JBC Papers in Press.

Published on December 1, 2006 as Manuscript M610536200

The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M610536200
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NON-ENZYMATIC GLYCATION OF BONE COLLAGEN MODIFIES OSTEOCLASTIC

ACTIVITY AND DIFFERENTIATION
Ulrich Valcourt1,3*, Blandine Merle1*, Evelyne Gineyts1, Stéphanie Viguet-Carrin1, Pierre D.
Delmas1, and Patrick Garnero2.
1
INSERM Research Unit 403 and Université Lyon 1, Lyon, France. 2Molecular Markers, Synarc,
Lyon, France.

Running title: AGEs modify osteoclastic activity and differentiation

Key words: Collagen, advanced glycation end products, bone resorption, osteoclast, cell
differentiation
3
Address all correspondence and reprint request to the present address: Ulrich Valcourt, Institut de
Biologie et Chimie des Protéines (IBCP), UMR 5086 CNRS - Université Lyon 1, IFR128 Biosciences
Lyon-Gerland, Lyon, France. Tel. 33 4 72 72 26 59; Fax: 33 4 72 72 26 04; E-mail:
u.valcourt@ibcp.fr.
* Both authors contributed equally to this work.

Type I collagen, the major organic unknown, AGEs might interfere with
component of bone matrix, undergoes a series osteoclastic differentiation and activity

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of post-translational modifications that occur
with aging, such as the non-enzymatic
glycation. This spontaneous reaction leads to
the formation of advanced glycation end
products (AGEs) which accumulate in bone
tissue and affect its structural and mechanical
properties. We have investigated the role of
matrix AGEs on bone resorption mediated by
mature osteoclasts and the effects of
exogenous AGEs on osteoclastogenesis. Using
in vitro resorption assays performed on
control- and AGE-modified bone and ivory
slices, we showed that the resorption process
was markedly inhibited when mature
osteoclasts were seeded on slices containing
matrix pentosidine, a well-characterized
AGE. More specifically, the total area
resorbed per slice, and the area degraded per
resorption lacuna created by osteoclasts, were
significantly decreased in AGE-containing
slices. This inhibition of bone resorption was
confirmed by a marked reduction of the
release of type I collagen fragments generated
by the collagenolytic enzymes secreted by
osteoclasts in the culture medium of AGE-
modified mineralized matrices. This effect is
likely to result from decreased solubility of
collagen molecules in the presence of AGEs,
as documented by the reduction of pepsin-
mediated digestion of AGE-containing
collagen. We found that AGE-modified BSA
totally inhibited osteoclastogenesis in vitro,
most likely by impairing the commitment of
osteoclast progenitors into pre-osteoclastic
cells. Although the mechanisms remain
through their interaction with specific cell-
surface receptors, because we showed that
both osteoclast progenitors and mature
osteoclasts expressed different AGEs
receptors, including RAGE (receptor for
AGEs). These results suggest that AGEs
decreased osteoclast-induced bone resorption,
by altering not only the structural integrity of
bone matrix proteins, but also the osteoclastic
differentiation process. We suggest that AGEs
may play a role in the alterations of bone
remodelling associated with ageing and
diabetes.
INTRODUCTION
Non-enzymatic glycation is a common
posttranslational modification of proteins
induced by the spontaneous condensation of
reducing sugars (e.g. glucose) and metabolic
intermediates (e.g. triose phosphates, glyoxal
and methylglyoxal) with free amino groups in
lysine or arginine residues. The early step of the
so-called Maillard reaction is the formation of a
Schiff base adduct to protein. This early
glycation product undergoes a reversible
rearrangement to form an Amadori product
adduct to protein (i.e. an intermediate glycation
product). Schiff bases and Amadori products
then undergo a complex series of
rearrangements, oxidations and/or
dehydratations along different chemical
pathways to produce a class of irreversible
adducts to proteins, the advanced glycation end
products (AGEs) [reviewed in (1,2)]. A direct

Copyright 2006 by The American Society for Biochemistry and Molecular Biology, Inc.
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consequence of these highly diverse reaction
pathways leading to AGE formation is that a
wide variety of AGEs with different chemical
structures is formed. Some AGEs are adducts to
the protein (e.g. carboxymethyllysine and
carboxyethyllysine), whereas others present
protein-protein crosslinks (e.g. pentosidine,
glyoxal-derived lysine dimer and methylglyoxal-
derived lysine dimer). Since the initial
characterization of the pentosidine crosslink by
Sell and Monnier (3,4), many AGEs (and more
recently advanced lipoxidation end products)
have been identified. However, it is unclear
which, if any, of these AGEs can be designated
as the most functionally relevant, and new AGEs
are continuously being discovered. Once formed,
AGEs are removed from the tissue only when
the protein involved is degraded. Consequently,
the most extensive accumulation of AGEs will
occur in tissues characterized by low turnover
and containing consequently long-lived proteins,
such as collagen in the extracellular matrix of
connective tissues (e.g. cartilage, bone, tendon
and skin) (5).
Formation and accumulation of AGEs are
characteristic features of tissues of aged people,
but also occur in patients with diabetes mellitus,
mainly those with poorly controlled
hyperglycemia. AGEs have also been strongly
implicated in the pathogenesis of AGE-related
and diabetic complications, such as retinopathy,
glomerulopathy, neuropathy and diabetic
atherosclerosis (6,7). It has recently been
reported that AGEs are involved in skeletal
diseases such as osteoarthritis, which is a
chronic disabling disorder of aged people.
Accumulation of AGEs in articular cartilage
affects cellular characteristics and increases
stiffness and brittleness of the tissue, thereby
rendering it more prone to mechanical damage
(8,9). AGEs modify the structure and the
subsequent functions of proteins by creating
intra- or intermolecular crosslinks (10); these
could partially explain the deleterious effects of
AGEs on the biomechanical properties of
connective tissues. AGEs also interfere with the
susceptibility of matrix proteins towards
proteolytic degradation. Degradation of AGE-
modified cartilage collagen by matrix
metalloproteinases is impaired compared with
unmodified collagen (11), and pepsin mediated-
solubilization of collagen is decreased in AGE-
modified collagen (10,12). AGEs have also been
found in bone tissue (4,13); in particular,
pentosidine, has been shown to accumulate with
age in cortical bone of human femur (14).
Accumulation of AGEs in bone collagen matrix
has been shown to alter the mechanical
properties of bone -decreasing its toughness- and
could therefore contribute to skeletal fragility
(15-17). Indeed, decreased bone strength and
osteopenia have been shown in rodent models of
diabetes (18,19). Osteopenia and osteoporosis
are often observed in patients with type I
diabetes, and recent cohort studies indicate that
diabetes itself is associated with increased risk of
fractures (20). However, the role of AGEs in
decreased bone strength in diabetes and
subsequent contribution to fracture risk remain
unclear.
Eventually, AGEs modify cellular behaviour by
interacting with specific cellular receptors, such
as RAGE (receptor for AGEs). In a craniotomy
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model in type I diabetic mice, the degree of bone
healing was reported to be 40% of that of non-
diabetic animals, and RAGE is expressed at
higher levels in the healing bone tissue of
diabetic animals compared to control animals. In
non-diabetic animals, AGE-modified BSA
application to the cranial defect reduced bone
healing by 40-60% (21). It has been shown in
vitro that AGEs accumulation in type I collagen
interfere with the proliferation and the
differentiation of osteoblastic cells, at different
stages of their development (22-25). AGE-
modified collagen also interferes with integrin-
mediated osteoblastic attachment (26). RAGE
has been shown to be expressed in osteoblasts
(21,27) and may modulate AGE-dependent
signaling in osteoblasts (27). This observation
could explain in part the decreased expression of
transcription factors responsible for osteoblastic
differentiation in type I diabetic mice model
(28).
Taken together, these findings suggest that
AGEs may play a role in the pathogenesis of
diabetic osteopenia as well as that of age-related
decreased bone strength and increased risk of
fracture; however the details of such deleterious
mechanisms remain to be clarified. Bone mass is
tightly regulated by the differentiation and
activity of bone-resorbing cells (osteoclasts) and
bone-forming cells (osteoblasts) through a
process called bone remodelling. The
contribution of non-enzymatic glycations to
bone remodelling has been poorly investigated
in vivo, mainly due to a lack of suitable animal
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models; for instance, insulin deficiency or
resistance in diabetic animal models might
interfere with cell differentiation or function
processes. Although the role of AGEs in
osteoblasts differentiation and function has been
well characterized in vitro (23,26,27,29), their
contribution to osteoclast activity and
differentiation is unknown. In the present study
we first investigated the effects of matrix AGEs
on bone resorption by osteoclasts, using an in
vitro resorption assay on AGE-modified
mineralized matrices. We then extended our
analysis to the contribution of exogenous AGEs
to osteoclastogenesis.
EXPERIMENTAL PROCEDURES
In vitro AGE-matrix and AGE-BSA formation.
Elephant ivory slices (6 mm-diameter) (kindly
donated by the Centre de conservation et d'étude
des collections, Lyon, France) and bone cortical
slices (3 mm-diameter) from 3-month-old calves
were used in these experiments. Human cortical
bone slices (3 mm-diameter) were prepared from
a 40-year-old male femur obtained from
necropsy (Faculté de Médecine Laennec, Lyon,
France). 0.3 mm-thick slices were cut with an
Isomet low speed saw (Buehler, Lake Bluff, IL),
cleaned by sonication, sterilized for 2 min in
70% ethanol and stored dried at -20°C until
AGE-matrix formation or in vitro resorption
assay. In vitro AGEs formation in mineralized
tissues was performed according to adapted
protocols of those previously described for
tendon or cartilage collagens (30,31). Ivory and
bone slices were incubated with 0.2 M D-Ribose
in 10 ml of phosphate-buffered saline solution
(PBS), containing 100 units/ml penicillin,
100 µg/ml streptomycin, and 1.5 mM PMSF, for
45 and 60 days at 37°C, respectively.
Alternatively, the slices were incubated with
0.05 M to 0.6 M D-Ribose in 10 ml Tris-
buffered Saline solution (TBS) (pH 7.4),
100 units/ml penicillin, 100 µg/ml streptomycin,
1.5 mM PMSF, for 2 to 7 days at 37°C, in order
to modulate the level of AGEs formed. Control
incubations were also performed with PBS or
TBS. Incubation solutions were replaced every
day and the pH was controlled routinely to
ensure optimal buffering activity during the
glycation process. AGE-BSA was prepared
according to a modified version of methods
previously described (32,33). Briefly, 50 mg/ml
of BSA (Fraction V, sterile filtered; Sigma-
Aldrich, Saint Quentin Fallavier, France) was
incubated at 37°C, under sterile conditions, with
0.6 M D-Ribose for 1 week or with 0.5 M D-
glucose and 0.3 M Lysine for 4 weeks in PBS
(pH 7.4) containing 100 units/ml penicillin, and
100 µg/ml streptomycin. The unincorporated
sugars were removed by dialysis against PBS.
These products were designated as AGE-1 and
AGE-2 respectively. Non-AGE-modified BSA
was incubated in the same conditions except for
the absence of reducing sugars. Glycated-BSA
(1-5 mol fructosamine per mol albumin, Sigma-
Aldrich) was used as a negative control. BSA
derivatives were quantified using the Bio-Rad
protein assay system (Bio-Rad, Marnes la
Coquette, France) and the concentrations of
pentosidine were determined as described
hereafter (Table III).
Rabbit osteoclast isolation and resorption
assay. Osteoclasts were isolated from the long
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bones of 4-day-old New Zealand rabbits. Bones
were removed, cleaned of soft tissues and bone
marrow, split, and scraped into M199 medium
supplemented with 10% fetal calf serum (FCS;
Invitrogen, Carlsbad, CA) and 20 mM HEPES
(Invitrogen). Cells were centrifuged at low
speed, resuspended in DMEM medium
containing 2 mM glutamine, 100 units/ml
penicillin, 100 µg/ml streptomycin and 2% FCS
(all products from Invitrogen), and seeded on
ivory or bone slices in 48-well or 96-well culture
plates, respectively, for 3 h at 37°C and 5% CO2.
After this setting period, non adherent cells were
removed, slices were washed in PBS, and the
remaining cells were incubated for 3 to 8 days in
complete DMEM medium containing 2% FCS
and at pH ~7.0 (34). At the end of culture, cells
were removed in 1 M NaOH for 20 min, and the
slices were rinsed 3 times in water and stained
with acid hematoxylin for 30 sec and then with
1% (wt/vol) toluidine blue in 1% (wt/vol)
sodium borate for 30 sec. The number and the
area of resorption lacunae were then measured
by light microscopy using LUCIA® Software
(Nikon, France), as previously described (35).
Results are expressed as the number of lacunae
and total area resorbed per slice, as well as the
area resorbed per lacuna per slice.
Biochemical assessment of bone collagen
degradation in culture media. The release of
type I collagen fragments, subsequent to
resorption of bone and dentin slices was also
quantified in the culture supernatants using two
immunological methods: (i) the concentrations
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of crosslinked and isomerised C-terminal
fragments of type I collagen were determined
using the CrossLaps for Culture ELISA kit
(Nordic Bioscience Diagnostics, Herlev,
Denmark) as recommended by the manufacturer;
and (ii) the concentrations of the helical peptide
corresponding to the 620-633 fragment of
human type I collagen were measured using the
METRA® Helical Peptide EIA kit (Quidel Corp.,
San Diego, CA). Collagen fragments released
into the culture media were also analyzed by
measuring the hydroxyproline concentration
using the Hydroxyproline by HPLC reagent
from Bio-Rad, after acid hydrolysis of the
conditioned culture media.
Murine osteoclast differentiation. Bone marrow
cells of 6-8-wk-old male NMRI mice were
seeded at 5000 cells/mm² and cultured for 8 days
in differentiation medium: DMEM medium
containing 10% FCS (Hyclone Laboratories,
Perbio Science, Cheshire, United Kingdom),
2 mM glutamine, 100 units/ml penicillin,
100 µg/ml streptomycin, 30 ng/ml recombinant
mouse M-CSF, and 30 ng/ml soluble
recombinant RANK-L mixed with 2.5 µg/ml
mouse anti-polyhistidine antibody, according to
the manufacturer’s recommendations (R&D
Systems Europe, France). After the
differentiation process on plastic dishes, mature
osteoclasts were removed after four washes with
PBS by using 0.25 mM EDTA in PBS during
30 min, and after centrifugation osteoclasts were
seeded on dentin slices and incubated in
differentiation medium for 8 days, as described
(36).
RAW264.7 cell culture and differentiation. The
murine monocytic cell line RAW264.7 (ATCC,
Manassas, VA) was cultured at 37°C and 5%
CO2, and maintained on 9-cm diameter uncoated
plastic dishes in D-MEM containing 10% FCS
(Invitrogen) with 2mM glutamine, 100 units/ml
penicillin, and 100 µg/ml streptomycin.
Osteoclastogenesis experiments were performed
as described previously (37); 100 cells/mm²
were seeded on a 96-well tissue culture plate
with the differentiation medium, in the presence
or absence of 30 ng/ml soluble recombinant
RANK-L (mixed with 2.5 µg/ml mouse anti-
polyhistidine antibody) and BSA derivatives.
For RT-PCR analysis, the cells were seeded at
500 cells/mm² and incubated or not with 50
µg/ml of BSA or AGE-1 for 48h.
Human CD-14-positive monocytes isolation
and Osteoclast Differentiation. Human
monocytes were isolated from peripheral blood
of healthy donors (Etablissement Français du
Sang, Lyon). Blood, diluted 1:1 with 4°C PBS,
was carefully layered on Ficoll-Paque™ Plus
(GE Healthcare, Sweden) and centrifuged at 700
×g for 20 minutes. Lymphocytes interface was
collected, washed twice with 4°C PBS followed
by centrifugation at 700 ×g for 12 minutes, and
resuspended in cold PBS containing 2% FBS.
CD-14 positive cells were isolated using
magnetic beads coated with a mouse monoclonal
anti-CD14 antibody according to the
recommendation of the manufacturer
(Dynabeads CD14 monocytes/macrophages,
Dynal Biotech, Invitrogen, Carlsbad, CA). CD-
14 positive cells were resuspended in DMEM
medium containing 10% of fetal calf serum
(Hyclone Laboratories, Perbio Science,
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Cheshire, United Kingdom), 25 ng/ml
recombinant human M-CSF, and 25 ng/ml
soluble recombinant RANK-L, mixed with 2.5
µg/ml mouse anti-polyhistidine antibody.
Culture media were replaced every second days
with growth and differentiation factors up to 15
days. For the bone resorption assay,
differentiating cells (at day 10) were detached
and seeded on control- and ribose-incubated
bovine bone slices and cultured for 8 days in the
presence of growth and differentiation factors.
Tartrate-Resistant Acid Phosphatase (TRAP)
activity assay and cytochemistry. TRAP activity
in the culture media was measured using a
colorimetric assay (38,39). The reaction buffer
(412 mM acetic acid, 0.209% Triton X-100, 412
mM NaCl, 4.12 mM EDTA, 10.6 mM ascorbic
acid, 10.1 mM 4-nitrophenylphosphate, 41.6
mM Na2 tartrate at pH 5.5) was added to
conditioned media, incubated for 1 hour at 37°C
in the dark, and then stopped with 100 µl of
300 mM NaOH. Colorimetric changes were
measured at 405 nm with 650 nm as the
reference using an ELISA reader. Cellular TRAP
activity was measured using the Leukocyte Acid
Phosphatase kit (Sigma-Aldrich). Briefly, the
cells were washed twice with PBS, fixed for 5
minutes with 2% glutaraldehyde in PBS and
stained according to the manufacturer’s
instructions.
Biochemical analyses of bone collagen. The
amount of pyridinoline (PYD),
deoxypyridinoline (DPD) and pentosidine was
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measured after hydrolysis of the bone or ivory
slices. Briefly, slices (~7 mg wet weight) were
hydrolyzed by HCl at 110°C during 20 hours.
Collagen crosslinks were extracted from
hydrolysates using Solid Phase Extraction (SPE)
crosslinks Chromabond column (Macherey-
Nagel, France). Separation of the different
crosslinks was performed by HPLC on an
Alliance 2695 separation module using an
Atlantis dC18 (3µm; 4.6 × 100 mm) column
protected by an Atlantis dC18 (3µm; 4.6 × 20
mm) guard cartridge (Waters Corporation,
Milford, MA). Molecules were separated by
using a gradient solution. Solvent A consisted of
0.06 % of HBFA, and solvent B was 50% of
solvent A and 50% of acetonitrile. The flow rate
was 1.2 ml/min and the column temperature
40°C. The separation of PYD and DPD was
performed during the first 12 minutes of an
isocratic step at 14% of solvent B, and
pentosidine was eluted during the following 24
min of gradient from 14 to 31% solvent B. PYD
and DPD were monitored for fluorescence at an
emission of 395 nm and an excitation of 297 nm
and then wavelengths were shifted to 385 nm
and 335 nm respectively for determination of
pentosidine (multi O fluorescence detector
Waters 2475). Pyridinium crosslinks were
quantified against a calibrator supplied by Metra
Biosystems Ltd. Pentosidine standard was
synthesized and calibrated with a standard of
pentosidine generously gifted by Dr Takahashi
(University of Saitama, Japan). Hydroxyproline
content was measured by HPLC using the
Hydroxyproline by HPLC reagent from Bio-
Rad.
Collagen solubility. After demineralization in
0.5 M EDTA 0.05 M Tris, pH 7.5, at 4°C for 72
hours, bone slices were rinsed and submitted to
limited pepsin digestion in 0.5 mg/ml pepsin
(Sigma-Aldrich) in 0.5 M acetic acid for 4 h at
37°C with constant shaking. The reaction (1 ml)
was stopped by the addition of 2 µl of 1 mM
pepstatin and centrifuged for 10 min at 12000
rpm. Collagen concentration was determined in
the supernatant and the pellet by measuring the
amount of hydroxyproline (as described above)
after acid hydrolysis in 6 N HCl for 20 h at
110°C. Collagen solubility was expressed as the
collagen concentration in the supernatant after
pepsin digestion as a percentage of total
recoverable collagen in pellet and supernatant.
RT-PCR. Total RNA was extracted from
RAW264.7 cells using the RNeasy kit (Qiagen,
Courtaboeuf, France), and digested with RQ1
RNase-free DNase (Promega, Charbonnière-les-
Bains, France) to remove any contaminating
genomic DNA. Reverse transcriptions were
performed as described previously (40), using 1
µg of RNA and 12.5 ng of anchored oligo-dT23
primers (5’-dT23VVV-3’, where V represents A,
C or G nucleotides) per µl, in the presence of
200 U of SuperScipt II-RNase H- (Invitrogen).
Polymerase chain reactions were carried out as
described (40), using specific primers designed
according to sequences available in the
databanks or published by other authors
(Table I). Primers for mouse glyceraldehyde-3’-
phosphate dehydrogenase (Gapdh) were used to
ascertain that an equivalent amount of cDNA
was synthesized. Controls where reverse
transcriptase was omitted and where cDNAs
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were replaced with water were also performed in
order to demonstrate the specificity of the
reactions (data not shown). Quantitative real-
time PCR reactions were performed as described
previously (40) using an iCycler (Biorad). Gene
expression levels were determined using the
comparative Ct method using Gapdh as
reference. The ground condition was set to 1 and
expression data are presented as bar graphs of
mean values ± SD.
Indirect immunofluorescence and direct
fluorescence. For actin cytoskeleton direct
fluorescence microscopy, cells seeded on glass
coverslips were fixed, permeabilized and stained
with Cy3-labeled phalloidin (Molecular Probes,
Interchim, France). For immunofluorescence
microscopy, fixed cell preparations were
processed as described (41). Rabbit polyclonal
anti-RAGE (H-300) was purchased from Santa
Cruz Biotechnology, Inc (Santa Cruz, CA). The
fluorescein isothiocyanate (FITC)-conjugated
rabbit anti-rabbit-IgG antibody was from DAKO
(Carpinteria, CA) and Cy3-conjugated goat anti-
rabbit-IgG antibody was from Jackson
ImmunoResearch Laboratories (West Grove,
PA). All photomicrographs were obtained by a
Leica DMRB microscope equipped with a Nikon
DXM1200 digital camera, using a Leica PL
Fluotar 20×/0.50 objective lens and
photographing at ambient temperature in the
absence of immersion oil. Image contrast was
adjusted using Adobe Photoshop after image
acquisition with the camera's LUCIA® software
(Nikon, France). Control experiments for which
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the primary antibody was omitted were also
performed (data not shown).
Statistical analyses. Data are presented as means
± SD, unless otherwise noted. Comparison
between experimental conditions for bone
resorption activity and osteoclastic
differentiation assays were assessed using the
nonparametric Mann-Whitney U test.
RESULTS
In vitro AGE-matrix formation
In order to analyze the effects of collagen AGEs
on bone resorption we used cortical bone slices
from 3-month-old calves, in which the formation
of AGEs was induced in vitro, by incubating the
slices with D-ribose in PBS at 37°C for various
times. After 60 days of incubation in the
presence of 0.2 M ribose, there was a browning
of the bone matrix, which characterizes the
Maillard reaction (Figure 1A). The formation of
AGEs was confirmed by the marked induction of
pentosidine concentration in the 3-month-old
cortical bone slices (Figure 1B). In contrast,
there was no change in the content of the mature
enzymatic collagen crosslinks PYD (Figure 1C)
and DPD (Figure 1D) in slices incubated at 37°C
either with or without ribose.
Effects of AGEs on osteoclastic bone resorption
We assessed the ability of mature osteoclasts to
degrade bone tissue by performing an in vitro
bone resorption assay using unfractionated bone
cells from rabbit long bones cultured for 3 or 8
days on control and AGE-containing slices. This
cell preparation is enriched in mature TRAP-
positive osteoclasts (Figure 2A) which are able
to degrade a mineralized matrix, as documented
by the formation of resorption lacunae (or pits)
(Figure 2B). After 3 days in culture, the total
area resorbed per slice did not change
significantly (Figure 2C), but we observed a
significant increase in the number of resorption
pits (Figure 2D) in ribose-incubated slices
compared to untreated ones. Consequently, the
mean area resorbed per pit per slice markedly
decreased (Figure 2E). We next extended the
resorption period to up to 8 days. After this
longer culture period, the area resorbed per slice
(-86%) (Figure 3A), the number of lacunae (-
51%) (Figure 3B) and the area degraded per
lacuna (-72%) (Figure 3C) were markedly
decreased in ribose-incubated slices compared to
control bones.
To ascertain that the effects of matrix AGEs on
bone resorption that we observed for rabbit
osteoclasts were not species-specific features but
representative of a more general phenomenon,
we performed similar experiments using in vitro
differentiated mouse osteoclasts. Fully
differentiated osteoclasts were detached from
plastic dishes and seeded on control- and ribose-
incubated ivory slices and incubated for 8 days.
As expected, incubation of ivory slices with
ribose increased markedly their pentosidine
content (up to 240 mmol/mol collagen) (Figure
4A), whereas the amount of PYD and DPD
crosslinks did not change significantly (Data not
shown). Consistent with the findings obtained
with rabbit osteoclasts, there was a marked
reduction of the total area resorbed per slice and
the area degraded per lacuna (-44% and -42%,
respectively) (Figure 4B and 4D) when the cells
were seeded on ribose-incubated slices.
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However the number of resorption lacunae was
not altered (Figure 4C).
With both rabbit and mouse osteoclast
experiments, there was no significant difference
of TRAP activity in the culture medium between
control-incubated and ribose-incubated slices,
indicating that the effects observed on bone and
ivory matrix resorption were not due to
differences in osteoclast seeding (data not
shown).
The effects of AGEs on bone resorption were
also investigated directly by measuring the
release of collagen fragments in the conditioned
media of rabbit and mouse osteoclast cultures.
We used two immunological assays, the
Crosslaps for Culture ELISA kit and the Helical
Peptide EIA kit, which respectively assess the
crosslinked and isomerised C-telopeptide
fragment (ECTX) and the helical region of the
D1 chain of type I collagen (42,43). There was a
marked reduction in the concentration of ECTX
released into the culture media of osteoclasts
seeded on ribose-incubated slices, not only after
8 days of culture (Figure 3D and 4E), but also
after 3 days of resorption (Figure 2F). In a
similar manner, the concentration of helical
peptide detected in the culture media from AGE-
modified slices diminished markedly compared
to those from control slices (Figure 3E and 4F).
The reduction of bone (and ivory) matrix
degradation was also confirmed by the decrease
of the hydroxyproline concentration (Figure 3F
and data not shown), which provides a global
read-out of the collagen release in the
conditioned media.
 7/28
Dose-dependent inhibition of bone resorption
by matrix AGEs
In human bone, pentosidine has been shown to
accumulate in an exponential manner with
ageing (14). The maximal range of pentosidine
found in bone tissues varies from 50 mmol/mol
of collagen in femora (14) to 140 mmol/mol of
collagen in vertebrae (44). In order to analyze
the dose-dependent effects of matrix AGEs on
bone resorption mediated by osteoclasts using
concentrations of collagen AGEs closer to the
physiological ranges, protocols for in vitro
matrix AGEs formation were modified. In a first
attempt, slices were incubated in Tris-buffered
saline buffer in the presence of increasing
concentrations of D-ribose (0.2 M, 0.4 M and
0.6 M) for a shorter time (up to 6 days). By this
method, we could not obtain a dose-dependent
formation of pentosidine, as a plateau was
rapidly reached even when lower molarities
(down to 0.05 M) of D-ribose were used (data
not shown). However, when slices were
incubated with the same molarities of D-ribose
(0.2 M), but during various times (2, 4 or 6
days), we could observe a slight dose-dependent
effect for the formation of pentosidine for 4 and
6 days of incubation, although not significant
(Figure 5A). In contrast, the content of the PYD
crosslink did not change (Figure 5B). The dose-
dependent increase of pentosidine was
associated with a dose-dependent reduction in
the concentration of the collagen products
helical peptide (Figure 5C) and hydroxyproline
(Figure 5D) released in the conditioned media of
osteoclasts
. helical peptide product (Figure 6F) released in
To further modulate the concentrations of matrix
AGEs, bone slices were also incubated with
0.3 M aminoguanidine (AMG), -an inhibitor of
AGEs formation (45)- in the presence or absence
of D-ribose (0.6 M) for 6 days. AMG alone did
not affect the concentration of PYD (Figure 5F)
and had no influence on the release of helical
peptide in the culture media (Figure 5G).
However, the presence of AMG partially
inhibited the browning of the bone matrix
induced by the incubation with D-ribose (data
not shown). In the presence of ribose, AMG
markedly inhibited (> 7-fold) the formation of
pentosidine molecules in bone slices (Figure 5E)
and the release of helical peptide (Figure 5G)
and hydroxyproline (Figure 5H) after 8 days of
resorption was significantly higher than in slices
incubated with ribose alone. When PBS was
used instead of TBS during incubation, we found
very similar results both for the formation of
pentosidine in bone slices (Supplemental Figure
1A) and the inhibition of osteoclast-mediated
bone resorption in AGE-containing matrices
(Supplemental Figure 1B).
Because sugars are present in low amounts in
connective tissues (46), we also tested whether
we could induce in vitro the formation of AGEs
using lower concentration of reducing sugars.
We found that incubating bone slices with only
0.05 M ribose for 6 days in PBS induced the
formation of pentosidine (up to 80 mmol/mol
collagen) (Supplemental Figure 2A). This
increase of pentosidine in bone matrix was
associated with a marked decrease of the
concentration of helical peptide product
measured in the culture media of rabbit
osteoclasts after 8 days of culture (Supplemental
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Figure 2B).
Inhibition of bone resorption by matrix AGEs
in human cortical bone
To confirm that the accumulation of pentosidine
in human bone matrix also interferes with bone
resorption mediated by osteoclasts, rabbit
unfractionated bone cells were seeded on
control- and ribose-incubated bone slices and
cultured for 8 days. The increase in pentosidine
content (80 mmol/mol of collagen) (Figure 6A) -
comparable to the physiological levels of
pentosidine observed in vivo- was associated
with a significant reduction of the area resorbed
per slice (Figure 6C), the area degraded per
lacuna (Figure 6E) and the concentration of the
the osteoclast culture media.
Dose-dependent decrease of collagen solubility
by matrix AGEs
AGEs may alter the function of proteins by
creating intramolecular and/or intermolecular
crosslinks. We investigated whether the
inhibition of collagen release in the culture
media of osteoclasts seeded on AGE-modified
mineralized slices could be due to an increase in
matrix insolubility by AGEs. We assessed the
ability of collagen molecules in bone slices
incubated or not with D-ribose to be digested by
pepsin. Pepsin digestion is commonly used to
determine in vitro the solubility of collagen
molecules (47). The presence of AGEs in bone
slices diminished the solubility of collagen
 8/28
molecules in a dose-dependent manner (Table
II).
AGEs receptors are expressed by osteoclasts
We first analyzed by RT-PCR the expression of
the different receptors of AGEs that are
expressed by undifferentiated (osteoclast
progenitors) and fully-differentiated osteoclasts.
We used monocyte/macrophage RAW264.7
cells that have the potential to differentiate into
osteoclast-like cells in vitro in the presence of
recombinant RANK-L (Receptor of Activator of
Nuclear Factor NB-Ligand). We showed that
undifferentiated RAW264.7 cells expressed
genes encoding several AGEs receptors,
including AGE-R1, AGE-R2, AGE-R3 (also
called Galectin-3), and RAGE (Figure 7A).
RAW264.7 cells differentiated in the presence of
RANK-L expressed not only the calcitonin
receptor and the TRAP encoding genes, two
established markers of osteoclasts, but also the
genes coding for the different AGE receptors
(Figure 7A). However, we could not see any
difference in the level of expression of these
receptors between undifferentiated and
differentiated osteoclasts, except for AGE-R2
encoding gene whose expression slightly
increased in mature osteoclasts (Figure 7A). We
then investigated the expression and localization
of RAGE in human and murine osteoclasts by
indirect immunofluorescence (Figure 7B and
Supplemental Figures 3C and 3D). RAGE was
expressed in multinucleated and TRAP-positive
cells that exhibited actin podosomes in vitro
(Figure 7B and Supplemental Figures 3C and
3D), three characteristic features of mature
osteoclasts (36,48). In non permeabilized cells,
RAGE was localized all over the cell surface
(Figure 7B), whereas it localized mainly to
punctuate structures in permeabilized cells
(Supplemental Figures 3C and 3D), most likely
corresponding to Golgi apparatus and the
secretory vesicles.
AGEs inhibit osteoclastic differentiation in a
dose-dependant manner
We then investigated whether exogenous AGEs
could interfere with osteoclastogenesis. We
performed in vitro differentiation assays using
mouse primary and immortalized osteoclast
precursor cells in the presence of AGE-
containing BSA (Figure 8). BSA was used as a
carrier protein for the in vitro formation of
AGEs, performed by its incubation either with
D-ribose (AGE-1), or with D-glucose and lysine
(AGE-2). We also used a commercial glycated-
BSA as a negative control, as this modified BSA
contains only immature glycation products. The
content of pentosidine in the different
preparations of modified BSA is shown in Table
III. We first analyzed the ability of AGE-
modified BSA to stimulate RAGE expression.
We treated undifferentiated RAW264.7 cells
with 50 µg/mL of AGE-1 for 48 hours and
showed that RAGE gene expression was
stimulated (2.5-fold), compared to BSA alone
(Figure 7C and 7D), whereas the level of AGE-
R3 expression level remained unchanged (Figure
7C). We then analyzed the differentiation
process from primary osteoclast progenitors by
assessing the TRAP activity in the different
conditions as well as the number of TRAP-
positive cells with more than 3 nuclei (Figure 8A
and 8B). The presence of two increasing
concentrations of control BSA, as well as of
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glycated-BSA, did not interfere with the
differentiation process. However, the two AGE-
modified BSA inhibited in a dose-dependant
manner the osteoclastic differentiation process,
as the TRAP activity and the number of
multinucleated cells markedly decreased in the
presence of AGE-1 and AGE-2 (Figure 8A and
8B). Similar results were obtained in the
monocytic cell line RAW264.7 (Figure 8C) and
in human primary monocytes (data not shown).
AGE-1 seemed to have a more potent inhibitory
effect than AGE-2. We then determine the
kinetic of the effects of AGEs along the
differentiation process. We used the RAW264.7
cells whose differentiation process is well
documented. The cells were stimulated with
RANK-L (at day 0), then AGE-1 or BSA was
added at different days (from day 0 to day 3) and
the cell phenotype was analyzed at the end of the
differentiation (day 4). We determined the
TRAP activity (Figure 8D) as well as the fusion
process, by counting the cells with more than 3
nuclei (Figure 8E). AGE-1 inhibited totally the
differentiation process when added at day 0 and
partially at day 1, but not significantly thereafter.
These results showed that the first step of
osteoclastogenesis was inhibited, mainly the
commitment of osteoclasts progenitors into pre-
osteoclastic cells.
DISCUSSION
Despite the growing identification of new AGEs
molecules in vitro, few have been characterized
in vivo, and very few have been identified in
 9/28
bone. Pentosidine, the best characterized AGE,
has been shown to accumulate with ageing in
human femora (14) and vertebrae (44). Recently,
imidazolone and NH-carboxymethyllysine (CML)
have been detected in human bone by
immunohistochemistry and the intensity of the
staining has been shown to correlate with the age
of the patients (13). AGEs accumulation may
have deleterious effects in bone tissue as they
modulate the functional properties of target
tissue. Indeed, in different ex vivo models, using
either untreated bone samples (16,17) or bone
specimens where matrix AGEs formation was
induced in vitro (15,49), collagen AGEs have
been demonstrated to have deleterious effects on
bone mechanical properties. Here, we
investigated the role of collagen AGEs on
resorption by osteoclasts and demonstrated that
matrix AGEs inhibited the resorption of bone
and ivory by mature osteoclasts of different
species (Figures 2-6), including human
(Supplemental Figure 3B).
Different methods were used to induce the
formation of matrix-AGEs. In particular, we
compared the effect of incubation of D-ribose
either in phosphate- and Tris-buffered saline
solutions and found that the concentration of
pentosidine obtained was similar in the two
conditions (Supplemental Figure 1A). These
results differ from those of Acharya and
colleagues who found that Tris inhibited the
glycation process of the globular RNase protein,
most likely by trapping the reducing sugar
aldotriose (50). Although the reasons for these
discrepant results are unclear, it may be possible
that differences of reducing sugars and/or the
substrate used in the two studies can be
involved. Indeed, collagen is organized into
fibrils that are embedded in a mineralized matrix
in bone tissue. The use of either type of buffers
also did not influence the degradation of bone
collagen by osteoclasts in vitro (Supplemental
Figure 1B), suggesting that in our experimental
settings data obtained with Tris-buffered saline
solution are valid.
To our knowledge, only one study has reported
the effects of AGEs on bone resorption. Miyata
et al. (51) showed that bone resorption was
enhanced using mouse unfractionated cell
containing osteoclasts when cultured on AGE-
modified dentin slices. However, this conclusion
was mainly drawn by counting the number of
resorption lacunae formed by osteoclasts on the
slices after 4 days in culture. In our study, we
also observed an increase in the number of
resorption lacunae when rabbit unfractionated
bone cells were cultured for 3 days on bone and
dentin slices (Figure 2 and data not shown).
However, when bone resorption was assessed
using specific biochemical markers of collagen
breakdown, we observed a decrease of dentin
collagen fragments released in the culture media
(Figure 2F). The mechanism responsible for the
increase in the number of resorption lacunae
obtained at 3 days of culture is presently
unknown. It is possible that the modification of
bone matrix with AGEs could create a
microenvironment favourable for the adherence
of osteoclasts in the early step of bone
resorption. However, our experiments clearly
indicate that AGEs have an overall inhibitory
effect on bone matrix degradation as we
observed a decrease of type I collagen fragments
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released in the conditioned media after 3 (Figure
2F) and 8 days (Figures 3 and 4) of culture.
AGEs may interfere with the collagenolytic
activity of proteases secreted by osteoclasts,
either by masking the cleavage sites or by
rendering them less accessible to proteases.
Alternatively, AGEs could trap collagen
fragments into the cross-linked bone matrix, by
creating intermolecular crosslinks between
matrix proteins. This hypothesis seems to be
supported by our findings showing that the
presence of AGEs into the bone matrix
decreased the solubility of bone collagen, as
documented by the pepsin digestion experiment
(Table II). Importantly, when the culture period
was extended for up to 8 days, there was a
consistent decrease in both the number and the
area of resorption lacunae formed by osteoclasts
seeded on AGE-modified slices. When seeded
on mineralized slices, osteoclasts alternate
between resorption and migration phases (36).
However, the cellular and molecular
mechanisms regulating or triggering either of
those two processes are still unknown. It is
possible that AGEs contained in collagen
molecules modulate the switch between
resorption and migration states, by inhibiting the
migratory process or by impairing the
completion of the resorption phase. Eventually,
it is also tempting to speculate that AGEs could
accelerate the rate of apoptosis in mature
osteoclasts, as it has been shown that exogenous
AGEs or matrix AGEs could increase cell death
in fibroblasts (52,53). These mechanisms would
explain the decrease of the number of resorption
 10/28
pits and/or in the area resorbed per lacuna for
rabbit cells in AGE-containing slices compared
to control slices. For the experiments where
mouse mature osteoclasts were used, the
resorption assay was performed in the presence
of RANK-L, a factor known to prolong
osteoclasts survival by inhibiting apoptosis (54).
In this context, we did not observe a difference
in the number of lacunae (Figure 4C), but did
find a decrease of the area resorbed per slice
(Figure 4B) and of the area degraded per lacuna
(Figure 4D), supporting a cellular effect of
AGEs on osteoclast survival.
Miyata and colleagues also observed an increase
in dentin resorption -i.e. an increase of the
number of resorption lacunae- when the cells
were cultured for 4 days in the presence of AGE-
modified BSA or AGE-containing E2-
microglobulin (51). We performed similar
experiments using AGE-1 and AGE-2 and rabbit
unfractionated bone cells, and bone resorption
was assessed after 8 days using biochemical
assays. In these conditions, we were not able to
detect any significant differences on collagen
release when osteoclasts were cultured in the
presence of BSA or AGE-modified BSA (data
not shown), indicating that exogenous AGEs did
not interfere directly with the resorption activity
of osteoclasts per se, and more specifically with
the degradation of type I collagen occurring in
the sub-osteoclastic compartment. The same
authors showed that AGEs enhanced
demineralised bone resorption in vivo, when
AGE-modified bone particles were implanted
subcutaneously in rat (51). However, we
previously demonstrated in our laboratory that
this subcutaneous degradation process involves
macrophages but not osteoclasts (55).
Altogether, our findings demonstrate that matrix
AGEs inhibited osteoclast-mediated bone
resorption in vitro.
We demonstrated for the first time that
osteoclastogenesis was totally inhibited in vitro
in the presence of the AGEs, AGE-1 and AGE-
2, but not the intermediate glycation products,
such as glycated-BSA (Figure 8). The inhibitory
effect involves not only the pentosidine
molecule (present in AGE-1), but also
uncharacterized AGEs, as pentosidine was not
formed in AGE-2 (Table III), as expected from
the source of reducing sugars used (32). This
finding indicates that osteoclast progenitors
express specific receptors for AGEs. There are
several molecules reported to interact with
AGEs, among those the AGE-R1/AGE-
R2/AGE-R3 complex of receptors (56), CD36
(57), scavenger receptors class AI/AII (SR-A)
(58), scavenger receptors class B type I (SR-BI)
(59) and RAGE (60). Using RT-PCR, we found
that undifferentiated RAW264.7 cells expressed
AGE-R1, AGE-R2, AGE-R3 and RAGE
encoding genes, and that the level of expression
remained unchanged with the osteoclastic
differentiation (except for AGE-R2). We also
found that AGE-1 up-regulated RAGE
expression (Figure 7). It has been shown that
AGEs themselves have the ability to stimulate
RAGE expression in osteoblastic cells (27), as
well as AGE-R3 (Galectin-3) expression (61).
However, we could not see any change in the
level of expression of AGE-R3 in RAW264.7
cells, probably due to cell-type specificity. This
suggests that AGEs may inhibit
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osteoclastogenesis mainly by a RAGE-
dependent manner, although RAGE-independent
pathways can not be excluded. Further studies
are needed to determine the involvement of the
different AGEs receptors and their downstream
effectors in the inhibition of osteoclastogenesis
induced by AGEs.
We showed that AGEs affect osteoclastogenesis
by targeting directly the osteoclast precursor
cells in vitro. This finding should be confirmed
in vivo, where the osteoclast differentiation takes
place in a microenvironment involving the
presence of different cellular actors, including
stromal cells/osteoblasts, and the regulatory
molecules, including RANK-L and
osteoprotegerin. Collagen AGEs have been
shown to impair osteoblast proliferation and to
inhibit osteoblastic differentiation in a cell-stage
dependent manner (22-25). Knowing that
osteoclastogenesis is intimately linked to
osteoblast differentiation and function in vivo
(62), it is tempting to speculate that AGEs could
also inhibit osteoclastic differentiation in an
indirect manner, by inhibiting the differentiation
of osteoblasts and the subsequent expression of
RANK-L by those cells in vivo. This could
explain the discrepancy in the number of pits
observed using rabbit unfractionated bone cells
and mouse mature osteoclastic cells. The former
is a heterogenous cell population also containing
osteoclast precursors and stromal/osteoblastic
cells able to perform in vitro osteoclastogenesis
when seeded on slices, whereas the latter
contains mainly fully differentiated osteoclasts.
 11/28
Consequently, the decrease in the number of
lacunae observed could be due to an inhibition
of cell differentiation as well.
Whether or not AGEs inhibit osteoclast
differentiation and function in vivo remains an
open issue. Hein et al. (2006) demonstrated that
in bone iliac crest biopsies from osteoporotic
patients, the trabecular bone surface covered by
osteoblasts negatively correlated with the
staining of imidazolone and CML staining in
bone specimens. However, no correlation was
observed with the histomorphometrical markers
of bone resorption, i.e., the eroded surface/bone
surface and osteoclast covered surface/bone
surface, and the intensity of the staining (13),
although this may due to a lack of precision of
bone resorption evaluation. However, bone
specimens were obtained from patients suffering
from osteoporosis, where other parameters
regulate bone resorption -mainly hormonal
factors- than the presence of AGEs alone.
AGEs accumulate with ageing in bone tissue and
may act on bone remodelling at different levels:
through the creation of intramolecular or
intermolecular crosslinks in bone matrix
components AGE may (i) impair the
biomechanical properties of bone collagen and
thus increase the brittleness of the tissue, as it
has been demonstrated for articular cartilage
(8,63) and (ii) decrease the solubility of bone
collagen and impair the normal bone turnover,
thus leading to a decline in AGEs removal in
bone tissue. Concomitantly, through the
interaction with cellular membrane receptors,
AGEs could modulate not only the osteoblastic
differentiation and function but also
osteoclastogenesis, and consequently decrease
bone remodelling and collagen turn-over,
enhancing the accumulation of more AGEs in
bone matrix. Consequence of such effects would
be deleterious for the overall bone strength and
could partially contribute to the increase of
fracture risk that is observed in aging people
(64) and patients suffering from diabetes (20).
In conclusion, we demonstrated for the first time
that bone resorption by osteoclasts is impaired
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by bone and dentin matrix AGEs, likely by
decreasing the solubility of collagen. In addition,
we showed that exogenous AGEs inhibited
osteoclastogenesis in vitro, most likely by
interacting with the cell-surface specific receptor
RAGE. All together these results indicated that
AGEs could contribute to some of the
physiopathological mechanisms of altered bone
remodelling observed with aging and diabetes.
 12/28

ACKNOWLEDGMENTS
We thank Cindy Bertholon for enzymatic and non-enzymatic collagen crosslinks assays and
the determination of hydroxyproline concentrations. We also thank Fabien Lavocat for technical help
with osteoclastogenesis. This work was supported in part by an unrestricted educational grant from Eli
Lilly to INSERM.

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 13/28

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56. Vlassara, H. (2001) Diabetes Metab Res Rev 17(6), 436-443
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K., Shichiri, M., and Horiuchi, S. (1995) Eur J Biochem 230(2), 408-415
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FIGURE LEGENDS

Figure 1. In vitro matrix AGEs formation in cortical bone slices. (A) 3-month-old bovine bone
slices (3 mm-width, 300 µm-thick) were incubated for 60 days in PBS buffer alone (Control) or with
0.2 M D-ribose at 37°C. AGEs formation in the presence of ribose is observed by the browning of the
bone matrix. Pentosidine (B), pyridinoline (C) and deoxypyridinoline (D) concentrations were
determined in control- and ribose-incubated bone slices. Pentosidine formation is induced in the
presence of ribose, whereas the concentrations of enzymatic crosslinks, pyridinoline and
deoxypyridinoline, did not change during the incubation. Statistical non significance (n.s.) for the
differences between control- and ribose-incubated slices using the nonparametric Mann-Whitney U
test are indicated (n=3 slices). n.d., not detected.

Figure 2. Assessment of bone resorption after 3 days of culture with rabbit unfractionated bone
cells. (A) Staining for the TRAP activity of unfractionated bone cells seeded on glass coverslips and
cultured for 3 days. Bar, 10 µm. (B) Lacunae of resorption created by unfractionated bone cells
cultured for 3 days on ivory slices and stained with toluidine blue and hematoxilin. Bar, 80 µm. (C-E)
Unfractionated bone cells, enriched in mature osteoclasts, were seeded on control- or ribose-incubated
bone slices and cultured for 3 days. The area resorbed (C) and the number of resorption lacunae (D)
per slice as well as the area resorbed per lacuna per slice (E) were determined microscopically using
the LUCIA® software. The results are shown as box plots showing the median, and inferior and

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superior quartiles (n=10 slices). (F) Unfractionated bone cells were seeded on control- or ribose-
incubated ivory slices (6 mm-width) and cultured for 3 days. Bone resorption was assessed
biochemically using the Crosslaps Culture ELISA determining the concentration of ECTX released in
the culture media. The results are expressed as mean ± SD for 5 slices. A representative experiment
from at least 3 independent experiments is shown in (C-F). n.s., not significant. ***, p<0.01.

Figure 3. Assessment of bone resorption after 8 days of culture with rabbit unfractionated bone
cells. Unfractionated bone cells were seeded on control- or ribose-incubated bone slices and cultured
for 8 days. Bone resorption was assessed microscopically (A-C) and biochemically (D-F). The area
resorbed (A) and the number of resorption lacunae (B) per slice as well as the area resorbed per
lacuna per slice (C) were determined with the LUCIA® software. The degradation of type I collagen
and its release in the osteoclasts culture media was determined using Crosslaps Culture Elisa (D) and
Helical Peptide EIA (E) assays, and by analysing the concentration of hydroxyproline after media
hydrolysis (F). The results are expressed as mean ± SD for 5 slices. A representative experiment from
at least 3 independent experiments is shown in (A-F). *, p<0.05; **, p<0.02; ***, p<0.01.

Figure 4. Assessment of ivory resorption by mouse mature osteoclasts. (A) Concentration of

pentosidine was determined in ivory slices (6 mm-width) incubated for 60 days in PBS buffer alone
(Control) or with 0.2 M D-ribose at 37°C. The results are expressed as mean ± SD for 3 slices.
Microscopic (B-D) and biochemical (E-F) assessments of resorption mediated by mature mouse
osteoclasts cultured for 8 days on control- or ribose-incubated slices. The area resorbed (B) and the
number of resorption lacunae (C) per slice as well as the area resorbed per lacuna per slice (D) were
determined using the LUCIA® software. The degradation of type I collagen and its release in the
osteoclast culture media was determined using Crosslaps Culture Elisa (E) and Helical Peptide EIA
(F) assays. The results are expressed as mean ± SD for 5 slices. A representative experiment from at
least 3 independent experiments is shown in (B-F). n.s., not significant. **, p<0.02; ***, p<0.01.

Figure 5. Matrix AGEs inhibit osteoclast-mediated bone resorption in a dose-dependant manner.

(A-D) Bone slices were incubated for 6 days in TBS buffer alone (-) or with 0.2 M ribose (Ribose) for
2, 4 or 6 days at 37°C. (E-H) Bone slices were incubated for 6 days in TBS buffer alone (-) or in the
presence of 0.3 M aminoguanidine (AMG) alone, 0.6 M D-ribose alone or both AMG and ribose at
37°C. The concentrations of pentosidine (A and E), and pyridinoline (B and F) were determined in
each condition of incubation. The results are expressed as mean ± SD for 3 slices. Rabbit
unfractionated bone cells were seeded on the different bone slices and cultured for 8 days and bone
resorption was assessed biochemically using Helical Peptide EIA assay (C and G), and by analysing
 17/28

the concentration of hydroxyproline after media hydrolysis (D and H). The results are expressed as
mean ± SD for 5 slices. A representative experiment from at least 3 independent experiments is
shown. Statistical significance or non significance (n.s.) of the differences between control-, AMG-
and/or ribose-incubated slices was determined using the nonparametric Mann-Whitney U test. *,
p<0.05;***, p<0.01. n.d., not detected.

Figure 6. Assessment of osteoclast-mediated resorption on human bone. Human bone slices (3 mm-
width, 300 µm-thick) were incubated for 6 days in TBS buffer alone (Control) or with 0.2M D-ribose
at 37°C and the concentrations of pentosidine (A) and pyridinoline (B) were determined in both
conditions. The results are expressed as mean ± SD for 3 slices. Microscopic (C-E) and biochemical
(F) assessments of resorption mediated by rabbit unfractionated bone cells cultured for 8 days on
control- or ribose-incubated slices. The area resorbed (C) and the number of resorption lacunae (D)
per slice as well as the area resorbed per lacuna per slice (E) were determined as described in the
material and methods section. Data are depicted as box showing medians, 25th and 75th quartiles. The
release of type I collagen in osteoclast culture media was determined using Helical Peptide EIA assay
(F). The results are expressed as mean ± SD for 5 slices. *, p<0.05;**, p<0.02. n.s., not significant.

Figure 7. Expression of receptors for AGEs and localization of RAGE in undifferentiated and
osteoclast-differentiated cells. (A) RT-PCR analysis of the expression of genes encoding the proteins
mentioned in the figure from RAW264.7 cells cultured in the absence (-) or presence (+) of

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recombinant RANK-L. (B) Staining for the TRAP activity of human CD-14 positive cells seeded on
glass coverslips and cultured for 15 days with recombinant RANK-L and M-CSF. Bar, 10 µm. RAGE
indirect immunofluorescence in human mature osteoclasts (non-permeabilized cells). Cell
preparations were counterstained with Hoescht 33258 to visualize the nuclei. (C) RT-PCR analysis for
RAGE and AGE-R3 gene expression in undifferentiated RAW264.7 cells cultured for 48h in the
presence of 50 µg/mL BSA or AGE-1. (D) Quantitative real-time RT-PCR analysis for RAGE gene
expression in undifferentiated RAW264.7 cells cultured as in (C).

Figure 8. AGEs inhibit osteoclast differentiation from primary and immortalized mouse osteoclast
precursor cells in vitro. (A) Mouse bone marrow cells were cultured in the absence (-) or presence
(+) of recombinant RANK-L either alone or with two increasing doses (50 and 100 µg/mL, triangle)
of BSA, glycated-BSA (G-BSA), AGE-1 or AGE-2. The cell phenotype was assessed by counting the
number of TRAP-positive osteoclasts with 3 or more nuclei. (B) Mouse bone marrow cells were
cultured as in (A) but with 50 µg/mL of each BSA derivatives. Osteoclatogenesis was determined by
measuring the TRAP activity in conditioned media. (C) RAW264.7 cells were cultured as in (A) but
with 25 and 50 µg/mL of each BSA derivatives (triangle). TRAP activity was measured in
conditioned media. (B and C) Data were expressed as the percentages of TRAP activity in cells
cultured with RANK-L only (-) and represent means ± SD of quadruplicates. (D and E) RAW264.7
cells were cultured in the presence of RANK-L (added at day 0) together with 50 µg/mL of BSA (-)
or AGE-1 (+) added at different days (from day 0 to day 3). Cells were harvested at day 4 and the
TRAP activity was measured in culture supernatant (D) and the number of TRAP-positive osteoclasts
with 3 or more nuclei was counted (E). Data are expressed as percentages relative to BSA alone for
each day and represent means ± SD of quadruplicates. *, p<0.05 compared to their corresponding
concentration of BSA.
 18/28

Table I. Oligonucleotide primers used for conventional RT-PCR and quantitative real-time RT-
PCR

Gene Primer sequence (strand) Product Temp PCR Reference or

size (bp) (°C) cycles accession no.

Primers used for conventional RT-PCR

RAGE 5’-AGCGGCTGGAATGGAAACTGAACA-3’ (+) 702 58 37 AB061668
5’-GAAGGGGCAAGGGCACACCATC-3’ (-)
AGE-R1 5’-CTCAGAGGGCGAGGACTATG-3’ (+) 440 60 32 NM_007838
5’-GGAGGCCACAAAAGGGCATC-3’ (-)
AGE-R2 5’-AGACTGTGGTGACCAGCACC-3’ (+) 331 60 32 NM_008925
5’-GGAATCCAAGGTAGGCGAGC-3’ (-)
AGE-R3 5’-GTCATGCCCCGCATGCTGAT-3’ (+) 263 60 26 X16834
5’-ACCGCAACCTTGAAGTGGTC-3’ (-)
CTR 5’-GTCTTGCAACTACTTCTGGATGC-3’ (+) 255 54 40 (37)
5’-AAGAAGAAGTTGACCACCAGAGC-3’ (-)
TRAP 5’-CTCTCTGACCACCTGTGCTTCCTC-3’ (+) 292 55 27 (37)
5’-GAACCTCTTGTCGCTGGCATCGTG-3’ (-)

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Gapdh 5’-ATCACTGCCACCCAGAAGAC-3’ (+) 443 57 27 (65)
5’-ACCGCAACCTTGAAGTGGTC-3’ (-)

Primers used for quantitative RT-PCR

RAGE 5’-CTGAAGGCTCTGTGGGTGAG-3’ (+) 162 58 40 NM_007425
5’-CCTCATCCTCCTGGCTTTCC-3’ (-)
Gapdh 5’-TGTGTCCGTCGTGGATCTGA-3’ (+) 76 58 40 (66)
5’-CCTGCTTCACCACCTTCTTGA-3’ (-)
 19/28

Table II. Bone matrix AGEs decreased pepsin-mediated collagen solubilization in a dose-dependent
manner

Experimental Condition TBS D-ribose (0.2 M)

Incubation time 6 days 2 days 4 days 6 days
Pentosidine (mmol/mol collagen) n.d. 7 ± 2* 168 ± 44* 209 ± 42*
Collagen solubility (%) 21.6 ± 1.3 18.5 ± 2.7 11.7 ± 2.3* 12.5 ± 0.5*

Bone slices were incubated for 6 days in TBS buffer alone or with 0.2 M D-ribose for 2, 4 or 6 days at
37°C. Collagen solubility was determined by a limited digestion with pepsin for 4 h at 37°C.
Pentosidine concentrations were also determined in bone slices incubated in the same conditions. The
results are expressed as mean ± SD of the percent of collagen solubility for 4 slices. n.d., not detected.
*, p<0.05 between control- and ribose-incubated slices.

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 20/28

Table III. Pentosidine concentrations in the different BSA derivatives

Experimental Condition BSA Glycated-BSA AGE-1 AGE-2

Pentosidine (pmol/mg of BSA) 5.1 ± 0.5 9.6 ± 2.3 1863.7 ± 10.6 6.0 ± 1.7

BSA was incubated as described in the experimental procedures section to produce AGE-1 and AGE-
2. Glycated-BSA is a commercially available modified-BSA that contains fructosamine, an
intermediate glycation product. Pentosidine concentrations were determined in each BSA derivative
and the results are expressed as mean ± SD (n=3).

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 21/28

Figure 1 Valcourt et al.

A B Pentosidine
1200

1000

mmol/mol collagen
Control Ribose
800

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600

400
3 mm
200
n.d.
0
Control Ribose

C Pyridinoline D Deoxypyridinoline
250 20

n.s. n.s.
mmol/mol collagen

mmol/mol collagen

200
15

150
10
100

5
50

0 0
Control Ribose Control Ribose
 22/28

Figure 2 Valcourt et al.

A B

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C Area resorbed/slice D Pit number/slice
0.20 55
0.18 50
n.s. ***
0.16 45
Area (mm²)

0.14 40
0.12 35
0.10 30
0.08 25
0.06 20
0.04 15
0.02 10
Control Ribose Control Ribose

E Area resorbed/pit/slice F βCTX (nM)

8000 100

7000
80
6000
Area (μm²)

60
5000

4000 *** 40
3000
20
2000 ***
1000 0
Control Ribose Control Ribose
 23/28

Figure 3 Valcourt et al.

A Area resorbed/slice B Pit number/slice C Area resorbed/pit/slice

1.4 400 4000

1.2 350 3500

300 3000
1.0
Area (mm²)

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Area (μm²)
250 2500
0.8
200 ** 2000
0.6
150 1500
0.4
100 1000 **
0.2 ** 50 500

0 0 0
Control Ribose Control Ribose Control Ribose

D βCTX (nM) E Helical Peptide (μg/mL) F Hydroxyproline (μg/mL)

25 1.6 3.5
1.4 3.0
20
1.2
2.5
15 1.0
2.0
0.8
1.5
10 0.6
1.0
*
0.4
5
0.2 *** 0.5
***
0 0 0
Control Ribose Control Ribose Control Ribose
 24/28

Figure 4 Valcourt et al.

A Pentosidine B Area resorbed/slice C Pit number/slice
300 4.5 180
4.0 160
250 n.s.
mmol/mol collagen

3.5 140
Area (mm²)

200 3.0 120

2.5 ** 100
150
2.0 80
100 1.5 60

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1.0 40
50
0.5 20
0 0 0
Control Ribose Control Ribose Control Ribose

D Area resorbed/pit/slice E βCTX (nM) F Helical Peptide (μg/mL)

35000 50 6

30000 5
40
25000
4
Area (μm²)

20000 *** 30
***
3
15000 20
2
10000
10
5000 1
***
0 0 0
Control Ribose Control Ribose Control Ribose
 25/28
Figure 5 Valcourt et al.
A 300
Pentosidine (mmol/mol Collagen)
E 180
Pentosidine (mmol/mol Collagen)
n.s. ***
160
250
140
200 120
100
150
80
100 60
40
50
20
n.d. n.d. n.d.
0 0

B 250
Pyridinoline (mmol/mol Collagen)
F 250
Pyridinoline (mmol/mol Collagen)

n.s. n.s.
200 200

150 150

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100 100

50 50

0 0

C Helical Peptide (μg/mL)

n.s.
G Helical Peptide (μg/mL)
1.0 1.4 n.s. n.s.
1.2
0.8
***
1.0
*** ***
0.6 0.8

0.4 0.6

0.4
0.2
0.2

0 0

D Hydroxyproline (μg/mL) H Hydroxyproline (μg/mL)

7 n.s. 8 n.s. *
6
*
5 6 *
*
4
4
3

2
2
1

0 0
Ribose - 2 days 4 days 6 days AMG - + - +
Ribose - - + +
 26/28

Figure 6 Valcourt et al.

A Pentosidine B Pyridinoline
90 200 n.s.
80

mmol/mol collagen
mmol/mol collagen

70 160

60
120
50
40
80
30
20 40
10
0 0
Control Ribose Control Ribose

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C Area resorbed/slice D Pit number/slice
1.2 275
250
1.0
225
200
Area (mm²)

0.8
175
0.6 150
125
0.4 n.s.
100

* 75
0.2
50
0 25
Control Ribose Control Ribose

E Area resorbed/pit/slice F Helical Peptide (ng/mL)

10000 200

8000 160
Area (μm²)

6000 120

4000 ** 80

2000 40 *

0 0
Control Ribose Control Ribose
 27/28

Figure 7 Valcourt et al.

A RANK-L
B Non-permeabilized Cells
- +

TRAP Activity Staining

AGE-R1

AGE-R2

AGE-R3

RAGE

CTR

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TRAP
H 2O
GAPDH
RAGE
AGE-1

C
BSA

RAGE

AGE-R3

GAPDH
Hoescht

D RAGE/GAPDH
3.0
(Arbitrary units)

2.0
Merge

1.0

0
BSA AGE-1
 28/28

Figure 8 Valcourt et al.

A 140
TRAP-positive osteoclasts (> 3 nuclei)
B 120
TRAP activity

120 100
100
Cell number

80

% Control
80 *
60
60 *
40
* 40

20
20 * *
*
0 0
- + - BSA G-BSA AGE-1 AGE-2
RANKL BSA G-BSA AGE-1 AGE-2

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C 120
TRAP activity

100

80
% Control

60 *
40 *
20 *
*
0
-
BSA G-BSA AGE-1 AGE-2

D 140
TRAP activity
E 140
TRAP-positive osteoclasts (>3 nuclei)

120 120

100 100
% Control

% Control

80 * 80

60 60 *
40 40

20 * 20
*
0 0
- + - + - + - + - + - + - + - +
Day 0 Day 1 Day 2 Day 3 Day 0 Day 1 Day 2 Day 3