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Dermatopontin Expression is Decreased in Hypertrophic Scar and Systemic Sclerosis Skin Fibroblasts and is Regulated by Transforming Growth Factor-β1, Interleukin-4, and Matrix Collagen

Kei Kuroda, Osamu Okamoto,* and Hiroshi Shinkai Department of Dermatology, School of Medicine, Chiba University, Chiba, Japan; *Department of Dermatology, Oita Medical University, Oita, Japan

Dermatopontin is a recently discovered extracellular investigated the effects of cytokines and matrix colla- matrix protein with proteoglycan and cell-binding gen on dermatopontin expression in normal cultured properties and is assumed to play important roles in fibroblasts. Transforming growth factor-β1 increased cell–matrix interactions and matrix assembly. In this dermatopontin mRNA and protein levels, while study we examined the expression of dermatopontin interleukin-4 reduced dermatopontin expression. Sub- mRNA and protein in skin fibroblast cultures from strate coated with type I collagen reduced dermato- patients with hypertrophic scar and patients with sys- pontin mRNA levels, the reduction being more temic sclerosis. Dermatopontin mRNA and protein prominent in three-dimensional collagen matrices. levels were reduced in fibroblast cultures from hyper- Our results suggest that the decreased expression of trophic scar lesional skin compared with fibroblasts dermatopontin is associated with the pathogenesis of from normal skin of the same hypertrophic scar fibrosis in hypertrophic scar and systemic sclerosis, patient. Fibroblast cultures from systemic sclerosis and that the effect of the cytokines and matrix collagen patient involved skin also showed significantly reduced on dermatopontin may have important implications expression of dermatopontin compared with normal for skin fibrosis. Key words: skin fibrosis. J Invest Dermatol skin fibroblasts from healthy individuals. We also 112:706–710, 1999

ypertrophic scar (HS) and systemic sclerosis (SSc) Although dermatopontin appears to be involved in the formation are fibrotic disorders characterized by excessive of some pathologic conditions of connective tissue, such as wound accumulation of collagen (Fleischmajer, 1993; healing, fibrosis, and tumor invasion, the regulation of dermato- Lapie`re, 1993). Although unregulated production pontin gene expression in these situations is poorly understood. In of collagen is considered to be a crucial process in this study we investigated the expression of dermatopontin in skin theH development of fibrosis in HS and SSc, other extracellular fibroblast cultures from patients with HS and SSc, and assessed the matrix molecules also play an important part in the pathogenesis effects of transforming growth factor-β (TGF-β), interleukin-4 of fibrosis (Vuorio et al, 1991; Xu et al, 1991; Lacour et al, 1992; (IL-4), and collagen matrices on gene expression. Tan et al, 1993). Dermatopontin (tyrosine-rich acidic matrix protein) is a recently MATERIALS AND METHODS discovered, 22 kDa extracellular matrix protein (Neame et al, 1989; Patients HS samples were obtained from scar lesions from five patients Cronshaw et al, 1993; Superti-Furga et al, 1993; Okamoto et al, undergoing reconstructive surgery, and from normal tissues adjacent to scar 1996). The protein displays a striking tendency to bind to the small lesions for use as control specimens. The age of HS patients was 18–42 y. dermatan sulfate proteoglycans (Neame et al, 1989; Okamoto et al, The disease duration after injury was from 4 mo to 3 y. Nine patients 1996). Furthermore, dermatopontin promotes cell attachment and with SSc, who fulfilled the criteria for diagnosis (American Rheumatism cytoplasmic spreading of dermal fibroblasts, where it is assumed Association, 1980), and nine sex-matched and age-matched normal indi- the adhesion activity is mediated by cell surface integrin binding viduals as controls were also studied. The age of normal skin donors ranged (Lewandowska et al, 1991). Recently, dermatopontin has been from 37 to 52 y (average 42 y), and the age of SSc patients was 35–54 y shown to bind type I collagen and accelerate the assembly of (average 44 y). SSc patients had disease durations of 6 mo to 12 y; seven of nine patients had the disease for less than 4 y. All SSc skin specimens collagen into fibrils (MacBeath et al, 1993). Thus, dermatopontin were obtained from the dorsal forearm. The fibrotic changes of HS and is thought to be a multifunctional protein of the extracellular matrix. SSc skin were confirmed histopathologically.

Cell cultures Establishment of human fibroblasts from skin samples was Manuscript received April 9, 1998; revised December 22, 1998; accepted performed as described by Hayflick and Moorhead (1961) with some for publication December 30, 1998. modifications. Full-thickness skin specimen from SSc and HS patients and Reprint requests to: Dr. Kei Kuroda, Department of Dermatology, normal individuals were cut into small pieces, placed on the culture Mount Sinai School of Medicine, 1425 Madison Avenue, Second floor, flask, and cultured in Dulbecco’s modified Eagle’s medium (DMEM) New York, NY, 10029. supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, Abbreviations: HS, hypertrophic scar; SSc, systemic sclerosis. 100 units per ml penicillin, and 50 units per ml streptomycin in a humid

0022-202X/99/$10.50 · Copyright © 1999 by The Society for Investigative Dermatology, Inc. 706 VOL. 112, NO. 5 MAY 1999 DERMATOPONTIN AND SKIN FIBROSIS 707

atmosphere containing 5% CO2 at 37°C. When outgrowth of fibroblasts from the pieces of skin samples reached some 75% of the culture surface, subculture was performed by 0.25% trypsin treatment. Fibroblasts at four to seven passages were used for experiments. Human recombinant TGF- β1 (R&D Systems, Minneapolis, MN) and human recombinant IL-4 (provided by Ono Pharmaceutical, Osaka, Japan) were used for cytokine studies. Viable cell number was determined by using a hemocytometer in a Trypan Blue stain. For three-dimensional collagen gels, collagen (bovine dermal type I collagen dissolved at 3 mg per ml in 50 mM acetic acid) was prepared by mixing type I collagen and 10% FBS in DMEM at pH 7.4. Skin fibroblasts from subconfluent cultures were mixed with the collagen solution to a final concentration of 5 ϫ 105 cells per ml, 1 mg collagen per ml, and 1% FBS. The collagen cell suspensions (15 ml) were immediately placed on to 100 mm tissue culture dishes and incubated for 2 h at 37°C. After polymerization, 15 ml of 1% FBS/DMEM was added to each dish and floating collagen gels were obtained by detaching the gel from the plate. For control monolayer cultures trypsinized fibroblasts from subconfluent cultures were seeded on plastic culture dishes and cultured in 1% FBS/ DMEM. For planar type I collagen coating, the bovine collagen preparation was diluted and added to plastic dishes at a final concentration of up to 125 µg per ml. Coated dishes were allowed to dry and rinsed with PBS before use. Figure 1. Reduced expression of dermatopontin mRNA in HS fibroblast cultures. (a) Confluent fibroblasts from scar lesions (S) and Northern blot analysis Total cellular RNA was extracted using the normal skin (N) of five patients (HS1–HS5) were incubated with serum- guanidinium isothiocyanate method (Chomczynski and Sacchi, 1987). An free DMEM for 24 h. After extraction of total RNA, northern blot analysis aliquot of total RNA (5 µg per lane) was size fractionated by 1% agarose were performed. (b) Levels of dermatopontin mRNA were quantitated by gel electrophoresis and transferred to a nylon filter. Equal loading was densitometric scanning and corrected for GAPDH mRNA. confirmed by ethidium bromide staining. Blots were prehybridized, hybrid- ized, autoradiographed, and stripped for rehybridization as previously described (Kuroda and Shinkai, 1997). Probes used for hybridization were a 0.7 kb dermatopontin human cDNA (Superti-Furga et al, 1993) (a generous gift from Dr. A. Superti-Furga, University Children’s Hospital, Switzerland) and, as a control of constitutive expression, a 0.8 kb human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (Arcrari et al, 1984). The intensities of specific mRNA were quantitated with a Fuji Bio-Image analyzer BAS2000 (Fuji Photo Film, Kawasaki, Japan).

Measurement of dermatopontin production Confluent fibroblasts were incubated in serum-free DMEM for 24 h, cell culture media collected and protein precipitated by the addition of trichloroacetic acid. Samples corresponding to the original volume of medium were electrophoresed on 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. For immunoblotting, proteins were electrophoretically transferred to poly- vinylidene fluoride membranes (Millipore, Bedford, MA) in transfer buffer (25 mM Tris, 200 mM glycin, 20% methanol) for 4 h at 60 V. Efficient transfer was ensured by amid black staining for the lanes of molecular weight markers. Dermatopontin was detected by incubation with anti- dermatopontin rabbit serum (Okamoto et al, 1996) and visualized by the biotin–avidin–peroxidase complex system (Zymed Laboratories, South San Francisco, CA) using diaminobenzidine as the chromogenic substrate. Dermatopontin band intensities were quantitated by densitometry. In a preliminary study serial dilutions of each sample were used to prepare immunoblotting analyses and amounts of samples within the linear range for this assay were determined. Total protein synthesis was measured by incubating cells in serum-free DMEM containing 5 µCi [3H]leucine per ml for 24 h. Total [3H]leucine incorporation into proteins was measured by trichloroacetic acid precipitation. Figure 2. Reduced dermatopontin protein levels in HS fibroblast RESULTS cultures. (a) Confluent fibroblasts from scar lesions (S) and normal skin (N) of five patients (HS1–HS5) were incubated with serum-free DMEM Reduced expression of dermatopontin mRNA and protein for 24 h. After the precipitation of cell culture media, the expression of in HS fibroblasts Northern blot analysis was performed on five dermatopontin was studied by immunoblotting analysis. (b) Dermatopontin pairs of fibroblast cultures established from lesional skin and normal protein levels were quantitated by densitometric scanning and corrected skin adjacent to scar lesions from the same patients (Fig 1a). for total radiolabeled protein. Dermatopontin mRNA levels, after correction with respect to GAPDH, were clearly reduced in fibroblast cultures from lesional skin compared with normal fibroblasts (Fig 1b). Immunoblotting fibroblasts was due to the specific reduction of dermatopontin analysis also revealed that the ratio of 22 kDa dermatopontin band biosynthesis. intensity to total protein synthesis was lower in fibroblast cultures from lesional skins compared with fibroblasts from normal skins Significant reduction of dermatopontin mRNA and protein (Fig 2). The relative decrease in dermatopontin synthesis was levels in SSc fibroblasts Dermatopontin mRNA and protein approximately coincident with reduction in dermatopontin mRNA levels were examined in cultured fibroblasts established from nine levels. The measurement of cell numbers at the end of the culture SSc patient involved skin samples and nine normal skin samples period revealed no significant differences between scar and normal from healthy individuals. Northern blot analysis demonstrated a fibroblasts. Thus, the reduction of dermatopontin synthesis in scar significantly lower level of dermatopontin mRNA in SSc fibroblasts 708 KURODA ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 5. Concentration-dependent and time-dependent regulation of dermatopontin mRNA levels by TGF-β1 and IL-4 in normal fibroblasts. Confluent normal fibroblasts were rinsed with PBS followed Figure 3. Significant reduction of dermatopontin mRNA levels in by synchronization with serum-free DMEM for 6 h, then incubated in SSc fibroblasts. (a) Confluent fibroblasts from normal healthy controls serum-free DMEM for 24 h with various cytokine concentrations or for and SSc patients were incubated with serum-free DMEM for 24 h. After various intervals with 2.5 ng per ml TGF-β1 or 5 units per ml IL-4. After extraction of total RNA, northern blot analysis was performed. (b) Levels extraction of total RNA, the expression of dermatopontin mRNAs was of dermatopontin mRNA were quantitated by densitometric scanning and studied by northern blot analysis. (a) TGF-β1; (b) IL-4. corrected for GAPDH mRNA. The results from control and SSc fibroblast groups were compared using the Mann–Whitney U test.

Figure 4. Reduction of dermatopontin protein levels in SSc fibroblasts. (a) Confluent fibroblasts of normal healthy controls and SSc Figure 6. Effect of TGF-β1 and IL-4 on dermatopontin protein patients were incubated with serum-free DMEM for 24 h. After expression in normal fibroblasts. (a) Confluent normal fibroblasts were precipitation of cell culture media, the expression of dermatopontin were rinsed with PBS followed by synchronization with serum-free DMEM for studied by immunoblotting analysis. (b) Dermatopontin protein levels were 6 h, then incubated in serum-free DMEM for 24 h with or without 5 ng quantitated by densitometric scanning and corrected for total radiolabeled per ml TGF-β1 or 5 units per ml IL-4. After precipitation of cell culture protein. The results from control and SSc fibroblast groups were compared media, the expression of dermatopontin were studied by immunoblotting using the Mann–Whitney U test. analysis. (b) Dermatopontin protein levels were quantitated by densitometric scanning and corrected for total radiolabeled protein. Data are mean Ϯ SD of triplicate cultures. TGF-β1; p Ͻ 0.001; IL-4, p Ͻ 0.001. compared to control fibroblasts (p Ͻ 0.01) (Fig 3), with dermato- pontin mRNA only faintly detectable in fibroblasts from two concentration-dependent manner up to 10 ng per ml (Fig 5a). SSc patients (patients 1 and 4). This was in agreement with β immunoblotting analysis, which revealed that the ratio of dermato- TGF- 1 response time-course experiments showed a steady increase in dermatopontin mRNA levels during the 48 h of the experiment. pontin band intensity to total protein synthesis was significantly β lower in SSc fibroblasts compared with control fibroblasts (p Ͻ 0.05) After 48 h incubation, 2.5 ng TGF- 1 per ml showed a 3.4- (Fig 4). Measurement of cell numbers at the end of the culture fold increase in dermatopontin mRNA compared with untreated period revealed no significant differences between SSc and normal samples. In contrast, incubation of cultured cells with IL-4 resulted fibroblasts. Thus, the reduction of dermatopontin synthesis in SSc in a significant decrease in dermatopontin mRNA levels in a fibroblasts was due to the specific reduction of dermatopontin concentration-dependent (up to 10 ng per ml) and time-dependent biosynthesis. manner (Fig 5b). After 48 h incubation, 5 units per ml IL-4 resulted in a 77% decrease in dermatopontin mRNA levels. Dermatopontin expression is differentially regulated by Immunoblotting analysis revealed that the ratio of dermatopontin TGF-β1 and IL-4 The effects of TGF-β1 and IL-4 on dermato- production to total protein synthesis was increased by TGF-β1 and pontin expression were examined on normal skin cultured decreased by IL-4 in normal skin cultured fibroblasts (Fig 6). fibroblasts. TGF-β1 enhanced dermatopontin mRNA levels in a Twenty-four hour incubation with TGF-β1 (5 ng per ml) or IL-4 VOL. 112, NO. 5 MAY 1999 DERMATOPONTIN AND SKIN FIBROSIS 709

assembly (MacBeath et al, 1993), while decorin markedly delays fibril formation (Vogel et al, 1984). These findings suggest that the two molecules may help regulate the assembly and turnover of collagen in the extracellular matrix through their diverse effect. In fibrosing skin diseases, the expression of decorin is also altered. Decorin mRNA levels vary in SSc cultured fibroblasts (Vuorio et al, 1991) and expression is decreased in keloid fibroblasts (Tan et al, 1993). Therefore, the altered expression of dermatopontin and decorin may greatly influence cell behavior and fibril formation in HS and SSc. In our study, not every SSc fibroblast expressed low levels of dermatopontin. Although we were unable to demonstrate a positive association between dermatopontin expression and clinical or histologic findings, there still remains a possibility that patient age or variability in disease duration of the fibrosis may affect the expression of dermatopontin. To date, regulation of dermatopontin expression by cytokines has not been studied in detail. Our results clearly indicate that TGF-β1 stimulates both dermatopontin mRNA and protein levels, whereas IL-4 reduces dermatopontin expression. TGF-β1 and IL-4 are assumed to be potential candidate molecules in the pathogenesis of fibrotic skin diseases (Roberts et al, 1986; Salmon et al, 1996). Figure 7. Matrix collagen downregulates dermatopontin gene Therefore, it is likely that these cytokines influence dermatopontin expression. (a) Normal fibroblasts from subconfluent cultures were seeded expression in the development of fibrosis in HS and SSc. SSc skin on to plastic culture dishes coated with various concentrations of type I fibroblasts express high levels of IL-4 in contrast to normal fibroblasts collagen, and incubated in serum-free DMEM for 24 h. After extraction (Salmon et al, 1996), suggesting that IL-4 is involved in the of total RNA, northern blot analysis were performed. (b) Normal fibroblasts persistent downregulation of dermatopontin expression. Thus, our from subconfluent cultures were grown on untreated plastic culture dishes results also suggest that reduced expression of dermatopontin in as monolayer cultures (ML) or in three-dimensional collagen gel (CG) for SSc fibroblasts may be due in part to the effects of IL-4. 24 h with 1% FBS/DMEM. Northern blot analysis were performed and In this study we have shown that dermatopontin expression was levels of dermatopontin mRNA were quantitated by densitometric scanning clearly reduced on the substrate coated with type I collagen and in and corrected for respective 28S rRNA levels. three-dimensional collagen matrices. Collagen matrices alter the expression of extracellular matrix components and collagenases (5 units per ml) resulted in a 2.5-fold increase and an 82% decrease, (Unemori and Werb, 1986; Eckes et al, 1993; Langholtz et al, respectively, as compared with untreated samples. Incubation with 1995). Decorin expression is also reduced in collagen matrices TGF-β1 and IL-4 slightly increased cell numbers to 1.2-fold each. (Greve et al, 1990). As dermatopontin is tightly bound to decorin Thus, TGF-β1 and IL-4 both modulate the specific biosynthesis in the extracellular space (Neame et al, 1989; Okamoto et al, of dermatopontin. 1996), the collagen matrix may decrease the accumulation of dermatopontin by reduced expression of both dermatopontin Downregulation of dermatopontin expression by matrix and decorin. collagen To evaluate the effects of matrix collagen on dermato- During the initiation and progression of fibrosis various soluble pontin expression, we cultured normal skin fibroblasts on type I mediators such as cytokines may induce the unusual accumulation collagen-coated dishes and in three-dimensional collagen gels. of collagen, and this marked alteration of collagen composition Substrata coated with type I collagen reduced dermatopontin may affect dermatopontin expression. TGF-β1 and IL-4 are potent mRNA levels in proportion to the concentration of coated collagen stimulators of collagen synthesis (Ignotz and Massague, 1986; (Fig 7a). A collagen concentration of 125 µg per ml reduced Postlethwaite et al, 1992) and therefore may affect dermatopontin dermatopontin mRNA levels by 40% compared with uncoated expression in the development of fibrosis in HS and SSc either dishes. When fibroblasts were cultured in three-dimensional colla- directly, or through increasing the collagen matrices around gen gels, the reduction in dermatopontin mRNA levels was more fibroblasts. prominent: µ80% reduction from levels observed in monolayer cultures on uncoated dished (Fig 7b). DISCUSSION We are grateful to Dr. A. Superti-Furga, University Children’s Hospital, Switzerland, for providing the cDNA clone of human dermatopontin. This work In the current study we have shown that dermatopontin mRNA was supported in part by the project research for progressive systemic sclerosis from and protein levels were significantly reduced in cultured fibroblasts the Ministry of Health and Welfare of Japan. established from fibrotic tissues of HS and SSc patients. Thus, the impaired expression of dermatopontin may be associated with the pathogenesis of fibrosis in HS and SSc. REFERENCES Dermatopontin plays an important part in cell–matrix interactions American Rheumatism Association Subcommittee for Scleroderma Criteria of and matrix assembly. These functions are thought to be greatly the American Rheumatism Association Diagnostic and Therapeutic Criteria affected by small dermatan sulfate proteoglycans. As originally Committee: Preliminary criteria for the classification of systemic sclerosis shown by Neame et al (1989), dermatopontin displays a striking (scleroderma). 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