Increased Expression of Heat-Shock Protein 47 is Associated with Overproduction of Type I Procollagen in Systemic Sclerosis Skin Fibroblasts

Increased Expression of Heat-Shock Protein 47 is Associated with Overproduction of Type I Procollagen in Systemic Sclerosis Skin Fibroblasts Kei Kuroda, Reiko Tsukifuji, and Hiroshi Shinkai Department of Dermatology, School of Medicine, Chiba University, Chiba, Japan

Heat-shock protein 47 (HSP47) is a collagen-binding stress protein that is thought to act as a collagenspecific molecular chaperon during the biosynthesis and secretion of procollagen. In this study we examined the expression of HSP47 mRNA and protein in systemic sclerosis (SSc) skin fibroblasts. HSP47 mRNA and protein levels were significantly higher in fibroblast cultures from SSc patient-involved skin samples than in fibroblasts from normal skin from healthy individuals, as assessed by northern blot and immunoblot analyses, respectively. SSc cultured fibroblasts with increased levels of HSP47 mRNA and protein showed high expression of type I procollagen. By in situ hybridization, SSc skin had a higher number of fibroblasts with high HSP47 and procollagen α1(I) mRNA levels than normal

skin, and the distribution of HSP47 mRNA was similar to that of procollagen α1(I) mRNA. We also investigated the effects of cytokines on the expression of HSP47 in normal cultured fibroblasts. Transforming growth factor-β1 and interleukin-4 increased HSP47 mRNA and protein levels, whereas interferon-γ reduced HSP47 expression. The same pattern of cytokine-regulated expression was observed for type I procollagen levels. These results indicate that HSP47 expression is closely associated with that of type I procollagen in skin fibroblasts, and that increased expression of HSP47 may be involved in the abundant production of type I procollagen by SSc fibroblasts. Key words: interferon-γ/ interleukin-4/transforming growth factor-β. J Invest Dermatol 111:1023–1028, 1998

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molecular chaperones in protein folding, assembling newly synthesized or reassembling malfolded proteins (Schlesinger, 1990). HSP47 binds to nascent single polypeptide chains of procollagen immediately after entry into the endoplasmic reticulum, and dissociates from procollagen chains in the Golgi apparatus (Satoh et al, 1996). When aberrant procollagen accumulates in the endoplasmic reticulum due to heat shock, depletion of ascorbic acid or after treatment with α, α9-dipyridyl, which inhibits the stable triple-helix formation of procollagen, HSP47 remains stably bound to the procollagen chains (Nakai et al, 1992; Satoh et al, 1996). Although it is thought HSP47 is a collagen-specific molecular chaperone, it is not yet clear whether HSP47 is associated with the overproduction of collagen by SSc fibroblasts. In this study we investigated the expression of HSP47 in SSc fibroblasts and examined the regulation of the HSP47 expression by cytokines believed to be involved in the pathogenesis or treatment of SSc tissue fibrosis; transforming growth factor-β (TGF-β), interleukin-4 (IL-4), and interferon-γ (INF-γ).

ystemic sclerosis (SSc) is a connective tissue disease characterized by fibrosis of the skin, subcutaneous tissue, and various internal organs (Fleischmajer, 1993). The most prominent pathologic manifestation of the disease is an excessive accumulation of extracellular matrix components, predominantly collagen types I and III. Many studies have shown increased amounts of type I collagen in tissue and fibroblast cultures derived from SSc patient involved skin (LeRoy, 1974; Rodnan et al, 1979; Uitto et al, 1979; Krieg et al, 1981). More recent data have also shown increased levels of type I collagen mRNA (Ka¨ha¨ri et al, 1984; Jimenez et al, 1986) and increased activity of α2(I) collagen promoter in SSc fibroblasts (Kikuchi et al, 1992), suggesting the enhancement of the type I collagen expression at the transcriptional level. In collagen biosynthesis, nascent single procollagen polypeptides, immediately after translation, undergo modification in the endoplasmic reticulum, form triple helical chains, and are secreted as procollagen into the extracellular space via the Golgi apparatus (Olsen, 1991). Therefore, the elucidation of the post-translational processing mechanisms in SSc fibroblasts, where abundant nascent procollagen polypeptides result in overproduction of collagen, may be important for understanding the precise mechanism of fibrosis in SSc. HSP47, a 47 kDa heat-shock protein, is a member of a group of heat shock proteins with unique collagen-specific binding characteristics (Nagata et al, 1986). Heat shock proteins play an important role as

Manuscript received March 17, 1998; revised June 4, 1998; accepted for publication August 27, 1998. Reprint requests to: Dr. Kei Kuroda, Department of Dermatology, School of Medicine, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-0856, Japan. Abbreviations: HSP47, heat-shock protein 47; SSc, systemic sclerosis.

MATERIALS AND METHODS Patients We studied nine patients with SSc who fulfilled the criteria for diagnosis (American Rheumatism Association, 1980), and nine sex- and agematched normal individuals. The seven patients were classified as having diffuse cutaneous SSc and two as having limited cutaneous SSc (LeRoy et al, 1988). The age of normal skin donors ranged from 37 to 52 y (average 42 y), and the age of SSc patients was 35–54 y (average 44 y). SSc patients had a disease duration of 6 mo to 12 y; seven of nine patients had the disease for less than 4 y. All skin specimens were obtained from the dorsal forearm, and changes in specimens from SSc patients were confirmed histopathologically. All the samples from SSc patients represented a considerable amount of fibrosis from the upper or middle dermis to the upper part of subcutaneous tissue, with relatively less numerous fibroblasts and minor inflammatory cell infiltrates.

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0022-202X/98/$10.50 Copyright © 1998 by The Society for Investigative Dermatology, Inc.

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Cell cultures Skin fibroblasts from patients with SSc and normal individuals were separated from tissue explant cultures. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units penicillin per ml, and 50 units streptomycin per ml. Incubation was performed at 37°C in 5% CO2. Confluent fibroblasts at five to seven passage were used for the experiments. Human recombinant TGF-β1 (R&D systems, Minneapolis, MN), human recombinant IL-4 (provided by Ono, Osaka, Japan), and human recombinant INF-γ (provided by Shionogi, Osaka, Japan) were used for the cytokine study. Northern blot analysis Total cellular RNA was extracted using the guanidinium isothiocyanate method (Chomczynski and Sacchi, 1987). An aliquot of total RNA (5 µg per lane) was size fractionated by 1% agarose gel electrophoresis and transferred to a nylon filter. Equal loading was confirmed by ethidium bromide staining. Blots were prehybridized, hybridized, autoradiographed, and stripped for rehybridization as previously described (Kuroda and Shinkai, 1997). Probes used for hybridization were a 1.5 kb HSP47 mouse cDNA (Takechi et al, 1992) (a generous gift from Dr. K. Nagata, Department of Cell Biology, Chest Disease Research Institute, Kyoto University) and a 1.5 kb procollagen α1(I) human cDNA (Chu et al, 1982). As a control of constitutive expression, a 0.8 kb human glyceraldehyde-3-phosphate dehydrogenase cDNA was used (Arcrari et al, 1984). The intensities of specific mRNA were quantitated with a Fuji Bio-Image analyzer BAS2000 (Fuji Photo, Kawasaki, Japan). Immunoblotting Cell extracts were prepared from lysis buffer (50 mM TrisHCl, pH 7.5, 150 mM NaCl, 1% NP-40, and 5 mM ethylenediamine tetraacetic acid). Equal amounts of cell extracts (2 µg protein per lane) were separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to polyvinylidene fluoride membranes. The membranes were stained with amido black before blocking to control for equal loading. HSP47 was detected by incubation with anti-HSP47 monoclonal antibody (1:1000 dilution; StressGen, Victoria, BC, Canada) and visualized by biotin-avidin-peroxidase complex system (Vector Laboratories, Burlingame, CA) using diaminobenzidine as the chromogenic substrate. Relative HSP47 protein levels were quantitated by densitometry. Measurement of newly synthesized collagen Fibroblast cultures were incubated in serum-free DMEM containing 5 µCi [3H]proline per ml in the presence of 50 µg L-ascorbic acid 2-phosphate per ml for 24 h. Labeled proteins secreted into culture media were precipitated by the addition of 5% trichloroacetic acid. Aliquots of equal amounts of trichloroacetic acid-precipitable radioactivity were dissolved in 50 mM acetic aid and digested with pepsin. Then SDS-polyacrylamide gel electrophoresis (PAGE) (5% gel containing 3.6 M urea) was performed as previously described (Kuroda and Shinkai, 1997). The radioactive bands were detected by fluorography, and the relative amounts of collagen were quantitated by densitometry. In situ hybridization For the preparation of RNA probes, a 0.7 kb mouse HSP47 XhoI-EcoRI cDNA fragment and a 1.5 kb human α1(I) procollagen BamHI-XbaI fragment were subcloned into pBluescript II and pGEM7Z, respectively. Labeled probes were synthesized by in vitro transcription with SP6, T7, or T3 polymerase in the presence of digoxigenin-labeled dUTP (Boehringer, Indianapolis, IN). Formalin-fixed, paraffin-embedded specimens from SSc patients and healthy controls were used for in situ hybridization. Sample pretreatment and hybridization was performed as described previously (Tsukifuji et al, 1997). Briefly, the deparaffined sections were incubated with proteinase K followed by chondroitinase ABC lyase. The slides were refixed in 4% paraformaldehyde, incubated with 2 mg glycine per ml, and dipped in 0.2N HCl. After prehybridization, sections were hybridized with anti-sense or sense RNA probes in a solution containing 50% formamide, 10% dextran sulfate, 13Denhardt’s solution, 200 µg yeast tRNA per ml, 600 mM NaCl, 1 mM ethylenediamine tetraacetic acid, 0.25% SDS, and 10 mM Tris-HCl (pH 7.6), and incubated for 18 h at 50°C. Sections were washed in 23sodium citrate/chloride buffer, treated with RNAase A, and washed with 0.23sodium citrate/chloride buffer at 50°C. Immunologic detection was performed with anti-digoxigenin antibody conjugated with alkaline phosphatase (Boehringer). After color reaction by Fast Red (BioGenex, San Ramon, IN) with naphthol phosphate, sections were counterstained with hematoxylin and mounted. Intensity of individual cell staining was expressed as no, weak, moderate, and strong staining. Semi-quantitation was based on the mean number of moderateand strong-staining cells per high-power field. A minimum of 10 high-power fields were assessed per section using a 10 eyepiece and 10 objective lens, and scored as follows: –, ,0.2 cells per high-power field; 1, 0.2–4 cells per high-power field; 11, 5–10 cells per high-power field; 111, .10 cells per high-power field.

Figure 1. Increased expression of HSP47 and procollagen α1(I) mRNA in cultured SSc fibroblasts. (a) Confluent fibroblasts of normal healthy controls and SSc patients were incubated with serum-free DMEM for 24 h. After extraction of total RNA, northern blot analysis was performed. (b) Levels of HSP47 and procollagen α1(I) mRNA were quantitated by densitometric scanning and corrected for glyceraldehyde-3-phosphate dehydrogenase mRNA. The results for the control and SSc fibroblast groups were compared by the Mann–Whitney U test.

Immunohistochemical staining Immunohistochemistry was performed on formalin-fixed, paraffin-embedded specimens. After blocking endogenous peroxidase activity with 1% H2O2, sections were incubated with anti-HSP47 monoclonal antibody (1:1600 dilution) overnight at 4°C. Slides were stained using the biotin-avidin-peroxidase complex system (Zymed Laboratories, San Francisco, CA) with diaminobenzidine as the chromogenic substrate. After development, slides were counterstained with hematoxylin and mounted. Semiquantitation was performed as described in in situ hybridization.

RESULTS Increased expression of HSP47 mRNA in cultured SSc fibroblasts Northern blot analysis for HSP47 and type I collagen was performed using total RNA isolated from cultures established from nine SSc patient-involved skins and nine normal skins of healthy individuals (Fig 1a). Scanning densitometory of mRNA transcripts, after correction by the value from glyceraldehyde-3-phosphate dehydrogenase mRNA, demonstrated a statistically higher level of HSP47 mRNA in fibroblast cultures from SSc patients than in those from healthy controls (Fig 1b). The level of procollagen α1(I) mRNA was also significantly higher in SSc fibroblasts than in normal fibroblasts. SSc fibroblasts with clearly high levels of procollagen α1(I) mRNA (No. 1–5) had high levels of HSP47 mRNA. Increased HSP47 protein expression in cultured SSc fibroblasts Immunoblot analysis with anti-HSP47 monoclonal antibody was performed on cell lysates from nine SSc and nine normal fibroblast cultures. The relative magnitudes of 47 kDa bands were assessed by densitometry, and demonstrated significantly higher levels of HSP47 protein in fibroblast cultures from SSc patients compared with healthy controls (Fig 2a). An increase in procollagen α1(I) was also observed in SSc fibroblasts, as estimated by densitometry on SDSPAGE fluorography. Increases in the relative amounts of type I procollagen in SSc fibroblasts were approximately coincident with increases in HSP47 (Fig 2b, c). Detection of HSP47 mRNA and protein in affected skin of SSc patients To determine the expression of HSP47 and type I

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and procollagen α1(I) also showed high expression of these molecules in resultant fibroblast cultures (Table I, Figs 1, 2). TGF-β1, IL-4, and INF-γ coordinately regulate HSP47 and type I collagen expression To further investigate the coexpression of HSP47 and type I collagen, we estimated the effects of TGF-β1, IL-4, and INF-γ on HSP47 and type I collagen expression in normal skin cultured fibroblasts. TGF-β1 and IL-4 enhanced HSP47 mRNA levels in a concentration-dependent manner up to 10 ng per ml each (Fig 5a, b). A time-response study of these cytokines showed a steady increase in HSP47 mRNA levels during the 48 h experiment. In contrast, incubation of the cultured cells with INF-γ resulted in a significant decrease in HSP47 mRNA levels in a dose-dependent (up to 1000 U per ml) and time-dependent manner (Fig 5c). TGFβ1, IL-4, and INF-γ regulated procollagen α1(I) mRNA levels synchronously to that of HSP47 mRNA; TGF-β1 and IL-4 increased procollagen α1(I) mRNA levels but INF-γ suppressed mRNA levels in a dose- and time-dependent manner. Protein levels of both HSP47 and type I collagen were increased by TGF-β1 and IL-4 and decreased by INF-γ (Fig 6). DISCUSSION

Figure 2. Increased expression of HSP47 and prollagen in cultured SSc fibroblasts. (a) For HSP47, confluent fibroblasts of normal healthy controls and SSc patients were incubated with serum-free DMEM for 24 h and immunoblot analysis was performed on extracted cell protein. For collagen, confluent fibroblasts were labeled with [3H]proline for 24 h and newly synthesized collagen secreted into the culture media was separated by SDSPAGE after pepsin digestion. Protein levels of HSP47 and procollagen α1(I) were quantitated by densitometric scanning of immunoblots and fluorographs, respectively. Values are expressed in relative densitometric units and results for control and SSc fibroblast groups were compared by the Mann–Whitney U test. (b) Immunoblotting of HSP47. (c) SDS-PAGE fluorography of types I procollagen.

procollagen in tissue, in situ hybridization was performed on skin specimens from SSc patients and healthy controls. The staining intensity of HSP47 and procollagen α1(I) varied between individual fibroblasts, particularly in SSc, but to some extent also in normal skin. SSc skin specimens contained greater numbers of fibroblasts with moderate or strong HSP47 staining compared with skin specimens from healthy controls (Fig 3, Table I). SSc skin samples also contained numerous fibroblasts with moderate or strong procollagen α1(I) staining. SSc skin specimens that showed high HSP47 mRNA expression (specimens 1–5) also contained high expression of procollagen α1(I) mRNA (Table I). Fibroblasts with moderate or strong HSP47 staining were found mainly in the deep reticular dermis and subcutaneous tissue in SSc patient samples, which was similar to the distribution of fibroblasts with moderate or strong procollagen α1(I) staining. Immunohistochemical analysis of HSP47 was performed on skin specimens from SSc patients and healthy controls. SSc skin samples contained more numerous fibroblasts with moderate or strong staining compared with normal skin (Fig 4, Table I). Strong-staining fibroblasts were found mainly in the deep reticular dermis and subcutaneous tissue. SSc skin specimens 1–5 that contained high expression of HSP47

In this study, we have shown that both HSP47 mRNA and protein levels were significantly increased in cultured SSc fibroblasts. Furthermore, SSc fibroblasts that contained increased levels of type I collagen mRNA and protein also exhibited high levels of HSP47 expression. These findings were confirmed by in situ hybridization with HSP47 and type I collagen anti-sense RNA probes and immunohistochemical analysis. Therefore the expression of HSP47 appears to be closely associated with type I collagen in SSc. The binding of HSP47 to nascent single procollagen polypeptides prevents premature folding or aggregation of the procollagen chains (Nagata et al, 1986; Satoh et al, 1996). A recent study showed that transfection of anti-sense HSP47 cDNA into mouse collagen-secreting embryonic cells causes a decrease in fully elongated nascent procollagen α1(I) (Sauk et al, 1994). HSP47 has also been reported to have an inhibitory effect on the degradation of procollagens in the endoplasmic reticulum (Jain et al, 1994). Therefore, in SSc fibroblasts with enhanced type I procollagen synthesis, an increased HSP47 synthesis may be necessary to maintain the stoichiometry of the interaction between the two molecules. Thus, HSP47 could play an important role in posttranslational processing of abundant type I procollagen chains, leading to overproduction of these procollagens in SSc. To date, the studies on HSP47 in pathologic conditions have been limited. Expression of HSP47 mRNA has been recently shown to increase in parallel with type I and III collagen mRNA during the progression of carbon tetrachloride-induced rat liver fibrosis (Masuda et al, 1994). In this study, not every fibroblast from SSc patients had high expression of HSP47 and type I collagen. Many studies have shown that the expression of type I collagen varies between different fibroblasts cultured from SSc patients (LeRoy, 1974; Uitto et al, 1979; Krieg et al, 1981). This may be explained by several factors, including the clinical or histologic stage of the disease and culturing conditions. In our study, SSc fibroblasts from patients with the shorter disease duration tended to show high expression of HSP47 and type I collagen. Our in situ hybridization and immunohistochemistry results showed considerable variation in HSP47 and type I procollagen expression between individual fibroblasts in the same skin specimen. Previous studies have also shown that SSc fibroblasts are heterogeneous in terms of collagen expression in vivo (Kulozik et al, 1990; Peltonen et al, 1990). The SSc skin specimens that had more numerous fibroblasts with high HSP47 and type I collagen mRNA levels in vivo, also contained higher levels in resultant fibroblast cultures. This indicates that the increased expression of both HSP47 and type I collagen by cultured SSc fibroblasts after serial passages in vitro reflects the expansion of high-expressing fibroblasts in skin. In addition to the strong association between HSP47 and type I procollagen expression in SSc fibroblasts, cytokine studies demonstrated coordinated regulation. TGF-β1 and IL-4 increased expression, and INF-γ decreased expression, of both HSP47 and type I procollagen in

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Figure 3. Increased expression of HSP47 and procollagen α1(I) mRNA in affected skin of SSc patients. In situ hybridization was performed with SSc and normal skin specimens. Hybridization with HSP47 anti-sense probes in SSc skin (a) and normal skin (b). Hybridization with procollagen α1(I) anti-sense probes in SSc skin (c) and normal skin (d). SSc skin contained greater numbers of fibroblasts with strong staining of HSP47 and procollagen α1(I) compared with normal skin. Scale bars: 20 µm.

Table I. Semi-quantitative analysis of HSP47 and procollagen α1(I) expression in the skin tissuea mRNA Case no. Controls 1 2 3 4 5 SSc patients 1 2 3 4 5 6 7

HSP47

procollagen α1(I)

HSP47 protein

1 2 1 2 2

1 1 1 1 1

1 1 1 1 1

11 11 11 1 11 2 1

11 111 111 11 111 1 1

11 111 11 11 111 1 1

aSemi-quantification for in situ hybridization and immunohistochemistry was based on the mean number of moderate- and strong-staining cells per high-power field, and scored as follows: 2, ,0.2 cells; 1, 0.2–4 cells; 11, 5–10 cells; 111, .10 cells.

Figure 4. Increased expression of HSP47 protein in affected skin of SSc patients. Immunohistochemistry was performed with SSc skin (a) and normal skin (b). Strong staining for HSP47 was found in SSc fibroblasts. Scale bars: 10 µm.

Figure 5. Dose- and time-dependent regulation of HSP47 and procollagen α1(I) mRNA levels by TGF-β1, IL-4, and INF-γ in normal fibroblasts. Confluent normal fibroblasts were incubated in serum-free DMEM for 24 h with various concentrations of cytokine and for various intervals with 2.5 ng TGF-β1 per ml, 5 units IL-4 per ml, or 10 units INF-γ per ml. After extraction of cellular total RNA, expression of HSP47 and procollagen α1(I) mRNA was studied by northern blot analysis. (a) TGF-β1; (b) IL-4; (c) INF-γ.

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We are grateful to Dr. K. Nagata, Department of Cell Biology, Chest Disease Research Institute, Kyoto University, Japan, for providing the cDNA clone of mouse HSP47. This work was supported in part by the project research for progressive systemic sclerosis from the Ministry of Health and Welfare of Japan.

REFERENCES

Figure 6. Effect of TGF-β1, IL-4, and INF-γ on the HSP47 and type I procollagen protein expression in normal fibroblasts. (a) Confluent normal fibroblasts were incubated in serum-free DMEM for 24 h with or without 5 ng TGF-β1 per ml, 5 units IL-4 per ml, or 100 units INF-γ per ml. After extraction of cell proteins, expressions of HSP47 were studied by western blot analysis. (b) Confluent normal fibroblasts were labeled with [3H]proline for 24 h in the absence or presence of 5 ng TGF-β1 per ml, 5 units IL-4 per ml, or 100 units INF-γ per ml, and newly synthesized collagen secreted into the culture media was separated by SDS-PAGE after pepsin digestion.

normal skin fibroblasts. Previous studies have also shown coregulation of HSP47 and collagen. HSP47 and type I collagen synthesis decreases after fibroblasts transformation (Nagata et al, 1988), whereas synthesis of both HSP47 and type IV collagen increases during differentiation of teratocartinoma F9 cells after treatment with retinoic acid (Takechi et al, 1992). TGF-β and epidermal growth factor are reported to coordinately regulate the expression of HSP47 and type I collagen in L6 myoblasts; TGF-β stimulating the expression of both HSP47 and type I collagen, and epidermal growth factor suppressing expression (Clarke et al, 1993). Exceptionally, HSP47 is not coregulated with collagen by heat induction. Treatment of L6 cells for a few hours at 42°C or 44°C leads to an increase in the levels of HSP47 mRNA and protein but procollagen type I levels remain unchanged (Clarke et al, 1993). The regulation of heat shock protein gene expression with respect to procollagen synthesis is not well understood. Co-regulation might be due to the sharing of common regulatory sequences in the promoter regions of the HSP47 and type I collagen genes, and heat induction may require the presence of heat shock elements found only in the promoter regions of HSP47. TGF-β and IL-4 stimulate the synthesis of collagen and other extracellular matrix components, and these cytokines are enhanced in SSc skin (LeRoy et al, 1989; Postlethwaite et al, 1992; Rudnicka et al, 1994; Salmon et al, 1996). Therefore, TGF-β and IL-4 are presumed to be potential candidate molecules in SSc pathogenesis. On the other hand, INF-γ is a potent inhibitor of collagen synthesis and is reported to be beneficial as an anti-fibrotic agent in the treatment of SSc (Hunzelmann et al, 1997). This means that the regulation of HSP47 expression by these cytokines, with respect to collagen production, may be responsible for the pathogenesis or treatment of tissue fibrosis of SSc.

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