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Activation of Tissue Inhibitor of Metalloproteinases-3 (TIMP-3) mRNA Expression in Scleroderma Skin Fibroblasts

1998; Elsevier BV; Volume: 110; Issue: 4 Linguagem: Inglês

10.1046/j.1523-1747.1998.00138.x

1523-1747

Laura Mattila, Kristiina Airola, Matti Ahonen, M. Hietarinta, Carol M. Black, Ulpu Saarialho‐Kere, Veli‐Matti Kähäri,

Autoimmune Bullous Skin Diseases

Excessive accumulation of fibrillar collagens is a hallmark of the cutaneous fibrosis in both systemic and localized scleroderma. Turnover of the collagenous extracellular matrix is dependent on the balance between collagenolytic matrix metalloproteinases and their specific inhibitors. We have examined the expression of the novel, matrix associated tissue inhibitor of metalloproteinases-3 (TIMP-3) in normal and scleroderma skin fibroblasts in culture and in vivo. The levels of TIMP-3 mRNA were elevated up to 2.5-fold in five of seven systemic sclerosis fibroblast strains, whereas TIMP-1 mRNA expression was elevated up to 1.8-fold in two and TIMP-2 mRNA expression up to 1.8-fold in two systemic sclerosis strains. Using in situ hybridization, TIMP-3 mRNA was detected in seven of 12 localized scleroderma skin samples, specifically in fibroblasts within fibrotic collagen fibers or in the vicinity of inflammatory cells. TIMP-1 mRNA was detected in three of eight scleroderma skin samples in fibroblasts adjacent to inflammatory cells. The expression of TIMP-3 mRNA by systemic sclerosis and normal skin fibroblasts was enhanced to a similar extent (by 8.6- and 8.1-fold, respectively) by transforming growth factor-β, and suppressed down to 34 and 54%, respectively, by tumor necrosis factor-α. Specific activation of TIMP-3 gene expression in scleroderma skin fibroblasts in culture and in vivo suggests a role for TIMP-3 in the pathogenesis of dermal fibrosis via inhibition of turnover of fibrotic dermal extracellular matrix, possibly due to upregulation of TIMP-3 expression by transforming growth factor-β. Keywords: fibrosis/matrix metalloproteinase. J Invest Dermatol 110:416–421 1998 Excessive accumulation of fibrillar collagens is a hallmark of the cutaneous fibrosis in both systemic and localized scleroderma. Turnover of the collagenous extracellular matrix is dependent on the balance between collagenolytic matrix metalloproteinases and their specific inhibitors. We have examined the expression of the novel, matrix associated tissue inhibitor of metalloproteinases-3 (TIMP-3) in normal and scleroderma skin fibroblasts in culture and in vivo. The levels of TIMP-3 mRNA were elevated up to 2.5-fold in five of seven systemic sclerosis fibroblast strains, whereas TIMP-1 mRNA expression was elevated up to 1.8-fold in two and TIMP-2 mRNA expression up to 1.8-fold in two systemic sclerosis strains. Using in situ hybridization, TIMP-3 mRNA was detected in seven of 12 localized scleroderma skin samples, specifically in fibroblasts within fibrotic collagen fibers or in the vicinity of inflammatory cells. TIMP-1 mRNA was detected in three of eight scleroderma skin samples in fibroblasts adjacent to inflammatory cells. The expression of TIMP-3 mRNA by systemic sclerosis and normal skin fibroblasts was enhanced to a similar extent (by 8.6- and 8.1-fold, respectively) by transforming growth factor-β, and suppressed down to 34 and 54%, respectively, by tumor necrosis factor-α. Specific activation of TIMP-3 gene expression in scleroderma skin fibroblasts in culture and in vivo suggests a role for TIMP-3 in the pathogenesis of dermal fibrosis via inhibition of turnover of fibrotic dermal extracellular matrix, possibly due to upregulation of TIMP-3 expression by transforming growth factor-β. Keywords: fibrosis/matrix metalloproteinase. J Invest Dermatol 110:416–421 1998 matrix metalloproteinase systemic sclerosis tissue inhibitor of metalloproteinases Excessive accumulation of extracellular matrix (ECM), especially fibrillar collagens of type I and type III, is a hallmark of cutaneous fibrosis in both systemic scleroderma (systemic sclerosis, SSc) and localized scleroderma (for reviews, see Kähäri, 1993Kähäri V.-M. Activation of dermal connective tissue in scleroderma.Ann Med. 1993; 25: 511-518PubMed Google Scholar; Varga and Jimenez, 1996Varga J. Jimenez S.A. Pathogenesis of scleroderma: cellular aspects.in: Clements P.J. Furst D.E. Systemic Sclerosis. Williams and Wilkins, Baltimore1996Google Scholar). In these two forms of dermal fibrosis an early perivascular inflammatory reaction is followed by enhanced deposition of collagenous ECM in the dermal layer (LeRoy et al., 1988LeRoy E.C. Black C.M. Fleischmajer R. et al.Scleroderma (systemic sclerosis): classification, subsets and pathogenesis.J Rheumatol. 1988; 15: 202-205PubMed Google Scholar). It has been shown that fibroblasts cultured from the affected skin of patients with both SSc and localized scleroderma produce increased amounts of several connective tissue components, mainly of type I collagen (LeRoy, 1974LeRoy E.C. Increased collagen synthesis by scleroderma skin fibroblasts in vitro.J Clin Invest. 1974; 54: 880-889Crossref PubMed Scopus (479) Google Scholar; Uitto et al., 1979Uitto J. Bauer E.A. Eisen A.Z. Scleroderma. Increased biosynthesis of triple-helical type I and type III procollagens associated with unaltered expression of collagenase by skin fibroblasts in culture.J Clin Invest. 1979; 64: 921-930Crossref PubMed Google Scholar) as a result of transcriptional activation of the corresponding genes (Kähäri et al., 1984Kähäri V.-M. Vuorio T. Näntö-salonen K. Vuorio E. Increased type I collagen mRNA levels in cultured scleroderma skin fibroblasts.Biochim Biophys Acta. 1984; 781: 183-186Crossref PubMed Scopus (60) Google Scholar; Kähäri et al., 1987Kähäri V.-M. Multimäki P. Vuorio E. Elevated proα2 (I) collagen mRNA levels in cultured scleroderma fibroblasts result from an increased transcription rate of the corresponding gene.FEBS Lett. 1987; 215: 331-334Abstract Full Text PDF PubMed Scopus (49) Google Scholar). It has also been documented that in the lesional skin of patients with localized scleroderma or SSc, a subpopulation of fibroblasts shows activation of type I collagen expression (Kähäri et al., 1988Kähäri V.-M. Sandberg M. Kalimo H. Vuorio T. Vuorio E. Identification of fibroblasts responsible for increased collagen production in localized scleroderma by in situ hybridization.J Invest Dermatol. 1988; 90: 664-670Abstract Full Text PDF PubMed Google Scholar; Scharffetter et al., 1988Scharffetter K. Lankat-buttgereit B. Krieg T. Localization of collagen mRNA in normal and scleroderma skin by in-situ hybridization.Eur J Clin Invest. 1988; 18: 9-17Crossref PubMed Scopus (176) Google Scholar), and that these activated fibroblasts often localize to the proximity of mononuclear inflammatory cells expressing transforming growth factor-β (TGF-β) (Kulozik et al., 1990Kulozik M. Hogg A. Lankat-buttgereit B. Krieg T. Co-localization of transforming growth factor β2 with α1 (I) procollagen mRNA in tissue sections of patients with systemic sclerosis.J Clin Invest. 1990; 86: 917-922Crossref PubMed Scopus (198) Google Scholar; Gruschwitz et al., 1990Gruschwitz M. Müller P.U. Sepp N. Hofer E. Fontana A. Wick G. Transcription and expression of transforming growth factor type beta in the skin of progressive systemic sclerosis: a mediator of fibrosis?.J Invest Dermatol. 1990; 94: 197-203Abstract Full Text PDF PubMed Google Scholar). The net accumulation of ECM in tissue is dependent on the balance between the synthesis and the degradation of matrix components by matrix metalloproteinases (MMP), a family of zinc-dependent endopeptidases collectively capable of degrading essentially all ECM components. At present, the MMP gene family contains 16 members, which according to their substrate specificity and structure can be divided into subgroups of collagenases, gelatinases, stromelysins, and membrane-type MMP (Kähäri and Saarialho-kere, 1997Kähäri V.-M. Saarialho-kere U. Matrix metalloproteinases in skin.Exp Dermatol. 1997; 6: 199-213Crossref PubMed Scopus (487) Google Scholar). Of these, the members of the collagenase subfamily, collagenase-1 (MMP-1), neutrophil collagenase (MMP-8), and collagenase-3 (MMP-13), are the principal secreted neutral proteinases capable of degrading fibrillar collagens of types I and III. The activity of MMP is specifically inhibited by tissue inhibitors of metalloproteinases (TIMP), which bind to activated MMP with 1:1 molar stoichiometry (Apte et al., 1995Apte S.S. Olsen B.R. Murphy G. The gene structure of tissue inhibitor of metalloproteinases (TIMP) -3 and its inhibitory activities define the distinct TIMP gene family.J Biol Chem. 1995; 270: 14313-14318Crossref PubMed Scopus (277) Google Scholar). At present, the TIMP gene family consists of four members (Greene et al., 1996Greene J. Wang M. Liu Y.E. Raymond L.A. Rosen C. Shi Y.E. Molecular cloning and characterization of human tissue inhibitor of metalloproteinase 4.J Biol Chem. 1996; 271: 30375-30380Crossref PubMed Scopus (466) Google Scholar; Kähäri and Saarialho-kere, 1997Kähäri V.-M. Saarialho-kere U. Matrix metalloproteinases in skin.Exp Dermatol. 1997; 6: 199-213Crossref PubMed Scopus (487) Google Scholar). Of these, TIMP-1 and TIMP-2 are produced in soluble form, but their expression is differently regulated. Production of TIMP-1 is enhanced by growth factors, e.g., TGF-β, whereas the expression of TIMP-2 is constitutive and not susceptible to regulation by most growth factors. In contrast, TIMP-3 is not soluble, but is associated with ECM in vitro and in vivo (Leco et al., 1994Leco K.J. Khokha R. Pavloff N. Hawkes S.P. Edwards D.R. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is an extracellular matrix-associated protein with a distinctive pattern of expression in mouse cells and tissues.J Biol Chem. 1994; 269: 9352-9360Abstract Full Text PDF PubMed Google Scholar). The expression of TIMP-3 in cultured cells is induced by mitogenic stimuli, e.g., serum, epidermal growth factor (EGF), and TGF-β specifically in the G1 phase of the cell cycle (Leco et al., 1994Leco K.J. Khokha R. Pavloff N. Hawkes S.P. Edwards D.R. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is an extracellular matrix-associated protein with a distinctive pattern of expression in mouse cells and tissues.J Biol Chem. 1994; 269: 9352-9360Abstract Full Text PDF PubMed Google Scholar; Wick et al., 1994Wick M. Bürger C. Brüsselbach S. Lucibello F.C. Müller R. A novel member of human tissue inhibitor of metalloproteinases (TIMP) gene family is regulated during G1 progression, mitogenic stimulation, differentiation, and senescence.J Biol Chem. 1994; 269: 18953-18960Abstract Full Text PDF PubMed Google Scholar; Wick et al., 1995Wick M. Härönen R. Mumberg D. et al.Structure of the human TIMP-3 gene and its cell cycle-regulated promoter.Biochem J. 1995; 311: 549-554Crossref PubMed Scopus (57) Google Scholar). Although TIMP-3 mRNA can be detected in several adult human tissues, e.g., placenta, heart, brain, lung, liver, skeletal muscle, kidney, and pancreas (Apte et al., 1994Apte S.S. Mattei M.G. Olsen B.R. Cloning of the cDNA encoding human tissue inhibitor of metalloproteinases-3 (TIMP-3) and mapping of the TIMP-3 gene to chromosome 22.Genomics. 1994; 19: 86-90Crossref PubMed Scopus (192) Google Scholar; Wick et al., 1995Wick M. Härönen R. Mumberg D. et al.Structure of the human TIMP-3 gene and its cell cycle-regulated promoter.Biochem J. 1995; 311: 549-554Crossref PubMed Scopus (57) Google Scholar), in normal adult human skin the expression of TIMP-3 mRNA is limited to growing hair follicles and stromal cells surrounding sweat glands (Airola et al., 1998Airola K. Ahonen M. Johansson N. Heikkilä P. Kere J. Kähäri V.-M. Saarialho-kere U.K. Human TIMP-3 is expressed during fetal development, hair growth cycle and cancer progression.J Histochem Cytochem. 1998Crossref Scopus (57) Google Scholar). A role for reduced turnover of the fibrotic ECM in the pathogenesis of cutaneous fibrosis has been suggested by studies showing that SSc skin fibroblasts in culture produce less MMP-1 (Takeda et al., 1994Takeda K. Hatamochi A. Ueki H. Nakata M. Oishi Y. Decreased collagenase expression in cultured systemic sclerosis fibroblasts.J Invest Dermatol. 1994; 103: 359-363Crossref PubMed Scopus (114) Google Scholar) and stromelysin-1 (MMP-3) and increased amounts of TIMP-1, as compared with control skin fibroblasts (Bou Gharios et al., 1994Bou Gharios G. Osman J. Black C. Olsen I. Excess matrix accumulation in scleroderma is caused partly by differential regulation of stromelysin and TIMP-1 synthesis.Clin Chim Acta. 1994; 231: 69-78Crossref PubMed Scopus (59) Google Scholar; Kirk et al., 1995Kirk T.Z. Mark M.E. Chua C.C. Chua B.H. Mayes M.D. Myofibroblasts from scleroderma skin synthesize elevated levels of collagen and tissue inhibitor of metalloproteinase (TIMP-1) with two forms of TIMP-1.J Biol Chem. 1995; 270: 3423-3428Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar; Kikuchi et al., 1997Kikuchi K. Kadono T. Furue M. Tamaki K. Tissue Inhibitor of Metalloproteinase 1 (TIMP-1) may be an autocrine growth factor in scleroderma fibroblasts.J Invest Dermatol. 1997; 108: 281-284Abstract Full Text PDF PubMed Scopus (89) Google Scholar). In addition, elevated TIMP-1 serum levels have been shown to correlate with the disease activity in SSc patients (Kikuchi et al., 1995Kikuchi K. Kubo M. Sato S. Fujimoto M. Tamaki K. Serum tissue inhibitor of metalloproteinases in patients with systemic sclerosis.J Am Acad Dermatol. 1995; 33: 973-978Abstract Full Text PDF PubMed Scopus (71) Google Scholar). In this study we show that activated scleroderma skin fibroblasts express elevated levels of TIMP-3 mRNA in culture and in vivo. In addition, the expression of TIMP-3 mRNA in systemic scleroderma and nonaffected skin fibroblasts was stimulated by TGF-β and suppressed by tumor necrosis factor-α (TNF-α). Activation of TIMP-1 expression was also detected in scleroderma skin in vivo, although to a lesser extent than TIMP-3. These results provide evidence that TIMP-3 may play a role in the pathogenesis of dermal fibrosis in systemic and localized scleroderma by inhibiting the turnover of fibrotic ECM. Skin fibroblast cultures were started from punch biopsies obtained from the margin of the fibrotic clinically affected skin in the forearm, wrist, or finger, and from symmetrically located nonaffected skin of seven patients (six females and one male), five with diffuse cutaneous SSc (dcSSc) and two with limited cutaneous SSc (lcSSc) ( Table 1), diagnosed according to the American Rheumatism Association criteria (American Rheumatism Association Subcommittee for Scleroderma Criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee, 1980American Rheumatism Association Subcommittee for Scleroderma Criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee Preliminary criteria for the classification of systemic sclerosis (scleroderma).Arthritis Rheum. 1980; 23: 581-590Crossref PubMed Scopus (4287) Google Scholar). Patients had a mean age of 43 y (range 30–52 y) and a mean disease duration of 38 mo (range 12–84 mo). For experiments, nonaffected and SSc fibroblasts were used in subcultures 3–5. Cells were maintained in Dulbecco's modification of Eagle's medium (Flow Laboratories, Irvine, U.K.) supplemented with 10% fetal calf serum (Flow Laboratories), 2 mM glutamine, 100 IU penicillin-G per ml, and 100 μg streptomycin per ml. For experiments, the cells were maintained in Dulbecco's modification of Eagle's medium supplemented with 1% fetal calf serum for 18 h. Thereafter, TNF-R55 specific human TNF-α (double mutant R32WS86T, Barbara et al., 1994Barbara J.A.J. Smith W.B. Gamble J.R. et al.Dissociation of TNF-α cytotoxic and proinflammatory activities by p55 receptor- and p75 receptor-selective TNF-a mutants.EMBO J. 1994; 13: 843-850Crossref PubMed Scopus (139) Google Scholar) (kindly provided by Dr. Walter Fiers, University of Gent, Belgium), human recombinant interleukin-1β (IL-1β, Boehringer, Mannheim, Germany), or bovine TGF-β1 (kindly provided by Dr. David R. Olsen, Celtrix, Santa Clara, CA) were added and the incubations were continued for 24 h.Table 1Expression of proα(I) collagen, TIMP-3, TIMP-2, and TIMP1 mRNA in SSc skin fibroblastsaThe levels ofproα(I) collagen, TIMP-3, TIMP-2, and TIMP-1 mRNA were determined by northern blot hybridizations, quantitated by densitometry, corrected for GADPH mRNA levels in each sample, and the ratio (affected/nonaffected) for each mRNA was calculated.PatientAge/sexType/duration of SSc (mo)bdcSSc, diffuse cutaneous systemic scleroderma; IcSSc, limited cutaneous systemic scleroderma.mRNA, ratio affected/nonaffectedproα(I) collagenTIMP-3TIMP-2TIMP-1SSc-152/MdcSSc/842.502.370.960.92SSc-247/FdcSSc/181.751.310.920.68SSc-335/FIcSSc/121.312.451.751.79SSc-440/FdcSSc/480.610.751.570.78SSc-547/FdcSSc/241.182.500.761.48SSc-630/FdcSSc/361.111.280.711.11SSc-748/FIcSSc/480.560.471.150.83a The levels ofproα(I) collagen, TIMP-3, TIMP-2, and TIMP-1 mRNA were determined by northern blot hybridizations, quantitated by densitometry, corrected for GADPH mRNA levels in each sample, and the ratio (affected/nonaffected) for each mRNA was calculated.b dcSSc, diffuse cutaneous systemic scleroderma; IcSSc, limited cutaneous systemic scleroderma. Open table in a new tab Total cellular RNA was isolated from cells using the single step method (Chomczynski and Sacchi, 1987Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62148) Google Scholar). Aliquots of total RNA (10–15 μg) were fractionated on 0.8% agarose gel containing 2.2 M formaldehyde, transferred to Zeta probe filter (Bio-Rad, Richmond, CA) by vacuum transfer (VacuGene XL; LKB, Bromma, Sweden) and immobilized by heating at 80°C for 30 min. The filters were prehybridized for 2 h and subsequently hybridized for 20 h with 32P-labeled cDNA. The filters were then washed, the final stringency being 0.1 × standard saline citrate-0.1% sodium dodecyl sulfate at 60°C (Thomas, 1980Thomas P.S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose.Proc Natl Acad Sci USA. 1980; 77: 5201-5205Crossref PubMed Scopus (5815) Google Scholar). For hybridizations, cDNA fragments corresponding to the coding regions of human TIMP-3 (636 bp) (Uría et al., 1994Uría J.A. Ferrando A.A. Velasco G. Freije J.M.P. López-otín C. Structure and expression in breast tumors of human TIMP-3, a new member of the metalloproteinase inhibitor family.Cancer Res. 1994; 54: 2091-2094PubMed Google Scholar), TIMP-2 (663 bp) (Stetler-stevenson et al., 1990Stetler-stevenson W.G. Brown P.D. Onisto M. Levy A.T. Liotta L.A. Tissue inhibitor of metalloproteinases-2 (TIMP-2) mRNA expression in tumor cell lines and human tumor tissues.J Biol Chem. 1990; 265: 13933-13938PubMed Google Scholar), and TIMP-1 (624 bp) (Carmichael et al., 1986Carmichael D.F. Sommer A. Thompson R.C. Anderson D.C. Smith C.G. Welgus H.G. Stricklin G.P. Primary structure and cDNA cloning of human fibroblast collagenase inhibitor.Proc Natl Acad Sci USA. 1986; 83: 2407-2411Crossref PubMed Scopus (241) Google Scholar) were generated by reverse transcriptase polymerase chain reaction with specific sense and anti-sense oligonucleotide primers using normal human skin fibroblast RNA as template, and their identity was verified by nucleotide sequencing. In addition, a 0.7 kb human proα1 (I) collagen cDNA (Mäkelä et al., 1988Mäkelä J.K. Raassina A. Virta A. Vuorio E. Human proα1 (I) collagen cDNA sequence for the C-propeptide domain.Nucl Acids Res. 1988; 16: 349Crossref PubMed Scopus (75) Google Scholar) and a 1.3 kb rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (Fort et al., 1985Fort P. Marty L. Piechaczyk M. El Sabrouty S. Dani C. Jeanteur P. Blanchard J.M. Various rat adults tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigenic family.Nucl Acids Res. 1985; 13: 1431-1442Crossref PubMed Scopus (1956) Google Scholar) were used. The 32P-cDNA-mRNA hybrids were visualized with autoradiography, quantitated with densitometry, and corrected for the levels of GAPDH mRNA in each sample. Formalin-fixed, paraffin-embedded skin specimens of 11 patients with localized scleroderma and one with systemic scleroderma (Table 2) were obtained from the Department of Dermatology, Helsinki University Central Hospital, Finland. Biopsies were taken from 3 mo to 3 y old lesions in different parts of the body and examined by an experienced dermatopathologist to confirm the clinical diagnosis by histopathology (Lever and Schaumburg-lever, 1990Lever W.F. Schaumburg-lever G. Histopathology of the Skin. 7th edn. JB Lippincott, Philadelphia1990: 511-516Google Scholar). The samples were divided into initial, middle, and late stage of the disease based on the degree of inflammation (the number of lymphocytic inflammatory infiltrates between collagen bundles and around blood vessels) and fibrosis (the presence of thickened collagen bundles in the dermal layer, atrophic eccrine glands).Table 2Expression of TIMP-3 and TIMP-1 mRNA in scleroderma skinaExpression of TIMP-3 and TIMP-1 mRNA in the lesional skin of 11 patients with localized scleroderma and one with SSc* was determined by in situ hybridization and estimated in range from – to ++. ND, not determined.Skin sampleAge/sexLocation and age (mo) of the lesionHistology and aspect of the lesionbHistology of the lesion: I, initial stage, slight fibrosis, strong inflammation; M, middle stage, fibrosis, slight inflammation; L, late stage, abundant fibrosis, moderate inflammation; estimated as described in the Materials and Methods.mRNATIMP-3TIMP-1143/Ftrunk, 6I, soft, lilac ring, indurated 3 mo after biopsy––222/Fback, 36M, atrophic, pigmented+ND327/Mabdomen, 4M, indurated–ND421/Fabdomen, 12M, soft, erythematous, pigmented+–512/Farm, 3M, indurated++645/Fupper back, 3M, atrophic, pigmented––744/Mtrunk, 6L, indurated, pigmented, lilac+ ++861/Fbreast, 7L, light yellow with lilac ring+ +ND961/Mwaist, 4L, livid, indurated–ND1085/Fabdomen, 24L, indurated++1130/Mback, 6L, indurated, hard, light-colored+–12*44/Ffoot, 6L, indurated, thick, light-colored––a Expression of TIMP-3 and TIMP-1 mRNA in the lesional skin of 11 patients with localized scleroderma and one with SSc* was determined by in situ hybridization and estimated in range from – to ++. ND, not determined.b Histology of the lesion: I, initial stage, slight fibrosis, strong inflammation; M, middle stage, fibrosis, slight inflammation; L, late stage, abundant fibrosis, moderate inflammation; estimated as described in the Materials and Methods. Open table in a new tab For in situ hybridizations, the human TIMP-3 cDNA fragment utilized for northern blot hybridizations was used (Airola et al., 1998Airola K. Ahonen M. Johansson N. Heikkilä P. Kere J. Kähäri V.-M. Saarialho-kere U.K. Human TIMP-3 is expressed during fetal development, hair growth cycle and cancer progression.J Histochem Cytochem. 1998Crossref Scopus (57) Google Scholar). The production and specificity of the TIMP-1 probe has been described previously (Carmichael et al., 1986Carmichael D.F. Sommer A. Thompson R.C. Anderson D.C. Smith C.G. Welgus H.G. Stricklin G.P. Primary structure and cDNA cloning of human fibroblast collagenase inhibitor.Proc Natl Acad Sci USA. 1986; 83: 2407-2411Crossref PubMed Scopus (241) Google Scholar; Sudbeck et al., 1992Sudbeck B.D. Jeffrey J.J. Welgus H.G. Mecham R.P. McCourt D. Parks W.C. Purification and characterization of bovine collagenase and tissue inhibitor of metalloproteinases.Arch Biochem Biophys. 1992; 293: 370-376Crossref PubMed Scopus (9) Google Scholar). RNA probes transcribed from TIMP-3 and TIMP-1 cDNA in sense orientation were used as controls for nonspecific hybridization. By FASTA alignment, highest similarities between the probes used and other members of the TIMP family were 53–55%, making cross-hybridization at high stringency unlikely. For detection of collagenase-1 (MMP-1) mRNA, a 550 bp EcoRV-SmaI fragment from the 5′-end of human MMP-1 cDNA (Goldberg et al., 1986Goldberg G.I. Wilhelm S.M. Kronberger A. Bauer E.A. Grant G.A. Eisen A.Z. Human fibroblast collagenase. Complete primary structure and homology to an oncogene transformation-induced rat protein.J Biol Chem. 1986; 261: 6600-6605Abstract Full Text PDF PubMed Google Scholar) was used, and for detection of collagenase-3 (MMP-13) mRNA, we used a 490 bp ApaI fragment from the coding region of human MMP-13 cDNA (Johansson et al., 1997aJohansson N. Westermarck J. Leppä S. et al.Collagenase-3 (matrix metalloproteinase-13) gene expression by HaCaT keratinocytes is enhanced by tumor necrosis factor-α and transforming growth factor-β.Cell Growth Differ. 1997; 8: 243-250PubMed Google Scholar). The specificities of these probes have been documented previously (Goldberg et al., 1986Goldberg G.I. Wilhelm S.M. Kronberger A. Bauer E.A. Grant G.A. Eisen A.Z. Human fibroblast collagenase. Complete primary structure and homology to an oncogene transformation-induced rat protein.J Biol Chem. 1986; 261: 6600-6605Abstract Full Text PDF PubMed Google Scholar; Johansson et al., 1997aJohansson N. Westermarck J. Leppä S. et al.Collagenase-3 (matrix metalloproteinase-13) gene expression by HaCaT keratinocytes is enhanced by tumor necrosis factor-α and transforming growth factor-β.Cell Growth Differ. 1997; 8: 243-250PubMed Google Scholar; Johansson et al., 1997bJohansson N. Saarialho-kere U. Airola K. et al.Collagenase-3 (MMP-13) is expressed by hypertrophic chondrocytes, periosteal cells, and osteoblasts during human fetal bone development.Dev Dynam. 1997; 208: 387-397Crossref PubMed Scopus (245) Google Scholar; Vaalamo et al., 1997Vaalamo M. Mattila L. Johansson N. et al.Distinct populations of stromal cells express collagenase-3 (MMP-13) and collagenase-1 (MMP-1) in chronic ulcers but not in normally healing wounds.J Invest Dermatol. 1997; 109: 96-101Abstract Full Text PDF PubMed Scopus (232) Google Scholar). In vitro transcribed anti-sense and sense RNA probes were labeled with α-35S-labeled UTP (Saarialho-Kere et al., 1993Saarialho-Kere U.K. Kovacs S.O. Pentland A.P. Olerud J.E. Welgus H.G. Parks W.C. Cell–matrix interactions modulate interstitial collagenase expression by human keratinocytes actively involved in wound healing.J Clin Invest. 1993; 92: 2858-2866Crossref PubMed Scopus (266) Google Scholar). Sections were hybridized with probes (2.5–4 × 104 cpm hybridization buffer per μl) and washed under stringent conditions, including treatment with RNase A, as described (Prosser et al., 1989Prosser I.W. Stenmark K.R. Suthar M. Crouch E.C. Mecham R.P. Parks W.C. Regional heterogeneity of elastin and collagen gene expression in intralobar arteries in response to hypoxic pulmonary hypertension as demonstrated by in situ hybridization.Am J Pathol. 1989; 135: 1073-1088PubMed Google Scholar; Saarialho-Kere et al., 1993Saarialho-Kere U.K. Kovacs S.O. Pentland A.P. Olerud J.E. Welgus H.G. Parks W.C. Cell–matrix interactions modulate interstitial collagenase expression by human keratinocytes actively involved in wound healing.J Clin Invest. 1993; 92: 2858-2866Crossref PubMed Scopus (266) Google Scholar). After autoradiography for 10–30 d, the photographic emulsion was developed and the slides were stained with hematoxylin and eosin. Samples of breast carcinomas were used as positive controls for the expression of TIMP-3 mRNA (Uría et al., 1994Uría J.A. Ferrando A.A. Velasco G. Freije J.M.P. López-otín C. Structure and expression in breast tumors of human TIMP-3, a new member of the metalloproteinase inhibitor family.Cancer Res. 1994; 54: 2091-2094PubMed Google Scholar). Statistical significance was determined by Student's t test. Increased TIMP-3 mRNA expression in systemic scleroderma skin fibroblastsIt has recently been shown that SSc skin fibroblasts produce increased amounts of TIMP-1, as compared with control skin fibroblasts (Kirk et al., 1995Kirk T.Z. Mark M.E. Chua C.C. Chua B.H. Mayes M.D. Myofibroblasts from scleroderma skin synthesize elevated levels of collagen and tissue inhibitor of metalloproteinase (TIMP-1) with two forms of TIMP-1.J Biol Chem. 1995; 270: 3423-3428Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar; Kikuchi et al., 1997Kikuchi K. Kadono T. Furue M. Tamaki K. Tissue Inhibitor of Metalloproteinase 1 (TIMP-1) may be an autocrine growth factor in scleroderma fibroblasts.J Invest Dermatol. 1997; 108: 281-284Abstract Full Text PDF PubMed Scopus (89) Google Scholar). To elucidate the role of TIMP-3 in the pathogenesis of dermal fibrosis in scleroderma, we first examined the abundance of TIMP-3 mRNA in dermal fibroblast cultures established from clinically affected and nonaffected skin of seven patients with SSc. As shown in Figure 1(a), both scleroderma and nonaffected skin fibroblasts express three specific TIMP-3 mRNA, the sizes of which (4.8, 2.8, and 2.4 kb) are in accordance with previous observations on other cell types (Wick et al., 1994Wick M. Bürger C. Brüsselbach S. Lucibello F.C. Müller R. A novel member of human tissue inhibitor of metalloproteinases (TIMP) gene family is regulated during G1 progression, mitogenic stimulation, differentiation, and senescence.J Biol Chem. 1994; 269: 18953-18960Abstract Full Text PDF PubMed Google Scholar; Airola et al., 1998Airola K. Ahonen M. Johansson N. Heikkilä P. Kere J. Kähäri V.-M. Saarialho-kere U.K. Human TIMP-3 is expressed during fetal development, hair growth cycle and cancer progression.J Histochem Cytochem. 1998Crossref Scopus (57) Google Scholar). In three of seven SSc fibroblast strains (SSc-1, SSc-3, and SSc-5) the expression of TIMP-3 mRNA was clearly elevated (2.4–2.5-fold), and in two additional SSc fibroblast strains (SSc-2 and SSc-6) the expression was slightly elevated (1.3-fold), as compared with the corresponding nonaffected skin fibroblasts (Figure 1a, Table 1). In two