Differential Effects of Transforming Growth Factor-β on the Expression of Collagenase-1 and Collagenase-3 in Human Fibroblasts

Artigo Acesso aberto Revisado por pares

Differential Effects of Transforming Growth Factor-β on the Expression of Collagenase-1 and Collagenase-3 in Human Fibroblasts

1998; Elsevier BV; Volume: 273; Issue: 16 Linguagem: Inglês

10.1074/jbc.273.16.9769

ISSN

1083-351X

Autores

José A. Urı́a, María Jiménez, Milagros Balbı́n, José M.P. Freije, Carlos López‐Otín,

Tópico(s)

Bone Metabolism and Diseases

Resumo

Collagenase-3 (MMP-13) is a matrix metalloproteinase (MMP) originally identified in breast carcinomas which is also produced at significant levels during fetal ossification and in arthritic processes. In this work, we have found that transforming growth factor β1 (TGF-β1), a growth factor widely assumed to be inhibitory for MMPs, strongly induces collagenase-3 expression in human KMST fibroblasts. In contrast, this growth factor down-regulated the expression in these cells of collagenase-1 (MMP-1), an enzyme highly related to collagenase-3 in terms of structure and enzymatic properties. The positive effect of TGF-β1 on collagenase-3 expression was dose- and time-dependent, but independent of the effects of this growth factor on cell proliferation rate. Analysis of the signal transduction mechanisms underlying the up-regulating effect of TGF-β1 on collagenase-3 expression demonstrated that this growth factor acts through a signaling pathway involving protein kinase C and tyrosine kinase activities. Functional analysis of the collagenase-3 gene promoter region revealed that the inductive effect of TGF-β1 is partially mediated by an AP-1 site. Comparative analysis with the promoter region of the collagenase-1 gene which contains an AP-1 site at equivalent position, confirmed that TGF-β1 did not have any effect on CAT activity levels of this promoter. Finally, by using electrophoretic mobility shift assays and antibody supershift analysis, we propose that c-Fos, c-Jun, and JunD may play major roles in the collagenase-3 activation by TGF-β1 in human fibroblasts. Collagenase-3 (MMP-13) is a matrix metalloproteinase (MMP) originally identified in breast carcinomas which is also produced at significant levels during fetal ossification and in arthritic processes. In this work, we have found that transforming growth factor β1 (TGF-β1), a growth factor widely assumed to be inhibitory for MMPs, strongly induces collagenase-3 expression in human KMST fibroblasts. In contrast, this growth factor down-regulated the expression in these cells of collagenase-1 (MMP-1), an enzyme highly related to collagenase-3 in terms of structure and enzymatic properties. The positive effect of TGF-β1 on collagenase-3 expression was dose- and time-dependent, but independent of the effects of this growth factor on cell proliferation rate. Analysis of the signal transduction mechanisms underlying the up-regulating effect of TGF-β1 on collagenase-3 expression demonstrated that this growth factor acts through a signaling pathway involving protein kinase C and tyrosine kinase activities. Functional analysis of the collagenase-3 gene promoter region revealed that the inductive effect of TGF-β1 is partially mediated by an AP-1 site. Comparative analysis with the promoter region of the collagenase-1 gene which contains an AP-1 site at equivalent position, confirmed that TGF-β1 did not have any effect on CAT activity levels of this promoter. Finally, by using electrophoretic mobility shift assays and antibody supershift analysis, we propose that c-Fos, c-Jun, and JunD may play major roles in the collagenase-3 activation by TGF-β1 in human fibroblasts. The matrix metalloproteinases (MMPs) 1The abbreviations used are: MMP, matrix metalloproteinase; IL-1β, interleukin 1β; TPA, 12-O-tetradecanoylphorbol-13-acetate; aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; TGF, transforming growth factor; CAT, chloramphenicol acetyltransferase; PKC, protein kinase C. 1The abbreviations used are: MMP, matrix metalloproteinase; IL-1β, interleukin 1β; TPA, 12-O-tetradecanoylphorbol-13-acetate; aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; TGF, transforming growth factor; CAT, chloramphenicol acetyltransferase; PKC, protein kinase C. or matrixins form a family of structurally related metalloendopeptidases that are collectively capable of degrading the different macromolecular components of the extracellular matrix. These enzymes play a major role in normal tissue remodeling processes such as embryonic development, bone growth, and resorption, ovulation, uterine involution, and wound healing (1Woessner J.F. FASEB J. 1991; 5: 2145-2154Crossref PubMed Scopus (3072) Google Scholar, 2Matrisian L.M. BioEssays. 1992; 14: 455-463Crossref PubMed Scopus (1324) Google Scholar, 3Birkedal-Hansen H. Moore W.G.I. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2629) Google Scholar, 4Stetler-Stevenson W.G. Aznavoorian S. Liota L.A. Annu. Rev. Cell Biol. 1993; 9: 541-573Crossref PubMed Scopus (1515) Google Scholar). In addition, abnormal expression of these proteinases may contribute to a variety of pathological processes such as rheumatoid arthritis (5Murphy G. Hembry R.M. J. Rheumatol. 1992; 19: 61-64Google Scholar), atherosclerosis (6Henney A.M. Wakeley P.R. Davies M.J. Foster K. Hembry R. Murphy G. Humphries S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8154-8158Crossref PubMed Scopus (533) Google Scholar), pulmonary emphysema (7Shapiro S.D. Am. J. Respir. Crit. Care Med. 1994; 150: 5160-5165Crossref Google Scholar), and tumor invasion and metastasis (8MacDougall J.R. Matrisian L.M. Cancer Met. Rev. 1995; 14: 351-362Crossref PubMed Scopus (400) Google Scholar). At present, the family of human MMPs is composed of 16 members that, according to structural and functional considerations, can be classified into four different families: collagenases, gelatinases, stromelysins, and membrane-type MMPs (1Woessner J.F. FASEB J. 1991; 5: 2145-2154Crossref PubMed Scopus (3072) Google Scholar, 2Matrisian L.M. BioEssays. 1992; 14: 455-463Crossref PubMed Scopus (1324) Google Scholar, 3Birkedal-Hansen H. Moore W.G.I. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2629) Google Scholar, 4Stetler-Stevenson W.G. Aznavoorian S. Liota L.A. Annu. Rev. Cell Biol. 1993; 9: 541-573Crossref PubMed Scopus (1515) Google Scholar), although there are some enzymes like macrophage metalloelastase (9Belaaouaj A. Shipley J.M. Kobayashi D.K. Zimonjic D.B. Popescu N. Silverman G.A. Shapiro S.D. J. Biol. Chem. 1995; 270: 14568-14575Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), stromelysin-3 (10Basset P. Bellocq J.-P. Wolf C. Stoll I. Hutin P. Limacher J.M. Podhajcer O.L. Chenard M.P. Rio M.C. Chambon P. Nature. 1990; 348: 699-704Crossref PubMed Scopus (1008) Google Scholar), MMP-19 (11Pendás A.M. Knäuper V. Puente X.S. Llano E. Mattei M.-G. Apte S. Murphy G. López-Otı́n C. J. Biol. Chem. 1997; 272: 4281-4286Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), and enamelysin (12Llano E. Pendás A.M. Knäuper V. Sorsa T. Salo T. Salido E. Murphy G. Simmer J. Bartlett J. López-Otı́n C. Biochemistry. 1997; 36: 15101-15108Crossref PubMed Scopus (184) Google Scholar) that do not belong to these groupings. Recently, we have cloned from a breast carcinoma a novel MMP that has been called collagenase-3 (MMP-13) (13Freije J.M. Dı́ez-Itza I. Balbı́n M. Sánchez L.M. Blasco R. Tolivia J. López-Otı́n C. J. Biol. Chem. 1994; 269: 16766-16773Abstract Full Text PDF PubMed Google Scholar, 14Pendás A.M. Matilla T. Estivill X. López-Otı́n C. Genomics. 1995; 26: 615-618Crossref PubMed Scopus (55) Google Scholar), since it represents the third member of the collagenase subfamily of human MMPs, the others being fibroblast and neutrophil collagenases. Biochemical characterization of recombinant human collagenase-3 has revealed that it degrades very efficiently the native helix of fibrillar collagens with preferential activity on type II collagen (15Knäuper V. López-Otı́n C. Smith B. Knight G. Murphy G. J. Biol. Chem. 1996; 271: 1544-1550Abstract Full Text Full Text PDF PubMed Scopus (782) Google Scholar, 16Welgus H.G. Jeffrey J.J. Eisen A.Z. J. Biol. Chem. 1981; 256: 9511-9515Abstract Full Text PDF PubMed Google Scholar). In contrast, fibroblast collagenase (MMP-1 or collagenase-1) is more active against type III collagen (17Hasty K.A. Jeffrey J.J. Hibbs M.S. Welgus H.G. J. Biol. Chem. 1987; 262: 10048-10052Abstract Full Text PDF PubMed Google Scholar) and neutrophil collagenase (MMP-8 or collagenase-2) preferentially cleaves type I collagen (18Fosang A.J. Last K. Knäuper V. Murphy G. Neame P.J. FEBS Lett. 1996; 380: 17-20Crossref PubMed Scopus (331) Google Scholar). Consequently, the three human collagenases characterized to date show distinct substrate specificities strongly suggesting that they have evolved as specialized enzymes to degrade tissues with different collagen composition. In addition to its degrading activity on fibrillar collagens, further analysis of the substrate specificity of collagenase-3 has revealed that this enzyme may also act as a potent gelatinase thus contributing to further degrade the initial cleavage products of collagenolysis to small fragments suitable for subsequent metabolism (15Knäuper V. López-Otı́n C. Smith B. Knight G. Murphy G. J. Biol. Chem. 1996; 271: 1544-1550Abstract Full Text Full Text PDF PubMed Scopus (782) Google Scholar). Furthermore, very recent studies have shown that collagenase-3 is also able to degrade the large cartilage proteoglycan aggrecan and other components of the extracellular matrix and basement membranes, including the large tenascin isoform, fibronectin, and type IV collagen (15Knäuper V. López-Otı́n C. Smith B. Knight G. Murphy G. J. Biol. Chem. 1996; 271: 1544-1550Abstract Full Text Full Text PDF PubMed Scopus (782) Google Scholar, 18Fosang A.J. Last K. Knäuper V. Murphy G. Neame P.J. FEBS Lett. 1996; 380: 17-20Crossref PubMed Scopus (331) Google Scholar, 19Knäuper V. Cowell S. Smith B. López-Otı́n C. O'Shea M. Morris H. Zardi L. Murphy G. J. Biol. Chem. 1997; 272: 7608-7616Crossref PubMed Scopus (294) Google Scholar).Analysis of the expression of collagenase-3 in human tissues has revealed that in addition to its presence in breast carcinomas, this enzyme is produced during fetal ossification (20Stahle-Bäckdahl M. Sandstedt B. Bruce K. Lindahl A. Jiménez M.G. Vega J.A. López-Otı́n C. Lab. Invest. 1997; 76: 717-728PubMed Google Scholar, 21Johansson N. Saarialho-Kere U. Airola K. Herva R. Nissinen L. Westermarck J. Vuorio E. Heino J. Kähäri V.M. Dev. Dyn. 1997; 208: 387-397Crossref PubMed Scopus (252) Google Scholar), and in degenerative joint diseases including osteoarthritis and rheumatoid arthritis (21Johansson N. Saarialho-Kere U. Airola K. Herva R. Nissinen L. Westermarck J. Vuorio E. Heino J. Kähäri V.M. Dev. Dyn. 1997; 208: 387-397Crossref PubMed Scopus (252) Google Scholar, 22Mitchell P.G. Magna H.A. Reeves L.M. Lopresti-Morrow L.L. Yocum S.A. Rosner P.J. Geoghegan K.F. Hambor J.E. J. Clin. Invest. 1996; 97: 761-768Crossref PubMed Scopus (820) Google Scholar, 23Reboul P. Pelletier J.P. Tardif G. Cloutier J.M. Martel-Pelletier J. J. Clin. Invest. 1996; 97: 2011-2019Crossref PubMed Scopus (421) Google Scholar, 24Borden P. Solymar D. Sucharczuk A. Lindman B. Cannon P. Heller R.A. J. Biol. Chem. 1996; 271: 23577-23581Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 25Wernicke D. Seyfert C. Hinzmann B. Gromnica-Ihle E. J. Rheumatol. 1996; 23: 590-595PubMed Google Scholar, 26Heller R.A. Schena M. Chai A. Shalon D. Bedilion T. Gilmore J. Wooley D.E. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2150-2155Crossref PubMed Scopus (664) Google Scholar, 27Lindy O. Konttinen Y.T. Sorsa T. Ding Y.L. Santavirta S. Ceponis A. López-Otı́n C. Arthritis Rheum. 1997; 40: 1391-1399Crossref PubMed Scopus (198) Google Scholar). At present, very little information is available on the mechanisms controlling collagenase-3 expression in both normal and pathological conditions. Thus, although several groups have reported that human collagenase-3 gene expression can be induced by IL-1β and tumor necrosis factor-α in chondrocytes from normal and osteoarthritic cartilage (22Mitchell P.G. Magna H.A. Reeves L.M. Lopresti-Morrow L.L. Yocum S.A. Rosner P.J. Geoghegan K.F. Hambor J.E. J. Clin. Invest. 1996; 97: 761-768Crossref PubMed Scopus (820) Google Scholar, 23Reboul P. Pelletier J.P. Tardif G. Cloutier J.M. Martel-Pelletier J. J. Clin. Invest. 1996; 97: 2011-2019Crossref PubMed Scopus (421) Google Scholar, 24Borden P. Solymar D. Sucharczuk A. Lindman B. Cannon P. Heller R.A. J. Biol. Chem. 1996; 271: 23577-23581Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), much less is known on the mechanisms modulating its expression in tumor processes. In this regard, and based on in situ hybridization experiments and on co-cultures of fibroblast and epithelial breast cancer cells, we have recently proposed that collagenase-3 is predominantly expressed within fibroblasts adjacent to the invasive tumor cells in response to diffusible factors released from the breast cancer cells (28Urı́a J.A. Stahle-Bäckdahl M. Seiki M. Fueyo A. López-Otı́n C. Cancer Res. 1997; 57: 4882-4888PubMed Google Scholar). A preliminary search of putative factors with ability to induce collagenase-3 expression in human fibroblasts revealed that only IL-1 and TPA were able to up-regulate the expression of this gene in KMST fibroblastic cells. By contrast, a series of cytokines and growth factors factors including aFGF, bFGF, platelet-derived growth factor, epidermal growth factor, tumor necrosis factor-α, and TGF-α, previously found to play important roles in up-regulating expression of other MMPs, did not show any effect on collagenase-3 expression by human fibroblasts. Since these findings strongly suggested that the mechanisms regulating collagenase-3 expression could be distinct to those operating in the control of other MMPs, we have extended our preliminary search for factors that could act as mediators of collagenase-3 expression in breast carcinomas. In this work, we show that TGF-β, a growth factor widely assumed to be inhibitory for MMPs like collagenase-1, strongly up-regulates collagenase-3 expression in KMST fibroblasts. We also analyze the mechanisms mediating this induction with the finding that this growth factor acts through a signaling pathway involving tyrosine kinase and protein kinase C activities. Finally, we perform a functional characterization of the promoter region of the collagenase-3 gene looking for the putative elements with ability to mediate the induction of this gene by TGF-β.DISCUSSIONIn this work we have shown that expression of human collagenase-3, a matrix metalloproteinase produced by breast carcinomas and arthritic cartilage, is induced by TGF-β1 in cultured human fibroblasts. This up-regulatory effect of TGF-β1 on the production of a potent proteolytic enzyme like collagenase-3 is in marked contrast with the widely assumed role of this growth factor as an inducer of anabolic responses in mesenchymal cells. In fact, TGF-β has been implicated in the induction of connective tissue formation by stimulating the synthesis of several extracellular matrix components such as fibronectin, thrombospondin, and types I and III collagen (35Ignotz R.A. Massagué J. J. Biol. Chem. 1986; 261: 4337-4345Abstract Full Text PDF PubMed Google Scholar, 48Roberts A.B. Sporn M.B. Assoian R.K. Smith J.M. Roche N.S. Wakefield L.M. Heine U.I. Liotta L.A. Falanga V. Kehrl J.H. Fauci A.S. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4167-4171Crossref PubMed Scopus (2399) Google Scholar, 49Fine A. Goldstein R.H. J. Biol. Chem. 1987; 262: 3897-3902Abstract Full Text PDF PubMed Google Scholar, 50Ignotz R.A. Endo T. Massagué J. J. Biol. Chem. 1987; 262: 6443-6446Abstract Full Text PDF PubMed Google Scholar, 51Raghow R. Postlethwaite A.E. Keski-Oja J. Moses H.L. Kang A.H. J. Clin. Invest. 1987; 79: 1285-1288Crossref PubMed Scopus (366) Google Scholar, 52Penttinen R.P. Kobayashi S. Pornstein P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1105-1108Crossref PubMed Scopus (351) Google Scholar). In addition, TGF-β suppresses the overall degrading activity on these matrix components through a concerted dual action involving the reduction in the production of a wide diversity of proteolytic enzymes including MMPs (53Edwards D.R. Murphy G. Reynolds J.J. Whitham S.E. Docherty A.J.P. Angel P. Heath J.K. EMBO J. 1987; 6: 1899-1904Crossref PubMed Scopus (1030) Google Scholar, 54Kerr L.D. Miller D.B. Matrisian L.M. Cell. 1990; 61: 267-278Abstract Full Text PDF PubMed Scopus (371) Google Scholar) and plasminogen activators (55Keski-Oja J. Blasi F. Leof E.B. Moses H.L. J. Cell Biol. 1988; 106: 451-459Crossref PubMed Scopus (93) Google Scholar), as well as the concomitant increase in the synthesis of their respective inhibitors (52Penttinen R.P. Kobayashi S. Pornstein P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1105-1108Crossref PubMed Scopus (351) Google Scholar, 56Laiho M. Saksela O. Andreasen P.A. Keski-Oja J. J. Cell Biol. 1986; 103: 2403-2410Crossref PubMed Scopus (324) Google Scholar, 57Lund L.R. Riccio A. Andreasen P.A. Nielsen L.S. Kristensen P. Laiho M. Saksela O. Blasi F. Dano K. EMBO J. 1987; 6: 1281-1286Crossref PubMed Scopus (194) Google Scholar, 58Overall C.M. Wrana J.L. Sodek J. J. Biol. Chem. 1989; 264: 1860-1869Abstract Full Text PDF PubMed Google Scholar). The ability of TGF-β family members to induce collagenase-3 expression is not exclusive of fibroblasts, since a recent report has shown that TGF-β2 up-regulates its expression in transformed keratinocytes (59Johansson N. Westermarck J. Leppä S. Häkkinen L. Koivisto L. López-Otı́n C. Peltonen J. Heino J. Kähäri V.M. Cell Growth & Differ. 1997; 8: 243-250PubMed Google Scholar). By contrast, another recent report has shown that TGF-β1 inhibits collagenase-3 expression in osteoblast cultures (60Rydziel S. Varghese S. Canalis E. J. Cell. Physiol. 1997; 170: 145-152Crossref PubMed Scopus (55) Google Scholar), suggesting that the effects of these growth factors are markedly dependent of the cell type. Nevertheless, the ability of TGF-β to stimulate the production of a proteolytic enzyme by human fibroblastic cells is not unprecedented, since Overall et al. (58Overall C.M. Wrana J.L. Sodek J. J. Biol. Chem. 1989; 264: 1860-1869Abstract Full Text PDF PubMed Google Scholar, 61Overall C.M. Wrana J.L. Sodek J. J. Biol. Chem. 1991; 266: 14064-14071Abstract Full Text PDF PubMed Google Scholar) have reported that levels of gelatinase A (MMP-2) are increased in gingival fibroblasts, although the magnitude of this up-regulating effect is lesser than that observed in the present work for collagenase-3. The possibility that collagenase-3 and gelatinase A are coordinately regulated in fibroblasts is of interest in light of recent findings demonstrating that both enzymes form part of an activation cascade which can generate the extracellular collagenolytic activity requested for the connective tissue degradation occurring in both normal and pathological conditions (62Knäuper V. Will H. López-Otı́n C. Smith B. Atkinson S.J. Stanton H. Hembry R.M. Murphy G. J. Biol. Chem. 1996; 271: 17124-17131Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar). In this context, it is specially noteworthy that collagenase-1 (MMP-1), despite sharing with collagenase-3 its unique ability to initiate degradation of the native helix of fibrillar collagens, shows a completely opposite response to TGF-β1 treatment. In fact, and as a consequence of the structural complexity of the extracellular matrix and basement membranes which must be proteolytically degraded by MMPs, these enzymes are often coregulated by the same cells in response to the same stimuli. In the case of collagenase-1 and collagenase-3 production by human fibroblasts, this statement seems to be true for cytokines like IL-1α and IL-1β and for tumor promoters like TPA, all of them displaying a marked up-regulatory effect on both MMP genes (Fig. 1). However, their divergent responses to TGF-β1 clearly indicate that in addition to common regulatory elements, these genes have distinct transcriptional elements that determine their specific expression patterns. Functional studies of the promoter region of the collagenase-3 gene performed in this work have shown that the AP-1 site present in its 5′-flanking region is responsible, at least in part, for the TGF-β1 mediated induction of this gene. AP-1 sites have also been reported to mediate the response to this growth factor of other genes such as type-1 plasminogen activator inhibitor (63Keeton M.R. Curriden S.A. van Zonneveld A.J. Loskutoff D.J. J. Biol. Chem. 1991; 266: 23048-23052Abstract Full Text PDF PubMed Google Scholar), osteocalcin (64Banerjee C. Stein J.L. Van Wijnen A.J. Frenkel B. Lian J.B. Stein G.S. Endocrinology. 1996; 137: 1991-2000Crossref PubMed Scopus (61) Google Scholar), retinoic acid and retinoid X receptors (65Chen Y. Takeshita A. Ozaki S. Kitanor S. Hanazawa S. J. Biol. Chem. 1996; 271: 31602-31606Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), as well as the autoinduction of the TGF-β1 gene itself (66Kim S.J. Angel P. Lafyatis R. Hattori K. Kim K.Y. Sporn M.B. Karin M. Roberts A.B. Mol. Cell. Biol. 1990; 10: 1492-1497Crossref PubMed Google Scholar). Nevertheless, the extent of stimulation of collagenase-3 gene expression by TGF-β1, as detected by Northern blot, was higher than the induction of promoter activity observed in transient cell transfection experiments with AP-1 containing constructs. Similar observations have been previously reported during the functional analysis of AP-1 sites present in other MMP genes (67Gaire M. Magbanua Z. McDonnell S. McNeill L. Lovett D.H. Matrisian L.M. J. Biol. Chem. 1994; 269: 2032-2040Abstract Full Text PDF PubMed Google Scholar,68Benbow U. Brinckerhoff C.E. Matrix Biol. 1997; 15: 519-526Crossref PubMed Scopus (433) Google Scholar). Therefore, it seems likely that this AP-1 site present in the collagenase-3 gene is necessary but not sufficient for mediating its response to TGF-β1 in human fibroblasts. The participation of additional elements which could be located further upstream in the 5′-flanking region of the collagenase-3 gene could contribute to explain the differences observed between data derived from Northern blot analysis and those from determination of relative CAT activity of reporter gene constructs. In addition, it is remarkable that other MMP genes, including collagenase-1, which are down-regulated by TGF-β, also contain AP-1 consensus sequences at approximately the same position as that present in the collagenase-3 gene (Fig. 7), thus supporting the idea that sequences other than AP-1 influence the responsiveness of the different MMP genes to this growth factor. Finally, it is also likely that TGF-β1, in addition to activating the collagenase-3 promoter, may increase the expression of this gene by post-transcriptional mechanisms such as stabilization of the corresponding mRNAs, which have been previously described to operate in the case of the collagenase-1 gene (69Vincenti M.P. Coon C.I. Lee O. Brinckerhoff C.E. Nucleic Acids Res. 1994; 22: 4818-4827Crossref PubMed Scopus (71) Google Scholar).In an attempt to determine the molecular basis of the somewhat paradoxical effect of TGF-β1 on collagenase-3 expression in human fibroblasts, we have further investigated the mechanistic aspects underlying this up-regulatory effect. In this work, and by using a series of specific inhibitors for different signaling pathways, we have found that the TGF-β1 action on collagenase-3 is mediated by PKC and tyrosine kinase signal transduction pathways. Since TGF-β receptors are Ser/Thr kinases, it is tempting to speculate that the activity affected by PKC inhibitors could be that intrinsic to the TGF-β receptors themselves. However, the observation that TGFβ-RI and -RII autophosphorylation is not affected by PKC inhibitors (70Miettinen P.J. Ebner R. López A.R. Derynck R. J. Cell Biol. 1994; 127: 2021-2036Crossref PubMed Scopus (784) Google Scholar) suggests that additional kinase activities acting downstream from the TGF-β receptor are involved in mediating the induction of collagenase-3 by TGF-β1. In addition, we have tried to correlate the TGF-β1 positive effect on collagenase-3 expression with some of the pleiotropic effects elicited by this multifunctional growth factor. It is well known that TGF-β displays a wide variety of actions even in the same cell type, depending on a number of factors including the specific target, conditions of cell culture, and presence of other growth regulators. Consistent with this, the observation that collagenase-3 induction by TGF-β1 in fibroblasts is accompanied by a weak stimulation of cell growth, together with data showing that in HaCaT keratinocytes TGF-β mediated up-regulation of this enzyme is accompanied by a potent inhibition of cell growth, 2J. A. Urı́a and C. López-Otı́n, unpublished results. strongly suggest that induction of collagenase-3 by this growth factor is independent of its effects on cell proliferation.Finally, in this work we have examined the possibility that cell specific induction of distinct members of the Fos/Jun family of transcription factors is responsible for the divergent regulation of collagenase-1 and collagenase-3 genes by TGF-β1 in human fibroblasts. Electrophoretic mobility shift assays and antibody supershift analysis revealed that c-Fos, c-Jun, and JunD are found in complexes formed with nuclear extracts prepared from KMST cells treated with TGF-β1. Analysis of the pattern of expression of these Fos/Jun proto-oncogenes in KMST cells treated with TGF-β1 confirmed that collagenase-3 induction is preceded by an increase in levels of expression of c-Fos and JunD. According to these results, it is conceivable that the induction of high levels of c-Fos and JunD favors the formation of specific complexes which bind and transactivate the collagenase-3 promoter, thus resulting in the observed up-regulation of this gene. Nevertheless, the participation of c-Jun in the process, after post-transcriptional mechanisms of regulation induced by TGF-β such as phosphorylation mediated by c-Jun N-terminal kinase (47Wang W. Zhou G. Hu M.C.-T. Yao Z. Tan T.-H. J. Biol. Chem. 1997; 272: 22771-22775Crossref PubMed Scopus (162) Google Scholar), cannot be ruled out. In this regard, it is well known that the preferential binding to AP-1 sites exhibited by different Fos/Jun proteins is dependent upon specific flanking and core nucleotide sequences, thus allowing fine regulation of expression of the diverse AP-1 driven genes. c-Jun and c-Fos have been proposed to be fundamental intermediates for collagenase-1 and stromelysin-1 gene activation by tumor necrosis factor-α in fibroblasts, and by TGF-β in keratinocytes, whereas inhibitory effects have been reported to be mediated by transient elevation of JunB (42Mauviel A. Chen Y.Q. Dong W. Evans C.H. Uitto J. Curr. Biol. 1993; 3: 822-831Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 45Mauviel A. Chung K.-Y. Agarwal A. Tamai K. Uitto J. J. Biol. Chem. 1996; 271: 10917-10923Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). However, in KMST cells, TGF-β had little if any effect on the expression of c-Jun or JunB, thus indicating that at least in these cells, other mechanisms should be involved in TGF-β1 elicited down-regulation of collagenase-1.In summary, we have provided evidence that TGF-β dissociates production of collagenase-1 and collagenase-3 by human fibroblasts. The opposite response of both enzymes to TGF-β1 in KMST cells makes them an appropriate model system for studying the molecular mechanisms controlling the expression of these two highly related enzymes in terms of structure and enzymatic properties, but displaying marked differences in their pattern of tissue expression. Further studies will be also required to evaluate the putative role of TGF-β1 as anin vivo inducer of human collagenase-3 in those conditions in which this enzyme has been found at high levels, including breast carcinomas and arthritic processes. The matrix metalloproteinases (MMPs) 1The abbreviations used are: MMP, matrix metalloproteinase; IL-1β, interleukin 1β; TPA, 12-O-tetradecanoylphorbol-13-acetate; aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; TGF, transforming growth factor; CAT, chloramphenicol acetyltransferase; PKC, protein kinase C. 1The abbreviations used are: MMP, matrix metalloproteinase; IL-1β, interleukin 1β; TPA, 12-O-tetradecanoylphorbol-13-acetate; aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; TGF, transforming growth factor; CAT, chloramphenicol acetyltransferase; PKC, protein kinase C. or matrixins form a family of structurally related metalloendopeptidases that are collectively capable of degrading the different macromolecular components of the extracellular matrix. These enzymes play a major role in normal tissue remodeling processes such as embryonic development, bone growth, and resorption, ovulation, uterine involution, and wound healing (1Woessner J.F. FASEB J. 1991; 5: 2145-2154Crossref PubMed Scopus (3072) Google Scholar, 2Matrisian L.M. BioEssays. 1992; 14: 455-463Crossref PubMed Scopus (1324) Google Scholar, 3Birkedal-Hansen H. Moore W.G.I. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2629) Google Scholar, 4Stetler-Stevenson W.G. Aznavoorian S. Liota L.A. Annu. Rev. Cell Biol. 1993; 9: 541-573Crossref PubMed Scopus (1515) Google Scholar). In addition, abnormal expression of these proteinases may contribute to a variety of pathological processes such as rheumatoid arthritis (5Murphy G. Hembry R.M. J. Rheumatol. 1992; 19: 61-64Google Scholar), atherosclerosis (6Henney A.M. Wakeley P.R. Davies M.J. Foster K. Hembry R. Murphy G. Humphries S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8154-8158Crossr

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Differential Effects of Transforming Growth Factor-β on the Expression of Collagenase-1 and Collagenase-3 in Human Fibroblasts