Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2011/03/11/M110.179762.DC1.html
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 17, pp. 14861–14869, April 29, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Dermatopontin Interacts with Fibronectin, Promotes
Fibronectin Fibril Formation, and Enhances Cell Adhesion*□
S

Received for publication, September 1, 2010, and in revised form, February 23, 2011 Published, JBC Papers in Press, March 11, 2011, DOI 10.1074/jbc.M110.179762

Aiko Kato‡, Osamu Okamoto§1, Kazushi Ishikawa§, Hideaki Sumiyoshi¶, Noritaka Matsuo¶, Hidekatsu Yoshioka¶,Motoyoshi Nomizu储, Tatsuo Shimada**2, and Sakuhei Fujiwara§
From the Departments of ‡Plastic Surgery, §Dermatology, and ¶Biochemistry and the **Faculty of Medicine, Oita University,
1-1 Idaigaoka, Hasama-machi, Yufu-shi, Oita 879-5593, Japan and the 储Laboratory of Clinical Biochemistry, School of Pharmacy,
Tokyo University of Pharmacy and Life Sciences, 1432-1, Horinouchi, Hachioji, Tokyo 192-0392, Japan

Dermatopontin (DP)3 is a 22-kDa protein located in theextracellular matrix (ECM), comprising about 12 mg/kg wet
dermis (1, for a review, see Ref. 2). It was initially purified frombovine dermis together with decorin (3). To date, over 10 dermatopontin homologues have been identified in five different
mammalian species (2, 4 –7) and in 12 different invertebrates(2).

* This work was supported in part by a grant from the Ministry of Education,
Culture, Science, Sports, and Technology of Japan (to A. K.).
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. 1–3.
1
To whom correspondence should be addressed. Tel.: 81-97-586-5882; Fax:
81-97-586-5889; E-mail: ookamoto@oita-u.ac.jp.
2
Professor Emeritus, Oita University.
3
The abbreviations used are: DP, dermatopontin; ECM, extracellular matrix;
Fn, fibronectin; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; TF, transcription factor; sulfo-NHS-LC-biotin, sulfo-N-hydroxysuccinimide-long chain-biotin.
□S

APRIL 29, 2011 • VOLUME 286 • NUMBER 17

DP has multiple activities. It induces fibroblast cell adhesion
probably via integrin-type receptors (8). DP accelerates collagen fibrillogenesis and modifies the newly formed collagen
fibrils (9). DP-knock-out mice demonstrate an Ehlers-Danlos
phenotype (10), showing an abnormal ECM architecture andtissue flexibility in response to mechanical forces. Previously,
we have shown that DP interacts with decorin and with transforming growth factor-␤1 (11). Thus, it is suggested that DP hasan ability to interact with multiple proteins, thereby forming afunctional supramolecular complex in the ECM. Recently, we
showed that DP promotes strong epidermal cell adhesion via
␣3␤1 integrin and syndecan (12). Considering this together
with the fibroblast adhesion findings, we hypothesize that DP
plays multiple roles in wound healing.
Just after wounding, extravasated blood forms a provisional
matrix, which is an ECM composed mainly of fibrin and fibronectin (Fn) and a liquid component known as the wound fluid. The
Fn in the provisional matrix serves as an adhesion and migration scaffold for fibroblasts in the surrounding ECM and forcirculation-derived cells (13, 14).
In tissues, Fn is activated and assembled into a fibrillar structure known as the Fn matrix, which is found during both
embryonic development and wound healing (15, 16). The only
authentic molecular species known to promote Fn fibril formation are integrins (17–19), among which ␣5␤1 integrin would
be the most recognized (20). A recombinant peptide, called
anastellin, is the only non-integrin molecule that can promote
Fn fibril formation (21). The Fn fibrils produced by anastellin
have properties similar to those formed by integrins, and thefibrils are termed superfibronectin (21).
During an investigation of the biological activities of DP invivo, we found that DP was present in the provisional matrix.
We further found that DP interacted with Fn and fibrin and thatDP enhanced the cell adhesion activity of Fn. We then demonstrated that DP promoted Fn fibril formation. This report
describes the possible biological functions of DP in dermal
wound healing. We demonstrate that DP is the first non-integrin ECM molecule that can activate Fn and initiate the formation of Fn fibrils.

EXPERIMENTAL PROCEDURES
Materials—DP was purified from newborn calf dermis, andan anti-DP carboxyl-terminal peptide antibody was produced
as reported previously (1). This antibody does not cross-reactwith fibronectin, fibrinogen, fibrin, or albumin. Human plasma
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We report that dermatopontin (DP), an abundant dermal
extracellular matrix protein, is found in the fibrin clot and in thewound fluid, which comprise the provisional matrix at the initialstage of wound healing. DP was also found in the serum but at alower concentration than that in wound fluid. DP co-localizedwith both fibrin and fibronectin on fibrin fibers and interactedwith both proteins. Both normal fibroblast and HT1080 celladhesion to the fibrin-fibronectin matrix were dose-dependently enhanced by DP, and the adhesion was mediated by ␣5␤1
integrin. The cytoskeleton was more organized in the cells thatadhered to the fibrin-fibronectin-DP complex. When incubatedwith DP, fibronectin formed an insoluble complex of fibronectin fibrils as visualized by electron microscopy. The interacting
sites of fibronectin with DP were the first, thirteenth, and fourteenth type III repeats (III1, III13, and III14), with III13 and III14
assumed to be the major sites. The interaction between III2–3
and III12–14 was inhibited by DP, whereas the interaction
between I1–5 and III12–14 was specifically and strongly enhanced
by DP. Because the interaction between III2–3 and III12–14 isinvolved in forming a globular conformation of fibronectin, andthat between I1–5 and III12–14 is required for forming fibronectin
fibrils, DP promotes fibronectin fibril formation probably bychanging the fibronectin conformation. These results suggestthat DP has an accelerating role in fibroblast cell adhesion to theprovisional matrix in the initial stage of wound healing.

Dermatopontin Interaction with Fibronectin

FIGURE 1. Schematic representation of plasma Fn monomer structure and Fn fragments. Type I, II, and III repeats are represented by diamonds, filled ovals,
and boxes, respectively. A V region is denoted by a circle. Numbers of each repeat are shown within or near each repeat. Positions of commercially available Fn
fragments are shown at the bottom.

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sections were observed using a fluorescent Optiphot-2 microscope (Nikon, Tokyo, Japan).
For detection of DP, crude samples of the serum and thewound fluid taken from the surgical wound of a patient werefirst fractionated using a gel filtration column, TSK gel G-3000
SWXL (7.5 ⫻ 300 mm) (Tosoh, Tokyo, Japan), in 50 mM Tris/
HCl, 0.5 M NaCl, 6 M urea, pH 7.5. The flow was 0.5 ml/min, and
0.5-ml fractions were collected. The fractions were separatedusing a 12% T, 0.3% C Tricine SDS-PAGE (22) under reducing
conditions and were Western blotted onto a PVDF membrane.
DP was identified with an anti-DP rabbit antibody, followed byincubation with an HRP-conjugated anti-rabbit IgG antibody.
For Fn detection, the crude samples were separated by 5% SDSpolyacrylamide gels under reducing conditions, and after Western blotting, Fn was probed with an anti-Fn mouse antibody
and HRP-conjugated anti-mouse antibody. Bands were visualized using an ECL reagent.
Immunoprecipitation—The fibrin clot was extracted with 50
mM Tris/HCl, pH 7.5, containing 0.2, 0.5, and 1% deoxycholate
successively at 4 °C. Anti-Fn mouse IgG and nonimmune
mouse IgG were coated on gels of Dynabeads protein A, and theimmunoprecipitation was performed according to the manufacturer’s protocol, using PBS containing deoxycholate as awash buffer. The gel was extracted with sample buffer for electrophoresis, and the samples were run on Tricine SDS-polyacrylamide gels under reducing condition and Western blotted.
DP was visualized as described above.
Cell Adhesion Assay—Fibrinogen was coated on a 96-wellplate (Assist, Tokyo, Japan) at 5 ␮g/ml in 0.14 M NaCl, 30 mM
phosphate buffer, pH 7.3, overnight at room temperature. Thenthe wells were rinsed once with PBS, and thrombin in PBS wasadded at 1 unit/ml and was incubated for 1 h at room temperature. After rinsing with PBS, the wells were blocked by 1% BSA
in PBS for 6 h, followed by a wash with PBS. Then Fn and/or DP
in PBS was added to the wells and was incubated overnight atroom temperature. Then the wells were rinsed with PBS threetimes. A monolayer of normal fibroblasts was detached with 5
mM EDTA in PBS, and the cells were suspended in DMEM. The
cells were inoculated in the wells at 30,000 cells/100 ␮l/well andwere incubated at 37 °C in an incubator. After incubation,
the wells were rinsed once with warm PBS, and the attachedcells were fixed with 1% glutaraldehyde for 30 min and thenstained with 0.1% crystal violet for 1 h. After staining, the wells
were rinsed with running water, dried, and photographed.
Finally, the dye was eluted with 0.1% Triton X-100 for 30 min,and absorbance at 595 nm was measured with a UV spectrometer ELX808 (BioTek, Winooski, VT). Cell adhesion was measured at 1 h of incubation, and unless otherwise mentioned, theVOLUME 286 • NUMBER 17 • APRIL 29, 2011

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Fn, an anti-Fn mouse monoclonal antibody, 30-kDa and
70-kDa thermolysin fragments, a recombinant fragment of thefirst type III repeat (III1) of fibronectin known as anastellin, anda 120-kDa chymotryptic fragment of Fn were purchased from
Sigma. Retronectin, which is a bacterially expressed fragment
spanning between III9 and an amino-terminal part of the Vregion of Fn lacking III11, was purchased from TaKaRa Chemical (Tokyo, Japan). Fluorescein isothiocyanate (FITC)-conjugated phalloidin was purchased from Sigma-Aldrich. Gradientslab gel, Readygel J 5–15%, was purchased from Bio-Rad, and aprotein A-conjugated affinity gel, Dynabeads protein A, waspurchased from Invitrogen. A carboxyl-terminal fragment
beginning with a V region of Fn, namely fragment 4, was purchased from R & D Systems (Minneapolis, MN). A monomer
plasma Fn structure and positions of these fragments within Fn
are shown in Fig. 1. Function-blocking anti-integrin subunit
antibodies (FB12 for ␣1, P1E6 for ␣2, ASC-1 for ␣3, P1H4 for
␣4, P1D6 for ␣5, M9 for ␣v, NKI-GoH3 for ␣6, and 6S6 for ␤1)
and human fibrinogen were from Chemicon (Temecula, CA).
An ECL reagent, nickel-Sepharose CL-4B, thrombin, and antiHis6 antibody were purchased from GE Healthcare. Anti-fibrinogen antibody and anti-␣5 and ␤1 integrin subunit antibodies were purchased from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). Anti-vinculin antibody was purchased from
Enzo Life Sciences International (Plymouth Meeting, PA). A
rhodamine-labeled anti-rabbit IgG antibody, FITC-labeledanti-mouse and anti-goat IgG antibodies, and nonimmune
mouse IgG were purchased from Jackson ImmunoResearchLaboratories, Inc. (West Grove, PA). Sulfo-NHS-LC-biotin,
HRP-conjugated streptavidin, and BCA reagent were purchased from Pierce. Specimens from patients were taken withthe patients’ consent, according to the guidelines of the EthicalCouncil of Oita University.
Cell Culture—Normal human fibroblasts and cells of ahuman fibrosarcoma-derived fibroblast cell line, HT1080, were
cultured as a monolayer in 75-cm2 bottles at 37 °C in a humidified 5% CO2, 95% air atmosphere. The cells were maintained in
DMEM containing 10% (v/v) FCS.
Immunohistochemistry and Western Blotting—A fibrin clot
taken from a 2-day-old burn bulla in the forearm of a 24-yearold female was embedded in Tissue-Tek威 O.C.T. compound(Sakura Finetek, Torrance, CA). Four-␮m sections were made,rinsed in PBS, blocked with 1% defatted skim milk in PBS, andincubated with either anti-DP and anti-Fn antibodies oranti-DP and anti-fibrinogen antibodies for 30 min, followed byincubation with a rhodamine-labeled anti-rabbit IgG antibody
and an FITC-labeled anti-mouse IgG antibody, respectively,
with three 10-min intervals of rinsing with PBS. The incubatedDermatopontin Interaction with Fibronectin
TABLE 1
Primer sequences
Product

Amino acid position

Fn III1Fn III1–2
Fn III2
Fn III3
Fn III2–3
Fn III12
Fn III13
Fn III14
Fn III12–14
Fn V
Fn III15

609–700
609–809719–809810–905
719–905
1721–1812
1813–1901
1902–1991
1721–1991
1992–2102
2103–2182

Sense primer sequence

Antisense primer sequence

5⬘-GAATTCGGTGGTGGTGAAGTCAAAGCG-3⬘
5⬘-GAATTCTGTTGTTTGTGAAGTAGACAG-3⬘
5⬘-GAATTCTGTTGTTTGTGAAGTAGACAG-3⬘
5⬘-GAATTCATCTGAGCGTGGGGTGCCAGT-3⬘
5⬘-GAATTCATCTGAGCGTGGGGTGCCAGT-3⬘
5⬘-GAATTCCTCCAGAGTGGTGACAACTCC-3⬘
5⬘-GAATTCAGTGGAGGCGTCGATGACCAC-3⬘
5⬘-GAATTCTGTCTTTTTCCTTCCAATCAG-3⬘
5⬘-GAATTCTGTCTTTTTCCTTCCAATCAG-3⬘
5⬘-GAATTCACCGTGTGGGTACAGGTGATA-3⬘
5⬘-GAATTCCTTATGCCTCTGCTGGTCTTT-3⬘

5⬘-GGATCCCAGACCTATCCAAGCTCAAGT-3⬘
5⬘-GGATCCCAGACCTATCCAAGCTCAAGT-3⬘
5⬘-GGATCCTCTCCTCTTGTGGCCACTTCT-3⬘
5⬘-GGATCCGCGCCTGATGCCCCTCCTGAC-3⬘
5⬘-GGATCCTCTCCTCTTGTGGCCACTTCT-3⬘
5⬘-GGATCCGCTATTCCTGCACCAACTGAC-3⬘
5⬘-GGATCCAATGTCAGCCCACCAAGAAGG-3⬘5⬘-GGATCCGCCATTGATGCACCATCCAAC-3⬘
5⬘-GGATCCGCTATTCCTGCACCAACTGAC-3⬘
5⬘-GGATCCGACGAGCTTCCCCAACTGGTA-3⬘
5⬘-GGATCCCCGGGACTCAATCCAAATGCC-3⬘

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in Table 1. PCR was performed using combinations of the primers, and the products were ligated into a TEasy vector (Promega, Madison, WI) and then were cloned into E. coli XL-1.
The vectors carrying the correct inserts were digested with
BamHI and EcoRI, and the fragments of the inserts were thenligated to corresponding sites of pCOLD I vectors and weretransfected into E. coli BL-21. A construct of Fn V was madeusing a pCOLD-TF vector because the construct in the
pCOLD-I vector did not express any fusion protein forunknown reasons.
Expression of fusion proteins of type III repeats and a V
region of Fn was performed at 15 °C for 24 h according to the
manufacturer’s instructions using a pCOLD bacterial expression system. The bacteria were collected by centrifugation. The
pellet was digested with 1 mg/ml lysozyme in 50 mM Tris/HCl,
pH 8.0, containing 0.14 M NaCl (TBS) for 1 h on ice and thenwas extracted by adding Nonidet P-40 at a final concentrationof 0.2% and sonificated. After the extract was cleared by centrifugation, the supernatant was applied to a nickel-Sepharose CL
4B column; washed with 20 mM sodium phosphate buffer, 0.5 M
NaCl, and 40 mM imidazole, pH 7.4 (starting buffer); and eluted
with 20 mM sodium phosphate buffer, 0.5 M NaCl, and 0.5 M
imidazole (elution buffer), pH 7.4, according to the manufacturer’s instructions. The expression of recombinant proteins
was determined by Western blotting probed with an anti-His6
antibody. The recombinant proteins were extensively dialyzed
against PBS. The concentration of the proteins was determinedby a BCA reagent, and BSA was used as a standard.
A Fn V region expressed using a pCOLD-TF vector appears
as a fusion protein with His6-tagged transcription factor (TF).
The fusion protein was adsorbed to a nickel-Sepharose CL-4B
column as described above, and the gel was rinsed with PBS.
Thrombin was added to the gel in PBS and incubated for 2 h atroom temperature. After digestion, the gel was rinsed with PBS.
Thrombin was present in an unbound fraction, and the liberated recombinant Fn V from the TF was associated with the gel.
The recombinant Fn V was eluted from the gel with a starting
buffer containing 1 M NaCl and was processed as describedabove. SDS-PAGE patterns of these recombinant Fn fragments
are shown in the supplemental Fig. 1.
Biotinylation of Fragments—A 30-kDa thermolysin fragmentof Fn and recombinant Fn type III repeats were labeled withsulfo-NHS-LC-biotin according to the manufacturer’s instructions. Briefly, the fragments in PBS were incubated with a
20-fold excess molar concentration of sulfo-NHS-LC-biotin in
PBS for 2 h at room temperature. The reaction was terminated
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volume of solution was 100 ␮l/well except for blocking by BSA
and elution by Triton X-100, where 300 ␮l/well and 50 ␮l/wellwere used, respectively. For HT1080 cell adhesion, the fibrinogen concentration was 20 ␮g/ml.
Immunofluorescent Staining—All of the procedures weredone at room temperature, except for the cell adhesion, which
was performed at 37 °C. Spots (about 5 mm in diameter) on thesurface of a siliconized glass plate (Dako Japan, Kyoto, Japan)
were coated with 10 ␮l of fibrinogen at 20 ␮g/ml in PBS overnight. Then, after rinsing with PBS, the coated fibrinogen wasconverted to fibrin by incubating with 10 ␮l of thrombin at 1
unit/ml in PBS for 1 h and then blocked by 1% BSA in PBS for6 h. Ten ␮l of Fn and DP mixture (20 ␮g/ml and 10 ␮g/ml,
respectively) in PBS was applied to the spot of immobilized
fibrin and was incubated overnight. In control experiments, DP
was replaced by the same concentration of BSA and identicallyprocessed. Fibroblasts at a density of 30,000 cells/20 ␮l of
DMEM were applied to the spot, and the cells were allowed toadhere for 1 h. After rinsing with PBS, the adhered cells were
fixed with 3.7% formaldehyde in PBS for 5 min, rinsed in PBS,
and then dehydrated in acetone for 5 min followed by rehydration in PBS. Permeabilization was performed in 0.1% Triton
X-100 for 10 min, followed by rinsing in PBS. Actin filaments
were stained with FITC-conjugated phalloidin in PBS. Vinculin
was stained with anti-vinculin antibody followed by rhodamine-labeled anti-rabbit antibody. The buffer was PBS containing 10% nonfat dried milk. An ␣5 integrin subunit was stainedwith anti-␣5 integrin antibody. The secondary antibody and thebuffer were the same as described above. Finally, the samples
were embedded in immersion oil. For HT1080 cell adhesion,
the concentrations of Fn, DP, and BSA were 10 ␮g/ml,
respectively.
Production of Recombinant Type III Repeats and V Region of
Fn—All Fn domains were expressed using a pCOLD威 bacterial
expression system (TaKaRa Bio, Tokyo, Japan) using Escherichia coli BL-21 as a host. Using this system, recombinant proteins were expressed in principle as the amino-terminally His6tagged form. The His6 tag is connected with enzyme cleavage
sequences and with additional amino acids originating frommultiple cloning sites. A reverse transcription product of cultured human fibroblasts was used as a template for PCR amplification of a DNA sequence encoding Fn domains. Antisense
primers were designed to have a BamHI sequence, and sense
primers were designed to have an EcoRI sequence at the5⬘-ends, respectively. The DNA sequences and positions ofprimers within DNA/amino acid sequences of Fn are indicatedDermatopontin Interaction with Fibronectin

by incubation with triethanolamine in Tris/HCl, pH 7.5, at a
final concentration of 10 mM for 1 h at room temperature. The
labeled proteins were dialyzed against PBS, and labeling was
confirmed by Western blotting probed by HRP-conjugatedstreptavidin. The labeled protein concentrations were determined as described above.
Solid-phase Assay for Protein Interaction—All procedures
were carried out at room temperature. Fn and the recombinantdomains in 30 mM sodium phosphate buffer, 0.14 M NaCl, pH
7.3 (PB-Na), were immobilized on a 96-well plate overnight. For
fibrin immobilization, fibrin was formed as described above.
The wells were then rinsed with PBS and were blocked with 1%
BSA in PBS for 6 h. After rinsing with PBS, either DP, Fn,labeled recombinant Fn domains, or the mixture dissolved in
PB-Na containing 0.05% Tween 20 (PB-Na-Tw) was added into
the wells and was incubated overnight. In the following procedure, incubation was done in PB-Na-Tw for 2 h, and rinsing
was done with the same buffer. DP was probed with ananti-DP antibody, followed by HRP-conjugated anti-rabbit
IgG antibody. Fn was probed with an anti-Fn monoclonal
antibody followed by a HRP-conjugated anti-mouse IgG
antibody. Biotinylated Fn domains were probed by HRPconjugated streptavidin. Color was allowed to develop byadding 0.057 M citric acid, 0.086 M disodium hydrogen phosphate containing 10 mM 2,2⬘-azido-bis (3-ethylbenzothiazoline-6-sulfonic acid) and 0.03% hydrogen peroxide in thedark. The color reaction was terminated by adding aliquotsof 0.1 M citric acid containing 1.5 mM sodium azide, after
which absorbance at 405 nm was determined.
Fibril-forming Assay—Fn fibril formation was assessed
according to a previous report (23). Briefly, 0.5 ␮M Fn in 50 mM
Tris/HCl, pH 8.0, containing 0.14 M NaCl, was centrifuged, andany insoluble material was removed. Then DP at 30 ␮M in thesame buffer was added to the Fn solution and incubated for 16 h
at room temperature. The reaction volume was 10 ␮l. Afterincubation, the mixture was centrifuged, and the pellets wererinsed once with the Tris buffer. Pellets and supernates wereapplied to 5–15% gradient gels (Bio-Rad) under reducing con-

14864 JOURNAL OF BIOLOGICAL CHEMISTRY

ditions and were stained by Coomassie Brilliant Blue R-250. For
control, BSA at 30 ␮M was mixed with Fn and was identicallyprocessed.
Electron Microscopy—One ␮M fibronectin was incubatedwith 8 ␮M DP in PBS at room temperature for 8 h. The samples
were placed on a poly-L-lysine-coated coverglass and were
immobilized at room temperature for 10 h in a wet chamber.
The samples were then treated with Karnovsky solution,
washed with cacodylic acid, stained with 1% OsO4 and 1% tannic acid, dehydrated, dried, and coated with OsO4. All of theobservations and photographs were performed with a scanningelectron microscope (S-4800, Hitachi, Japan) operated at anacceleration voltage of 15 kV.

RESULTSDP Is Present in the Provisional Matrix and in the Wound
Fluid—Double immunofluorescent staining of DP and Fn in afibrin clot revealed that DP was present in a net-like structure offibrin (Fig. 2A). Fn was also stained in a similar pattern (Fig. 2B)
as demonstrated in a merged image (Fig. 2C). The staining ofDP overlapped with Fn in the periphery of the fibrillar structure. Thus, DP partially overlaps with Fn. DP was also stained ina similar partial overlap pattern with fibrin, which was probedby anti-fibrinogen antibody (Fig. 1, D–F).
The fibrin clot was extracted with TBS containing deoxycholate. Fn and DP were extracted by TBS containing 1%
deoxycholate, but the yield was low (data not shown). Immunoprecipitation by anti-Fn antibody and anti-fibrinogen antibodies probed with anti-DP antibody did not demonstrate apositive DP band (data not shown). Therefore, whether DPinteracts with Fn or fibrin could not be confirmed by thisapproach.
Wound fluid, which is a soluble fraction of the provisional
matrix, was electrophoresed on a Tricine SDS-polyacrylamide
gel and Western blotted. DP as well as Fn were present in thewound fluid (Fig. 3, lanes 2 and 5). Interestingly, DP was also
found in the serum (lanes 3). Although the staining intensity of
Fn was similar between the wound fluid and the serum (lanes 5
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FIGURE 2. Immunofluorescent images of Fn and DP in a fibrin clot. A fibrin clot was double stained with an anti-DP rabbit antibody (A) and anti-Fn mouse
antibody (B), an anti-DP rabbit antibody (D), and an anti-fibrinogen mouse antibody (E). These antibodies were detected by a rhodamine-labeled anti-rabbit
antibody and an FITC-labeled anti-mouse antibody, respectively. In the insets in A, B, D, and E, the primary antibodies were replaced with either preimmune
serum or IgG, followed by incubation with the secondary antibodies. C and F, merged images of A and B and of D and E, respectively. Bars, 20 ␮m.

Dermatopontin Interaction with Fibronectin

and 6), the relative intensity of DP in the wound fluid was muchgreater than that in the serum (Fig. 3, lanes 2 and 3), suggestingthat the DP concentration is elevated in the wound.
DP Interacts with Fn and Fibrin and enhances the interaction—
DP showed a dose-dependent interaction with immobilized Fn,
and immobilized DP also interacted with Fn (Fig. 4, A and B).
DP interacted with immobilized fibrin in a manner similar tothat of Fn (Fig. 4C), whereas immobilized DP did not show
evident interaction with fibrin in a liquid phase (data notshown). DP demonstrated much weaker interaction withimmobilized fibrinogen (data not shown), suggesting that ahigher order structure of fibrin is required for an interaction
with DP, as is the case for the Fn-fibrinogen/fibrin interaction(24, 25).
Next, the interaction between Fn and fibrin was examined in
the presence of DP. Fn interacted with fibrin as reported previously (data not shown) (24). The interaction between fibrin andFn was strongly enhanced by DP in a dose-dependent manner(Fig. 4D). From these results, DP is hypothesized to form aternary complex with Fn and fibrin.
DP Enhances Fibroblast Adhesion to the Fn-Fibrin Complex—
We examined if the DP-Fn-fibrin ternary complex enhancescell adhesion. When increasing concentrations of DP form acomplex with Fn and fibrin, fibroblast adhesion to the complex
was greatly enhanced in a dose-dependent manner (Fig. 5A). In
comparison, BSA did not induce this enhancement, and thelevel of adhesion remained near background level. When background cell adhesion was subtracted, the enhancement of celladhesion by DP was about 10-fold.
Next, a panel of antibodies was examined for inhibition ofcell adhesion. Fibroblast adhesion to the ternary complex offibrin-Fn-DP was significantly inhibited by anti-␣5 and -␤1
integrin subunit antibodies (Fig. 5B). Thus, ␣5␤1 integrin was
the cell surface receptor involved in fibroblast cell adhesion to
the fibrin-Fn-DP complex.
Many of the adhered fibroblasts to the fibrin-Fn-DP complex
were well spread (Fig. 6A), whereas most of those that adhered
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FIGURE 4. Interaction profiles of DP with Fn and/or fibrin. A, interaction of
DP with Fn. Fn was immobilized in wells at 5 ␮g/ml, DP was added at theconcentrations indicated on the x axis, and bound DP was detected as
described under “Experimental Procedures.” B, interaction of Fn with DP. DP
was immobilized at 5 ␮g/ml, and Fn was added and detected. C, fibrin was
immobilized at 5 ␮g/ml, and DP was added and detected. In A–C, rectangles
indicate nonspecific interaction of DP or Fn at the indicated concentrations
with the plate where ligand immobilization was omitted. D, enhancement ofinteraction between Fn and fibrin by DP. Fibrin was immobilized at 10 ␮g/ml,
and a mixture of Fn at 5 ␮g/ml and DP at the indicated concentrations wasapplied to the fibrin (〫). Fn and BSA were mixed and applied to the fibrin (⽧).
Bound Fn was detected as described. A rectangle indicates the nonspecificreaction in a condition where Fn was omitted from the fibrin-Fn-DP interaction and probed by anti-Fn antibody. A representative absorbance of thehighest DP concentration is shown. In A–D, experiments were performed
three times in triplicate, and means ⫾ S.D. are shown.

to the fibrin-Fn complex were not (Fig. 6B). The fibroblasts thatadhered to the ternary complex developed an organized linear
actin cytoskeleton and vinculin in a dot-like pattern (Fig. 6, C
and E), whereas those that adhered to the fibrin-Fn complex
showed a poor organization of actin cytoskeleton and vinculin(Fig. 6, D and F). The integrin ␣5 and ␤1 staining showed a finegranular pattern, and the staining appeared slightly enhancedin the cells that adhered to the ternary complex (Fig. 6, G–J).
Another cell line, HT1080, demonstrated an almost identicalprofile of cell adhesion enhancement by DP (supplemental Fig.
2). The cytoskeleton and integrin profiles were similar to thosein the case of fibroblasts, although the organization of the actin
cytoskeleton was less developed than that of fibroblasts (supplemental Fig. 3).
Upon gross examination, fibroblast adhesion patterns to theternary complex of fibrin-Fn-DP and to the binary complex offibrin-Fn were not greatly different (photographs in Fig. 5A).
Interestingly, HT1080 cells demonstrated different patterns ofcell adhesion to the ternary and the binary complexes. When
DP is present, HT1080 cells adhered to the ternary complex ina homogenous, sheetlike pattern (photographs in supplemental
Fig. 2A), whereas in the absence of DP, the pattern of the cells
that adhered to the Fn-fibrin complex was much less organized.
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FIGURE 3. Western blotting of the wound fluid and the serum. Lanes 1–3,
detection of DP. Lane 1, a DP standard. Lanes 2 and 3, DP in the wound fluid
and in the serum, respectively. Lanes 4 – 6, detection of Fn. Lane 4, a Fn standard. Lanes 5 and 6, Fn in the wound fluid and in the serum, respectively.
Molecular mass values are indicated at the left of lanes 1 and 4.

Dermatopontin Interaction with Fibronectin

From these results, we concluded that DP not only enhances
the amount of Fn that interacts with fibrin but also induces astructural change in Fn.
DP Induces Formation of Superfibronectin—The enhancement of the cell adhesion to Fn by DP is consistent with theformation of fibronectin fibrils. Therefore, we tested for Fn
fibril formation. When Fn is activated and forms fibrils, it
becomes insoluble (23). We next determined whether an insoluble pellet of Fn was formed in the presence of DP. As shown in
Fig. 7A, in the presence of DP, most of the Fn forms an insoluble
large pellet that migrates above the Fn monomer, indicating theformation of Fn fibrils. The larger band as well as the Fn monomer reacted with the anti-Fn antibody; therefore, the larger
band was proved to be a polymer of Fn (Fig. 7A, lanes 7 and 8).
The major portion of DP remained in the supernatant fraction,
as shown by a band around 22 kDa (Fig. 7A, lane 3), and a minor
fraction of DP is associated with the pellet fraction (lane 4). In

14866 JOURNAL OF BIOLOGICAL CHEMISTRY

FIGURE 6. Morphologies of the adhered fibroblasts and cytoskeletalorganization. A and B, crystal violet staining of the fibroblasts. Appearance ofthe cells from Fig. 5A. A, cells that adhered to a ternary complex of fibrin-FnDP; B, cells that adhered to a complex of fibrin-Fn-BSA. C, E, G, and I, fluorescence staining of cells that adhered to the fibrin-Fn-DP complex. D, F, H, and J,
corresponding staining of cells that adhered to the fibrin-Fn-BSA complex.
C and D, phalloidin staining. E and F, vinculin staining. The insets in C–F represent a higher magnification of the staining. Negative controls of vinculin
staining demonstrated a background staining (data not shown). G and H,
integrin ␣5 subunit staining. I and J, integrin ␤1 subunit staining. In E–J, theinset in each panel represents a negative control as described under “Experimental Procedures.” Bars, 20 ␮m.

contrast, incubation of Fn by itself or with BSA formed a faintlarger molecular mass pellet, but the amount was much lessthan that formed in the presence of DP (lanes 2 and 6).
To confirm the formation of Fn fibrils, the insoluble pellet
was examined by scanning electron microscopy. As expected,
Fn formed numerous fibrils when incubated with DP (Fig. 7B,
1). The fibrils were 50 –200 nm in diameter, irregular, and torVOLUME 286 • NUMBER 17 • APRIL 29, 2011

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FIGURE 5. A, enhancement of cell adhesion to the fibrin-Fn-DP ternary complex. Fibrin was immobilized, and then mixtures of Fn at 1 ␮g/ml and DP (F) at
the concentrations indicated on the x axis were applied to the wells. Normal
fibroblasts were incubated, and cell adhesion was quantified as describedunder “Experimental Procedures.” For a control, DP was replaced with BSA (Œ)
and identically processed. A rectangle indicates the cell adhesion to fibrin-DP
complex at the indicated concentration. Experiments were performed threetimes in triplicate, and means ⫾ S.D. are shown. The appearance of the cells isshown in the panel. Photographs were taken after adhered cells were stainedand dried. The top panel indicates a well in which a ternary complex wasformed at DP concentration of 1.25 ␮g/ml. The bottom panel represents a wellin which a Fn-fibrin-BSA complex at the same concentration is formed. B, inhibition of cell adhesion to the fibrin-Fn-DP complex by anti-integrin functionblocking antibodies. Antibodies were mixed with fibroblasts in suspension,and the cells were allowed to adhere. PBS was added to the cell suspension asa control. Antibody concentrations were 10 ␮g/ml. *, p ⬍ 0.01. Data areexpressed as mean ⫾ S.D. of triplicate results. Experiments were performed
three times and gave similar results.

Dermatopontin Interaction with Fibronectintuous. These features were similar to those of the fibronectin
fibrils formed on the cell surface as reported previously (26). In
contrast, in the absence of DP, Fn formed some short aggregates, but it never formed the long fibrillar structures seen in
the presence of DP (Fig. 7B, 2). The small aggregates may reflect
the faint Fn band in the pellet fraction on SDS-PAGE in Fig. 7A(lane 2). DP alone did not form fibrils except for sparse tiny
aggregates (data not shown). These results suggest that DP
induces Fn fibril formation.
Major Interaction Sites of Fn with DP are III13 and III14—We
investigated the binding sites on Fn for DP, using Fn fragments(Fig. 1). Two fragments, anastellin and retronectin, stronglyinteracted with DP (Fig. 8A). Other fragments demonstrated
statistically significant but relatively weak interactions (Fig.
7A). DP interacted more strongly with retronectin than with anoverlapping 120-kDa fragment, indicating that the binding sites
locate within a stretch between III12 and V. To further identify
the binding sites, type III repeats around the candidate region
were expressed in E. coli, and their binding to DP was examined. As shown in Fig. 8B, DP demonstrated strong interaction
with III1, III13, and III14, but it did not bind to other domains.
Because complex domains of III1–2 as well as III12–14 are
known to be important for Fn fibril formation (27–29), we also
APRIL 29, 2011 • VOLUME 286 • NUMBER 17

expressed these domains in tandem fashion and examined theinteraction with DP. DP failed to interact with III1–2, but itinteracted with III12–14 at a similar level as it did with III14 (datanot shown). Functional and structural analyses of III1–2 revealthat this domain has a specific structure and is primarily cryptic(30, 31); nevertheless, the domain is involved in Fn fibril formation. Considering this together with the fact that DP failed tointeract with III1–2, we conclude that III1 is a potential or acryptic DP interaction site and that the main DP interaction
sites are III13 and III14.
DP Modifies Interdomain Interactions of Fn—For Fn fibril
formation, interactions between I1–5 and other domains(within which are III1–2 and III12–14) are necessary (27–29).
Interestingly, these type III repeats were also DP binding sites,
as shown in Fig. 8B. Therefore, the influence of DP on the interaction of the 30-kDa fragment (I1–5) with these domains was
examined. DP did not enhance the interaction between I1–5 andIII1–2, whereas an interaction between I1–5 and III12–14 wasgreatly enhanced by the presence of DP (Fig. 9A). As a control,
an interaction between I1–5 and III15 was relatively unaffectedby DP. Another important interaction between III2–3 andIII12–14, which is crucial for maintaining a compact conformation of native Fn (32), was partially disrupted by DP (Fig. 9B).
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FIGURE 7. Fn fibril formation by DP. A, lanes 1 and 2, the supernatant and thepellet of Fn alone. Lanes 3 and 4, supernatant and pellet of a mixture of Fn with
DP. Lanes 5 and 6, supernatant and pellet of a mixture of Fn with BSA. Lanes 7
and 8, bands in lanes 3 and 4 were Western blotted and probed with anti-Fn
antibody. Lanes 9 and 10, negative controls of lanes 7 and 8 probed by nonimmune mouse IgG. Molecular masses are indicated on the left. B, scanningelectron microscopy of the Fn fibrils. 1, Fn incubated with DP; 2, Fn incubatedwithout DP. Bars, 5 ␮m.

FIGURE 8. Determination of the binding sites of Fn with DP. A, screening ofFn fragments that interact with DP. Fn and Fn fragments indicated on the xaxis were immobilized in wells at 2 ␮g/ml. DP was added to the wells at 2
␮g/ml, and the interaction was monitored as described under “Experimental
Procedures.” background, absorbance of 2 ␮g/ml DP added to noncoatedwells. *, p ⬍ 0.01 compared with a value of background absorbance. **, p ⬍0.01 compared with that of a 120-kDa fragment. B, determination of Fn
domains that interact with DP. Recombinant Fn domains were immobilized inwells at 10 ␮g/ml. Fn was immobilized at 2 ␮g/ml. The following procedures
are the same as those described above. In A and B, experiments were
performed three times in triplicate, and means ⫾ S.D. (error bars) are
shown. background, absorbance of noncoated wells incubated with thesame concentration of DP. *, p ⬍ 0.01 compared with a value of background absorbance.

Dermatopontin Interaction with Fibronectin

These data show that DP would change Fn conformation bymodifying inter- and intramolecular interactions.

DISCUSSION
In this study, we found that DP is present in the provisional
wound matrix, the wound fluid, and in the serum in addition to
the ECM. The presence of DP in the serum was unexpected, andthe function of DP in the serum is not known. It is possible that
DP functions as a coagulation factor because an 18-kDa invertebrate homologue of DP from blood cells of a crab shell, Limulus polyphemus, is an agglutination factor (33). DP in the provisional matrix may derive from either the serum or theinterstitial fluid. The presence of DP in the provisional matrix
and in the wound fluid is a novel finding, and this is the firstobservation that DP may play a role in wounds.
DP-kockout mice exhibit an Ehlers-Danlos syndrome phenotype (10). The previous report focused on the change of the
ECM architecture. Because patients with Ehlers-Danlos syndrome demonstrate delayed wound healing and abnormal scarformation (34, 35), the knock-out mice would be expected tohave a similar phenotype, but this has not been examined. Froma functional aspect, DP not only makes a ternary complex with
Fn and fibrin but also enhances the interaction. On the other
hand, immobilized DP showed less and no interaction with liq-14868 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 286 • NUMBER 17 • APRIL 29, 2011

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FIGURE 9. Modification of interdomain interactions of Fn by DP.
A, enhancement of the interaction by DP. Recombinant Fn domains shown on
the x axis were immobilized in wells at 20 ␮g/ml. A biotinylated 30-kDa fragment of Fn was added at 5 ␮g/ml in the presence or absence of 10 ␮g/ml DP.
B, inhibition of the interaction by DP. Recombinant III12–14 was immobilized at
1 ␮g/ml, and biotinylated III2–3 was added at 0.2 ␮g/ml in the presence orabsence of 2 ␮g/ml DP. The bound 30-kDa fragment and the III2–3 wereprobed by a streptavidin-conjugated HRP as described under “Experimental
Procedures.” *, p ⬍ 0.01. Experiments were performed three times in triplicate, and means ⫾ S.D. (error bars) are shown.

uid phase Fn and fibrin, respectively. These findings indicate apossibility that the binding site of DP is partially or completely
cryptic in the solid phase. The enhancement of the Fn interaction with fibrin in the presence of DP contributes to an increasein cell adhesion as shown in Fig. 5. The cells that adhered to theternary complex demonstrated increased spreading and a moreorganized cytoskeleton, indicating that the cells undergo morphological changes beyond adhesion on the complex.
Koblinski et al. (36) reported that co-immobilization of Fn
with nonadhesive proteins, such as BSA and cytochrome c, onan artificial surface induced a strong cell adhesion enhancement. This study importantly elucidated that Fn conformation
can be changed when it is simultaneously immobilized withseveral nonadhesive proteins, even with BSA. In the present
study, fibrin was immobilized first, and then complexes wereformed on the fibrin. In our system, a mixture of Fn and BSA did
not enhance cell adhesion, whereas a mixture with DP did.
Therefore, we confirmed that the observed cell adhesion
enhancement was specifically induced by DP, and the mechanism of the enhancement was different from that in the previous report (36).
In this study, we found that DP promoted Fn fibril formation.
An interaction between III2–3 and III12–14 is reported to be necessary for maintaining the compact and inactive conformationof Fn (32). Once activated by integrins, multiple interdomaininteractions are created between conformationally transformed Fn molecules, and Fn fibrils are formed (37). For Fn
fibril formation, the presence of I1–5 is essential (37, 38), andinteractions between I1–5 and III1–2 (27, 28) and/or I1–5 andIII12–14 (29) are reported to play very important roles.
The major interaction sites of Fn with DP were determined to
be III13 and III14, and a potential or cryptic site was III1. All ofthese domains are involved in a formation of Fn fibrils. The
finding that an interaction between I1–5 and III12–14 wasenhanced strongly and that between III2–3 and III12–14 was considerably inhibited by DP supports the idea that the change ofthe interaction between these domains of Fn and DP promotes
Fn fibril formation. Because the interaction between I1–5 andIII1–2 was not enhanced by DP, it is likely that the III1–2 iscryptic as previously reported (30, 31). However, it is possiblethat these domains unfold as other interdomain interactions
are disrupted as described below (23).
Apart from integrins, anastellin is the only known non-integrin type protein that can induce superfibronectin formation(21). The binding site of Fn to anastellin is III3, and a conformational change of cryptic III1–2 follows the interaction (23). The
binding site on Fn for anastellin locates within an interaction
site between III2–3 and III12–14, as in the case for DP, indicatingthat domain unfolding of a Fn molecule is important for Fn
fibril formation.
Anastellin is an artificial III1 domain of Fn that lacks A and B
␤-sheets covering one-third of the amino terminus of thedomain (21, 39). On the other hand, DP is an endogenous protein that is found in many types of tissues, with the dermis being
the most abundant source. Therefore, it is possible that DP
plays a role as a natural activator of Fn in a provisional matrix
before fibroblasts or endothelial cells migrate and interact with
Fn via ␣5␤1 integrin.

Dermatopontin Interaction with Fibronectin
Although DP is an abundant protein within the ECM, itsbiological functions remain largely unknown. In this report, DP
was shown to be present in the provisional wound matrix, and,more importantly, DP was found to be the first non-integrin
protein that can activate Fn and induce Fn fibril formation.
Thus, in a context of Fn activation, DP may help a wound to healby accumulating in the wound either from the surrounding
ECM or from the serum.
Acknowledgment—We thank Aiko Yasuda for technical assistance.
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