(inhibitory)'
Ac8ve'SMADs' accumulate'in' the'nucleus'

B.'Schmierer,'C.'S.'Hill,'Nat+Rev+Mol+Cell+Biol+8,#970&82#(2007).'

8'SMADS'
(receptor? ac8vated)'

TGF?' Ac8vin? linked' BMP? linked'

MH1' MH2'

MH1' Learn about the different classes of SMADs (Receptor activated, co-SMAD, Inhibitory SMAD). All in all there are 8 SMADS, which are divided into three types: (1) receptor activated (R-SMAD), (2) common mediator SMAD (co-SMAD), and (3) inhibitory (I-SMAD). The R-SMADs are involved in the activation of transcription upon ligand binding. Co-SMADs do not get
A.'Moustakas,'C.'H.'Heldin,'Development+136,#3699&714#(2009).'

Lack'

25

phosphorylated, and allow for increasing the affinity of the binding of the DNA to the R-SMAD. I-SMADs are involved in the inactivation of repression. They lack MH1 domains. BeSMAD'proteins'consist'of'MH1'and'MH2' domains of SMADs (MH1, MH2, linker region, -hairpin loop, L3 familiar with the different functional loop). domains'and'a'linker'region' SMAD proteins consist of MH1 and MH2 domains and a linker MH1' Linker' MH2' TGF?'type'I' region. MH1 is going to bind to the SMAD box, and allows receptor receptor' specificity to binding. MH2 is phosphorylated by the Type I receptor. Phosphoryla8on'site' cytoplasmic' The MH2 domain contains the L3 loop region that actually interacts domain' with the Type I receptors. The L3 loop is responsible for the SMAD' specificity of the SMAD to the DNA binding site. The small sequence differences of the L3 loop residues can determine whether the Specicity' protein is in the right structure. MH1 domains are involved in DNA binding and interaction with transcription factors. The MH2 domain DNA' is involved in receptor activation, SMAD oligomerization, transcriptional activation, interaction with CBP/p300, and interaction with transcription factors. The phosphorylation will occur at the candidate target sites. The linker region is a short, synthetic strand of DNA that allows introduction of restriction and binding sites.
'J.'Massagu,'Nature+Reviews+Molecular+Cell+Biology+1,#169&78#(2000).'

SMADs bind DNA through a secondary structure known as the -hairpin loop. They bind to the SMAD box, which has the sequence GTCT, and makes hydrogen bonds with unpaired groups, allowing binding in a sequencespecific manner. One can be able to demonstrate the presence of the binding site for a particular protein by a gel shift assay. An individual can take a DNA sequence with the SMAD box and label fragment and run a gel electrophoresis. The Quan8ta8ve'eects:''The'type'of'coopera8ng' bound complex cannot move as easily as separated proteins. At high enough concentrations, the binding sites will then be occupied. The DNA binding domain sequences are conserved in different transcrip8on'factor'can'also'aect'the'threshold'of' SMADs, and especially in SMAD?mediated'responses.' all receptor activated SMADs.
In'this'example,' Know the basics about how the'target'gene' SMADs cooperate with other is'either'on'or' transcription factors to regulate o'(at'low'signal' specific target genes. intensity)' Different SMADs have specific depending'on' whether'the' effects in which the R-SMADcoopera8ng' SMAD4 complexes often cooperate transcrip8on' with other sequence-specific factor'is'X'or'Y' These'kinds'of'gene'network'mo8fs'are' transcription factors. The high affinity binding of SMADs typically requires cooperation with other factors. important'to'make'a'complex'system'work' Transcriptional complexes involving SMADs involve repetition of the recognition site or a SMAD box next to a site next to a binding site. Different target genes are affected depending on the combination of SMAD and cooperating transcription factor (cofactor). A lot of cooperative transcription factors might be cell-specific. Such cooperation has also quantitative effects. The type of cooperating transcription factor can also affect the threshold of SMAD-mediated responses. A target gene can be either on or off at low signal intensity depending on the cooperating transcription factor. It is also important in sequential and self-modifying signaling. SMAD signaling may induce the expression of another transcription factor that can then cooperate with the SMAD to regulate other targets. This is exemplary of a feed-forward loop. It is also a time-dependent loop because it takes time to develop. A feed-forward loop allows the cell to be sensitive to the duration In'addi8on'to'post?transla8onal'modica8on,'another' of a signal. These kinds of gene network motifs are important to make a complex system avenue'for'regulatory'input'are'Inhibitory'SMADs,'which' work. MH1 and MH2 domains are important for cooperativity. The differences in the inhibit'R?SMAD'ac8va8on' amino acid sequence in the MH1 and MH2 domains of Growth' Figure 7-69 Molecular Biology of the Cell ( Garland Science 2008) Figure 7-71 Molecular Biology of the Cell ( Garland Science 2008) Growth' TGF?' the SMAd family members will determine with which factor' factor'R' SMAD'6'and'SMAD'7' transcription factors they can interact. The linker are'inhibitory'SMADs' Because'SMADS'can'be' region is considered the target for regulation via postphosphorylated'by'these' translational modification, which allows for biological other'kinases,'crosstalk' cross talk.
B.'Schmierer,'C.'S.'Hill,'Nat+Rev+Mol+Cell+Biol+8,#970&82#(2007).'

MAP'kinase' for'binding'to'Type'I' Know how SMADs are regulated. receptor'and'preven8ng' SMADs can receive regulatory input from other phosphoryla8on,'or'by' signaling pathways through post-translational binding'to'SMAD'4' modification in the following manner: (1) (preven8ng'R?SMAD' phosphorylation, (2) dephosphorylation, and (3) binding)'' ubiquitination. Phosphorylation can occur at various sites by kinases other than Type I receptors. For R.'Derynck,'Y.'E.'Zhang,'Nature+425,#577&84#(2003).' example, phosphorylation by Erk MAP kinase can impair nuclear translocation of the SMAD complex, while phosphorylation by P38 MAP kinase or JNK can

Act'either'by'compe8ng'

can'occur'between'the' TGF?/SMAD'pathway' and'other'signaling' pathways,'such'as'those' induced'by'growth' factors'(e.g.'mitogen? ac8vated'protein'(MAP) kinase'pathways)'

J.'Massagu,'Nature+Reviews+Molecular+Cell+Biology+1,#169&78#(2000).'

26

enhance SMAD activity. Because SMADs can be phosphorylated by other kinases, crosstalk can occur between the TGF-/SMAD pathway and other signaling pathways, such as those induced by growth factors. Dephosphorylation occurs by SMAD phosphatases, which is one way to terminate SMAD signaling. Ubiquitination is the last way, utilizing ubiquitin ligases to target SMAD for degradation by the proteosome. In addition to post-translational modification, another avenue for regulatory input are inhibitory SMADs, which inhibit R-SMAd activation. SMAD6 and SMAD7 are inhibitory SMADs, and act either by competing for binding to Type I receptor and preventing phosphorylation, or by binding to SMAD 4, which ultimately prevents R-SMAD binding. Inhibitory input can come from cytokine and growth factor signaling. Several signaling pathways can induce SMAD 6 and SMAD 7 gene expression. These SMADs can then inhibit TGF- signaling.

Lecture XXVII
A Look at Som e Other Signaling System s that Affect Transcription

Terminology
Term Cytokine Cytokine Receptor VEGF (Vascular endothelial Growth Factor) JAK (Janus-Associated Kinase) STAT (Signal Transducer and Activator of Transcription, or Signal Transduction And Transcription) NF-B (Nuclear factor kappa-light-chain-enhancer of activated B cells) I-B (Nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor) Steroid Steroid Receptor Definition Small cell-signaling protein molecules that are secreted by numerous cells and are a category of signaling moleucles used extensively in intercellular communication. Receptors that are specific in binding to cytokines. A signal protein produced by cells that stimulate vasculogenesis and angiogenesis. Family of intracellular, nonreceptor tyrosine kinases that transduce cytokinemediated signals via the JAK-STAT pathway. Protein involved in regulation of growth, survival, and differentiation in cells.

Protein complex that controls the transcription of DNA, found in all animal cell types and involved in cellular responses such as stress, cytokines, free radicals, UV radiation, oxidized LDL, and bacterial or viral antigens. Family of cellular proteins that inhibit the NF-B transcription factor.

Class of organic compounds that contain cycloalkane rings joined to each other. They are small hydrophobic signaling molecules. Found in the plasma membrane, cytoplasm, and nucleus, they are intracellular receptors that can change gene expression over time period of hours to days by initiating signal transduction when a steroid hormone binds to the stated receptor.

Lecture Objectives
Take a brief look at a few other signal transduction systems that cells use to regulate gene transcription. Remember the TGF- signaling pathway, and that there are other pathways that are involved. R-SMAD-SMAD4 complexes often cooperate with other sequence specific transcription factors. The high affinity binding of SMADs typically requires cooperation with other factors and can act with coactivators or corepressors. From this, we know also that different target genes are affected depending on the combination of SMAD and cooperating transcription factor (cofactor). Thus, the regulatory input can occur at many levels. There are factors that can potentiate signaling of Type I and II receptors, and ligands that can affect expression of cofactors. The TGF- pathway is one of several that can affect transcription. There are others. There is cytokinase signaling through the JAK/STAT pathway. Remember that cytokines are small proteins released by cells that have specific effects on cells that have the appropriate cytokine receptor. There are many, but cytokines include interleukins, lymphokines, tumor necrosis factors, and the interferons. This is particularly helpful in

27

immune response. The JAK/STAT pathway has three components: (1) the cytokine receptor (with no intrinsic enzymatic activity), (2) the Janus Kinase or JAK) (which associates with the receptor), and the Signal Transducer and Activator of Transcription or STAT (which is the latent cytoplasmic transcription factor). STAT is not bound to the receptor. There is a large family of specific cytokine receptors selectively expressed in different types of cells. There are four types of JAKs, and seven types of STATs that are either homodimers or heterodimers). The pathway of the Cytokine JAK/STAT signaling pathway is executed in a certain manner. Ligand binding to the receptor causes transphosphorylation (in the form of autophosphorylation), with STAT binding to phosphotyrosine. Tyrosine phosphorylation allows the release of STATs. At the same time, secondary STAT is phosphorylated via the growth factor receptor tyrosine kinase pathway. The two pathways converge with the reciprocal binding of SH2 to the phosphotyrosine, yielding a STAT dimer, called P-STAT. P-STAT dimer then enters the nucleus, causing STAT activating the expression of various genes including SOCS1. The expression product SOCS1 then exits the nucleus and then binds to the cytokine-JAK/STAT complex and inducing a negative feedback mechanism, stopping the pathway. The JAK/STAT pathway uses particular strategies, such as dimer formation, phosphorylation, and subcellular localization. With cytokine signaling through the JAK/STAT pathway, an activated JAK kinase phosphorylates a STAT, which then dimerizes and goes to the nucleus. There can be crosstalk between the TGF- signaling and the cytokine JAK/STAT signaling. Several signaling pathways can induce SMAD6 and/or SMAD7 expression, including those in the JAK/STAT pathway. Another pathway of particular interest is the NF-B pathway, in which signaling is triggered ProToll-Like by bacterial cell wall products (a form of innate inammatory Receptors Activation of cytokine genes immunity), inflammatory cytokines, viral (Innate the NF-kappaB Bacterial Cell are turned on Signaling Wall Products Immunity by NF-kappaB infection, UV irradiation, B or T cell activation, Receptors) System transcription which are forms of adaptive immunity. They factor are a family if five related transcription factors. In innate immunity there is a process. In innate immunity, involved in a certain manner. Signaling through the NF-B pathway involves NF-B being kept in the cytoplasm until the signaling pathway is activated. I-B is the inhibitory subunit that binds to NF-B. When the pathway is activated, I-B gets phosphorylated, which causes it to be targeted for destruction by the proteosome. Now NF-B can go into the nucleus. VEGF (vascular endothelial growth factor signaling) is the third example of another pathway cells use to regulate cell signaling. Mesodermal cells with VEGFR-2 utilize VEGF to develop into angioblasts, and then (with more proteins and VEGF) to develop into the endothelial cell tube. It utilizes a form of receptor tyrosine kinase signaling. VEGF binds to the receptor allowing the opening of extracellular domains. These extracellular domains dimerize and kinase domains phosphorylate each other. The receptor then binds and activates PLC and then other effectors with SH2 or PTB domains bind to the phosphotyrosines, and Ras and Raf activation. These in turn catalyze MEK activation, which then catalyzes ERK activation. This pathway of activation is known as the MAP kinase pathway. From there, the kinase from ERK activation moves to the nucleus, allowing activation of the transcription factor and binding to the cells DNA causing the cellular proliferation and differentiation. With VEGF signaling, an activated kinase goes into the nucleus and activates a latent transcription factor by phosphorylating it. We can also utilize steroids as a signaling tool. Steroids are small hydrophobic (lipid-soluble) signaling molecules. They can be found in the cytoplasm or nucleus, and acts as the receptor for the ligand and transcription factor. This superfamily of signaling molecules contains a DNA binding domain, a ligand binding domain (for direct binding) and transcriptional activation domains. Steroids can pass through the plasma membrane and bind either to a receptor in the cytoplasm or a receptor already in the nucleus (depending on the

28

type of steroid). Steroid binding allows the receptor to bind DNA and activate (or repress) the transcription of specific genes. They are essentially free from the inhibitory state and can allow transcription. This can allow a context-dependent response to steroids. Certain receptors can coordinate the expression of several different genes, depending on the presence of different cooperating transcription factors. Compare and contrast these pathways with the TGF-. Pathway Strategies Similarities JAK/STAT Heterodimer Induced proximity model Formation, applies Phosphorylation, and Subcellular Phosphorylation is the Localization key molecular switch in this system. Invoves a change in the localization of the transcription factor from the cytoplasm to the nucleus. There is a latent transcription factor that accumulates in the nucleus when the signaling pathway is activated. Induced proximity model applies. Phosphorylation is a key molecular switch.

Differences The kinase in this system (JAK) is a separate protein from the receptor (associated instead of bound) Autophosphorylation (transphosphorylation) mechanism with JAK rather than one receptor type phosphorylating a different receptor type, as with TGF- receptors Phosphorylation of the receptor is used to recruit the binding of the transcription factor to the receptor (which is not true of SMADs). Removal of an inhibiting subunit is required for activation instead of addition of a cooperating partner. The inhibitory protein gets degraded in the process. Autophosphorylation (transphosphorylation) mechanism with VEGFR rather than one receptor type phosphorylating a different receptor type as with TGF- receptors. Involves a change in the localization of the kinase from the cytoplasm to the nucleus (where it phosphorylates gene-specific transcription factors). Ligand passes directly through the membrane instead of binding to a receptor in the membrane. The steroid receptor is itself the gene-specific transcription factor. Ligand binding directly affects the transcription factors and is the primary switch mechanism, not phosphorylation. (Some receptors are further regulated by phosphorylation.)

NF-B

Dimer Dissociation, Phosphorylation, and Subcellular Localization Phosphorylation

VEGF Signaling

Steroid Signaling

Ligand Binding, Subcellular Localization

Change in the transcription factor localization from cytoplasm to nucleus (in some cases). Steroid receptors also participate in cooperative interactions, like SMADs.

Understand the variety of ways that cells regulate transcription factors. From these pathways, we can note that there is a common theme of latency of the transcription factors. In order for these signaling pathways to start having an effect, the cell need only to activate a latent transcription factor that is already present and ready to go. If the cell had to first make the transcription factor from scratch, the responses would not start as quickly and this could be deleterious and inefficient. Giving the only function of activating the latent transcription factor is such a boon that the cell can regulate these several transcription factors simultaneously and in response to intracellular to extracellular demands.

Lecture XXVIII
Experim ental Tool: Transgenic Approaches in Physiology

Terminology
Term Genotype Phenotype Definition Genetic makeup of a cell, specific to a character in consideration. Observable characteristic or trait

29

Wild-Type Mutant Constitutively Active Transgenic Mouse Transgene Random Insertion Founder Mouse Chromosomal DNA Promoter Constitutive Promoter Inducible Promoter Coding Sequence Protein of Interest Reporter Gene Pronucleus Zygote Surrogate Mother Polymerase Chain Reaction (PCR) Agarose Gel Electrophoresis

Phenotype of the typical form of an organism occurring in nature. Individual, organism, or genetic character arising from mutation. Protein whose activity is constant and active, one that transcribed from the DNA all the time. Mouse that has had its genome altered by genetic engineering. Gene or genetic material that has been transferred naturally or by genetic engineering. Describes a segment of DNA containing a gene sequence isolated from one organism and introduced into a different organism. Insertion of DNA segment into an unpredictable portion of the chromosome. Laboratory mice that have been produced from a genetically manipulated egg or embryo. DNA compacted into a chromosome. In prokaryotes, it is compacted into a circular form known as the plasmid. Regulatory region of DNA usually located upstream of a gene, providing a control point for regulated gene transcription. An unregulated promoter that allows for continual transcription of its associated gene. Type promoter in which their activity is induced by the presence or absence of biotic or abiotic factors. Portion of a genes DNA or RNA, composed of exons that codes for protein. Protein under study by a researcher. Gene that researchers attach to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants. Nucleus of a sperm or egg cell during the process of fertilization, after the sperm enters the ovum, but before they fuse. Initial cell formed when two gamete cells are joined by means of sexual reproduction. Mother that is carrying the embryo of that does not have the mothers DNA, a foreign embryo. Scientific technique to amplify a single or a few copies of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Method used to separate proteins by charge or size in a medium consisting of agarose.

Lecture Objectives
Learn about transgenic and knockout mouse approaches in physiological studies. Unfortunately, because the use of human subjects in experiments is ethically questionable though the motives are typically altruistic, scientists have to model human diseases in animal subjects. It can be done by studying the physiology of a normal animal or tissue and then altering the system in some way to gain insight into the normal physiology. We can do this in several ways: (1) by exposing the animal to different physiological Suppose'you'think'that'protein'B'ts'into'a' situations, (2) treating the animal with agents that affect protein function (inhibitors or activators) or agents that affect gene expression, or (3) altering the genetic makeup (genotype) of an animal. We can do this with two physiological'pathway'as'follows:' different types of mouse subjects: transgenic and knockout mice. For example, suppose there is a pathway, and a scientist is investigating One*way*to*test*the*hypothesis.* A' B' C' D' the pathway that signals in a sequential order, with the signal causing expression of A, then B, the C, etc. The scientist can run two Signal' experiments, either by manipulating the genetic makeup so that one of the coding proteins is inactivation (causing downstream inactivation) or by increasing the gene expression of one of the proteins, so that the A' B' C' D' Hypothesis:''Signal'acFvates'A'which'acFvates'B'etc.''' upstream sequence would be inactivated. To explain the downstream Signal' inactivation, the genetic makeup is altered so that B is no longer present, and (predictable) C and D should not be active even when the signal is Another*way*to*test*the*hypothesis.* even when A is still activated byExperimental'test'#1:''If'we'alter'the'geneFc' A' B' C' D' present, the signal. The upstream makeup'of'the'animal'such'that'B the inactivation can be done by altering the genetic makeup of the animal so'is'no'longer' Signal' B is expressed in a form that is active all present,'C'and'D'should'not'be'acFve'even'when' the time (constitutively) and then C and D remain active, but not A, even with the absence of the signal. These the'signal'is'present.''A To'perform'this'test,'we'need'to'be'able'to' A' B' C' D' two strategies work, but require the introduction of the altered'should'sFll'be'acFvated'by' product into eliminate'the'funcFon'of'B.''One'way'is'to'make'a' the'signal.'' B'*' Signal' the animal. Introduction of a foreign gene into the animal is necessary to knockout'animal'in'which'the'gene'encoding'B' a transgenic animal. The transgenic mouse has altered gene make has'been'inacFvated.''More'on'this'in'the'next' expression into the animal, and the knockout mouse has elimination of function in one of the proteins. Experimental'test'#2:''If'we'alter'the'geneFc'makeup' lecture.' of'the'animal'such'that'B'is'expressed'in'a'form'that' It is also important to remember what are a genotype, a phenotype, and the difference between wild-type and is'acFve'all'the'Fme'(consFtuFvely'acFve),'then'C'and' of an individual cell or organsm and the associated alleles at one or mutant. Genotype is the genetic constitution D,'but'not'A'should'also'be'acFve'all'the'Fme'(even' in'the'absence'of'the'signal).''

30

more specific loci. The phenotype is the observable characteristics of a cell or organism. A wild-type organism is the normal and common form other organism while the mutant is a changed or abnormal form. Know about the basics about how transgenic mice are produced. Microinject#DNA# Transgenic and knockout mice are utilized as research into#ferBlized# tools. To understand a gene of interest, the gene is egg# Plasmid'DNA' Fo,#Founder#transgenic#mouse# injected into transgenic or knockout mice with the Embryos# genotype manipulated. From there, a phenotype is generated, and thus the function of a gene is learned. A Transfer# InserBon*of*transgene*into*a* Ospring# transgenic mouse is a mouse with foreign DNA known as a embryos# Chromosome*in*the*ferBlized*egg* transgene (engineered by the experimenter) inserted into Cooper,'Figure'3.37' Transgene'DNA' the chromosomal DNA. It is typically occurring by random insertion into a inserts'into'the' chromosome of the fertilized egg. Making a transgenic mouse is beneficial because it chromosomal' allows the experimenter to express an altered gene product or a normal one that is DNA,'but'not'at' expressed at the wrong time, place, or amount. It can allow interpretation on any'parFcular'site.'' It'goes'in' function, phenotype of the transgene expression as well the physiological pathways Transgene# randomly.'' DNA# affected in the subject, and what parts of the protein important in the function. InserFon'does'not' These interpretations can allow application into disease models. This can not only be require'matching' up'of'sequences.' done in mice, but also bacteria, flies, fish, plants, yeast, birds, cows, worms, frogs, Chromosomal# and pigs.
' DNA#

Making*a*transgenic*mouse*

You can get a transgene into every cell of a mouse by microinjection of the transgene DNA into the pronucleus of a mouse zygote. After fertilization, there is a brief period before the nucleus fuses. The predecessor to this newly developed nucleus is the pronucleus. The injected mouse zygote from there is implanted into a surrogate mother that carries the organism to term. This has a fairly good success rate with stable integration of How*can*we*control*when*and*where*the* transgene observed in 10-40% of mice and each founder mouse can be used to establish an independent line of transgene*is*expressed*in*the*animal?* transgenic mice.
Transgene'
Promoter' Plasmid' Cut'out' linear' fragment' Gene'X'

Know the basics about the composition of transgenes. Inject'DNA' A transgenic mouse has foreign DNA (transgene) present in the chromosomal DNA of into'zygote' every cell. The DNA is injected and inserted randomly. Transgene DNA inserts into a chromosomal DNA, but not at any particular site. It goes in randomly. Insertion does not require matching up of sequences. Once in a chromosome, a transgene is usually passed on to successive generations. The question is now how the experimental can control when and where the transgene is expressed in the animal. An experimenter and cut out a fragment from the plasma and then place it into the transgene with promoters and inject it into the zygote. The promoter is the sequence in the DNA to which RNA polymerase binds to begin PCR*method*for*detecBng*transgene** transcription. The promoter is used to influence where and when the transgene is Mouse'tail'DNA' Tg' Primer' expressed and at what level of expression. The promoter can be a wellPrimer' characterized promoter region from a gene that directs a specific pattern of gene Polymerase'Chain'ReacFon' PCR' expression, a constitutive promoter, an inducible promoter, a gene promoter region under study. The gene can be a sequence encoding the protein of interest, a sequence encoding a reporter gene, Cre Run'on'an'agarose'gel' recombinase, or other possibilities.
'

'

Understand how the PCR method can be used to detect the presence of transgenes. This falls into the question of how to determine which mice exhibit the transgene. The general idea of the PCR method is to amplify the region of the transgene with boundaries known as primers. From there, an experimenter can run it on an agarose gel and observe the presence of the isolated copies of DNA segment.

Polymerase' chain'reacFon' (PCR)'

Lecture XXIX
Pollard,'Fig.'6`7'

Experim ental Tool: Knockout M ice (Targeted Gene Inactivation)

Terminology
Term Knockout Mouse Forward Genetics Definition A mouse whose DNA has been genetically engineered so that it does not express particular proteins Approach that encompasses several means of identifying the gene or set of genes that are responsible for a particular phenotype within an organism. Forward genetics can be thought of as a counter to reverse genetics, which seeks to alter genes in order to illuminate their multiple phenotypes

31

Reverse Genetics

Allele Embryonic Stem Cells Pluripotent Homologous Recombination Gene Targeting Vector Region of Homology Drug Resistance Gene Selection Strategy Screening Strategy Positive and Negative Selection

An approach to discovering the function of a gene by analyzing the phenotypic effects of specific gene sequences obtained by DNA sequencing. This investigative process proceeds in the opposite direction of so-called forward genetic screens of classical genetics. Simply put, while forward genetics seeks to find the genetic basis of a phenotype or trait, reverse genetics seeks to find what phenotypes arise as a result of particular genes. One or two more forms of a gene or a genetic locus. Pluripotent stem cells derived from the inner cell mass of the blastocyst, an earlystage embryo. Distinguished by their pluripotency and their ability to replicate indefinitely. Refers to the ability to differentiate into any of the three germ layers: endoderm, mesoderm, and ectoderm. Type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. A bacteriophage, plasmid, or other agent that transfers genetic material from one cell to another. Traits that are common among organisms due to sharing a common ancestor, and such traits have similar embryological origins and development. Genes in an microorganism which confer resistance to antibiotics, through manipulation of surface proteins or the antibiotics target. Methods to enrich for correctly targeted cells. Diagnostic tests to identify correctly targeted cells. Positive Selection: Select for ES cell that have taken up a drug resistance gene included in the targeting vector. Negative Selection: Select against ES cells that have randomly integrated the targeting vector. Gene that codes for neomycin resistance in cells. Gene that codes for the phosphotransferase (kinase) that is found in most living cell, which catalyze the phosphorylation of deoxythymidine to deoxytymidine 5phosphate. Use of restriction enzymesto cut or digest DNA at specific recognition nucleotide sequences known as restriction sites. Method utilized for detection of a specific DNA sequence in DNA samples. It combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. Method used to separate proteins by charge or size in a medium consisting of agarose. Strcture formed in the early embryogenesis of mammals, after the formation of the morula. Mother that is carrying the embryo of that does not have the mothers DNA, a foreign embryo. Single organism that is composed of two or more different populations of genetically distinct cells that originated from different zygotes involved in sexual reproduction. Transmission of the targeted gene copy to the offspring if the embryonic stem cells contributes to the germline (gonads). Gene is inactivated in all cells at all times. Gene ablation is cell type-specific or inducible. Specific sites in a DNA molecule for Cre protein that surround a directional core sequence where recombination can occur. Sandwiching of a DNA sequence between two lox P sites and is a contraction of the phrase flanked by LoxP. Tyrosine recombinase enzyme derived from the P1 Bacteriophage that carries out site-specific recombination events. Expression of certain genes in specific tissue, with effects only occurring in cells expressing certain genes.

Neo Gene (Neomycin Resistance Gene) TK Gene (Thymidine Kinase Gene) Restriction Enzyme Digest Southern Blot Agarose Gel Electrophoresis Blastocyst Surrogate Mother Chimera Germline Transmission Conventional Gene Targeting Conditional Gene Targeting LoxP (Locus of Crossover in P1) Floxed Gene Cre Recombinase Tissue Specific Recombination

Lecture Objectives
Know why knockout mice are useful in physiological studies. Knockout mice have an endogenous gene of interest that is inactivated (knocked out). In variations of this technique, a gene can be modified or replaced with a different gene. Why do researchers make knockout mice? It allows the determination: (1) which physiological functions are lost when the specific gene is altered, (2)

32

which functions are not affected, (3) the kinds of adaptive or maladaptive changes result when the gene is altered, (4) whether the phenotype mimics a disease of interest. In a bigger picture, knockout mice utilize an element of reverse genetics, in which there is a gene attained and the researcher wants to determine what are the effects of a mutation (genotype to phenotype), as opposed to forward genetics, in which there is a mutant phenotype to determine which gene was mutated (phenotype to genotype). It allows the determination of whether it fits the hypothesis or not. Learn how knockout mice are generated. Knockout mice are generated from embryonic stem cells in culture. Embryonic stem cells are pluripotent stem cells that can be maintained in an undifferentiated cell culture. An altered version of the target gene constructed by genetic engineering is introduced into each embryonic stem cell, and them each cell proliferates to form a colony. The colony is then tested for whether each cell has had the DNA fragment replace one copy of the normal gene. Then we need to prepare the female mouse for uptake. The female mouse mates, waits 3 days and harvest early embryos. The embryonic stem cells are then Gene!Targe;ng! Region!of! injected into the early embryo. The hybrid early embryo is partially Region!of! homology! formed from embryonic stem cells. The hybrid is then introduced into homology! the pseudopregnant mouse until birth. The somatic cells of the offspring Targe;ng!vector! are then tested for presence of the altered gene and then selected mice Drug&resistance&gene& are bred to test for gene in germ-line cells. This process can yield a transgenic mouse with one copy of the target gene replaced by altered...! ...! Gene&X& gene in germ line. Chromosome! Know about homologous recombination. Gene targeting (knockout) technology depends on pairing up and recombination between homologous sequences of DNA, known as homologous recombination. The recombination involves switching (a crossing over) of a chromosome between certain gene targeting vectors. Basically, there is a crossing over into the targeting vector and then a return to the homologous sequence. The crossing must be done twice or face the consequence of incomplete crossover. A major challenge of gene targeting is that mammalian cells undergo a high frequency of random integration events in addition to the more rare targeted events. If random insertion occurs, then the gene of interest is not knocked out. Random insertion occurs more frequently than homologous recombination.
!

...!

Drug&resistance&gene&

Posi;ve!and!nega;ve!selec;on!
Cells!are!resistant!to!drug!A! if!neo!gene!present!

Chromosome! aler!targe;ng! !

...!

Drug!B!kills!cells!if! tk!gene!present!

T!

exon!

exon!

exon!

Homologous! recombina;on!

Tk!gene!should! not!be!added!in! if!correct! targe;ng!occurs!!

Torres!pg.!5!

ES Cells

Introduce targeting vector by electroporation

Select for colonies that survive both positive and negative selection drugs

Expand those rare surviving colonies into separate cell lines

Screen the candidate cell lines for those that really do have the gene of interest correctly targeted

Understand the strategy of positive/negative selection. This leads to the question how scientists can separate the correctly targeted embryonic stem cells from the ones that result from random integration. We can utilize selection strategies (ways to enrich for correctly targeted cells) or screening strategies (diagnostic tests to identify correctly targeted cells. One selection involves positive and negative selection. Positive selection selects for embryonic stem cells that have taken up a gene included in the targeting vector. Negative selection selects against embryonic stem cells that have randomly integrated the targeting vector. Learn about Southern blotting as a method to test for correct gene targeting. Restriction enzyme digestion and Southern blotting can be utilized to check if the gene of interest has been correctly targeted. Southern Blotting simply looks at the structure of DNA at the gene of interest before and after correct targeting has occurred. It can be used to analyze the DNA of embryonic stem cells ot check if the correct gene has been targeted. The DNA from individual embryonic stem cell clones is digested with an enzyme. The digest samples are then run by agarose

33

gel electrophoresis to separate these digested proteins. From there, a researcher can determine which cells have a correctly targeted embryonic stem cell clone. The overall summary is that through a combination of restriction enzyme digestion, transfer to nitrocellulose paper, and DNA probe hybridization, one can check for the expected structural changes in the gene of interest. Basically, this is to confirm that the changes in DNA that were expected to occur (when gene targeting is done correctly) actually happen? If a scientist did get embryonic stem cells that are correctly targeted, what will he or she do with it? From there, the embryonic stem cells that have been correctly targeted are then injected into a normal mouse embryo (at the blastocyst stage). Several embryos are then placed into a surrogate mother. The embryonic stem cells mix together with the normal cells and then contribute to various tissues, producing chimeras. Chimeras are altered foreign cells that are incorporated into the normal embryo. If the embryonic stem cells contribute to the germline (gonads) of the chimeric mouse, then the targeted gene copy How!to!get!homozygous!knockout!mice! might be transmitted to the offspring. From there, we can get two types of knockout mice, either homozygous or heterozygous. Heterozygous mice will have only one of the copies that are knocked out, while homozygous will have both copies knocked out.
KpnI&Digests&

Types!of!gene! targe;ng!

wildtype&

Torres!Fig!2!

Conven;onal!gene! ConvenJonal&gene&targeJng& targe;ng:!!gene!is! inac;vated!in!!all!cells!at! all!;mes!

Cell&typeCspecic&gene&targeJng& ! Condi;onal!gene! targe;ng:!!gene!abla;on! is!cell!typeTspecic!or! Inducible&gene&targeJng& inducible.! +inducer!

+/T!

+/T!

=!area!of!gene!KO!
!

This is a form of conventional gene targeting. However, there is a small problem with conventional gene targeting. It may not be T/T! +/+! +/T! that useful. The gene of interest can be expressed in multiple cell types, and will not ! allow the determination of whether the phenotype is due to ablationInterbreed!mice!carrying!Floxed!gene!with!transgenic! of the gene function in one cell type versus mice!expressing!Cre!recombinase!in!specic!;ssue! the other. Eliminating the gene everywhere could be lethal to the embryo during development, and it is irreversible. It does not allow the deletion of the gene in the adult form, when the tissue is already fully formed. Know the basics about conditional gene targeting sing the cre-IoxP system. Conditional gene targeting involves gene ablation that is cell type-specific or inducible. One can utilize conditional targeting in the Cre/LoxP system. LoxP is in the chromosome, and is involved in the removal of the gene segment from the chromosome. Cre recombinase is expressed only in specific tissue. The recombination occurs in a tissue specific manner. The new mouse strain should have the floxed gene and the Cre transgene. The LoxP sites recombine only in cells expressing Cre recombinase.

Southern!blot!of! mouse!tail!DNA!

Cre&transgene& oxed&gene& specic!manner! New&mouse&strain& Note:!loxP!sites!in!this! Interbreed! contains&oxed&gene& example!are!placed!in! lines! and&Cre&transgene&

Recombina;on!occurs!in!a!;ssueT

introns!anking!a!cri;cal! exon.!
!

Lodish,!Fig!8T35!

loxP&

loxP&

The Cre-LoxP system is a three-step process that consists of: loxP!sites!recombine! 1. Making mice in which the gene of interested is floxed with the recombination sites. Using convention only!in!cells! gene targeting methods does this. expressing!Cre! 2. Make a different mouse line (transgenic mice) that expresses the Cre recombinase enzyme in specific recombinase! Lodish,!Fig!8T35! target tissue. 3. Interbreed mice carrying Floxed gene with transgenic mice expressing Cre recombinase. This yields mice that have the floxed gene (meaning that it has LoxP) and transgene.

Lecture XXX
TGF- Signaling Knockout M ice, Disease M odels, and Case Conclusion

Terminology
Term Hyperdilation Balance Model Knock in versus Knockout Definition Excessive dilation of the blood vessels. Regulation in the transition from one phase to another of a process. Assumed that ALK1 and ALK5 were both expressed in endothelial cells. Knock-in: Genetic engineering method that involves the insertion of a protein coding cDNA sequence at a particular locus in an organisms chromosome. Knockout: Genetic engineering of an existing gene by replacement or disruption with an artificial piece of DNA. Knock-in is similar to knockout, but replaces a

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Reporter Gene LacZ or -gal Reporter Gene Luciferase Reporter Gene Endothelial-Specific Knockout Homozygous Knockout Heterozygous Knockout BMP (Bone Morphogenic Protein) Modifier Genes

gene with another instead of deletes it. Gene that researchers attach to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants. Reporter gene that encodes -galactosidase, which cause bacteria that express the gene to appear blue in a media containing X-gal. Gene utilized as a laboratory reagent that yields a class of oxidative enzymes utilized in bioluminescence and distinct from a photoprotein. Strategy that involves making mice that lack ALK1, ALK5 or TGFTII gene and to observe effects in vascular endothelial cells. Genotype where both alleles have the same inactivated gene. Genotype where one of the alleles have the inactivated gene. Group of growth factors that orchestrate tissue architecture throughout the body. Segment of DNA that is involved in producing a polypeptide chain. It can include regions preceding and following the coding DNA as well as introns between the exons.

Lecture Objectives
Several!mutaEons!that!cause!HHT!are!part! Know which genes are mutated in HHT and how these genes fit into the TGF- pathway. of!the!TGF9!signaling!pathway! Remember that hereditary hemorrhagic telangiectasia was covered from a Ligand! Endoglin! systemic to a molecular level, and the mutations that cause HHT are SMAD4* !juvenile! attributed to the TGF- signaling pathway, and affecting gene targeting.. polyposis/HHT! syndrome There is most likely a mutation either in the ALK1 receptor or the Co-SMAD. ALK1! There are several types of HHT, but HHT type 2 is the one involved in the Co9SMAD! mutations in the Activin receptor-like kinase 1 (ALK-1) gene, which is a Type 1 TGF- superfamily receptor. How does the defect in the signaling pathway result in specific vascular defects in hereditary hemorrhagic telangiectasia? We know that the genetic defects most likely result in aberrant endothelial cell responses to specific signals, including dysregulation of a variety of genes in endothelial cells. We can know where the problem is via a microarray. We can isolate the mRNAs from normal and HHT cells and then compare with probes.

X!

X!

X!

!!

The current ideas about HHT involve dysregulation of the specific genes that could result in abnormal production of the extracellular matrix, altering cell adhesion and migration in angiogenesis. This can result in irregular vessel formation, impaired recruitment and ifferentiation of mesenchymal cells into smooth muscle cells. Understand how knockout mice and transgenic mice can help us to understand and mimic disease. Knockout mice were utilized in with two methods: conventional/global knockout and conditional/tissue-specific knockout. Conventional gene targeting was utilized to make a global knockout. The conventional (homozygous) knockout of ALK-1 resulted in the vascular abnormalities with embryo death at mid-gestation. The ALK-1 Knockout spurred vascular abnormalities. It yielded the following findings: (1) excessive fusion of capillary plexes into cavernous vessels, (2) hyperdilation of large vessels, (3) deficient differentiation and recruitment of vascular smooth muscle cells, and (4) enhanced expression of angiogenic factors and proteases. Initially, HHT was attributed to homozygous phenotypes for HHT, but scientists made a more thorough investigation to show that mice that were heterozygous for the mutation in ALK-1 exhibit age-dependent vascular lesions similar to those in humans. Transgenic methods were used to test and counter the assumption of the Balance Model in mice. Mice were also utilized as a media for a tissue-specific knockout strategy that was discussed in the previous strategy. This utilized an endothelial cell-speciic knockout strategy, in which mice are made or obtained with a floxed verison of ALK1, ALK5, or TGFRII genes, all in separate experiments. Scientists would make a transgenic mouse that would express Cre recombinase only in vascular endothelial cells, and interbreed the transgenic Cre expressing mice with each of the floxed mouse lines, and obtained the mice with floxed gene as well as the Cre transgene.

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Hypothesis:!!Balance!model!for!TGF9! signaling!in!the!regulaEon!of!angiogenesis!
Learn about the In!endothelial!cells,! evolution of ideas describing TGF- pathway signaling during vascular maturation and in HHT. ALK1!might!normally!! regulate!the!transiEon! The first model that was presented was the Balance Model. Initially it was thought from!the!acEvaEon!phase! in blood vessels express both ALK1 and ALK5, essentially two that endothelial cells to!the!resoluEon!phase!of! different type I receptors. In endothelial cells, ALK1 might normally regulate the angiogenesis.!from the activation phase to the resolution phase of angiogenesis. In ALK1 transition
In!ALK1!KO!mice,!the! activation phase. Essentially, based on that model, HHT was from too much activation Revised!model!of!TGF9!signaling!during! defects!would!result!from! and not enough resolution. The balance model assumed that ALK1 and ALK5 were vascular!maturaEon! the!abnormal!persistence! both expressed in Signaling!between!ECs! of!the!acEvaEon!phase.!endothelial cells.
and!SMCs!might!also! be!aected!in!HHT!

knockout mice, the defects would result from the abnormal persistence of the

AcEvaEon! ResoluEon! S.!P.!Oh,!et#al.,#PNASs#97,$2626$(2000).! To test the expression pattern of ALK5 using a reporter gene approach, one copy of the ALK5 gene was knocked out and the lacZ gene was inserted into that ALK5 locus as a reporter. The same experiment was done to replace the ALK1. Experimental performance yielded results that stated that ALK1 was expressed in vascular endothelial cells, but ALK5 is actually expressed in vascular smooth muscle cells, countering the assumption made by the Balance Model.

Endothelial!cell! (has!ALK1)!

Smooth!muscle!cell! (has!ALK5)!
Laboratory!InvesEgaEon!(2006)!86,!116129!

With the newly revised model of TGF- signaling during vascular maturation, we know that ALK1 was expressed in the endothelial cell while ALK5 receptors are in the smooth muscle. Scientists can predict that conditionally knocking out ALK1, ALK5, and TGFRII in endothelial cells would yield the following results: Knockout of ALK1 results in severe malformations mimicking the pathologic features of HHT (expected). Knockout of ALK5 resulted in not exhibiting vascular defects (expected). The!new!model!incorporates!BMP9/BMP10!and!their! Type!II!receptors!that!can!interact!with!ALK1!in!vascular! not show these vascular defects (which was not expected). Knockout of TGFRII did
endothelial!cells!

The conclusion generated from this is that ALK1, not ALK5 and TGFRII, is necessary in endothelial cells for the signaling that is pertinent to the pathogenesis of HHT. Since the TGFRII knockout did not mimic HHT, this suggests that the ligand for ALK1 signaling might The!Point:!!There!are!now!some!good!mouse!models!of! not actually be a member of the TGF- subfamily. Other experiments with endothelial cells in culture indicated that BMP9 and HHT!and!these!can!be!used!to!test!ideas!about!the! BMP10 were actually better candidates to be the ligand for ALK1 than TGF-1. factors!that!inuence!the!formaEon!of!vascular!defects! So far, the most recent model shows incorporation of BMP9 and BMP 10 and their Type II receptors that can interact with ALK1 in vascular endothelial cells. Realize that we do not yet understand everything about HHT. There are still, unfortunately, lots of questions about HHT. Why are the lesions in HHT so localized in the affected person? Is a second hit event necessary to initiate a local lesion? Could malformations be related to abnormal responses to local injury? We also dont know why is the severity of the disease so variable between affected P.!Mogakhar!et!al..!2009! individuals? Are there modifier genes that can influence how severely the disease presents itself? Why are the key target genes that are abnormally expressed in HHT? Do non-transcriptional effecs also contribute? How do defects like arteriovenous malformations actually form? To address the plethora of questions and plausible hypotheses, HHT researchers have made some inducible knockouts, when the gene is only knockout when an inducer is given. One group made an inducible knockout of ALK1 so they could get adult mice and then induce ALK1 knockout. They found that wounding can induce the new formation of arteriovenous malformation in the ALK-1 deleted mice. The dorsal skinfold chamber model can be used to follow the same blood vessels over time. Using the dorsal skinfold window chamber system the development of an arteriovenous malformation in response to wounding was followed over time. This may help to understand how the lesions develop. So whats the point? There are some good mouse models of HHT and these can be used to test ideas about the factors that influence the formation of vascular defects. From what we understand so far, we also have potential therapeutic targets. There has been success with drugs that inhibit the VEGF signaling pathway, with VEGF elevation is present in the skin telagiectatic lesions of HHT patients. Scientists have remembered that notch signaling along with VEGF-A signaling, is one of the key regulators of tip cell formation during angiogenesis. Studies have also shown that Notch4 normalization reduces the blood vessel size in arteriovenous malformations. Thus, in conclusion, there is still much to be learned and researched about hereditary hemorrhagic telangiectasia. Studies such as those previously stated unraveled more possible causes for hereditary hemorrhagic telangiectasia.

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