Dermatopontin promotes epidermal keratinocyte adhesion via α3β1 integrin and a proteoglycan receptor

Article abstract of DOI:10.1021/bi901066f

Dermatopontin, an extracellular matrix component initially purified from bovine dermis, promoted cell adhesion of the human epidermal keratinocyte cell line (HaCaT cells). HaCaT cells spread on dermatopontin and formed actin fibers. Adhesion of HaCaT cells to dermatopontin was inhibited by both EDTA and heparin and was mediated in part by α3β1 integrin. A synthetic peptide (DP-4, PHGQVVVAVRS; bovine dermatopontin residues 33-43) specifically inhibited adhesion of cells to dermatopontin, and when the DP-4 peptide was coated on the well, it promoted cell adhesion in a dosedependent manner. An active core sequence of the DP-4 peptide was localized to an eight-amino acid sequence (GQVVVAVR). These results indicate that dermatopontin is a novel epidermal cell adhesion molecule and suggest that the DP-4 sequence is critical for the cell adhesive activity of dermatopontin. Adhesion of cells to DP-4 was strongly inhibited by heparin. When HaCaT cells were treated with heparitinase I, the cells failed to adhere to DP-4 but chondroitinase ABC treatment did not influence the adhesion activity. DP-4 specifically interacted with biotinylated heparin, and this interaction was inhibited by unlabeled heparin. DP-4 peptide significantly promoted the adhesion of cells overexpressing syndecans, and syndecan bound to a DP-4 peptide affinity column. These results suggest that HaCaT cells adhere to dermatopontin through α3β1 integrin and a heparan sulfate proteoglycan-type receptor, which is likely a syndecan. We conclude that dermatopontin plays a role as a multifunctional adhesion molecule for epidermal cells.

Full text of DOI:10.1021/bi901066f

Biochemistry 2010, 49, 147–155 147
DOI: 10.1021/bi901066f
Dermatopontin Promotes Epidermal Keratinocyte Adhesion via R3β1 Integrin and
a Proteoglycan Receptor†
Osamu Okamoto,*,‡ Kentaro Hozumi, Fumihiko Katagiri, Naoya Takahashi, Hideaki Sumiyoshi,§ Noritaka Matsuo,§
Hidekatsu Yoshioka,§ Motoyoshi Nomizu, and Sakuhei Fujiwara‡
§
‡Department of Dermatology, and Department of Biochemistry, Faculty of Medicine, Oita University, 1-1 Idaigaoka, Hasama-machi,
Yufu-shi, Oita 879-5593, Japan, and Laboratory of Clinical Biochemistry, School of Pharmacy, Tokyo University of Pharmacy
and Life Sciences, 1432-1, Horinouchi, Hachioji, Tokyo 192-0392, Japan
Received June 24, 2009; Revised Manuscript Received November 23, 2009
ABSTRACT: Dermatopontin, an extracellular matrix component initially purified from bovine dermis,
promoted cell adhesion of the human epidermal keratinocyte cell line (HaCaT cells). HaCaT cells spread
on dermatopontin and formed actin fibers. Adhesion of HaCaT cells to dermatopontin was inhibited
by both EDTA and heparin and was mediated in part by R3β1 integrin. A synthetic peptide (DP-4,
PHGQVVVAVRS; bovine dermatopontin residues 33-43) specifically inhibited adhesion of cells to
dermatopontin, and when the DP-4 peptide was coated on the well, it promoted cell adhesion in a dose-
dependent manner. An active core sequence of the DP-4 peptide was localized to an eight-amino acid sequence
(GQVVVAVR). These results indicate that dermatopontin is a novel epidermal cell adhesion molecule and
suggest that the DP-4 sequence is critical for the cell adhesive activity of dermatopontin. Adhesion of cells to
DP-4 was strongly inhibited by heparin. When HaCaT cells were treated with heparitinase I, the cells failed to
adhere to DP-4 but chondroitinase ABC treatment did not influence the adhesion activity. DP-4 specifically
interacted with biotinylated heparin, and this interaction was inhibited by unlabeled heparin. DP-4 peptide
significantly promoted the adhesion of cells overexpressing syndecans, and syndecan bound to a DP-4 peptide
affinity column. These results suggest that HaCaT cells adhere to dermatopontin through R3β1 integrin and a
heparan sulfate proteoglycan-type receptor, which is likely a syndecan. We conclude that dermatopontin
plays a role as a multifunctional adhesion molecule for epidermal cells.
Dermatopontin is a small 22 kDa extracellular matrix (ECM)1
protein (1), which comprises ∼0.001% of the wet weight
of the dermis (2, 3). It was initially isolated from a bovine dermal
extract during the purification of decorin (1). So far, more than
10 dermatopontin homologues have been identified in five
different mammalian species (1, 4-7) and in 12 different inver-
tebrates (8-14). Mammalian dermatopontins are composed of
183 amino acids, containing five disulfide loop structures (1).
Further, the molecule is tyrosine-rich, especially in the amino-
terminal region (1, 5).
Dermatopontin appears to have multiple and diverse func-
tions. It accelerates collagen fibrillogenesis, makes the diameters
of newly formed collagen microfibrils smaller (15), and forms a
supramolecular complex with both decorin and TGF-β1 (2, 16).
Overexpression of dermatopontin in V16 cells reduced the extent
of cell proliferation, and dermatopontin is expressed in quiescent
cells (6). Another report indicated that dermatopontin enhanced
human prostate cancer cell growth and that transgenic mice
developed prostatic neoplasia (17). These findings suggest that
dermatopontin regulates the cell cycle in a cell-type specific
manner.
Dermatopontin promotes fibroblast and neurogenic cell adhe-
sion (18). The adhesion requires high concentrations of derma-
topontin, and the adhesion is inhibited by decorin, biglycan, or
RGD-containing peptides (18). However, cell surface receptors
for dermatopontin have not been identified. In invertebrates,
dermatopontin from amoebocytes in a Limulus polyphemus crab
shell induces amebocyte aggregation (8). Similarly, dermatopon-
tin from demosponge Suberites domuncula was shown to have
aggregation activity of the demosponge cells at low concentra-
tions (9). These findings from invertebrates suggest that derma-
topontin is an adhesive molecule for diverse cells.
Several reports indicate a change in the expression level of this
protein in progressive systemic sclerosis (19), keloids, leiomyo-
ma (20, 21), and myocardial infarction (7), in which a rearrange-
ment of collagen fibers is observed (22). Since the expression level
of dermatopontin is increased in the myocardial infarct zone (7)
and dermatopontin promotes fibroblast adhesion, we hypothe-
sized that dermatopontin plays a role in wound healing. The
process of wound healing is complex, and several interconnected
steps occur, such as ECM deposition, cell proliferation, and cell
migration.
†This work was supported in part by a grant from the Ministry of
Education, Culture, Science, Sports, and Technology of Japan to O.O.
*To whom correspondence should be addressed: Department of
Dermatology, Faculty of Medicine, Oita University, 1-1 Idaigaoka,
Hasama-machi, Yufu-shi, Oita 879-5593, Japan. Telephone: þ81 97 586
5882. Fax: þ81 97 586 5889. E-mail: ookamoto@med.oita-u.ac.jp.
1Abbreviations: ECM, extracellular matrix; SDS-PAGE, sodium
dodecyl sulfate-polyacrylamide gel electrophoresis; EDTA, ethylene-
diaminetetraacetic acid; PBS, phosphate-buffered saline; BSA, bovine
serum albumin; PVDF, polyvinylidene fluoride; SD, standard devia-
tion.
Wound healing requires many different cell types, growth
factors, and ECM proteins (23-25). Interaction between ECM
proteins and cell surface receptors, such as integrins, syndecans,
and growth factor receptors, is important for the healing
r
2009 American Chemical Society
Published on Web 11/24/2009
pubs.acs.org/Biochemistry

148 Biochemistry, Vol. 49, No. 1, 2010
Okamoto et al.
process (26-28). These receptors are ubiquitously expressed in
adult tissues, suggesting that ECM components, such as collagen,
laminin, and proteoglycans, bind to various types of cells through
the receptors.
incubator. After incubation, the wells were rinsed once with
warm PBS, and the attached cells were fixed with 1% glutar-
aldehyde for 30 min and then stained with 0.1% crystal violet for
1 h. After staining had been completed, the wells were rinsed with
running water and dried. Finally, the dye was eluted with 0.1%
Triton X-100 for 30 min, and the absorbance at 595 nm was
measured with an ELX808 UV spectrometer (BioTek, Winooski,
VE). In inhibition experiments, dermatopontin was coated at a
concentration of 5 μg/mL and EDTA, heparin, antibodies, and
peptides were included during the cell adhesion assay.
For the enzyme treatment of the cells, HaCaT cells were first
treated with cycloheximide (CHx) at 2 μg/mL at 37 °C for 2 h,
and then the cells were detached with EDTA. The same con-
centration of CHx was included in all the following steps. The cell
suspension was treated with either 10 mIU/mL heparitinase I
or 50 mU/mL chondroitinase ABC at 37 °C for 30 min. For
control, PBS was added to the cell suspension. After the enzyme
treatment, cell adhesion was assessed for 1 h.
Although dermatopontin is mainly expressed in the dermis, its
biological activity may not be limited to the dermis since dermal
components can regulate the behavior of epidermal cells during
skin repair (29-31). Here, we examined the biological role of
dermatopontin with keratinocytes. We found that dermato-
pontin promoted keratinocyte adhesion, and active sequences
in dermatopontin for keratinocyte adhesion were identified
using synthetic peptides. We also characterized the cellular
receptors for dermatopontin. Together with its role in the dermis,
these new findings suggest that dermatopontin plays a role in skin
repair.
MATERIALS AND METHODS
Materials. Dermatopontin was purified from newborn calf
dermis as reported previously (2). Biotin-conjugated heparin
(molecular mass of approximately 15 kDa) was purchased from
Sigma (St. Louis, MO). Heparitinase I and chondroitinase ABC
were purchased from Seikagaku Kogyo (Tokyo, Japan). Porcine
intestinal heparin and fluorescamine were obtained from Wako
(Osaka, Japan). Functional blocking anti-integrin subunit anti-
bodies (FB12 for R1, P1E6 for R2, ASC-1 for R3, P1D6 for R5,
NKI-GoH3 for R6, and 6S6 for β1) were obtained from
Chemicon (Temecula, CA). Antibodies used for Western blotting
(I-19 for R3, H-104 for R5, H-87 for R6, and N-20 for β1) and
anti-syndecan-1 antibody (DL-101) were obtained from Santa
Cruz Biotechnology (Santa Cruz, CA). An affinity support, Affi-
gel 10, was obtained from Bio-Rad (Hercules, CA). CNBr-
activated Sepharose 4B and enzyme chemiluminescent (ECL)
reagent were obtained from GE Healthcare (Buckinghamshire,
U.K.). Horseradish peroxidase (HRP)-conjugated streptavidin
was obtained from Pierce (Rockford, IL). ARH-77 human
lymphoid cell lines transfected with syndecan constructs were
provided by Y. Yamada from the National Institutes of Health
(Bethesda, MD).
Cell Culture. The human epidermal keratinocyte cell line
(HaCaT cells) was cultured as a monolayer in 75 cm2 bottles at
37 °C in a humidified 5% CO2/95% air atmosphere. The cells
were maintained in Dulbecco’s modified Eagle’s medium
(DMEM) containing 10% (v/v) fetal calf serum (FCS). Two cell
lines were used, namely, ARH-77- Synd-1 and ARH-77- Cont
which express recombinant syndecan-1 and an empty vector,
respectively (32, 33). These cells are derivatives of an ARH-77
human lymphoid cell line that expresses small amounts of
heparan sulfate proteoglycan (HSPG) on the cell surface but
does not express syndecan (34, 35). These cell lines were main-
tained in a suspension culture in RPMI 1640 medium supple-
mented with 5% FCS and antibiotics (penicillin at 100 U/mL and
streptomycin at 100 μg/mL).
Affinity Chromatography and Western Blotting. HaCaT
cells were extracted in 50 mM Tris-HCl and 0.15 M NaCl (pH
7.40) containing 100 mM n-octyl β-D-glucoside (n-OG), 1 mM
CaCl2, and MgCl2 for 1 h on ice. Dermatopontin was immobi-
lized on Affi-gel 10 in 0.1 M MOPS buffer containing 80 mM
CaCl2 and 6 M urea (pH 7.5) at a protein (μg):bead slurry
(μL) ratio of 1:1, according to the manufacturer’s instructions.
BSA was immobilized in 0.1 M MOPS buffer in the same
manner. The beads were added to the cell extract and incu-
bated at 4 °C for 2 h. The beads were placed in a column, and
they were rinsed with buffer A [50 mM Tris-HCl (pH 7.40)
containing 25 mM n-OG, 1 mM CaCl2, and 1 mM MgCl2]. The
column was eluted with buffer A containing 10 mM EDTA.
Aliquots of each fraction from the affinity columns were sub-
jected to 7.5% SDS-PAGE under reducing conditions and
blotted onto a PVDF membrane. Integrins were identified with
anti-integrin antibodies, followed by the HRP-conjugated anti-
mouse IgG antibody. Images were visualized using an ECL
reagent. Under nonreducing conditions, the integrin R3 subunit
was detected at a position near 150 kDa both in the cell extract
and in the gel-bound fraction. Little reactivity with antibodies
against R5 and β1 subunits was found under the nonreducing
conditions. Therefore, the detection of integrin subunits was
conducted under reducing conditions. For peptide affinity
chromatography, DP-4 peptide with additional glycine at the
N-terminus (GPHGQVVVAVRS) was immobilized on CNBr-
activated Sepharose 4B. The n-OG extract of HaCaT cells was
added and the mixture was incubated overnight at 4 °C. Then,
the beads were treated with heparitinase I (0.1 mIU/mL) and
with chondroitinase ABC (0.5 mU/mL) for 18 h at room
temperature in 40 μL of buffer containing 40 mM Tris-HCl
(pH 8.0), 40 mM NaOAc, 5 mM Ca(OAc)2, 0.01% BSA, and
complete protease inhibitor cocktail, and the supernatant frac-
tion of each sample was collected. Eluates were subjected to 7.5%
SDS-PAGE under reducing conditions, and syndecan was
detected as described above by Western blotting.
Cell Adhesion Assay. Dermatopontin and synthetic peptides
were coated on a 96-well plate in 0.14 M NaCl and 30 mM
phosphate buffer (pH 7.3) overnight at room temperature. After
immobilization, the wells were rinsed once with PBS and then
blocked with 1% BSA in PBS for 1 h, followed by three washes
with PBS. A monolayer of cultured cells was detached with 5 mM
EDTA in PBS, and the cells were suspended in DMEM. The
ARH-77 cell lines were collected by centrifugation and then
suspended in RPMI 1640. The cells were inoculated in the wells at
a density of 30000 cells/well and were incubated at 37 °C in an
Cytoskeletal Staining. Dermatopontin (10 μg/mL) was
coated on a silan-coated glass plate (Dako Japan, Kyoto, Japan),
and cell adhesion for 1 h was performed as described above. The
adherent cells were fixed with 70% methanol for 10 min and then
blocked with 10% skim milk in PBS for 30 min. Actin filaments
were stained with fluorescein isothiocyanate-conjugated phalloi-
din (Sigma-Aldrich, St. Louis, MO), with 3 ꢀ 5 min intervals of
rinsing with PBS. In some experiments, synthetic peptides were

Article
Biochemistry, Vol. 49, No. 1, 2010 149
coated on the dish instead of dermatopontin and the dishes were
processed identically as described above.
Synthetic Peptides of Dermatopontin. Peptides were gen-
erally were 12 amino acid residues in length and overlapped
with neighboring peptides by four amino acids as described
previously (36). If the N-terminal amino acid was either gluta-
mine or glutamic acid, one amino acid was extended to avoid
pyroglutamine formation. Cysteine residues were omitted to
avoid the influence of disulfide bonds. Peptides were synthesized
manually by the Fmoc-based solid-phase method with a C-
terminal amide form (36). N,N-Dimethylformamide (DMF)
was used as a solvent. Diisopropylcarbodiimide/N-hydroxyben-
zotriazole and 20% piperidine in DMF were used for condensa-
tion and deprotection of Fmoc groups, respectively. Respective
amino acids were condensed using 4-(20,40-dimethoxyphenyl-
Fmoc-aminomethyl)phenoxy or NovaSyn TGR resin in a step-
wise fashion. Side chains of aspartic acid, glutamic acid, serine,
threonine, and tyrosine were protected by tert-butyl. Asparagine,
glutamine, and histidine were protected by trityl. Lysine was
protected by tert-butoxycarbonyl. Arginine was protected by
2,2,5,7,8-pentamethyldihydrobenzofuran-5-sulfonyl. The pro-
tected peptide resins were cleaved from the resin and deprotected
via incubation in a trifluoroacetic acid/thioanisole/m-cresol/
ethanedithiol/H2O mixture (80:5:5:5:5) for 3 h at room tempera-
ture. Purification was performed using HPLC on a Mightysil RP-
18 (5 mm ꢀ 250 mm, Kanto Chemical, Tokyo, Japan) column
with a water/acetonitrile gradient containing 0.1% trifluoroacetic
acid. The purity and identity of the peptides were confirmed with
an analytical HPLC and an electrospray ionization mass spectro-
meter at the Central Analysis Center, Tokyo University of
Pharmacy and Life Sciences.
Interaction between the Peptides and Heparin. The inter-
action between the peptides and heparin was analyzed as
previously reported (37). Synthetic peptides were coated on the
wells at 10 μg/mL and blocked by BSA as described in the section
on cell adhesion. Biotinylated heparin was dissolved in PBS
containing 0.1% BSA at 10 μg/mL and added to the wells, and
the wells were incubated at room temperature for 1 h. The wells
were rinsed with PBS containing 0.1% BSA three times, and then
the bound biotinylated heparin was detected by HRP-conjugated
streptavidin according to the manufacturer’s instructions.
Finally, the color was allowed to develop via addition of
0.057 M citric acid and 0.086 M disodium hydrogen phosphate
containing 10 mM 2,20-azidobis(3-ethylbenzothiazoline-6-sulfo-
nic acid) and 0.03% hydrogen peroxide in the dark. The color
reaction was terminated by adding aliquots of 0.1 M citric acid
containing 1.5 mM sodium azide, after which the absorbance at
405 nm was determined. For inhibition experiments, heparin was
included during the assay.
F
IGURE 1: Adhesion of HaCaT cells to dermatopontin. In the top
panel, dermatopontin was coated with various concentrations and
HaCaT cells were incubated for 30 min (O) or 1 h (4). Data are
expressed as means ( SD of triplicate results. (A-E) Stains of
adhered HaCaT cells after a 1 h incubation on dermatopontin.
Dermatopontin concentrations were (A) 0.625, (B) 1.25, (C) 2.5,
(D) 5, and (E) 10 μg/mL. The magnification was 200ꢀ. (F) Staining
of phalloidin. The magnification was 400ꢀ. Experiments were
performed three times and gave similar results.
RESULTS
Dermatopontin Is a Potent Adhesion Molecule for Ha-
CaT Cells. Cell adhesive activity of dermatopontin was exam-
ined using the human epidermal keratinocyte cell line (HaCaT).
Dermatopontin induced strong HaCaT cell adhesion in a dose-
dependent manner (Figure 1, top panel). At 30 min, an adhesion
plateau was observed at 5 μg/mL dermatopontin, and at 1 h, an
adhesion plateau was reached at 2.5 μg/mL. A large fraction of
the adhered HaCaT cells showed spreading at 1 h on coated
dermatopontin at concentrations of >2.5 μg/mL (Figure 1C-E).
On the basis of the results, cell adhesion was conducted with a 1 h
incubation in the following experiments unless otherwise men-
tioned. Upon adhesion to dermatopontin, actin filaments were
organized (Figure 1F), but the level of organization was lower
than the levels of those formed in the cells which adhered to type I
collagen (data not shown). Fibroblasts also adhered to derma-
topontin in a dose-dependent manner; however, >20 μg/mL
dermatopontin was needed to reach a plateau, and the final level
of adhesion (0.35) was lower than that of HaCaT cells (1.4) (data
not shown). These results suggested that dermatopontin pro-
motes cell adhesion in a cell-type specific manner.
Calculation of Immobilization Efficiency. After immobi-
lization of peptides on the plate and rinsing as described above,
150 μL of PBS containing 3% Triton X-100 was added to each
well, and the wells were incubated for 15 min. Then aliquots
of the same buffer containing 20 μL of fluorescamine in acetone
(1 mg/mL) were added. Fluorescence emission at 465 nm was
determined using a Spectra Fluor Plus microplate fluorometer
(Tecan, Salzburg, Austria) with excitation at 405 nm. Coating
efficiencies (percent) were determined relative to corresponding
peptide standards made in PBS containing 3% Triton X-100.
Statistical Analysis. Statistical analysis was conducted using
the Student’s t test, and a P value of <0.05 was considered to be
statistically significant.
Next, we examined the effect of EDTA and heparin on
adhesion of cells to dermatopontin (Figure 2). Adhesion of
HaCaT cells to dermatopontin was completely inhibited by
EDTA at concentrations of >2 mM (Figure 2A). In con-
trast, adhesion was partially inhibited by heparin (Figure 2B).
These results suggest that the adhesion of HaCaT cells to

150 Biochemistry, Vol. 49, No. 1, 2010
Okamoto et al.
F
IGURE 2: Effect of EDTA and heparin on adhesion of cells to
dermatopontin. EDTA (A) and heparin (B) solutions in DMEM
were applied to the dermatopontin-coated wells and were incubated
for 5 min at room temperature. HaCaT cells were added to the wells
and incubated for 1 h. The horizontal lines represent the final
concentrations of EDTA (A) and heparin (B). Data are expressed
as mean ( SD of triplicate results. Experiments were performed three
times and gave similar results.
F
IGURE 3: Identification of an integrin-type cell surface receptor for
dermatopontin. (A) Inhibition of adhesion of cells to dermatopontin
by anti-integrin functional blocking antibodies. Six antibodies
against different integrin subunits were mixed with HaCaT cells in
suspension, and the cell adhesion assay was performed. PBS was
added to the cell suspension as a control. Antibody concentrations
were 10 μg/mL for anti-R3 and 50 μg/mL for other antibodies. The
incubation time was 30 min. Asterisks indicate p < 0.01. Data are
expressed as means ( SD of triplicate results. Experiments were
performed three times and gave similar results. (B) Dermatopontin
affinity chromatography. The n-OG extract of HaCaT cells was
passed over a dermatopontin column, and the eluted fractions were
probed with anti-integrin R3 (top panel), β1 (middle panel), and R5
(bottom panel) subunit antibodies. Lane 1 contained the whole cell
extract, and lanes 2-4 contained the antibody reaction mixtures in
the second to fourth fractions, respectively, eluted from the derma-
topontin column by 10 mM EDTA. Lane 5 contained the fraction
eluted from the BSA column. Molecular masses (kilodaltons) are
indicated at the left of each panel.
dermatopontin is cation- and heparin-dependent, and that
dermatopontin interacts with multiple receptors.
Dermatopontin Binds to R3β1 Integrin. Since the inhibi-
tion of cell adhesion by EDTA may indicate the involvement of
integrins as a receptor, a panel of functional blocking antibodies
against integrin subunits was tested for blocking adhesion of cells
to coated dermatopontin. Both anti-integrin R3 and β1 subunit
antibodies strongly inhibited adhesion of cells to dermatopontin,
whereas other anti-integrin subunit antibodies were inactive
(Figure 3A). We also confirmed the receptor interaction using
affinity chromatography. Chromatography of a cell extract
using a dermatopontin-conjugated gel revealed that both R3
and β1 integrin subunits were eluted by 10 mM EDTA
(Figure 3B), while trace amounts of these subunits were seen
in the eluted fractions from a BSA-conjugated gel (Figure 3B,
lane 5). In addition, the R5 integrin subunit, which is also
expressed on HaCaT cells, did not bind to the dermatopontin-
conjugated gel (Figure 3B). Taken together, these results suggest
that binding of R3β1 integrin is involved in adhesion of HaCat
cells to dermatopontin.
Peptide Screening of Cell Binding Sequences in Derma-
topontin. To identify the cell-binding sites on dermatopontin, 16
synthetic peptides covering most of dermatopontin sequence
were generated (Table 1). The 16 peptides were screened for their
ability to block adhesion of cells to dermatopontin. As shown in
Figure 4A, the DP-4 peptide significantly inhibited adhesion of
cells to dermatopontin to the background level, whereas the rest
of the 15 peptides were inactive (Figure 4A). Next, these synthetic
peptides were coated on wells, and their cell adhesion activity was
examined (Figure 4B). DP-4 peptide induced strong cell adhe-
sion. We also found that DP-8 and DP-13 peptides had cell
adhesion activity, but these activities were lower than that of the
DP-4 peptide (Figure 4B). The remainder of the peptides were
inactive in this assay. These results suggested that the DP-4
sequence is involved in adhesion of cells to dermatopontin.
Characterization of Adhesion of HaCaT Cells to the DP-
4 Peptide. Next, we characterized adhesion of cells to the DP-4
peptide. A scrambled peptide DP-4S (AVRVSQVPHGV) was
used as a control. The DP-4 peptide exhibited a dose-dependent
cell adhesion with a plateau at 3-5 μg/mL (Figure 5, top panel),
while DP-4S did not exhibit any activity. The scrambled peptide
also did not inhibit adhesion of cells to dermatopontin (data not
shown). These data suggest that the activity of DP-4 is sequence
specific. As observed with dermatopontin, some of the cells that
adhered to DP-4 at concentrations of >3 μg/mL were spread, but
the cells remained round when attached to the DP-4 peptide at
lower concentrations (Figure 5A-E). The attached and spread
cells had fewer actin fibrils as shown in Figure 5F, compared to
those that attached to dermatopontin.
Determination of the Essential Amino Acid Sequence in
the DP-4 Peptide Required for Cell Adhesion. To determine
the minimum active sequence of the DP-4 peptide needed
to induce cell adhesion, deletion peptides of DP-4, named

Article
Biochemistry, Vol. 49, No. 1, 2010 151
Table 1: Synthetic Dermatopontin Peptidesa
aPeptide sequences are expressed in the one-letter code, and the locations of the peptides are expressed as the numbers of amino acids within the bovine
dermatopontin sequence. Cysteine residues have been omitted, and short peptides interrupted with cysteines were not synthesized (from amino acid 114 to 125).
F
IGURE 4: Determination of the cell adhesion site on dermatopontin.
(A) Inhibitory activity of synthetic peptides for adhesion of cells to
dermatopontin. HaCaT cells were incubated with the synthetic pep-
tides (200 μg/mL) listed in Table 1 on a dermatopontin-coated plate,
and adhesion was measured. (B) Cell adhesion activity of synthetic
peptides. Synthetic peptides werecoated at 4 μg/mL, and cell adhesion
was measured. The coating efficiencies of the peptides were 6-10%
(DP-1-5, DP-10, DP-11, DP-15,and DP-16), 11-15% (DP-6, DP-7,
DP-9,and DP-13), and 18% (DP-12). Because DP-4 was poorly
reactive with fluorescamine, the calculation was done using DP-4a
as shown in Figure 6. Thecoating efficiencies of DP-8 and DP-14 were
not available because of a poor reactivity with fluorescamine. In
panels A and B, the incubation time was 30 min, and asterisks indicate
p < 0.01. Data are expressed as means ( SD of triplicate results.
Experiments were performed three times and gave similar results.
F
IGURE 5: Adhesionof cells to the DP-4 peptide. Inthe top panel, the
DP-4 peptide was coated as indicated on the horizontal line, and cell
adhesion was performed. For a control, scrambled peptide DP-4S
was coated. Experiments wereperformed three times and gavesimilar
results. (A-E) Stains of adhered cells. DP-4 concentrations were (A)
1.56, (B) 3.13, (C) 6.25, (D) 12.5, and (E) 25 μg/mL. The magni-
fication was 200ꢀ. (F) Staining of phalloidin. The magnification
was 400ꢀ.

152 Biochemistry, Vol. 49, No. 1, 2010
Okamoto et al.
DP-4a-4i, were produced as indicated in Figure 6. The inhibi-
tory ability of the deletion peptides with respect to adhesion
of cells to dermatopontin was examined (Figure 6, left panel).
DP-4a, which lacks one amino-terminal amino acid, retained the
inhibitory activity. DP-4b, which lacks two amino acids, exhib-
ited a reduced inhibitory effect. DP-4c, which lacks four amino
acids, was inactive. DP-4e, which lacks one carboxyl-terminal
amino acid, retained its inhibitory ability, whereas DP-4f, which
lacks two amino acids, was inactive. The inhibition experiments
using deletion peptides revealed that the GQVVVAVR sequence
is critical for cell adhesion activity.
DP-4e, were active for cell adhesion when coated directly on the
wells (Figure 6, right panel). These data show that peptides active
in blocking adhesion of cells to dermatopontin are also active for
direct cell adhesion. Thus, the minimum amino acid sequence in
the cell binding domain of dermatopontin was determined to be
GQVVVAVR, which corresponds to amino acids 35-42 of
bovine dermatopontin.
Effect of EDTA and Heparin on Adhesion of Cells
to DP-4. Adhesion of cells to DP-4 was inhibited by EDTA
(Figure 7A), but the inhibition was not as strong as that with
dermatopontin. In contrast, cell adhesion was strongly inhibited
by heparin (Figure 7B), suggesting that adhesion of cells to DP-4
is largely heparin-dependent. Treatment of HaCaT cells with
heparitinase I completely abolished adhesion to DP-4, while
chondroitinase ABC treatment did not have any effect on
adhesion (Figure 7C). In addition, adhesion of HaCaT cells to
DP-4 was not inhibited by anti-integrin R1, R2, R3, R5, R6, or β1
subunit functional blocking antibodies (data not shown). These
results strongly suggest that the receptor for DP-4 is a cell surface
HSPG.
We also determined direct cell attachment activity using
the deletion peptides. Only three peptides, DP-4a, DP-4b, and
The DP-4 Peptide Interacts with Heparin and Synde-
cans. The binding of DP-4 with heparin was examined using
biotinylated heparin. Biotinylated heparin bound to the DP-
4-coated wells, while the scrambled peptides, DP-4S, did not
interact (Figure 8A). Similarly, the DP-5 peptide that partially
overlaps with DP-4 did not interact with heparin (Figure 8A).
The interaction between the DP-4 peptide and biotinylated
heparin was blocked by unlabeled heparin in a dose-dependent
manner (Figure 8B). These data demonstrate that the dermato-
pontin-derived active peptide sequence binds to heparin, suggest-
ing that a cell surface HSPG is the cellular receptor.
Syndecans are the most common receptor among cell surface
HSPGs. Adhesion of syndecan-1-overexpressing ARH-77 cells to
DP-4 was compared with that of cells transfected with an empty
vector, because ARH-77 human lymphoid B cells express a low
level of cell surface proteoglycans (34, 35). The DP-4 peptide by
itself induced cell adhesion of control vector-transfected ARH-77
cells; however, the extent of adhesion of syndecan-1-expressing
ARH-77 cells to DP-4 was significantly greater than that of the
control cells (Figure 8C). The other ARH-77 cells expressing
syndecan-2 and -4 showed less adhesion to DP-4 (data not
shown). On the other hand, neither the syndecan-expressing
nor the control ARH-77 cells demonstrated adhesion to DP-5.
Further, both DP-4 and DP-4S Sepharose 4B beads were
F
IGURE 6: Determination of the minimal active sequence of derma-
topontin needed for cell adhesion. Inhibition of adhesion of cells to
dermatopontin by deletion peptides of DP-4 (left). Deletion peptides
of DP-4 shown in the center were mixed with suspended HaCaT cells
at a concentration of 200 μg/mL, and the mixtures were incubated for
30 min on a dermatopontin-coated plate. The DP-4 peptide and PBS
were used as positive and negative controls, respectively. Cell adhe-
sion activities of deletion peptides (right). Deletion peptides were
coated at a concentration of 4 μg/mL, and cells were incubated for
1 h. Asterisks indicate p < 0.01. Data are expressed as means ( SD of
triplicate results. Experiments were performed twice and gave similar
results.
F
IGURE 7: Inhibition of adhesion of cells to the DP-4 peptide by EDTA, heparin, and enzymatic treatment of cells. DP-4 was coated at a
concentration of 4 μg/mL and incubated with EDTA (A) and heparin (B) in the same manner as described in the legend of Figure 2. Data are
expressed as means ( SD of triplicate results. Experiments were performed three times and gave similar results. (C) HaCaT cells were treated with
enzymes and added to the peptide-coated wells. PBS was added to the cells as a control. Data are expressed as means ( SD of triplicate results.
The asterisk indicates p<0.01.

Article
Biochemistry, Vol. 49, No. 1, 2010 153
actin filaments. These findings indicate that the cells underwent
morphological changes beyond adhesion on dermatopontin.
The cell adhesion sequence of dermatopontin was localized to
eight amino acids in the DP-4 peptide in bovine dermatopontin
and was located on the first loop structure. On the basis of
hydrophobicity analysis using ProtParam of ExPasy, this peptide
demonstrates a high hydrophobic score of 0.418. Although the
DP-4 peptide is hydrophobic, when combined with adjacent
peptides, DP-3-4 and DP-4-5 peptides possess hydrophilic
scores (-0.304 and -0.216, respectively). The minimum active
sequence of DP-4 was GQVVVAVR, and the arginine residue
may play a critical role for the biological activity. While a precise
analysis by X-ray diffraction is required for the determination of
the location of the DP-4 peptide, it is possible that the DP-4
region containing the arginine residue is exposed on the molec-
ular surface. We also examined the adhesion of HaCaT cells to
the human DP-4 peptide (PQGQVIVAVRS), which differs by
two amino acids from bovine DP-4 (PHGQVVVAVRS). The
human DP-4 peptide promoted cell adhesion when coated on a
dish and inhibited adhesion of cells to dermatopontin in a fashion
similar to that of bovine DP-4 (data not shown). These data
suggest that the biological activity of the DP-4 sequence is
conserved in both human and bovine dermatopontin, and that
this site is important for dermatopontin.
One of the cell surface receptors for dermatopontin was
determined to be R3β1 integrin (Figure 3). Because the cell
adhesion domain was identified as the DP-4 peptide, we exam-
ined if this peptide is the integrin binding site. Unexpectedly,
adhesion of cells to DP-4 was not inhibited by anti-integrin
antibodies (data not shown). These findings indicate that there is
an integrin binding site somewhere in the dermatopontin mole-
cule. It is likely that the integrin binding site requires more
complex, longer peptides, or a domain with three-dimensional
conformations. This site was not identified by the approaches
using short peptides in this study.
We found via several approaches that a HSPG-type cell
surface receptor is involved in adhesion of cells to the DP-4
peptide (Figures 7 and 8). Other studies have also suggested
syndecans as cell surface receptors for several ligands (38-40). In
fact, syndecan-expressing ARH-77-Synd-1 cells demonstrated a
small but statistically significant enhancement of adhesion of cells
to DP-4 (Figure 8C). On the other hand, ARH-77-Cont cells that
do not express syndecan showed weak adhesion to the DP-4
peptide, but not to DP-5. Pull-down analysis using DP-
4-conjugated beads also supported the direct binding activity
of syndecan to DP-4 (Figure 8D). These findings suggest that the
cells specifically adhere to DP-4 through HSPG and syndecan is a
potential HSPG receptor, but it also suggests that other mole-
cules on the ARH-77 cells may participate in the adhesion. The
possible receptor molecular species was not examined further in
this study. Syndecan-knockout mouse analysis revealed that this
receptor plays important roles in wound healing via promoting
cell adhesion and migration (41). With all the data taken together,
we suggest that syndecan binding of dermatopontin via DP-4
may affect various biological events, such as wound healing.
In this study, it was revealed that HaCaT cells utilize multiple
receptors for adhesion to dermatopontin, that is, R3β1 integrin
and HSPG-type receptors. Similar types of multireceptor systems
composed of integrins and syndecans, or other receptors, are
known (38, 42-45). For example, syndecan and R2β1 integrin
are required for fibroblast spreading after adhesion to the laminin
R1 LG4 module (38). Syndecan-4 and R5β1 integrin promote
F
IGURE 8: Identification of the HSPG-type cell surface receptor for
the DP-4 peptide. (A) Peptides indicated under the horizontal line
were coated on wells, and biotinylated heparin was added. Bound
biotinylated heparin was probed with HRP-conjugated streptavidin.
(B) Effect of heparin on binding of biotinylated heparin to the DP-4
peptide. Biotinylated heparin was mixed with unlabeled heparin and
added to the DP-4-coated wells. Data are expressed as means ( SD
of triplicate results. Asterisks indicate p<0.01. (C) Adhesion of
syndecan-expressing and control ARH-77 cells to DP-4. DP-4 and
DP-5, partially overlapped withDP-4, were coated at a concentration
of 4 μg/mL, and the cells were incubated for 1 h. Asterisks indicate
p < 0.01. Data are expressed as means ( SD of triplicate results.
(D) Affinitybinding ofsyndecan-1 to DP-4-conjugated Sepharose 4B
(lane 1, DP-4; lane 2, DP-4S) was examined by incubation of peptide
beads with the HaCaT cell lysate. The beads were treated with
heparitinase I and chondroitinase ABC for syndecan-1 elution and
then were subjected to 7.5% SDS-PAGE and Western blotting. The
arrow shows syndecan-1, and molecular masses (kilodaltons) are
indicated at the left.
prepared for examination of the direct binding of DP-4 to
syndecan (Figure 8D). The protein bound to the peptide beads
was treated with heparitinase I and chondroitinase ABC to elute
proteoglycans by sugar chain digestion, and syndecan-1 was
detected by Western blotting of the DP-4 bead fraction. These
data demonstrate that DP-4 binds to the syndecan-1 proteogly-
can directly. We conclude that syndecan is involved in adhesion
of cells to DP-4.
DISCUSSION
In this study, we found that dermatopontin promoted strong
epidermal cell adhesion via an eight-amino acid active site. We
also identified the cell surface receptors for dermatopontin.
Although cell adhesion activity of dermatopontin had been
described for fibroblasts (18), fibroblast cell adhesion required
much higher concentrations of dermatopontin than the epider-
mal cells for maximal adhesion. These findings suggest that
dermatopontin demonstrates stronger cell adhesion activity for
epidermal HaCaT cells than for dermal fibroblasts. HaCaT cells
not only adhere to dermatopontin but also spread on it, forming

154 Biochemistry, Vol. 49, No. 1, 2010
Okamoto et al.
focal adhesion of melanoma cells and migration of melanoma
cells on fibronectin (42). Although the mode and the fate of
cooperation between syndecans and integrins on adhesion of cells
to dermatopontin are not known, it is feasible that the coopera-
tion results in certain biological activities beyond adhesion.
R3β1 integrin is expressed on the keratinocyte cell surface (46),
and it is known to participate in keratinocyte adhesion and
migration on an unprocessed form or on a specific module of
laminin-332 (47, 48). Syndecan is also expressed in keratinocytes,
and the expression is enhanced around the wound edge (49). A
growing body of evidence indicates that syndecan is involved in
cell migration (41, 50). Recently, it was reported that the
interaction of syndecan-1 and the globular domain of laminin-
332 is indispensable for keratinocyte migration on the unpro-
cessed form of laminin-332 (47). Likewise, the involvement of
both R3β1 integrin and syndecan in adhesion of cells to derma-
topontin implies that dermatopontin has some roles in a wide
range of biological events, including wound healing.
Dermatopontin is one of the noncollagenous components in
the dermis, and it localizes around the collagen fibers (2, 3).
Dermatopontin-deficient mice exhibit a phenotype of Ehlers-
Danlos syndrome, demonstrating poorly organized collagen
microfibrils and susceptibility in the skin for mechanical shear
stress (51). Thus, dermatopontin plays an indispensable role in
maintaining functional tissue integrity. In skin injury, keratino-
cytes at the wound edge have direct contact with dermal
components. At this time, dermatopontin in the newly exposed
dermis may influence the behavior of epidermal cells by supply-
ing a scaffold for the cells through its cell adhesion activity. In
addition, the identification of the cell adhesion domain implies a
possibility that this synthetic peptide may be used as a therapeutic
to wounds.
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ACKNOWLEDGMENT
We thank Ralph D. Sanderson and Yoshihiko Yamada for the
lymphoid cell lines.
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