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Methylglyoxal Inhibits the Binding Step of Collagen Phagocytosis

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Bacterial infection-induced fibrosis affects a wide variety of tissues, including the periodontium, but the mechanisms that dysregulate matrix turnover and mediate fibrosis are not defined. Since collagen turnover by phagocytosis is an important pathway for matrix remodeling, we studied the effect of the bacterial and eukaryotic cell metabolite, methylglyoxal (MGO), on the binding step of phagocytosis by periodontal fibroblasts. Type 1 collagen was treated with various concentrations of methylglyoxal, an important glucose metabolite that modifies Arg and Lys residues. The extent of MGO-induced modifications was authenticated by amino acid analysis, solubility, and cross-linking. Cells were incubated with fluorescent beads coated with collagen, and the percentage of phagocytic cells was estimated by flow cytometry. MGO inhibited collagen binding (20% of control for 10 mm MGO) in a time- and concentration-dependent manner. MGO-induced inhibition of binding was prevented by aminoguanidine, which blocks the formation of collagen cross-links. MGO reduced collagen binding strength and blocked intracellular calcium signaling. MGO modified the Arg residue in the critical alpha2beta1 integrin-binding recognition sequence of triple helical collagen peptides, whereas MGO-induced cross-linking of Lys residues played only a small role in binding inhibition. Thus, MGO modifications of Arg residues in collagen could be a key factor in the impaired degradation of collagen that promotes fibrosis in chronic infections, such as periodontitis.
MGO effects on collagen receptors. A, cells were incubated with PBS or MGO (1 or 10 mM) collagen-coated magnetite beads. Cells were lysed, and bead-associated proteins were isolated and immunoblotted for integrin 2 subunit or for DDR1. In some lanes, as indicated, increased numbers of beads were analyzed to adjust for the reduced numbers of bound beads caused by MGO treatment. For the top panel, integrin 2 subunit immunoblot, the preparations were as follows. Lane 1, PBS collagen beads, no adjustment; lane 2, 10 mM MGO collagen beads, no adjustment; lane 3, 1 mM MGO collagen beads, no adjustment; lane 4, 10 mM MGO collagen beads, 2-fold adjustment; lane 5, 1 mM MGO collagen beads, 2-fold adjustment; lane 6, 1 mM MGO collagen beads, 4-fold adjustment; lane 7, 10 mM MGO collagen beads, 4-fold adjustment; lane 8, BSA-coated beads; lane 9, 10 mM MGO-treated BSA-coated beads. For the bottom panel, DDR1 immunoblot, the preparations were as follows. Lane 1, PBS collagen beads, no adjustment; lane 2, 1 mM MGO collagen beads, no adjustment; lane 3, 10 mM MGO collagen beads, no adjustment; lane 4, PBS collagen beads, 2-fold adjustment; lane 5, 10 mM MGO collagen beads, 4-fold adjustment; lane 6, 1 mM MGO, 4-fold adjustment; lane 7, PBS collagen beads, no adjustment; lane 8, 1 mM MGO collagen beads, 2-fold adjustment; lane 9, 10 mM MGO collagen beads, 2-fold adjustment. Data are representative of three independent samples analyzed for integrin 2 subunit and DDR1. B, collagencoated beads were incubated with cells in the presence of irrelevant, isotype control antibody (bar 1) or in the presence of 1 g/ml 4B4 antibody (bar 2; to block integrins containing 1 subunits) or with 10 mM MGO collagen beads (bar 3) or with 10 mM MGO collagen beads followed by 4B4 antibody (bar 4) or with collagen beads followed by 2 g/ml P1E6 antibody (bar 5; to block integrin 2 subunit) or with 10 mM MGO collagen beads followed by P1E6 antibody (bar 6). Data are mean S.E. of cells binding fluorescent beads. 4B4 and P1E6 antibody and MGO inhibited collagen bead binding (p 0.001 versus control collagen beads). Data were computed from three replicate cultures for each condition and are representative of three independent experiments. In each replicate, 10,000 cells were counted. C, immunostaining for ligand binding-induced epitope on integrin 1 subunit using the antibody 12G10 in human gingival fibroblasts. Cells were incubated with collagen-coated beads (CCB) (top panel) or MGO-treated beads (lower panel). Block arrows point to staining in the upper left panel and in bound beads for phase-contrast images (right panels). This experiment was repeated three times with similar results.
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Methylglyoxal Inhibits the Binding Step of
Collagen Phagocytosis
*
Received for publication, October 19, 2006, and in revised form, January 16, 2007 Published, JBC Papers in Press, January 17, 2007, DOI 10.1074/jbc.M609859200
Sandra A. C. Chong
, Wilson Lee
, Pam D. Arora
, Carol Laschinger
, Edmond W. K. Young
§
, Craig A. Simmons
§
,
Morris Manolson
, Jaro Sodek
, and Christopher A. McCulloch
‡1
From the
Canadian Institutes of Health Research Group in Matrix Dynamics, University of Toronto, Toronto, Ontario M5S 3E2,
Canada and the
§
Department of Mechanical and Industrial Engineering, University of Toronto, Toronto, Ontario M5S 3E2, Canada
Bacterial infection-induced fibrosis affects a wide variety of
tissues, including the periodontium, but the mechanisms that
dysregulate matrix turnover and mediate fibrosis are not
defined. Since collagen turnover by phagocytosis is an impor-
tant pathway for matrix remodeling, we studied the effect of the
bacterial and eukaryotic cell metabolite, methylglyoxal (MGO),
on the binding step of phagocytosis by periodontal fibroblasts.
Type 1 collagen was treated with various concentrations of
methylglyoxal, an important glucose metabolite that modifies
Arg and Lys residues. The extent of MGO-induced modifica-
tions was authenticated by amino acid analysis, solubility, and
cross-linking. Cells were incubated with fluorescent beads
coated with collagen, and the percentage of phagocytic cells was
estimated by flow cytometry. MGO inhibited collagen binding
(20% of control for 10 m
M MGO) in a time- and concentration-
dependent manner. MGO-induced inhibition of binding was
prevented by aminoguanidine, which blocks the formation of
collagen cross-links. MGO reduced collagen binding strength
and blocked intracellular calcium signaling. MGO modified the
Arg residue in the critical
2
1
integrin-binding recognition
sequence of triple helical collagen peptides, whereas MGO-in-
duced cross-linking of Lys residues played only a small role
in binding inhibition. Thus, MGO modifications of Arg residues
in collagen could be a key factor in the impaired degradation of
collagen that promotes fibrosis in chronic infections, such as
periodontitis.
Connective tissue homeostasis is maintained by fibroblasts
that can synthesize and degrade the collagenous matrix in
response to changes in physiological and pathological condi-
tions. Disruptions to the balance of matrix remodeling may lead
to net loss of collagen or to disorganized overgrowth of collagen
in several pathological conditions (1). In chronic infections of
connective tissues, such as periodontitis, bacterial cell metabo-
lites (2) disrupt matrix homeostasis and promote collagen loss
and fibrosis in collagen-rich tissues (1). At least part of this loss
of homeostasis has been attributed to infection-induced
increase of collagen synthesis and impaired collagen degrada-
tion (3, 4). Although an intracellular phagocytic pathway of col-
lagen degradation by fibroblasts is known to be important for
maintaining homeostasis in mature connective tissues (5), the
effect of bacterial and eukaryotic cell metabolites on collagen
phagocytosis has not been defined.
The recognition and binding of collagen molecules by cell
surface receptors is the initial, rate-limiting step in collagen
phagocytosis by fibroblasts. The principal receptor for type I
fibrillar collagen is the
2
1
integrin (6 –10), which is a critical
mediator of the binding step of collagen phagocytosis (11, 12).
Integrins are composed of transmembrane
and
subunits.
Each subunit exhibits a large extracellular domain, a single-pass
transmembrane domain, and a small cytoplasmic tail. The N
terminus of the integrin
2
-subunit encompasses a 7-fold
repeat. Between repeats II and III, as well as in several other
-subunits, are an additional 200 amino acids designated as the
A (or I) domain (13). The A domain can regulate cell binding to
collagen by recognizing the Gly-Phe-hydroxyproline (Hyp)
2
-
Gly-Glu-Arg motif in collagen, which is considered the princi-
pal collagen binding site for the
2
1
integrin (14 –16).
Prolonged exposure of collagen to bacterial and eukaryotic
cell glucose metabolites affects collagen structure by forming
intermolecular cross-links that can reduce the flexibility of con-
nective tissues and reduce matrix turnover (17). Notably, MGO
has been detected in the fluid draining from periodontitis infec-
tions (18), but the effect of MGO-induced modifications on the
binding and internalization steps of collagen phagocytosis
(which rely on the
2
1
integrin) has not been investigated. To
define a potential role for phagocytosis in periodontitis-in-
duced pathologies of connective tissues, we assessed the effect
of MGO treatment of collagen on the
2
1
integrin-dependent
binding step of collagen phagocytosis. Since collagen turnover
in periodontal connective tissues is faster than in any other
human tissue yet studied (19) and since cultured periodontal
fibroblasts primarily express the
2
1
integrin (20), we used
cells from the periodontium as a model system for defining the
critical, rate-limiting steps in collagen phagocytosis. Here we
provide evidence that MGO-induced modifications of the Gly-
Phe-Hyp-Gly-Glu-Arg motif in collagen inhibit the collagen bind-
ing step of phagocytosis. These data suggest a mechanism that
* This work was supported by the Canadian Institutes of Health Research and
Alpha Omega. The costs of publication of this article were defrayed in part
by the payment of page charges. This article must therefore be hereby
marked advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
1
To whom correspondence should be addressed. Rm. 244, Fitzgerald Bldg.,
150 College St., Toronto, Ontario M5S 3E2, Canada. Tel.: 416-978-1258; Fax:
416-978-5956; E-mail: christopher.mccculloch@utoronto.ca.
2
The abbreviations used are: Hyp, hydroxyproline; MGO, methylglyoxal;
TRITC, tetramethylrhodamine isothiocyanate; PBS, Mg
2
-, Ca
2
-free phos-
phate-buffered saline; DAPI, 4,6-diamidino-2-phenylindole; APG, p-azido-
phenyl glyoxal monohydrate; BSA, bovine serum albumin; PIPES, 1,4-
piperazinediethanesulfonic acid.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 11, pp. 8510 –8520, March 16, 2007
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
8510 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282NUMBER 11 MARCH 16, 2007
at Univ of Toronto - OCUL on August 24, 2007 www.jbc.orgDownloaded from
explains how chronic bacterial infection in close proximity to con-
nective tissues dysregulates collagen degradation, thereby leading
to loss of connective tissue homeostasis.
EXPERIMENTAL PROCEDURES
Reagents—Latex (2-
m diameter) beads were purchased
from Polysciences (Warrington, PA). Antibodies to
-actin
(clone AC-15), TRITC-phalloidin, 4,6-diamidino-2-phenylin-
dole (DAPI),
D-ribose, MGO (Sigma catalogue number
M0252), aminoguanidine, and maleic anhydride were from
Sigma. Bovine serum albumin was purchased from Calbio-
chem. Carboxyl ferromagnetic beads (2–2.9
m) were pur-
chased from Spherotech (Libertyville, IL). Triple helical colla-
gen peptides (36 amino acids) mimicking the
2
1
integrin
binding site of collagen were obtained from Dr. G. Fields (Flor-
ida Atlantic University, Boca Raton, FL). Bacterial collagenase
was from Worthington. Activating
2
integrin antibody (JBS2)
and antibody to
2
integrin (AB1936) were purchased from
Chemicon International (Temecula, CA). Activating
1
inte-
grin antibody (CD29 mouse anti-human) was obtained from
Serotec (Raleigh, NC). Antibody to the discoidin domain recep-
tor 1 (c-20/sc-532) was from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). Antibody to
1
integrin (4B4) was from Beck-
man-Coulter (Burlington, Ontario, Canada). Antibody to
2
integrin (P1E6) was from Chemicon. Antibody to methylg-
lyoxal-AGE (Arg-pyrimidine) was from Biologo (Germany).
The protein cross-linker p-azidophenyl glyoxal monohydrate
(APG) was obtained from Pierce.
Cell Culture and Human Gingival Tissues—Human gingival
fibroblasts (passages 4 –10) were derived from primary explant
cultures as described previously (21). Cells were grown in
-minimal essential medium, 15% (v/v) fetal bovine serum, and
10% antibiotics, maintained at 37 °C in a humidified incubator
containing 5% CO
2
, and were passaged with 0.01% trypsin.
Rat-2 fibroblasts (ATCC CRL 1764; American Type Culture
Collection) were subcultured from frozen stocks and incubated
in Dulbecco’s modified Eagle’s medium (high glucose) contain-
ing 10% fetal bovine serum and antibiotics. Discoidin domain
receptor 1
/
(DDR1
/
) and DDR1
/
fibroblasts (kindly
donated by W. Vogel (University of Toronto) were passaged
with 0.25% trypsin plus 1 m
M EDTA and grown in similar
medium as above. For examination of MGO-induced alter-
ations of tissues in situ, biopsies of human gingiva from healthy
and periodontitis-affected human subjects were obtained (n
3 subjects each) and fixed with paraformaldehyde, and histolog-
ical sections were prepared for immunostaining. The biopsies
were obtained with informed consent according to the specifi-
cations of the Health Sciences Research Ethics Board at the
University of Toronto.
Collagen Beads—Collagen-coated latex beads were prepared
as previously described (20). Briefly, beads were incubated with
1 ml of an acidic solution of bovine type I collagen (2.9 mg/ml;
Vitrogen, Cohesion Technologies), neutralized with 100
lof
1
N NaOH to pH 7.4 to facilitate fibril formation on the beads,
vortexed, and incubated at 37 °C for 10 min. This time period is
known to allow stable fibril formation on the beads (22). BSA-
coated beads were prepared by incubating latex beads with 1 ml
of 1% (w/v) BSA for 30 min at 37 °C. The beads were pelleted,
incubated with control or MGO-containing medium (see
below), sonicated in solution for 10 s, and incubated overnight
at 37 °C in a humidified incubator containing 5% CO
2
.
Collagen was treated with 0.1, 1, or 10 m
M MGO diluted in
PBS overnight or up to 5 days at 37 °C. In some experiments,
collagen was glycated with 0.5
MD-ribose for up to 5 days at
37 °C. Control collagen was prepared in parallel in Mg
2
-,
Ca
2
-free phosphate-buffered saline (PBS) for both methods.
After incubation, the beads were pelleted, washed with PBS,
resuspended in PBS, and counted prior to incubation with cells.
Evaluation of equal loading of collagen on beads was done by
immunoblotting of bead-associated collagen as described (23).
For assessment of bead-associated proteins, identical proce-
dures were used, except proteins were coated on magnetite
beads. Inhibition of MGO-induced cross-linking was obtained
by aminoguanidine (20 m
M) co-incubation of collagen-coated
beads overnight.
Analysis of Cross-linked Collagen—The relative abundance
of cross-linking after MGO was estimated by analysis of
hydroxyproline content after solubilization of collagen by pep-
sin digestion (50
g of pepsin/ml in 0.5 N acetic acid, 23 °C, 16 h)
followed by amino acid analysis using high pressure liquid chro-
matography (Alliance 1690; Waters; Milford, MA; Hospital for
Sick Children, Toronto, Canada). For SDS-PAGE, sample
buffer (50
l; 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol,
50 m
M dithiothreitol) was added to control, collagen-coated
beads or MGO-treated beads. Samples were prepared by heat-
ing beads to 50 °C for 30 min, and the eluates were analyzed by
SDS-PAGE (7.5% acrylamide) followed by silver staining.
Adhesion Assays and Flow Cytometry—Cells were counted
electronically, and collagen beads (yellow-green fluorescent
beads; Ex
max
485 nm, Em
max
515 nm) were loaded on to
the top (i.e. dorsal) surfaces of cells at a 6:1 bead/cell ratio. After
incubation of beads with cells for defined time periods, cells
were washed (PBS), detached with 0.01% trypsin from culture
dishes, and resuspended in PBS, a process that removes loosely
attached, nonspecifically bound beads (24, 25). Samples of cells
were analyzed for bead binding by flow cytometry (Beckman-
Coulter Altra, Miami, FL) to estimate the percentage of cells
with bound fluorescent beads and the relative number of beads
per cell. For
1
or
2
integrin-blocking experiments, cells were
preincubated with 4B4 antibody (1
g/ml) or P1E6 (2
g/ml),
respectively, incubated with beads for 1 h, washed with PBS,
trypsinized, and analyzed by flow cytometry. Similarly, for
2
and
1
integrin activation, JBS2 mouse anti-human monoclonal
antibody (10
g/ml) or CD29 mouse anti-human (10
g/ml)
was added prior to collagen-coated bead exposure and flow
cytometry analysis as described (4).
Evaluation of collagen bead internalization was conducted as
previously described (11). Briefly, fluorescein isothiocyanate/
collagen-coated beads were incubated for specific times with
cells, and subsequent internalization was blocked by cooling on
ice. Fluorescence from extracellular beads was quenched by
trypan blue, thereby permitting fluorescence microscopic dis-
crimination and quantification of external (quenched) and
internalized (nonquenched) beads.
Binding Strength Assay—The relative binding strength of col-
lagen beads to cells was estimated by a shear wash assay (4, 26)
MGO Inhibits the Binding Step of Collagen Phagocytosis
MARCH 16, 2007 VOLUME 282 NUMBER 11 JOURNAL OF BIOLOGICAL CHEMISTRY 8511
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in which binding of beads to cells was examined in situ. Cells
grown in monolayer culture (24-well plates) were incubated
with control or glycated collagen-coated fluorescence beads for
1 h and subjected to increasing numbers of washes with PBS
from 1 to 32 washes with a repeating pipettor (Eppendorf). Cells
were fixed with 3.7% formaldehyde for 15 min, permeabilized
with Triton X-100, washed with PBS, stained with DAPI (5
g/ml), and air-dried. DAPI-stained nuclei were used to iden-
tify the location of each cell, and the number of bound beads
associated with each cell was counted by fluorescence micros-
copy in a 5-mm wide circular zone in the middle of the well. For
each experiment, at least 50 cells were counted in each well, and
four wells were evaluated for each experiment. The experiment
was repeated three times. During washing with the pipette, cells
adhered to the bottom of the culture dish experience local shear
stress due to radial outflow of the ejected fluid over the cell
layer. The shear stress on the cells can be approximated for wall
shear stress produced by the normal impingement of an axi-
symmetric jet on a planar surface. According to Phares et al.
(27), the wall shear stress varies radially outward from the stag-
nation point (the point at which the jet initially hits the cell
surface), increasing to a maximum value (
max
) governed by the
following.
max
44.6
u
0
2
Re
0
1/2
H
D
2
(Eq. 1)
In this equation,
represents the fluid density, H is the jet
height from the planar surface, D is the nozzle diameter, u
0
is
the jet exit velocity, and Re
0
u
0
D/
is the jet Reynolds num-
ber, where
is the fluid kinematic viscosity. This maximum
shear stress occurs at a radial distance of r
max
0.09H from the
jet axis (27). In these experiments, the pipette tip diameter was
D 0.8 mm, the volumetric flow rate was Q 250
l/s, jet
height was estimated to be H 10 mm, and fluid properties
were approximately those of water. Thus, u
0
0.5 m/s and Re
0
400, resulting in a maximum shear stress of
max
3.5 pas-
cals (or 35 dynes/cm
2
)atr
max
0.9 mm.
Cell Spreading—Type I fibrillar collagen-coated plates (Bio-
coat Cell Environments; BD Biosciences) were treated over-
night with MGO or with control vehicle. Cells were plated and
spread for 1, 2, and 4 h, washed with PBS, fixed with 3.7% form-
aldehyde, permeabilized with 0.2% Triton X-100, and stained
with rhodamine-phalloidin (5
M). Cells were washed and air-
dried, and coverslips were mounted with anti-fade solution.
Cells were photographed, and projected surface areas were
measured by digital morphometry.
[Ca
2
]
i
Measurements—Nonfluorescent latex beads were
coated with collagen and glycated overnight with MGO. Beads
were applied to cells that were loaded with fura 2/acetoxym-
ethyl ester (3
M). Intracellular calcium concentration ([Ca
2
]
i
)
was measured with a microscope-based, ratio fluorimeter over
time (28). Only cells that exhibited bound collagen-coated
beads were measured.
Immunoblotting—Cells were spread over type I collagen or
MGO-treated collagen-coated plates for specific time periods.
After spreading, cells were washed with PBS and extracted with
SDS-sample buffer. Protein concentrations were measured
with the Bio-Rad assay using BSA as a standard. The relative
amounts of proteins were normalized for all samples. Proteins
were separated on 7.5–10% gradient SDS-polyacrylamide gels
under nonreducing conditions and transferred to nitrocellu-
lose. Blots were probed with appropriate antibodies for
2
inte-
grin or DDR1. Immunoblots were developed by chemilumines-
cence (ECL; Amersham Biosciences).
Bead-associated proteins including integrins and DDR1 were
analyzed as previously described (29). Briefly, control and
MGO-treated collagen-coated magnetite beads were added to
cells for 1 h. After incubation, medium was removed, and the
cells were washed to remove loosely bound beads. Cells and
beads were scraped with cytoskeletal extraction buffer (0.5%
Triton X-100, 50 m
M NaCl, 300 mM phenymethylsulfonyl fluo-
ride, 10 m
M PIPES, and 1
M phalloidin, pH 6.8). Magnetite
beads were isolated with a magnet (Dynal) at 4 °C, washed with
cytoskeletal extraction buffer, and pelleted, and bead-associ-
ated proteins were released during 20-s sonication in SDS-sam-
ple buffer. Beads were removed and counted to estimate rela-
tive protein concentrations in samples. Differences in the
relative amounts of total proteins were normalized between
samples, and proteins were analyzed by immunoblotting as
described above.
Cross-linking of Collagen—Collagen-coated beads were incu-
bated overnight at 37 °C with APG (10 m
M; pH 7). Beads were
incubated on ice, and cross-linking was activated by UV light (8
min; 2 15 watts at a distance of 5 cm) and verified by SDS-
PAGE. The beads were pelleted and washed. Incubation of col-
lagen beads with cells was performed as described above prior
to flow cytometry analysis.
Inhibition of MGO-induced Lysine Cross-linking of Collagen
Collagen-coated beads were treated with maleic anhydride to
protect lysine sites from MGO-induced cross-linking. Collag-
en-coated beads were incubated overnight with MGO (10 m
M;
37 °C). In some experiments, maleic anhydride protection was
reversed by incubating beads at pH 9 prior to MGO treatment.
Collagen bead binding methods were used as described above,
followed by analysis with flow cytometry. Collagen beads
treated with maleic anhydride alone served as controls.
Mass Spectrometry—Triple helical collagen peptides encom-
passing the
2
1
integrin binding site of collagen (0.5 mg of
peptide at 50 n
M) were dissolved in PBS and treated overnight at
37 °C with 10 m
M MGO. Excess reagent was removed using
protein-desalting spin columns (Pierce) and equilibrated in 25
m
M ammonium bicarbonate. MGO-treated peptide (25 nM)
was vacuum-dried prior to mass spectrometry (Hospital for
Sick Children). The remaining 25 n
M peptide was digested with
trypsin (in 25 m
M NH
4
HCO
3
) to give an E/S ratio of 1:500. The
digestion was allowed to proceed overnight at 37 °C, evaporated
to dryness, and analyzed on a Micromass electrospray ioniza-
tion quantitative time-of-flight mass spectrometer coupled
with a Waters CapLC high pressure liquid chromatography sys-
tem (Hospital for Sick Children).
Statistical Analyses—For all data sets, experiments were
repeated at least three times, and each repeat contained three
replicates. For quantitative data, means and S.E. were calcu-
lated. Comparisons between samples were performed using
either Student’s t test or single factor analysis of variance fol-
MGO Inhibits the Binding Step of Collagen Phagocytosis
8512 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282NUMBER 11 MARCH 16, 2007
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lowed by Scheffe’s test for individual differences. Statistical sig-
nificance was set at p 0.05.
RESULTS
Collagen Model System—We incubated collagen with MGO
(30) for 19 h or with
D-ribose for time periods up to 5 days (31,
32). Treatment of collagen with
D-ribose (5 days of treatment)
inhibited collagen binding to cells (38.0 3.5% of controls; n
4; p 0.01), but overnight treatments with MGO were more
inhibitory (23.6 11.1% of controls for 1 m
M MGO; 14.4
1.7% of controls for 10 m
M MGO; n 4; p 0.001). All further
experiments were performed with MGO-treated collagen,
since the treatments were more efficacious and required less
time.
MGO-treated Collagen—The efficacy of the MGO treat-
ment of collagen bound to beads was examined by amino
acid analysis of eluted collagen from beads. With increasing
concentrations of MGO, there were reduced ratios of Arg to
hydroxyproline (Table 1). Since MGO treatment of Arg
forms imidazolones that hydrolyze to ornithine (33), we also
measured ornithine/hydroxyproline ratios, which increased
with higher concentrations of MGO. The solubility of MGO-
treated collagen samples was tested by pepsin digestion, fol-
lowed by measurement of hydroxyproline content in the solu-
ble fraction. Complete solubilization of control collagen was
obtained after 16-h pepsin treatment, whereas MGO-treated
collagen (1 and 10 m
M) showed limited solubilization, even
after 24 h of pepsin digestion. Of the material that was solubi-
lized in pepsin-digested fractions, there was much less
hydroxyproline content after treatment with higher concentra-
tions of MGO (Fig. 1A). From SDS-PAGE analysis, we also
found evidence for higher molecular mass aggregates of colla-
gen molecules after MGO treatment, which is suggestive of
increased collagen cross-linking (Fig. 1B) or possibly altered
charge profile due to MGO derivatization.
We examined human gingival tissue samples removed from
either healthy (n 3) or periodontitis-affected lesions (n 3)
and, following paraformaldehyde fixation, immunostained
these samples for MGO adducts with a monoclonal antibody to
methylglyoxal-AGE (Arg-pyrimidine). These samples showed
strong staining of the connective tissues adjacent to infected
periodontal pockets but not in healthy tissues (Fig. 1C).
Binding Step of Phagocytosis—MGO treatment of collagen
reduced collagen bead binding to cells to less than one-third of
control values (Fig. 2A), and this effect was more pronounced at
higher MGO doses (Fig. 2B; p 0.01). These effects were not
attributable to a loss of collagen from beads, since measure-
ments of bead-bound collagen by dot blotting (23) showed
equivalent amounts of bound collagen, independent of MGO
treatment (0.026 pg of collagen/bead after incubation in 3
M
collagen solutions; 0.024 pg of collagen/bead after incubation in
3
M collagen solutions followed by MGO treatment). Cells
were also incubated with BSA-
coated beads to assess the specificity
of the MGO treatment effect. There
were low percentages (10%) of
cells that bound BSA-coated beads
(Fig. 2A); however, MGO treat-
ments of BSA-coated beads exerted
no effect on the percentages of cells
with bound BSA beads. Reduced
collagen bead binding was also
observed in Rat-2 fibroblasts at
equivalent reductions as human
gingival fibroblasts (Fig. 2B).
We estimated collagen adhesion
strength by comparing the relative
proportion of cells that bound mul-
tiple beads when cells were incu-
bated with beads at fixed bead/cell
ratios. As reported earlier (11), cells
exhibited a range of collagen bead
binding in that most cells bound one
bead, but a small population could
bind four or more beads, presum-
ably reflecting intrinsic heterogene-
ity in the affinity of collagen recep-
tors in the cell population. Notably,
FIGURE 1. Validation of MGO treatment of collagen. A, collagen solubility after MGO treatment assessed by
hydroxyproline content. Data are mean S.E. of hydroxyproline content. ***, p 0.001 versus control (PBS).
B, SDS-PAGE analysis of collagen gels after MGO treatment (10 m
M). MGO, particularly at 10 mM, promotes
cross-linking of collagen monomers into larger aggregates. C, healthy and periodontitis-affected human gin-
gival connective tissues immunostained for MGO. The arrow points to lateral surface of periodontitis pocket
wall that is stained heavily for MGO.
TABLE 1
MGO-induced modification of collagen
Latex beads were coated with collagen and then treated with MGO at the indicated
concentrations as described under “Experimental Procedures.” Collagen was eluted
from beads and subjected to amino acid analysis. The relative amounts of Arg,
hydroxyproline (OH-Pro), and ornithine were quantified from three independent
samples. MGO treatment of Arg residues forms imidazolones, which hydrolyze to
ornithine.
MGO Arg/OH-Pro ratio Ornithine/OH-Pro ratio
mM
0 0.56 0.07 0.003 0.001
1 0.30 0.04 0.015 0.004
10 0.09 0.03 0.031 0.009
MGO Inhibits the Binding Step of Collagen Phagocytosis
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cultures of human gingival fibroblasts incubated with control
collagen-coated beads showed higher percentages of cells with
multiple bound beads compared with cultures exposed to
MGO-treated collagen-coated beads, which usually exhibited
binding of only one bead (Fig. 2C). These data indicated that
MGO treatment may inhibit the binding strength of high affin-
ity collagen receptors or that collagen beads may bind to each
other under control conditions; conceivably, this interbead
binding is inhibited by MGO. To examine these possibilities in
more detail, we determined if beads adhered to each other
under control and MGO conditions. At concentrations of
beads that replicated the conditions used here but without cells,
the distribution of bead interactions for untreated collagen
beads was measured by flow cytometry: single beads, 94%; dou-
blets, 5%; triplets, 0.3%; quadruplets, 0.06%. For 10 m
M MGO-
treated collagen beads, the distribution was as follows: single
beads, 95%; doublets, 4%; triplets, 0.4%; quadruplets, 0.08%. We
also examined the ability of the cells to bind collagen beads at
high bead concentrations. We incubated beads with cells at
bead/cell loading ratios of 4:1, 6:1, 8:1, 12:1, and 16:1. There was
no observable plateau of bead binding when bead ratios were
increased, even at bead/cell ratios of 16:1 (data not shown),
indicating that under these experimental conditions, collagen
receptors are not limiting.
With a more direct approach to measure the effect of MGO
modifications on collagen binding, we estimated the strength of
cell binding to collagen (4, 26). After an increasing number of jet
washes, cells incubated with control beads showed progressively
lower numbers of beads bound per cell, until reaching a plateau
after four washes (Fig. 2D). In contrast, virtually all MGO-treated
collagen beads were dislodged after one wash. BSA-coated beads
were also washed off almost entirely after a single wash.
We determined whether the effect of MGO treatment on
collagen binding could be increased by prolonged incubations
of collagen with MGO. Only small differences of the percentage
of cells binding collagen beads were evident between the differ-
ent time periods (16 –122 h; Table 2), indicating that the effect
of MGO on collagen binding is maximized within 16 h. Inhibi-
FIGURE 2. Effect of MGO on cell binding to collagen. Collagen-coated bead binding (6 beads/cell; 1-h incubation for all experiments; 3 independent
samples/group). Data are mean S.E. A, cells were incubated with collagen (control or 1 m
M MGO-treated) or BSA-coated fluorescent beads (control or
MGO-treated). For collagen beads, 1 m
M MGO treatment reduced binding 3-fold compared with PBS control (***, p 0.001). Binding of BSA-coated beads was
unaffected by MGO treatment. B, Rat-2 cells and human gingival fibroblasts were incubated with PBS (control)- or MGO-treated collagen-coated beads. Data
are percentages of cells binding MGO-collagen beads compared with PBS-treated collagen beads (***, p 0.001 reduction compared with PBS control). C, cells
were incubated with PBS (control) or MGO (10 m
M) collagen-coated beads and assayed by flow cytometry for the percentage of cells binding 1, 2, 3, or 4 or more
beads. ***, p 0.0001 MGO versus control; *, p 0.1 MGO versus control. D, binding strength was estimated with a jet wash assay. The total number of beads
bound to each DAPI-stained cell was counted and expressed as beads/cell. There were large reductions (less than one-tenth prior to washing) of binding for
MGO-treated collagen and BSA beads after one wash (p 0.001) but only minimal reductions for collagen beads.
TABLE 2
Effect of MGO incubation times on collagen bead binding
Collagen beads were treated with vehicle or MGO (1 mM) for the indicated times
and then incubated with Rat-2 cells for 1 h at 6 beads/cell. The percentage of cells
with bound beads was estimated by flow cytometry. Data are mean S.E. of the
percentage of control cells for the indicated MGO treatment time. Between 16 and
122 h, there was no significant reduction of inhibition (p 0.2).
Duration of incubation 16 h 40 h 88 h 122 h
Collagen bead binding
(percentage of controls)
52.3 2.6% 68.1 5.6% 63.9 5.3% 46.6 3.9%
MGO Inhibits the Binding Step of Collagen Phagocytosis
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tion of collagen binding was affected by collagen bead loading
(Fig. 3A). Cells incubated with higher numbers of MGO-treated
collagen-coated beads exhibited significantly reduced bead
binding as a result of MGO treatment. The inhibition of colla-
gen bead binding by MGO was also dependent on the duration
of cell incubation times with beads, since there was greater inhi-
bition of binding at longer time periods (Fig. 3B).
We assessed whether inhibition of collagen binding was a
specific result of MGO-induced collagen cross-link formation.
Aminoguanidine is a nucleophilic hydrazine compound that
blocks the formation of glucose-derived collagen cross-links
(34, 35). After overnight co-incubations of collagen beads with
MGO and aminoguanidine, collagen bead binding was 90% of
control levels (195 25.1% for 1 m
M MGO and 323 14.0% for
10 m
M MGO; data are percentages of increases compared with
MGO treatment alone).
Cell Spreading and Intracellular Calcium—Lamellipodial
extension around fibrils is required for phagocytosis and intra-
cellular digestion of collagen (12), a component process of
phagocytosis that can be modeled by measuring cell spreading
in vitro (11). After 1, 2, and4hofcell attachment to control or
MGO-collagen, we quantified cell spreading by measuring pro-
jected cell surface area in cultures stained with rhodamine phal-
loidin (Fig. 4A). Cells were more rounded and exhibited 40%
of the surface area when plated on MGO-collagen compared
with control collagen. The reduced spreading of cells on MGO-
collagen was not due to cell death, since these cells spread nor-
mally when replated on control collagen.
During the initial phases of phagocytosis, when cells extend
pseudopodia around collagen beads, intracellular free calcium
ion concentration ([Ca
2
]
i
) is increased, or binding is strongly
inhibited (36). Since matrix glycation can impair intracellular
calcium signaling in endothelial cells (37), we measured [Ca
2
]
i
following collagen bead binding to cells. In experiments using
untreated control collagen beads (single cells in nine separate
cultures), we found a slow, steady increase of [Ca
2
]
i
with an
increase of 41 5n
M Ca
2
over 20 min (p 0.001 compared
with base line) (Fig. 4B), similar to a previous report (36). In
contrast, cells that had bound MGO-treated collagen beads
(single cells in nine separate cultures) showed no increase of
[Ca
2
]
i
, after 20 min (p 0.001 collagen compared with MGO-
collagen). This effect was not due to cell toxicity from the MGO
bound to the collagen, since cells plated on MGO-treated tissue
culture plastic exhibited increases of [Ca
2
]
i
that were similar
to controls (40 n
M increase). As a second control, cells that were
incubated with BSA-coated beads also showed no increase of
[Ca
2
]
i
(data not shown). Finally, to evaluate whether MGO-
treated collagen quenched fura 2 fluorescence, we measured
base line levels of [Ca
2
]
i
but found no effect of the MGO on
fura 2 fluorescence at the isosbestic point (356 nm excitation).
After binding to collagen, fibroblasts internalize the protein,
which is then degraded in phagolysosomes (5). The effect of
MGO on collagen internalization was estimated by incubating
FIGURE 3. MGO effect on collagen coated-bead binding. Three independ-
ent samples were analyzed by flow cytometry for each group. Data are
mean S.E. of MGO-treated beads as a percentage of PBS control bead bind-
ing. A, cells were incubated with PBS control or 10 m
M MGO collagen-coated
beads at increasing bead/cell ratios. MGO effect was enhanced at higher
bead/cell ratios. **, p 0.01 versus PBS control; ***, p 0.001 versus control.
B, cells were incubated with PBS (control) or 10 m
M MGO collagen-coated
beads for the indicated times and assayed for binding. The effect of MGO
treatment was dependent on bead incubation time with cells. ***, p 0.001
MGO treatment versus PBS control.
FIGURE 4. MGO treatment of collagen inhibits cell spreading and calcium
signaling. A, cell spreading after4honcontrol or MGO-treated collagen gels.
Actin filaments were stained with rhodamine phalloidin (500). The pro-
jected surface area of cells was analyzed by morphometry after 1, 2, and 4 h.
***, p 0.001 MGO versus PBS control; **, p 0.01 versus control. B, fura
2-loaded human gingival fibroblasts were incubated with PBS (control) colla-
gen-coated beads or MGO (treated) collagen-coated beads. Intracellular cal-
cium concentration ([Ca
2
]
i
) was estimated in single cells that bound collag-
en-coated beads. MGO collagen did not stimulate increased [Ca
2
]
i
during
lamellipodial extension. For both collagen and MGO-collagen beads, nine
different cell samples were analyzed, and the mean increase of [Ca
2
]
i
from
one cell in each sample was computed.
MGO Inhibits the Binding Step of Collagen Phagocytosis
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cells with nonfluorescent beads coated with fluorescein isothio-
cyanate-collagen. Trypan blue quenching of collagen bead flu-
orescence was used to discriminate internalized from external
beads. The number of internalized beads in 10 m
M MGO-
treated collagen was reduced to about one-quarter of control
levels (Table 3). To determine whether the very low number of
MGO-treated collagen beads that did bind to cells were indeed
capable of being internalized, we adjusted for the greatly
reduced binding of beads after MGO treatment and counted
internalized beads in only those cells with bound beads. For
cells that had bound one or more beads, there were no statisti-
cally significant differences (p 0.2) of internalized beads
between control and MGO-treated samples (Table 3).
Collagen Receptors—Since MGO evidently affects the bind-
ing step of collagen phagocytosis but not the subsequent inter-
nalization step, we examined collagen bead-associated proteins
to characterize the collagen receptors that may be affected by
MGO. After a 1-h period of collagen bead binding, cells were lysed,
and proteins associated with bound beads were immunoblotted to
examine integrin
2
subunit and DDR1, both of which are impor-
tant receptors for fibrillar collagen (6, 38). When bead-associated
proteins were examined, there was greatly reduced integrin
2
subunit and DDR1 protein on MGO-collagen beads, an effect that
was increased by higher concentrations of MGO (Fig. 5A). We also
examined whether the lower numbers of MGO-collagen beads
that bound to cells were indeed capable of interacting with these
receptors. Accordingly, increased amounts of bead-associated
proteins were loaded on gels to adjust for the lower numbers of
bound beads. Under these experimental conditions, no increased
integrin
2
subunit was detected, but there was detectable signal
for the DDR1 associated with 1 m
M MGO and 10 mM MGO-
treated collagen beads (Fig. 5A). These data indicated that the inte-
grin
2
subunit but not DDR1 is involved in MGO-reduced bind-
ing of collagen to cells.
We determined the functional contribution of the
2
1
inte-
grin in collagen binding by preincubating cells with either an
integrin
2
subunit (P1E6) or integrin
1
subunit-inhibitory
antibody (4B4) prior to exposure with collagen beads. After the
addition of 4B4 antibody, binding of collagen beads was
reduced to less than one-fifth of control values (Fig. 5B), in
agreement with earlier data (20), indicating the importance of
the integrin
1
subunit in fibrillar collagen binding by these
cells. Similarly, treatment of cells with P1E6 antibody also
reduced collagen bead binding to cells (one-fifth of control lev-
els). As shown above, 10 m
M MGO treatment of collagen
reduced bead binding to one-quarter of control values. Incuba-
tion of MGO-treated collagen beads with the P1E6 antibody did
not further reduce collagen binding. However, MGO treatment
of collagen beads followed by incubation with the 4B4 antibody
reduced binding further (Fig. 5B), indicating that in concert
with the integrin
1
subunit, other integrin
subunits are
involved; this is consistent with previous findings of measurable
but limited involvement of the
1
1
and
3
1
integrins in phag-
ocytosis by human gingival fibroblasts (20).
Since bead binding could also be mediated by other, nonin-
tegrin collagen receptors, we studied the effect of DDR1 in
MGO-induced inhibition of collagen binding. Binding inhibi-
tion levels of DDR1
/
cells were compared with background-
matched, DDR1
/
control cells. After1hofbead binding,
analysis by flow cytometry showed only a minimal difference of
MGO-mediated inhibition in DDR1 control and null cells
(DDR1
/
-MGO-treated, 30.0 2.9% of control collagen
beads; DDR1
/
-MGO-treated, 26.6 2.8% of control colla-
gen beads; n 3 replicates/group; p 0.2). Thus, DDR1 had no
measurable effect on the inhibitory effect of MGO on collagen
bead binding.
We next studied whether the binding inhibition mediated by
MGO could be reversed with activating antibodies to the inte-
grin
2
and
1
subunits. Human gingival fibroblasts were incu-
bated with activating antibodies (for the integrin
2
subunit
JBS2 and for the integrin
1
subunit CD29) as described (4)
prior to exposure to MGO-treated collagen beads. Although
this treatment enhanced binding of untreated collagen beads,
there was no enhancement of MGO-modified collagen binding
after activation of the integrin
2
and
1
subunits (1 collagen
bead/cell; collagen binding of PBS control, 4.9 0.7% of cell
population; JBS2, 5.2 0.9% of cell population; CD29, 4.4
0.1% of cell population; JBS2 and CD29, 5.2 0.8% of cell pop-
ulation), indicating that the MGO effect could not be reversed
by antibody-induced collagen receptor activation. This effect
was investigated further by analyzing human fibroblasts that
were incubated with collagen or MGO-treated collagen beads
and then immunostained with 12G10 antibody. This antibody
recognizes an epitope in
1
integrins that is exposed on ligand
binding (39). In cells incubated with collagen beads and then
immunostained, there was prominent 12G10 staining around
the collagen beads that was not present in cells incubated with
MGO-collagen beads (Fig. 5C).
Mechanism of Binding Inhibition—Since MGO evidently
increased cross-linking of collagen molecules (Fig. 1B), possibly
by virtue of its capacity to modify Arg and Lys residues (30), we
first explored the effect of collagen cross-linking by exposing
TABLE 3
Effect of MGO on collagen internalization
Fluorescein isothiocyanate-collagen was attached to beads and treated with vehicle or MGO (10 mM; overnight or 16 h). Beads were incubated with Rat-2 cells (6 beads/cell)
for 1, 2, or 4 h and examined by fluorescence microscopy. Fluorescence associated with external beads was quenched by trypan blue. The numbers of cells with internalized
(fluorescent) beads were counted (top row, uncorrected). To determine whether the very low number of MGO-treated collagen beads that did bind to cells were indeed
capable of being internalized, we adjusted for the greatly reduced numbers of cells binding MGO-treated collagen beads by counting only cells with bound beads (assessed
by phase-contrast microscopy). At 10 m
M MGO, this involved an 4-fold correction. Data are expressed as the percentage of cells with internalized beads. For the
uncorrected data (top row) there were significant reductions (p 0.01) at all time periods for the MGO effect. When corrections for binding were computed (bottom row),
there was no significant effect of MGO treatment on collagen bead internalization for all time periods (p 0.2).
Time of incubation 1 h control 1 h MGO 2 h control 2 h MGO 4 h control 4 h MGO
Percentage of cells with internalized collagen beads
(uncorrected for bead binding)
49 7.9% 12 3.6% 89 8.8% 23 4.4% 100 0% 25 6.6%
Percentage of cells with internalized collagen beads
(corrected for bead binding)
52 9.9% 68 4.8% 85 7.5% 92 6.3% 100 0% 100 0%
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collagen coated beads to a chemical cross-linker (APG), which
reacts with the guanidino group of Arg at pH 7 8. Cross-
linking with APG (10 m
M) inhibited collagen binding in
human gingival fibroblasts by nearly 2-fold although not as
effectively as MGO (PBS-treated collagen control 35.7
0.8% of cell population; 1 m
M MGO 12.5 0.4% of cell
population; 10 m
M APG 20.8 1.2% of cell population;
p 0.001). We next determined the effect of MGO-induced
cross-linking of Lys residues by treating collagen-coated
beads with maleic anhydride, a reagent that protects Lys res-
idues from reacting with MGO. With beads pretreated with
maleic anhydride and then treated with MGO overnight and
finally incubated with cells, we found that very little of the
MGO-induced inhibition of collagen binding was attributa-
ble to modifications of Lys residues (percentage of cells bind-
ing beads: control collagen 32.45 0.61%; 10 m
M MGO-
collagen 6.24 0.74%; maleic anhydride/MGO-treated
collagen 6.77 0.73%). Further, reversal of the maleyla-
tion by incubating beads at pH 3 had an insignificant effect
on the MGO effect on collagen binding.
FIGURE 5. MGO effects on collagen receptors. A, cells were incubated with PBS or MGO (1 or 10 mM) collagen-coated magnetite beads. Cells were lysed, and
bead-associated proteins were isolated and immunoblotted for integrin
2
subunit or for DDR1. In some lanes, as indicated, increased numbers of beads were
analyzed to adjust for the reduced numbers of bound beads caused by MGO treatment. For the top panel, integrin
2
subunit immunoblot, the preparations
were as follows. Lane 1, PBS collagen beads, no adjustment; lane 2,10m
M MGO collagen beads, no adjustment; lane 3,1mM MGO collagen beads, no
adjustment; lane 4,10m
M MGO collagen beads, 2-fold adjustment; lane 5,1mM MGO collagen beads, 2-fold adjustment; lane 6,1mM MGO collagen beads,
4-fold adjustment; lane 7,10m
M MGO collagen beads, 4-fold adjustment; lane 8, BSA-coated beads; lane 9,10mM MGO-treated BSA-coated beads. For the
bottom panel, DDR1 immunoblot, the preparations were as follows. Lane 1, PBS collagen beads, no adjustment; lane 2,1m
M MGO collagen beads, no
adjustment; lane 3,10m
M MGO collagen beads, no adjustment; lane 4, PBS collagen beads, 2-fold adjustment; lane 5,10mM MGO collagen beads, 4-fold
adjustment; lane 6,1m
M MGO, 4-fold adjustment; lane 7, PBS collagen beads, no adjustment; lane 8,1mM MGO collagen beads, 2-fold adjustment; lane 9,10
m
M MGO collagen beads, 2-fold adjustment. Data are representative of three independent samples analyzed for integrin
2
subunit and DDR1. B, collagen-
coated beads were incubated with cells in the presence of irrelevant, isotype control antibody (bar 1) or in the presence of 1
g/ml 4B4 antibody (bar 2; to block
integrins containing
1
subunits) or with 10 mM MGO collagen beads (bar 3) or with 10 mM MGO collagen beads followed by 4B4 antibody (bar 4) or with
collagen beads followed by 2
g/ml P1E6 antibody (bar 5; to block integrin
2
subunit) or with 10 mM MGO collagen beads followed by P1E6 antibody (bar 6).
Data are mean S.E. of cells binding fluorescent beads. 4B4 and P1E6 antibody and MGO inhibited collagen bead binding (p 0.001 versus control collagen
beads). Data were computed from three replicate cultures for each condition and are representative of three independent experiments. In each replicate,
10,000 cells were counted. C, immunostaining for ligand binding-induced epitope on integrin
1
subunit using the antibody 12G10 in human gingival
fibroblasts. Cells were incubated with collagen-coated beads (CCB)(top panel) or MGO-treated beads ( lower panel). Block arrows point to staining in the upper
left panel and in bound beads for phase-contrast images (right panels). This experiment was repeated three times with similar results.
MGO Inhibits the Binding Step of Collagen Phagocytosis
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To focus on the effects of MGO on the
2
1
integrin binding
site of fibrillar collagen, we used a triple helical collagen peptide
that encompasses the
2
1
integrin binding site (Gly-Phe-Hyp-
Gly-Glu-Arg) (Table 4) (40). We have shown previously the
utility of this reagent for study of collagen phagocytosis by the
2
1
integrin (23). This peptide contains 3 Arg residues, one of
which is located within the
2
1
integrin recognition sequence.
Fluorescent beads coated with this peptide (1 m
M incubation
solution) bound only weakly to Rat 2 cells (10% of cells bound
collagen peptide beads), and MGO treatment reduced this to
1%. Mass spectrometry analysis of the MGO-treated peptide
demonstrated that all Arg residues, including those within the
integrin recognition sequence, were altered by binding with
MGO (addition of one or two MGOs) and indicated the species
derived from the loss of H
2
O from the modified peptides. This
mass increment is consistent with the notion that these adducts
are probably Arg-derived hydroimi-
dazolone residues of MG-H1 (41).
However, it could not be ascer-
tained whether there was preferen-
tial modification of a particular Arg
residue within the peptide (Fig. 6,
Table 3). Nevertheless, the modifi-
cation of Arg residues was consist-
ent with our finding that MGO
treatment reduced the effectiveness
of tryptic digestion of the MGO-
treated peptide, as detected by mass
spectrometry. Since the peptide
contains no Lys residues, this find-
ing underlines the importance of
MGO-modified Arg residues in the
inhibitory effects on collagen bead
binding.
DISCUSSION
The principal finding of this
report is that MGO treatment of
collagen inhibits the binding step of
collagen phagocytosis, a critical
process in maintaining connective
tissue homeostasis (5, 19). Since the structure and remodeling
of collagen-rich tissues, such as the periodontium, is strongly
affected in periodontitis (1, 3, 5), we suggest that disruptions of
collagen binding in phagocytic fibroblasts (5), possibly by bac-
terial and eukaryotic metabolites, such as MGO (2), may affect
connective tissue homeostasis and contribute to pathology.
Notably, MGO is an important metabolite of eukaryotic and
bacterial cell metabolism (2) and is found in periodontal infec-
tions at increased concentrations (18). Since staining for MGO
was only detectable in tissues directly adjacent to bacterially
infected pockets, we suggest that a major contributor of MGO
in periodontitis lesions is likely to be pathogenic bacteria. The
concentrations of MGO used in the in vitro experiments
reported here are much higher than those found physiologi-
cally, and it will be important to determine in the future if MGO
FIGURE 6. Structural changes induced by MGO. Triple helical peptides (36 amino acids) containing the
Gly-Phe-Hyp-Gly-Glu-Arg sequence, the
2
1
integrin binding site of collagen, were treated with MGO (10 mM).
Digestion with endoproteinase Glu-C and mass spectrometry analysis indicate that MGO treatment selectively
modified arginine residues. No cross-linking of peptides was detected.
TABLE 4
MGO modification sites of collagen triple helical peptides
The sequence was as follows: X-GPOGPOGPOGPOGARGERGFOGERGPOGPOGPOGPO (from C to N terminus, where O represents Hyp, X is CH
3
-(CH
2
)
8
-CO-, and
Z is methylglyoxal).
Endoproteinase Glu-C digest
m/z (measured)
m/z (calculated) m Peptide sequence Modification sites
Da
678.324 678.321 0.003 18–23: (E)RGFOGE(R)
732.337 732.332 0.005 18–23: (E)RGFOGE(R) R
18
Z-H
2
O
750.347 750.342 0.005 18–23: (E)RGFOGE(R) R
18
Z
804.358 804.353 0.005 18–23: (E)RGFOGE(R) R
18
2Z-2H
2
O
822.365 822.363 0.002 18–23: (E)RGFOGE(R) R
18
2Z
1242.627 1242.623 0.004 24–36: (E)RGPOGPOGPOGPO()
1296.639 1296.634 0.005 24–36: (E)RGPOGPOGPOGPO()R
24
Z-H
2
O
1314.647 1314.644 0.003 24–36: (E)RGPOGPOGPOGPO()R
24
Z
1368.661 1368.655 0.006 24–36: (E)RGPOGPOGPOGPO()R
24
2Z-H
2
O
1386.673 1386.665 0.008 24–36: (E)RGPOGPOGPOGPO()R
24
2Z
1711.872 1711.866 0.006 1–17: X-GPOGPOGPOGPOGARGE(R) X-
1765.890 1765.877 0.003 1–17: X-GPOGPOGPOGPOGARGE(R) X-R
15
Z-H
2
O
1783.898 1783.887 0.011 1–17: X-GPOGPOGPOGPOGARGE(R) X-R
15
Z
1837.909 1837.898 0.011 1–17: X-GPOGPOGPOGPOGARGE(R) X-R
15
2Z-H
2
O
1855.917 1855.908 0.009 1–17: X-GPOGPOGPOGPOGARGE(R) X-R
15
2Z
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is indeed found at these concentrations in pathological situa-
tions (18), particularly since MGO at 1m
M can modify inte-
grin binding sites (42).
We found that overnight incubations of collagen with 1 or 10
m
M MGO decreased solubility and hydroxyproline content in
pepsin-solubilized fractions of MGO-treated collagen, indicat-
ing enhanced cross-link formation. The MGO-induced forma-
tion of cross-links strongly affected the binding step of collagen
phagocytosis and is consistent with previous findings showing
that after only 10 h of MGO treatment, cell attachment to col-
lagen is markedly reduced (30).
Collagen Binding—As described earlier (11, 12, 20), the
2
1
integrin is a critical receptor for mediating the binding step of
collagen phagocytosis; this specificity was shown here using an
integrin
2
subunit-blocking antibody. The effect of MGO on
collagen binding was apparently specific as measured in bind-
ing experiments using MGO-treated BSA beads, which showed
no differences in binding compared with untreated BSA beads.
Although we also considered that the discoidin domain recep-
tor 1 (43) may be involved in the MGO inhibition of collagen
binding, cells null for DDR1 showed no difference in the level of
inhibition when compared with DDR1-expressing cells. Con-
ceivably, other collagen receptors, including Endo 180 (44),
may also be involved in mediating phagocytosis but were not
examined here. Indeed, the role of Endo 180 as a gelatin receptor
may explain the small proportion of integrin-independent binding
that we found, possibly due to collagen denaturation in assays that
were conducted near collagen’s melting temperature.
Integrin interaction with ligands is dependent on both affin-
ity and avidity, which, in turn, govern integrin function (45).
Impaired adhesion strength and/or reduced receptor-ligand
interactions may contribute to the decreased collagen binding
observed with MGO-treated collagen. In measurements of
binding strength with a shear force assay (4, 26), similar to BSA
beads, MGO-treated collagen-coated beads were weakly bound
and readily dislodged by shear forces. These data were consist-
ent with studies of binding of multiple collagen-coated beads; in
cells incubated with glycated collagen samples, there was no
binding of multiple beads compared with control collagen.
Since MGO causes collagen to become more insoluble, it is
possible that MGO may enhance the association of collagen
monomers within insoluble collagen structures, possibly by
cross-linking. In this situation, available integrin binding sites
on the surface of the beads would be reduced, because the sur-
face to mass ratio of the collagen fiber is smaller than the mon-
omer. This is a possible explanation for the reduction of colla-
gen bead binding caused by MGO.
Cell spreading and lamellipodial extension around collagen
fibrils is an important step in phagocytosis and subsequent
intracellular digestion of collagen (19). We found that cells
spread poorly on MGO-treated collagen, as reported earlier
(30). Notably, the spreading processes that occur in early stages
of collagen phagocytosis are regulated by intracellular calcium
signaling (11, 36). Previous studies of endothelial cells have
demonstrated an impairment of calcium signaling due to gly-
cation of specific matrix proteins, but the mechanism that
mediates this disruption is unknown (37, 46). We found that
MGO-treated collagen blocked the increases of intracellular
calcium concentration seen in cells treated with control colla-
gen beads, consistent with the notion that MGO inhibits colla-
gen recognition and adhesion.
Since integrin activation is required for cell spreading (47, 48)
and for the collagen binding step of phagocytosis (4, 25), we
considered that antibody-induced activation of integrins could
overcome potential MGO collagen-induced inactivation. How-
ever, the use of integrin-activating antibodies showed no
enhancement of bead binding, indicating that independent of
integrin activation status, MGO collagen is poorly recognized
by collagen receptors.
Mechanism of Binding Inhibition—MGO-induced modifica-
tions of critical amino acids in collagen binding are the most
likely cause of the binding inhibition. Treatment with amin-
oguanidine, a nucleophilic hydrazine compound that blocks the
formation of glucose-derived collagen cross-links by derivatiz-
ing MGO (34), prevented MGO-induced inhibition of collagen
binding. To identify the possible mechanisms involved in inhi-
bition of collagen adhesion, we focused on the structural
changes caused by MGO, primarily the cross-linking of Lys
residues and modifications of Arg residues that are mediated by
MGO (30). Cross-linking of collagen by APG, a chemical cross-
linker that preferentially modifies Arg residues (49), also inhib-
ited the binding step of collagen phagocytosis, supporting the
notion that Arg modifications are important in MGO-induced
impairment of collagen binding. The effect on Arg residues was
investigated further in native collagen by protecting the Lys
residues with maleic anhydride treatment (50) prior to MGO
modification of Arg residues. The resulting Arg-specific alter-
ations to collagen showed effects on collagen binding that were
not statistically different from MGO-collagen in which both
Lys and Arg residues were available for modification.
The sequence Gly-Phe-Hyp-Gly-Glu-Arg is the principal
fibrillar collagen binding site for the
2
1
integrin (14–16) in
fibrillar collagens I and II and is therefore critical for the bind-
ing step of collagen phagocytosis (12). We used a triple helical
peptide (36 amino acids) that contains the
2
1
integrin bind-
ing site of type I collagen (40) and does not contain Lys residues.
By mass spectrometry, we found that Arg residues in this
sequence were modified by MGO, indicating that modification
of Arg residues within the critical
2
1
integrin binding site of
collagen may alter binding to cells. The modification to Arg
residues by MGO with a mass difference of 54 has been pre-
viously reported (41). MGO modifications involved all three
Arg residues in the peptide but with no apparent preference for
a specific Arg residue. These MGO modifications of Arg resi-
dues in the binding sequence of collagen may have a direct
effect on the binding capacity of the
2
1
integrin. Our data are
consistent with recent data showing that MGO-induced mod-
ifications of the Arg-Gly-Asp sequence of type IV collagen
inhibit integrin binding (51) and that MGO formed arginine-
derived hydroimidazolone residues at modification sites in
Arg-Gly-Asp and Gly-Phe-Hyp-Gly-Glu-Arg integrin-binding
sites of collagen, causing endothelial cell detachment, anoikis,
and inhibition of angiogenesis in endothelial cells (42).
Collectively, these findings demonstrate that MGO modifi-
cation of Arg residues in the
2
1
integrin binding region of
type I collagen molecules severely impairs collagen binding
MGO Inhibits the Binding Step of Collagen Phagocytosis
MARCH 16, 2007 VOLUME 282 NUMBER 11 JOURNAL OF BIOLOGICAL CHEMISTRY 8519
at Univ of Toronto - OCUL on August 24, 2007 www.jbc.orgDownloaded from
that is required for phagocytosis of collagen fibers. Since
phagocytosis is an important step for collagen degradation in
mature tissues (5), the binding inhibition resulting from the
modification of Arg residues could be responsible in part for
the fibrosis that occurs in periodontitis.
Acknowledgments—We thank Greg Fields (Florida Atlantic Univer-
sity) for provision of the triple helical collagen peptides and Wolfgang
Vogel (University of Toronto) for provision of the DDR1
/
and
DDR1
/
cells. We are grateful to Laura Silver for assistance with
preparation of the manuscript and the figures.
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MGO Inhibits the Binding Step of Collagen Phagocytosis
8520 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282NUMBER 11 MARCH 16, 2007
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Ethnopharmacological relevance Most Aristolochiaceae plants are prohibited due to aristolochic acid nephropathy (AAN), except Xixin (Asarum spp.). Xixin contains trace amounts of aristolochic acid (AA) and is widely used in Traditional Chinese Medicine. Methylglyoxal and D-lactate are regarded as biomarkers for nephrotoxicity. Aim of the study The use of Xixin (Asarum spp.) is essential and controversial. This study aimed to evaluate tubulointerstitial injury and interstitial renal fibrosis by determining urinary methylglyoxal and D-lactate after withdrawal of low-dose AA in a chronic mouse model. Materials and Methods C3H/He mice in the AA group (n = 24/group) were given ad libitum access to distilled water containing 3 μg/mL AA (0.5 mg/kg/day) for 56 days and drinking water from days 57 to 84. The severity of tubulointerstitial injury and fibrosis were evaluated using the tubulointerstitial histological score (TIHS) and Masson’s trichrome staining. Urinary and serum methylglyoxal were determined by high-performance liquid chromatography (HPLC); urinary D-lactate were determined by column-switching HPLC. Results After AA withdrawal, serum methylglyoxal in the AA group increased from day 56 (429.4 ± 48.3 μg/L) to 84 (600.2 ± 99.9 μg/L), and peaked on day 70 (878.3 ± 171.8 μg/L; p < 0.05); TIHS and fibrosis exhibited similar patterns. Urinary methylglyoxal was high on day 56 (3.522 ± 1.061 μg), declined by day 70 (1.583 ± 0.437 μg) and increased by day 84 (2.390 ± 0.130 μg). Moreover, urinary D-lactate was elevated on day 56 (82.10±18.80 μg) and higher from day 70 (201.10 ± 90.82 μg) to 84 (193.28 ± 61.32 μg). Conclusions Methylglyoxal is induced after AA-induced tubulointerstitial injury, so methylglyoxal excretion and metabolism may be a detoxification and repair strategy. A low cumulative AA dose is the key factor that limits tubulointerstitial injury and helps to repair. Thus, AA-containing herbs, especially Xixin, should be used at low doses for short durations (less than one month).
Thesis
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