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• W2047835997 abstract "Collagenase-1 is invariantly expressed by migrating basal keratinocytes in all forms of human skin wounds, and its expression is induced by contact with native type I collagen. However, net differences in enzyme production between acute and chronic wounds may be modulated by soluble factors present within the tissue environment. Basic fibroblast growth factor (bFGF, FGF-2) and keratinocyte growth factor (KGF, FGF-9), which are produced during wound healing, inhibited collagenase-1 expression by keratinocytes in a dose-dependent manner. However, KGF was >100-fold more effective than bFGF at inhibiting collagenase-1 expression, suggesting that this differential signaling is transduced via an FGF receptor that binds these ligands with different affinities. Reverse transcriptase-polymerase chain reaction analysis of human keratinocyte mRNA for fibroblast growth factor receptors (FGFRs) revealed expression of only FGFR-2 IIIb, the KGF-specific receptor, which also binds bFGF with low affinity, and FGFR-3 IIIb, which does not bind bFGF or KGF. FGFRs that bind bFGF with high affinity were not detected. Our results suggest that bFGF and KGF inhibit collagenase-1 expression through the KGF cell-surface receptor (FGFR-2 IIIb). Because bFGF induces collagenase-1 in most cell types, cell-specific expression of FGFR family members may dictate the regulation of matrix metalloproteinases in a tissue-specific manner. Collagenase-1 is invariantly expressed by migrating basal keratinocytes in all forms of human skin wounds, and its expression is induced by contact with native type I collagen. However, net differences in enzyme production between acute and chronic wounds may be modulated by soluble factors present within the tissue environment. Basic fibroblast growth factor (bFGF, FGF-2) and keratinocyte growth factor (KGF, FGF-9), which are produced during wound healing, inhibited collagenase-1 expression by keratinocytes in a dose-dependent manner. However, KGF was >100-fold more effective than bFGF at inhibiting collagenase-1 expression, suggesting that this differential signaling is transduced via an FGF receptor that binds these ligands with different affinities. Reverse transcriptase-polymerase chain reaction analysis of human keratinocyte mRNA for fibroblast growth factor receptors (FGFRs) revealed expression of only FGFR-2 IIIb, the KGF-specific receptor, which also binds bFGF with low affinity, and FGFR-3 IIIb, which does not bind bFGF or KGF. FGFRs that bind bFGF with high affinity were not detected. Our results suggest that bFGF and KGF inhibit collagenase-1 expression through the KGF cell-surface receptor (FGFR-2 IIIb). Because bFGF induces collagenase-1 in most cell types, cell-specific expression of FGFR family members may dictate the regulation of matrix metalloproteinases in a tissue-specific manner. Wound repair is a highly organized process that requires a series of spatially and temporally regulated events to heal a tissue defect. Among these, effective proteolytic degradation of extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; FGF, fibroblast growth factor; bFGF, basic FGF; MMP, matrix metalloproteinases; TGF, transforming growth factor; KGF, keratinocyte growth factor; FGFR, FGF receptor; ELISA, enzyme-linked immunosorbent assay; bp, base pair(s). 1The abbreviations used are: ECM, extracellular matrix; FGF, fibroblast growth factor; bFGF, basic FGF; MMP, matrix metalloproteinases; TGF, transforming growth factor; KGF, keratinocyte growth factor; FGFR, FGF receptor; ELISA, enzyme-linked immunosorbent assay; bp, base pair(s). macromolecules by various proteases is necessary to remodel the damaged tissue, promote neovascularization, and facilitate efficient migration of cells during re-epithelialization (1Mignatti P. Rifkin D.B. Welgus H.G. Parks W.C. Clark R.A.F. The Molecular and Cellular Biology of Wound Repair. 2nd Ed. Plenum Press, New York1996: 427-474Google Scholar). Yet, in chronic ulcers, the overproduction of matrix-degrading proteases and/or the lack of production of their natural inhibitors probably contributes to the underlying pathogenesis of the non-healing state by interfering with normal repair processes and by perpetuating matrix destruction. Matrix metalloproteinases (MMPs) constitute a family of zinc-dependent enzymes that collectively have the capacity to degrade virtually all components of the ECM (2Birkedal-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 (2631) Google Scholar). While most members of this family possess overlapping substrate specificities, the metallocollagenases, a subgroup of the MMP gene family, have the unique ability to initiate cleavage of fibrillar collagens I, II, and III at a specific locus in their triple helical domain. At physiologic temperature, cleaved collagen molecules denature into gelatin and become susceptible to further digestion by other proteases. Of the three known human collagenases, collagenase-1 (MMP-1) is the enzyme principally responsible for collagen turnover in most tissues and, in particular, the skin. Previous studies from our laboratories and others have shown that basal keratinocytes at the leading edge of migration in both normally healing wounds and chronic ulcers invariantly express collagenase-1 (3Saarialho-Kere U.K. Chang E.S. Welgus H.G. Parks W.C. J. Clin. Invest. 1992; 90: 1952-1957Crossref PubMed Scopus (130) Google Scholar, 4Stricklin G.P. Li L. Jancic V. Wenczak B.A. Nanney L.B. Am. J. Pathol. 1993; 143: 1657-1666PubMed Google Scholar, 5Saarialho-Kere U.K. Vaalamo M. Airola K. Niemi K.-M. Oikarinen A.I. Parks W.C. J. Invest. Dermatol. 1995; 104: 982-988Abstract Full Text PDF PubMed Scopus (66) Google Scholar). Signal for collagenase-1 is confined to the basal layer of epidermis, diminishes progressively away from the wound edge, and is absent in intact skin. Furthermore, collagenase-1 expression is rapidly induced in wound edge keratinocytes after injury, persists during the healing phase, and ceases following wound closure (6Inoue M. Kratz G. Haegerstrand A. Ståhle-Bäckdahl M. J. Invest. Dermatol. 1995; 104: 479-483Abstract Full Text PDF PubMed Scopus (112) Google Scholar). In chronic, non-healing wounds expression of this MMP is prominent and excessive, whereas in normally healing wounds its expression is transient and localized precisely to areas of active re-epithelialization (3Saarialho-Kere U.K. Chang E.S. Welgus H.G. Parks W.C. J. Clin. Invest. 1992; 90: 1952-1957Crossref PubMed Scopus (130) Google Scholar, 7Saarialho-Kere U.K. Kovacs S.O. Pentland A.P. Olerud J. Welgus H.G. Parks W.C. J. Clin. Invest. 1993; 92: 2858-2866Crossref PubMed Scopus (278) Google Scholar). We have demonstrated that collagenase-1 expression by basal keratinocytes is induced following contact with native type I collagen, 2B. D. Sudbeck, B. K. Pilcher, H. G. Welgus, and W. C. Parks, (1997) J. Biol. Chem. 272, in press. 2B. D. Sudbeck, B. K. Pilcher, H. G. Welgus, and W. C. Parks, (1997) J. Biol. Chem. 272, in press. and the activity of this enzyme is required for cell migration (9Pilcher B.K. Sudbeck B.D. Dumin J. Krane S.M. Welgus H.G. Parks W.C. J. Cell Biol. 1997; 137: 1-13Crossref PubMed Scopus (490) Google Scholar). Thus, expression of matrix-degrading enzymes by keratinocytes during cutaneous wound repair is a normal and programmed response to injury, and altered cell-matrix interactions may play a critical role in regulating this response. In addition to cell-matrix interactions, soluble mediators present in the ECM during wound repair may influence collagenase-1 expression. Keratinocyte collagenase-1 production is stimulated by several growth factors including transforming growth factor-α (TGF-α)/epidermal growth factor (10Lyons J.G. Birkedal-Hansen B. Pierson M.C. Whitelock J.M. Birkedal-Hansen H. J. Biol. Chem. 1993; 268: 19143-19151Abstract Full Text PDF PubMed Google Scholar), hepatocyte growth factor/scatter factor (11Dunsmore S.E. Rubin J.S. Kovacs S.O. Chedid M. Parks W.C. Welgus H.G. J. Biol. Chem. 1996; 271: 24576-24582Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), transforming growth factor β1 (TGF-β1) (12Garlick J.A. Parks W.C. Welgus H.G. Taichman L.B. J. Dent. Res. 1996; 75: 912-918Crossref PubMed Scopus (49) Google Scholar, 13Mauviel 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), and interferon-λ (14Tamai K. Ishikawa H. Mauviel A. Uitto J. J. Invest. Dermatol. 1995; 104: 384-390Abstract Full Text PDF PubMed Scopus (57) Google Scholar). Furthermore, several of these growth factors (e.g.epidermal growth factor and hepatocyte growth factor/scatter factor) can augment ECM-directed collagenase-1 expression by keratinocytes (11Dunsmore S.E. Rubin J.S. Kovacs S.O. Chedid M. Parks W.C. Welgus H.G. J. Biol. Chem. 1996; 271: 24576-24582Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar,15Sudbeck B.D. Parks W.C. Welgus H.G. Pentland A.P. J. Biol. Chem. 1994; 269: 30022-30029Abstract Full Text PDF PubMed Google Scholar). In effect, while cell contact with specific matrices establishes the primary “on and off” signals, soluble mediators may finely control the net output of collagenase-1 by keratinocytes. Basic fibroblast growth factor (bFGF, FGF-2) and keratinocyte growth factor (KGF, FGF-9) belong to a family of heparin-binding growth factors that exert a variety of effects on multiple cell types (16Basilico C. Moscatelli D. Adv. Cancer Res. 1992; 59: 115-165Crossref PubMed Scopus (1049) Google Scholar). bFGF is widely expressed in vivo, is a potent angiogenic factor, and induces collagenase-1 production by cultured fibroblasts (17Chua C.C. Chua B.H. Zhao Z.Y. Krebs C. Diglio C. Perrin E. Connect. Tissue Res. 1991; 26: 271-281Crossref PubMed Scopus (93) Google Scholar, 18Sasaki T. J. Dermatol. 1992; 19: 664-666Crossref PubMed Scopus (34) Google Scholar), endothelial cells (19Okamura K. Sato Y. Matsuda T. Hamanaka R. Ono M. Kohno K. Kuwano M. J Biol. Chem. 1991; 266: 19162-19165Abstract Full Text PDF PubMed Google Scholar, 20Cornelius L.A. Nehring L.C. Roby J.D. Parks W.C. Welgus H.G. J. Invest. Dermatol. 1995; 105: 170-176Abstract Full Text PDF PubMed Scopus (127) Google Scholar), and osteoblasts (21Hurley M.M. Marcello K. Abreu C. Brinkerhoff C.E. Bowik C.C. Hibbs M.S. Biochem. Biophys. Res. Commun. 1995; 214: 331-339Crossref PubMed Scopus (23) Google Scholar). In addition, bFGF stimulates growth and proliferation of human keratinocytes (22O'Keefe E.J. Chin M.L. Payne R.E.J. J. Invest. Dermatol. 1988; 90: 767-769Abstract Full Text PDF PubMed Google Scholar, 23Shipley G.D. Keeble W.W. Hendrickson J.E. Coffey R.J.J. Pittelkow M.R. J. Cell. Physiol. 1989; 138: 511-518Crossref PubMed Scopus (143) Google Scholar). In contrast, KGF is expressed exclusively by cells of mesenchymal origin, such as fibroblasts (24Rubin J.S. Osada H.-J. Finch P.W. Taylor W.G. Rudikoff S. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 802-806Crossref PubMed Scopus (735) Google Scholar) and microvascular endothelial cells (25Smola H. Thiekotter G. Fusenig N.E. J. Cell Biol. 1993; 122: 417-429Crossref PubMed Scopus (335) Google Scholar), yet it specifically influences epithelial cells by a paracrine signaling mechanism (24Rubin J.S. Osada H.-J. Finch P.W. Taylor W.G. Rudikoff S. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 802-806Crossref PubMed Scopus (735) Google Scholar, 26Finch P.W. Rubin J.S. Miki T. Ron D. Aaronson S.A. Science. 1989; 245: 752-755Crossref PubMed Scopus (813) Google Scholar, 27Miki T. Bottaro D.P. Fleming T.P. Smith C.L. Burgess W.H. Chan A.M. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 246-250Crossref PubMed Scopus (654) Google Scholar). Both bFGF and KGF are expressed during epidermal wound repair (28Kurita Y. Tsuboi R. Ueki R. Rifkin D.B. Ogawa H. Arch. Dermatol. Res. 1991; 284: 193-197Crossref Scopus (41) Google Scholar, 29Werner S. Peters K.G. Longaker M.T. Fuller-Pace F. Banda M. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6896-6900Crossref PubMed Scopus (532) Google Scholar), and topical application of bFGF to wounds accelerates healing (30Tsuboi R. Rifkin D.B. J. Exp. Med. 1990; 172: 245-251Crossref PubMed Scopus (271) Google Scholar). Likewise, inhibition of KGF signaling in basal keratinocytes of epidermis following injury impairs re-epithelialization, presumably by inhibiting keratinocyte proliferation (31Werner S. Smola H. Liao X. Longaker M.T. Krieg T. Hofschneider P.H. Williams L.T. Science. 1994; 266: 819-822Crossref PubMed Scopus (510) Google Scholar). In this report, we demonstrate that bFGF and KGF down-regulate collagenase-1 expression by keratinocytes in a cell type-specific manner. Additionally, we show that KGF is >100-fold more potent than bFGF in suppressing collagenase-1 production and that keratinocytes express only two fibroblast growth factor receptors (FGFRs): FGFR-3 IIIb, which does not bind bFGF or KGF, and FGFR-2 IIIb, which binds KGF with high affinity, but poorly to bFGF. Thus, bFGF and KGF inhibition of keratinocyte collagenase-1 expression probably occurs exclusively through the KGF (FGFR-2 IIIb) receptor. Recombinant human bFGF, recombinant human KGF, and a polyclonal neutralizing antiserum to bFGF were obtained from R & D Systems (Minneapolis, MN). Bovine type I collagen (Vitrogen-100) was purchased from Celltrix Laboratories (Palo Alto, CA). Human keratinocytes were harvested from healthy adult skin from reduction mammoplasties or abdominoplasties as described previously (15Sudbeck B.D. Parks W.C. Welgus H.G. Pentland A.P. J. Biol. Chem. 1994; 269: 30022-30029Abstract Full Text PDF PubMed Google Scholar, 32Pentland A.P. Needleman P. J. Clin. Invest. 1986; 77: 246-251Crossref PubMed Scopus (169) Google Scholar). Briefly, the subcutaneous fat and deep dermis were removed, and the remaining tissue was incubated in 0.25% trypsin in phosphate-buffered saline. After 16 h, the epidermis was separated from the dermis with forceps, and the keratinocytes were scraped into Dulbecco's modified Eagle's medium. The keratinocyte suspension was added to fresh Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum and 0.1% penicillin/streptomycin. A specified amount of keratinocyte suspension was then plated onto tissue culture dishes coated with 1 mg/ml Vitrogen. Under these culture conditions, the keratinocytes proliferate, migrate, differentiate, and cornify similar to cells in vivo. Growth on native type I collagen is necessary for induction of collagenase-1 and keratinocyte adhesion (5Saarialho-Kere U.K. Vaalamo M. Airola K. Niemi K.-M. Oikarinen A.I. Parks W.C. J. Invest. Dermatol. 1995; 104: 982-988Abstract Full Text PDF PubMed Scopus (66) Google Scholar,8Guo L. Yu Q.O. Fuchs E. Eur. Mol. Biol. Organ. 1993; 12: 973-986Crossref PubMed Scopus (233) Google Scholar, 15Sudbeck B.D. Parks W.C. Welgus H.G. Pentland A.P. J. Biol. Chem. 1994; 269: 30022-30029Abstract Full Text PDF PubMed Google Scholar). The amount of collagenase-1 accumulated in keratinocyte-conditioned medium was measured by indirect competitive ELISA (33Cooper T.W. Bauer E.A. Eisen A.Z. Collagen Relat. Res. 1982; 3: 205-211Crossref Scopus (60) Google Scholar). This ELISA is completely specific for collagenase-1, has nanogram sensitivity, and detects both active and zymogen enzyme forms, as well as collagenase-1 bound to tissue inhibitor of metalloproteases (TIMP) or bound to substrate. Results were obtained from triplicate determinations and were normalized to total cell protein as quantified by the BCA protein assay (Pierce) using bovine serum albumin as a standard. Postconfluent keratinocytes plated on type I collagen were cultured for 24 h in the presence of Dulbecco's modified Eagle's medium/fetal calf serum containing control or experimental solutions. The culture wells were then washed and replaced with methionine-free Dulbecco's modified Eagle's medium containing 5% dialyzed fetal calf serum (to remove free amino acids), 1 mm sodium pyruvate, 2 mml-glutamine, 0.1 mm each of nonessential amino acids, 50 μCi/ml [35S]methionine (ICN Radiochemicals, Irvine CA), and the identical concentrations of experimental reagents. Conditioned medium was collected after 24 h and stored at −70 °C for analysis by immunoprecipitation. Specific polyclonal antisera to collagenase-1 (11Dunsmore S.E. Rubin J.S. Kovacs S.O. Chedid M. Parks W.C. Welgus H.G. J. Biol. Chem. 1996; 271: 24576-24582Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), stromelysin-1 (34Saarialho-Kere U.K. Kovacs S.O. Pentland A.P. Parks W.C. Welgus H.G. J. Clin. Invest. 1994; 94: 79-88Crossref PubMed Scopus (203) Google Scholar), 92-kDa gelatinase (35Shapiro S.D. Fliszar C. Broekelman T. Mecham R.P. Senior R.M. Welgus H.G. J. Biol. Chem. 1995; 270: 6351-6356Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), or TIMP-1 (36Lacraz S. Nicod L. Welgus H.G. Dayer J.-M. J. Clin. Invest. 1995; 96: 2304-2310Crossref PubMed Scopus (376) Google Scholar) were used to immunoprecipitate the35S-labeled metalloproteinases from keratinocyte-conditioned medium as described (37Welgus H.G. Campbell E.J. Bar-Shavit Z. Senior R.M. Teitelbaum S.L. J. Clin. Invest. 1985; 76: 219-224Crossref PubMed Scopus (149) Google Scholar). Samples were precleared with protein A-Sepharose (Zymed, San Francisco, CA), and supernatants were incubated with antibody for 1 h at 37 °C and then overnight at 4 °C. Immune complexes were precipitated with protein A-Sepharose and washed extensively. Radiolabeled proteins were resolved by polyacrylamide gel electrophoresis and visualized by fluorography. Total incorporated radioactivity was determined from the same conditioned medium by trichloroacetic acid precipitation. Total RNA was isolated from cultured keratinocytes by phenol-chloroform extraction (38Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63019) Google Scholar). RNA (5 μg) was denatured and resolved by electrophoresis through a 1% formaldehyde-agarose gel, transferred overnight to Hybond N+ (Amersham Corp.), and hybridized with radiolabeled collagenase-1 (39Goldberg G.I. Wilhelm S.M. Kronberger A. Bauer E.A. Grant G.A. Eisen A.Z. J. Biol. Chem. 1986; 261: 6600-6605Abstract Full Text PDF PubMed Google Scholar) and GAPDH cDNA probes. The cDNA probes were labeled by random priming (Boehringer Mannheim, Mannheim, Germany) with [α-32P]dCTP (NEN Life Science Products). Following hybridization, the membranes were washed and exposed to x-ray film for an appropriate duration. To determine which FGFRs were expressed by both human keratinocytes and fibroblasts, total RNA was harvested as above. RNA was treated with RQ1 RNase-free DNase (Promega, Madison, WI) to remove any contaminating DNA as described (40Swee M.H. Parks W.C. Pierce R.A. J. Biol. Chem. 1995; 270: 14899-14906Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). DNase-treated RNA (5 μg) was reverse transcribed with random hexamers using kit reagents and under the manufacturer's recommended conditions (GeneAmp RNA PCR kit, Perkin-Elmer, Norwalk, CT). For each sample, a parallel reaction was run without reverse transcriptase as a control. Expression of FGFRs 1–4 in human keratinocytes was detected by polymerase chain amplification of cDNA using a single primer pair to amplify conserved sequences in the tyrosine kinase domain of all FGFRs (41McEwen D.G. Ornitz D.M. Biotechniques. 1997; 22: 1068-1070Crossref PubMed Scopus (11) Google Scholar). The primer sequences used for PCR were DO156 (5′-TCNGAGATGGGAGRTGATGAA-3′) and DO158 (5′-CCAAGTCHGCDATCCTTCAT-3′), which produce a 341-bp product. PCR was for 30 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, followed by a final extension step of 72 °C for 7 min. To determine which members of the FGFR family were expressed, PCR products were analyzed by restriction digestion analysis with PstI, BalI,ScaI, or NarI. Digested fragments were separated by nondenaturing polyacrylamide gel electrophoresis and visualized by silver staining. To determine the expression of FGFR-2 isoforms (IIIb and IIIc) by human keratinocytes and fibroblasts, we amplified random primed cDNA with specific primers as described (42Pekonen F. Nyman T. Rutanen E.-M. Mol. Cell. Endocrinol. 1993; 95: 43-49Crossref PubMed Scopus (52) Google Scholar). Briefly, the cDNA was amplified for FGFR-2 IIIb using the 5′S primer corresponding to a region within the FGFR-2 IIIb-specific exon K: 5′-CAATGCAGAAGTGCTGGCTCTGTTCAA-3′. FGFR-2 IIIc was amplified using the 5′S primer corresponding to a region within the FGFR-2 IIIc specific exon B: 5′-GTTAACACCACGGACAA-3′. The 3′AS primer used in both PCR reactions was from nucleotides 2093–2112 of the cDNA coding for FGFR-2 IIIb. The same 3′AS primer was used for amplification of both FGFRs, since the nucleotide sequence is identical for both isoforms in this region (27Miki T. Bottaro D.P. Fleming T.P. Smith C.L. Burgess W.H. Chan A.M. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 246-250Crossref PubMed Scopus (654) Google Scholar, 43Miki T. Fleming T.P. Bottaro D.P. Rubin J.S. Science. 1991; 251: 72-75Crossref PubMed Scopus (362) Google Scholar, 44Yayon A. Zimmer Y. Shen G.H. Avivi A. Yarden Y. Givol D. EMBO J. 1992; 11: 1885-1890Crossref PubMed Scopus (133) Google Scholar). PCR was for 40 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, followed by a final extension step of 72 °C for 7 min. The predicted fragment size was 822 bp for the FGFR-2 IIIb and 830 for FGFR-2 IIIc. The products were separated through a 2% agarose gel and visualized by ethidium bromide staining. Further specificity was determined by transfer to Hybond N+ followed by Southern hybridization with a radiolabeled product-specific oligonucleotide probe. The probe was labeled by terminal transferase (Boehringer Mannheim) with [α-32P]dCTP. Following hybridization, the membranes were washed and exposed to x-ray film for an appropriate duration. To determine the expression of FGFR-3 isoforms (IIIb and IIIc) by human keratinocytes and fibroblasts, we amplified random primed cDNA with specific primers as described (45Scotet E. Houssaint E. Biochim. Biophys. Acta. 1995; 1264: 238-242Crossref PubMed Scopus (42) Google Scholar). The 5′S primer used (5′-GCACCGGCCCCATCCTGCAGGCGG-3′) corresponds to nucleotides 789–811 of the human FGFR-3 gene, and the 3′AS primer used (5′-TACACACTGCCCGCCTCGTCAGC-3′) corresponds to nucleotides 1135–1158 of the FGFR-3 gene, generating a product with a predicted size of 369 bp. PCR was for 30 cycles of 94 °C for 1 min, 68 °C for 1 min, and 72 °C for 1 min followed by a final extension step of 72 °C for 7 min. Following amplification, products were analyzed by restriction digestion analysis with HaeII andTaqI, allowing subsequent identification of IIIb and IIIc isoforms. Products were separated by agarose gel electrophoresis, transferred to Hybond N+, and hybridized with a radiolabeled product-specific oligonucleotide probe. The probe was labeled by terminal transferase with α [32P]dCTP. Following hybridization, the membranes were washed and exposed to x-ray film for an appropriate duration. Previous reports have documented the capacity of bFGF to stimulate collagenase-1 production in cells of mesenchymal origin (18Sasaki T. J. Dermatol. 1992; 19: 664-666Crossref PubMed Scopus (34) Google Scholar, 19Okamura K. Sato Y. Matsuda T. Hamanaka R. Ono M. Kohno K. Kuwano M. J Biol. Chem. 1991; 266: 19162-19165Abstract Full Text PDF PubMed Google Scholar, 46Kennedy S.H. Qin H. Lin L. Tan E.M. Am. J. Pathol. 1995; 146: 764-771PubMed Google Scholar, 47Varghese S. Ramsby M.L. Jeffrey J.J. Canalis E. Endocrinology. 1995; 135: 2156-2162Crossref Scopus (63) Google Scholar). Consistent with these studies, we found that bFGF increased collagenase-1 production by human dermal fibroblasts in a dose-dependent manner (Fig.1 A). At 1.0 ng/ml bFGF, collagenase-1 production was augmented 5-fold over control levels. To assess if bFGF modulates keratinocyte collagenase-1 production, cells were exposed to increasing concentrations of growth factor for 72 h, and collagenase-1 accumulation in the medium was quantified by ELISA. In contrast to other cell types, bFGF potently inhibited keratinocyte collagenase-1 expression, with an ED50 of ∼1.0 ng/ml (Fig. 1 B). Preincubation with anti-bFGF neutralizing antiserum abolished collagenase-1 down-regulation (Fig. 1 C), thus demonstrating that the effect was due to the growth factor itself and not to a contaminant. Metabolic labeling and immunoprecipitation experiments confirmed that bFGF inhibited keratinocyte collagenase-1 production at the level of new enzyme synthesis (Fig. 2 A). Immunoprecipitation of the same conditioned media for stromelysin-1 showed similarly reduced expression of this MMP (Fig. 2 B), whereas the synthesis of 92-kDa gelatinase and TIMP-1 was unchanged (data not shown). Inhibition of collagenase-1 and stromelysin-1 expression was specific, since synthesis of total secreted proteins by keratinocytes increased slightly following bFGF treatment (TableI). The disparity in bFGF concentrations required to effectively inhibit keratinocyte collagenase-1 production in Figs. 1and 2 reflect the individual skin donors examined, whom we have found to exhibit variable sensitivities to the growth factor.Table ITotal protein synthesisConditionProtein synthesis1-aValues for protein synthesis are in trichloracetic acid-precipitable counts/min.Control12,258 ± 1068bFGF (1.0 ng/ml)13,279 ± 2167bFGF (25 ng/ml)16,622 ± 956KGF (1.0 ng/ml)16,220 ± 3723KGF (10 ng/ml)15,237 ± 676The same labeled cellular proteins used for immunoprecipitation of collagenase-1 (Fig. 2 and 4B) were instead precipitated with 20% trichloroacetic acid as described under “Experimental Procedures.” The data presented are the means ± S.D. of triplicate determinations from three separate wells per treatment.1-a Values for protein synthesis are in trichloracetic acid-precipitable counts/min. Open table in a new tab The same labeled cellular proteins used for immunoprecipitation of collagenase-1 (Fig. 2 and 4B) were instead precipitated with 20% trichloroacetic acid as described under “Experimental Procedures.” The data presented are the means ± S.D. of triplicate determinations from three separate wells per treatment. Total RNA was isolated from keratinocytes that had been treated for 24 h in the absence or presence of bFGF (25 ng/ml) and was analyzed by Northern hybridization. bFGF inhibited steady-state collagenase-1 mRNA levels, causing a 68% reduction when compared with untreated controls (Fig.3 A). Identically treated cells were cultured for 48 h, and collagenase-1 protein was quantified by ELISA (Fig.3 B). bFGF inhibited collagenase-1 protein expression (57%) proportionally to the drop in mRNA levels (68%), indicating pretranslational regulation. Although bFGF consistently inhibited keratinocyte collagenase-1 expression, we often had to use relatively high concentrations (≥10 ng/ml) of the growth factor to observe this activity (Fig. 2 and other data not shown). Because multiple FGFs bind to more than one FGFR with different affinities (48Partanen J. Makela T.P. Eerola E. Korhonen J. Hirvonen H. Claesson-Welsh L. Alitalo K. EMBO J. 1991; 10: 1347-1354Crossref PubMed Scopus (459) Google Scholar, 49Ornitz D.M. Leder P. J. Biol. Chem. 1992; 267: 16305-16311Abstract Full Text PDF PubMed Google Scholar, 50Mansukhani A. Dell'Era P. Moscatelli D. Kornbluth S. Hanafusa H. Basilico C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3305-3309Crossref PubMed Scopus (113) Google Scholar, 51Ornitz D.M. Xu J. Colvin J.S. McEwen D.G. MacArthur C.A. Coulier F. Gao G. Goldfarb M. J. Biol. Chem. 1996; 271: 15292-15297Abstract Full Text Full Text PDF PubMed Scopus (1415) Google Scholar), we postulated that other members of the FGF family might be more potent inhibitors of collagenase-1 production. KGF, a mesenchymal cell-derived cytokine that acts specifically on epithelial cells (24Rubin J.S. Osada H.-J. Finch P.W. Taylor W.G. Rudikoff S. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 802-806Crossref PubMed Scopus (735) Google Scholar), was chosen as a candidate because of its relevance to epidermal wound repair. Paralleling the effects of bFGF, treatment of cultured keratinocytes with increasing concentrations of KGF resulted in a dose-dependent inhibition of collagenase-1 expression (Fig. 4 A). Futhermore, KGF was more potent, consistently demonstrating an ED50 of ∼0.01 ng/ml, at least 100-fold lower than bFGF. As demonstrated by metabolic labeling and immunoprecipitation, KGF inhibited collagenase-1 production (Fig. 4 B). Again, KGF was effective at lower concentrations than bFGF (Fig. 4 A versusFig. 1 B). As observed for bFGF, stromelysin-1 biosynthesis was also inhibited by KGF treatment, whereas 92-kDa gelatinase and TIMP-1 were unaffected (data not shown). Total protein synthesis was mildly increased by KGF (Table I), indicating the specificity of its collagenase-related activity. Northern hybridization was performed to determine if KGF inhibited collagenase-1 production in a manner similar to bFGF. Keratinocytes treated with KGF (1.0 ng/ml) displayed a dramatic reduction in collagenase-1 mRNA compared with untreated cells (Fig. 5 A). To compare KGF inhibition of collagenase-1 mRNA with collagenase-1 protein, conditioned media samples from the same skin donor were analyzed by ELISA. Quantitation demonstrated that inhibition of secreted collagenase-1 protein closely paralleled decreased mRNA levels (Fig. 5 B; 69versus 70%, respectively). FGFs activate a fa" @default.
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• W2047835997 title "Cell Type-specific Inhibition of Keratinocyte Collagenase-1 Expression by Basic Fibroblast Growth Factor and Keratinocyte Growth Factor" @default.
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