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• W2036733178 abstract "Both cytokines and matrix metalloproteinases (MMPs) are active during physiologic and pathologic processes such as cancer metastasis and wound repair. We have systematically studied cytokine-mediated MMP regulation. Cytokine-mediated proteinase induction and activation were initially investigated in organ-cultured human skin followed by determination of underlying cellular and molecular mechanisms using isolated skin cells. In this report we demonstrate that tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) synergistically induce pro-MMP-9 in human skin as well as isolated dermal fibroblasts and epidermal keratinocytes. Furthermore, TNF-α promotes proteolytic activation of pro-MMP-9 by conversion of the 92-kDa pro-MMP-9 to the 82-kDa active enzyme. This activation occurred only in skin organ culture and not by either isolated fibroblasts or keratinocyte, although the pro-MMP-9 activation could be measured in a cell-free system derived from TNF-α-activated skin. The cytokine-mediated induction of pro-MMP-9 in dermal fibroblasts was evident by increased mRNA. At the transcription level, we examined the cytokine-mediated transactivation of the 5′-region promoter of the human MMP-9 in dermal fibroblasts. The results demonstrated that TNF-α and TGF-β could independently stimulate the 5′-flanking 670-base pair promoter. A TGF-β-response element (−474) and an NF-κB-binding site (−601) were identified to be the cis-elements for TGF-β or TNF-α activation, respectively. Taken together, these findings suggest a specific mechanism whereby multiple cytokines can regulate MMP-9 expression/activation in the cells of human skin. These results imply roles for these cytokines in the regulation of MMP-9 in physiologic and pathologic tissue remodeling. Both cytokines and matrix metalloproteinases (MMPs) are active during physiologic and pathologic processes such as cancer metastasis and wound repair. We have systematically studied cytokine-mediated MMP regulation. Cytokine-mediated proteinase induction and activation were initially investigated in organ-cultured human skin followed by determination of underlying cellular and molecular mechanisms using isolated skin cells. In this report we demonstrate that tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) synergistically induce pro-MMP-9 in human skin as well as isolated dermal fibroblasts and epidermal keratinocytes. Furthermore, TNF-α promotes proteolytic activation of pro-MMP-9 by conversion of the 92-kDa pro-MMP-9 to the 82-kDa active enzyme. This activation occurred only in skin organ culture and not by either isolated fibroblasts or keratinocyte, although the pro-MMP-9 activation could be measured in a cell-free system derived from TNF-α-activated skin. The cytokine-mediated induction of pro-MMP-9 in dermal fibroblasts was evident by increased mRNA. At the transcription level, we examined the cytokine-mediated transactivation of the 5′-region promoter of the human MMP-9 in dermal fibroblasts. The results demonstrated that TNF-α and TGF-β could independently stimulate the 5′-flanking 670-base pair promoter. A TGF-β-response element (−474) and an NF-κB-binding site (−601) were identified to be the cis-elements for TGF-β or TNF-α activation, respectively. Taken together, these findings suggest a specific mechanism whereby multiple cytokines can regulate MMP-9 expression/activation in the cells of human skin. These results imply roles for these cytokines in the regulation of MMP-9 in physiologic and pathologic tissue remodeling. tumor necrosis factor-α transforming growth factor-β base pair matrix metalloproteinases Dulbecco's modified Eagle's medium chloramphenicol acetyltransferase polymerase chain reaction polyacrylamide gel electrophoresis extracellular matrix TGF-β-response element platelet-derived growth factor epidermal growth factor interleukin Cytokines have been shown to be involved in many physiologic and pathologic processes. Tumor necrosis factor-α (TNF-α)1 is thought to be essential for macrophage-mediated normal wound healing (1Leibovich S.J. Polverini P.J. Shepard H.M. Wiseman D.M. Shively V. Nuseir N. Nature. 1987; 329: 630-632Crossref PubMed Scopus (1003) Google Scholar, 2Fajardo L.F. Kwan H.H. Kowalski J. Prionas S.D. Allison A.C. Am. J. Pathol. 1992; 140: 539-544PubMed Google Scholar). Whereas elevated levels of TNF-α have been linked to deficient wound healing (3Garner W.L. Karmiol S. Rodriguez J.L. Smith Jr., D.J. Phan S.H. J. Invest. Dermatol. 1993; 101: 875-879Abstract Full Text PDF PubMed Google Scholar, 4Cooney R. Iocono J. Maish G. Smith J.S. Ehrlich P. J. Trauma. 1997; 42: 415-420Crossref PubMed Scopus (54) Google Scholar), a lack of TNF-α was found to be associated with hypertrophic scars (5Kitzis V. Engrav L.H. Quinn L.S. J. Surg. Res. 1999; 87: 134-141Abstract Full Text PDF PubMed Scopus (20) Google Scholar, 6Peruccio D. Castagnoli C. Stella M. D'Alfonso S. Momigliano P.R. Magliacani G. Alasia S.T. Burns. 1994; 20: 118-121Crossref PubMed Scopus (22) Google Scholar). This suggests that misregulated TNF-α results in disordered wound healing. Similarly, transforming growth factor-β (TGF-β) is a component of normal wound healing (7Beck L.S. Chen T.L. Mikalauski P. Ammann A.J. Growth Factors. 1990; 3: 267-275Crossref PubMed Scopus (49) Google Scholar, 8Sporn M.B. Roberts A.B. J. Clin. Invest. 1993; 92: 2565-2566Crossref PubMed Scopus (90) Google Scholar). Increased TGF-β is associated with hypertrophic scar and fibrosis (9Ghahary A. Shen Y.J. Scott P.G. Gong Y. Tredget E.E. J. Lab. Clin. Med. 1993; 122: 465-473PubMed Google Scholar, 10Zhang K. Garner W. Cohen L. Rodriguez J. Phan S. J. Invest. Dermatol. 1995; 104: 750-754Abstract Full Text PDF PubMed Scopus (139) Google Scholar). In addition, accumulating evidence has suggested a role for inflammatory cytokines in promoting tumor metastasis, although the mechanism is not clarified (11Malik S.T. Semin. Cancer Biol. 1992; 3: 27-33PubMed Google Scholar, 12Roberts A.B. Thompson N.L. Heine U. Flanders C. Sporn M.B. Br. J. Cancer. 1988; 57: 594-600Crossref PubMed Scopus (164) Google Scholar). The roles for these mediators in the regulation of extracellular matrix may relate to the spectrum of genes they induce and appear to share a common theme of tissue remodeling. Compelling evidence has documented the role of matrix metalloproteinases in the remodeling of connective tissue during angiogenesis, tumor metastasis, and tissue repair (13Parks W.C. Wound Repair Regen. 1999; 7: 423-432Crossref PubMed Scopus (334) Google Scholar). Of the growing family of MMPs, MMP-2 (gelatinase A, 72-kDa type IV collagenase, EC3.4.24.24) and MMP-9 (gelatinase B, 92-kDa type IV collagenase, EC3.4.24.35) are unique for their fibronectin-like collagen binding domains (14Vu T.H. Werb Z. Parks W.C. Mecham R.P. Matrix Metalloproteases. Academic Press, New York1995: 115-148Google Scholar). MMP-2 and MMP-9 are thought to be responsible for detaching basal keratinocytes from the basement membrane and thus promote their migration to cover exposed connective tissue (15Salo T. Makela M. Kylmaniemi M. Autio-Harmainen H. Larjava H. Lab. Invest. 1994; 70: 176-182PubMed Google Scholar, 16Murphy G. Ward R. Hembry R.M. Reynolds J.J. Kuhn K. Tryggvason K. Biochem. J. 1989; 258: 463-472Crossref PubMed Scopus (196) Google Scholar). This notion is based on their restricted expression pattern at the wound edge and their substrate preference for basement membrane, type IV collagen. In addition, the type IV collagenases may also degrade type VII collagen, the major collagen component of anchoring fibrils essential for the attachment of the epidermis to the dermis, (17Karelina T.V. Bannikov G.A. Eisen A.Z. J. Invest. Dermatol. 2000; 114: 371-375Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 18Seltzer J.L. Eisen A.Z. Bauer E.A. Morris N.P. Glanville R.W. Burgeson R.E. J. Biol. Chem. 1989; 264: 3822-3826Abstract Full Text PDF PubMed Google Scholar). Although the actual functions of the type IV collagenases in normal physiologic processes are not clear, accumulated evidence has linked them to many diseases. Excessive type IV collagenase activity is associated with non-healing chronic wounds where it is thought that type IV collagen is over-digested during re-epithelialization (19Moses M.A. Marikovsky M. Harper J.W. Vogt P. Eriksson E. Klagsbrun M. Langer R. J. Cell. Biochem. 1996; 60: 379-386Crossref PubMed Scopus (110) Google Scholar, 20Tarlton J.F. Bailey A.J. Crawford E. Jones D. Moore K. Harding K.D. Wound Repair Regen. 1999; 7: 347-355Crossref PubMed Scopus (82) Google Scholar). Consistent with this fact, low levels of MMP-9 were found in hypertrophic scars where collagen is over-deposited (21Neely A.N. Clendening C.E. Gardner J. Greenhalgh D.G. Warden G.D. Wound Repair Regen. 1999; 7: 166-171Crossref PubMed Scopus (81) Google Scholar). The expression of pro-MMP-9 is regulated by many soluble mediators such as TNF-α, IL-1β, TGF-β, the ECM, oncogenes, and tumor promoters (22Okada Y. Tsuchiya H. Shimizu H. Tomita K. Nakanishi I. Sato H. Seiki M. Yamashita K. Hayakawa T. Biochem. Biophys. Res. Commun. 1990; 171: 610-617Crossref PubMed Scopus (112) Google Scholar, 23Lyons 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, 24Sehgal I. Thompson T.C. Mol. Biol. Cell. 1999; 10: 407-416Crossref PubMed Scopus (155) Google Scholar, 25Mauviel A. J. Cell. Biochem. 1993; 53: 288-295Crossref PubMed Scopus (397) Google Scholar). After secretion into the ECM, the activity of MMP-9 can be further regulated by specific tissue inhibitors and by proteolytic activation via removal of the amino-terminal inhibitory domain. Most of the previous investigations on cytokine-mediated MMP-9 expression utilized tumor or transformed cell lines in which the mitogenic signaling and the cell cycle machinery are constitutively active. In chronic wounds and some invasive cancer tissue the 92-kDa pro-MMP-9 is processed into the active 82-kDa form (26Wysocki A.B. Staiano-Coico L. Grinnell F. J. Invest. Dermatol. 1993; 101: 64-68Abstract Full Text PDF PubMed Google Scholar, 27Zeng Z.S. Guillem J.G. Br. J. Cancer. 1998; 78: 349-353Crossref PubMed Scopus (36) Google Scholar, 28Rha S.Y. Kim J.H. Roh J.K. Lee K.S. Min J.S. Kim B.S. Chung H.C. Breast Cancer Res. Treat. 1997; 43: 175-181Crossref PubMed Scopus (46) Google Scholar). Very little is known about the molecular regulation of expression and proteolytic activation of MMP-9 in these pathologic situations. Because multiple cytokines are coordinately present in wound sites, we have studied the interaction of multiple cytokines on MMP-9 induction and its proteolytic activation in organ-cultured human skin. In the second phase of the study we dissected the cytokine-mediated MMP-9 regulation at the cellular level. We isolated dermal fibroblasts and keratinocytes from human skin and examined the effect of cytokines on MMP-9 regulation. Finally, we investigated the cytokine-responsive cis-elements in the promoter of the human MMP-9 gene to identify the molecular target sites of such regulation. In a previous report we demonstrated that exposure of human skin to TNF-α led to activation of the pro-MMP-2, and such proteolytic activation could be reconstructed by embedding the dermal fibroblasts in collagen lattices (29Han Y.P. Tuan T.L. Wu H. Hughes M. Garner W.L. J. Cell Sci. 2001; 114: 131-139PubMed Google Scholar). In the present study we extend our findings by showing that the 92-kDa pro-MMP-9 is induced in human skin by TGF-β, and this induction is additively enhanced by a second signal from TNF-α. In addition, we found that the 92-kDa pro-MMP-9 is converted to the 82-kDa active form when organ-cultured skin is treated with TNF-α. Furthermore, we show here that the TNF-α-mediated pro-MMP-9 activation is caused by an unidentified factor that is tightly associated with skin tissue. Our cellular dissection experiments demonstrate that cytokine-mediated pro-MMP-9 induction in the human skin is due to dermal fibroblasts and epidermal keratinocytes. At the molecular level, we provide evidence showing that these two cytokines target their response elements in the 5′-promoter of the human MMP-9 gene. These findings represent the first demonstration for additive roles of TNF-α and TGF-β on the induction and activation of MMP-9 in human skin. Vitrogen containing 95% type I collagen was purchased from Cohesion Technologies (Palo Alto, CA). Cytokines were purchased from R & D Systems (Minneapolis, MN). The antibodies against MMP-9 (AB805) were purchased from Chemicon International (Temecula, CA). The Immobilon-P was purchased from Millipore (Bedford, MA). The enhanced chemiluminescence (ECL) was purchased from Amersham Pharmacia Biotech.The gelatin was from Sigma. Gelatin-Sepharose 4B was purchased fromAmersham Pharmacia Biotech. RNA was extracted by Trizol from Life Technologies, Inc. The reagents for reverse transcriptase-PCR were purchased from Promega (Madison, WI). The Platinum Taq DNA polymerase and oligodeoxynucleotide primers were from Life Technologies, Inc. KGM and KBM were from Clonetics. Quick-change site-directed mutagenesis kit was purchased from Stratagene (La Jolla, CA). DNA sequencing was carried out at the University of Southern California/Norris Comprehensive Cancer Center. Normal human skin was obtained from patients undergoing reconstructive or aesthetic surgery (University of Southern California IRB 999061). The full thickness skin was decontaminated by incubation in 2× antibiotic containing DMEM (200 units/ml penicillin G sodium, 200 units/ml streptomycin sulfate, and 0.5 μg/ml amphotericin B) at 4 °C overnight. Then the skin was cut into equal sizes with 0.5 cm on each side and incubated in DMEM at 37 °C with 5% CO2for 8 h. To decrease the effects of endogenous soluble factors in the skin induced by the harvesting process, the medium was changed three times during the 8-h incubation. Finally, the skin piece was immersed in 2 ml of DMEM with specific cytokines and was maintained at 37 °C with 5% CO2. The conditioned media were sampled at the indicated times for gelatinolytic zymogram assay and Western blot as mentioned below. Dermal fibroblasts and keratinocytes were isolated from full thickness human skin (3Garner W.L. Karmiol S. Rodriguez J.L. Smith Jr., D.J. Phan S.H. J. Invest. Dermatol. 1993; 101: 875-879Abstract Full Text PDF PubMed Google Scholar). The isolated fibroblasts were cultured in DMEM containing 10% fetal bovine serum with antibiotics. The keratinocytes were grown in complete KGM. Before exposure to cytokines the medium was replaced with serum-free DMEM for fibroblasts and KBM, the basal medium, for keratinocytes. For some experiments the fibroblasts were embedded in collagen lattices (29Han Y.P. Tuan T.L. Wu H. Hughes M. Garner W.L. J. Cell Sci. 2001; 114: 131-139PubMed Google Scholar). The transformed human keratinocytes (kindly provided by Dr. David Woodley, University of Southern California) were cultured in KGM to confluence. The cells were stimulated by 10 ng/ml TNF-α in KBM for 72 h in standard culture condition. In these conditions most of the gelatinase secreted in the medium is the 92-kDa pro-MMP-9. The conditioned media from 10 10-cm dishes were collected, and particulate debris was removed by centrifugation at 4,000 × g for 10 min. The conditioned media were passed through a 1-ml gelatin-Sepharose 4B column followed by washing with 400 mmNaCl in 50 mm Tris, pH 7.5. The bound gelatinase was eluted by 100 mm HCl and immediately neutralized by Tris base to pH 7.5. The explants of full thickness skin were stimulated with or without TNF-α at 10 ng/ml for 70 h in DMEM at 37 °C with 5% CO2. To test whether the TNF-α-mediated pro-MMP-9 activation occurred inside the skin tissue or by secreted factors, the explants were washed and re-cultured in fresh DMEM without cytokine. The original conditioned media from the 70-h stimulation and the conditioned media from the re-culture were incubated at 37 °C for additional 8–20 h and analyzed by gelatinolytic zymography. In another experiment, the TNF-α-treated skin explant was minced and then extracted using a buffer containing 2% Triton X-100, CaCl2at 0.2 g/liter, KCl at 0.4 g/liter, MgSO4 at 0.1 g/liter, NaCl at 6.4 g/liter, and 50 mm Tris, pH 7.5. After a 5-h incubation at room temperature the Triton-soluble and -insoluble fractions were obtained by centrifugation at 12,000 ×g for 10 min. Purified pro-MMP-9 was added to these fractions and incubated at 37 °C for 20 h followed by the zymogram analysis. The conditioned media were mixed with SDS-PAGE sample buffer in the absence of reducing agent and electrophoresed in 10% polyacrylamide gel containing 0.1% (w/v) gelatin. Electrophoresis was performed at 4 °C with 120 V for 16 h. After electrophoresis, SDS in the gel was removed by incubation with 2.5% Triton X-100. Gelatinolytic activities were developed in buffer containing 5 mm CaCl2, 150 mm NaCl, and 50 mm Tris, pH 7.5, for 16 h at 37 °C. The gelatinolytic activities were visualized by staining with Coomassie Blue R-250. The gelatinase from conditioned media was enriched by binding it to gelatin-conjugated Sepharose 4B. Briefly, 1 ml of conditioned media was incubated with 40 μl of gelatin-Sepharose 4B (Amersham Pharmacia Biotech) for 3 h. The beads were washed four times with 0.4m NaCl in 50 mm Tris, pH 7.5. The bound protein was eluted with 1× SDS-PAGE sample buffer at the reducing condition. After SDS-PAGE, the protein was transferred to Immobilon-P (Millipore). The protein blot was exposed to anti-human MMP-9 antibodies and followed by horseradish peroxidase-conjugated secondary antibodies that were detected by enhanced chemiluminescence. The dermal fibroblasts were grown in 10-cm dishes until subconfluent. To induce quiescence in the cells, the dishes were washed with DMEM and incubated in serum-free DMEM for 4 h. For the titration experiment, the dishes were stimulated with TNF-α (10 ng/ml) and TGF-β (1 ng/ml) individually or in combination in DMEM for 20 h. For the time course experiment the cells were stimulated by a combination of TNF-α with TGF-β and harvested 0, 2, 4, 6, 12, and 20 h later. After the dishes were stimulated for the indicated time, they were washed with phosphate-buffered saline, and the total RNA was extracted by Trizol. RNA was quantified by measuring the adsorption at 260 nm. 2 μg of total RNA was reverse-transcribed using avian myeloblastosis virus-reverse transcriptase with 1 μg of random deoxynucleotide hexamer in the presence of 4 mm MgCl2 and 2.5 mmdNTP. After completion of the 1-h incubation at 37 °C, the reaction was terminated via heating to 94 °C for 3 min. The annealing temperature for amplification of human MMP-9 and β-actin was 64 °C (ROBOCYCLER, Stratagene). The PCR amplification was performed with platinum Taq DNA polymerase for 30 cycles. The product was resolved in agarose gel (1.8%), followed by staining with ethidium bromide, and recorded by digital camera. The relative density of the products was quantitated by the Alpha imaging system. The oligonucleotide primers for PCR were adapted from a previous report (30Platzer C. Blankenstein T. Cytokines; A Practical Approach. IRL Press, New York1995: 57-68Google Scholar, 31Janowska-Wieczorek A. Marquez L.A. Matsuzaki A. Hashmi H.R. Larratt L.M. Boshkov L.M. Turner A.R. Zhang M.C. Edwards D.R. Kossakowska A.E. Br. J. Haematol. 1999; 105: 402-411Crossref PubMed Scopus (98) Google Scholar). The predicted PCR product size for β-actin and for MMP-9 is 548 and 479 bp, respectively. The identity of the PCR product for MMP-9 was confirmed by DNA sequencing (Norris Cancer Center, University of Southern California). DNA fragments containing the 5′-region of the humanMMP-9 gene and the NF-κB-response elements were inserted into pBLCAT2 (32Boshart M. Kluppel M. Schmidt A. Schutz G. Luckow B. Gene ( Amst. ). 1992; 110: 129-130Crossref PubMed Scopus (230) Google Scholar). The wild type NF-κB reporter plasmid, pNFκB3x-CAT, was constructed by inserting an enhancer element into pBLCAT2 at HindIII/BamHI sites. The insertion fragment consists of a triple tandem repeat of the NF-κB consensus binding site with the sequence of 5′-AGC TTG GGA CTT TCC GGG ACT TTC CGG GAC TTT CCG GAT CC-3′ (Promega). An NF-κB enhancer mutant, pNFκBm3x-CAT, was constructed by insertion of the adapter (5′-AGC TGT ACA CTT TCC TAC ACT TTCC TAC ACT TTC CG-3′) into pBLCAT2. The plasmid containing the wild type 670-bp upstream region of humanMMP-9 gene was a gracious gift of Drs. Sato and Boyd (33Sato H. Kita M. Seiki M. J. Biol. Chem. 1993; 268: 23460-23468Abstract Full Text PDF PubMed Google Scholar,34Gum R. Lengyel E. Juarez J. Chen J.H. Sato H. Seiki M. Boyd D. J. Biol. Chem. 1996; 271: 10672-10680Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). To facilitate reconstruction, the entire 670-bp promoter was inserted into pBLCAT2 and named as pM9–670-CAT. Briefly, the 670-bp promoter was amplified by PCR with the forward primer 5′-AAG CTT CTA GAG GCT ACT GTC CCC-3′ and the reverse primer 5′-TCT AGA GGT GTC TGA CTG CAG GTG-3′. The PCR product was cloned into pCR2.1-TOPO, an intermediate vector (Invitrogen). Finally, the insert from the TOPO vector was inserted into pBLCAT2. A p65 NF-κB consensus binding site at −601 was characterized previously (35Sato H. Seiki M. Oncogene. 1993; 8: 395-405PubMed Google Scholar). Scanning the 670-bp promoter for potential transcription factor binding sites, we noticed a consensus p50 NF-κB-binding site located at −328 from the transcription start site (MatInspector version 2.2, GSF-National Research Center for Environment and Health). A p65 NF-κB enhancer deletion construct, pM9–590-CAT, that contains the 590-bp region upstream from the transcription start site was created. This construct was generated by PCR with a forward primer 5′-AAG CTT AGC CTT GCC TAG CAG AGC CCA TTC-3′ and a backward primer 5′-TCT AGA GGT GTC TGA CTG CAG GTG-3′. Another deletion construct, pM9–460-CAT, was generated by deleting the p65 NF-κB and the potential TGF-β-response element (TRE) (−474). This plasmid was constructed by digesting pM9–670-CAT withHindIII and EcoR V, filling-in, and self re-ligation. A mutation construct in the TRE, pM9–670-mTRE-CAT, was generated by PCR-based mutagenesis. Briefly, the TRE mutation was generated by PCR using sequencing grade Taq DNA polymerase with a forward primer 5′-AAG CTT CTA GAG GCT ACT GTC CCC-3′ and reverse primer 5′-GTC AGA TAT CCT CCC CTG ATC ACT CCC CAC ACT-3′. In this TRE mutant the wild type sequence 5′-AGGTTTGGGGA-3′ was substituted by 5′-TGATCAGGGGA-3′ (the mutant bases are underlined). The PCR product was ligated to pCR2.1-TOPO vector. Then the 200-bp HindIII/EcoRV fragment from the wild type pM9–670-CAT was replaced by the mutant version. Finally, we created a site-directed mutant at the potential p50 NF-κB-binding region (−328/−319) and named it pM9–670-mp50-CAT. This was accomplished by the QuickChange Site-directed Mutagenesis Kit (Stratagene) with the following primer: 5′-TCA GAC CAA GGG ATGAAG GAT AAC TCC AGC TTC ATC CCC CTC CC-3′ (the mismatched four nucleotides are underlined). The insertion fragments of the wild type, the mutant pM9–670mTRE-CAT, and mutant pM9–670-mp50-CAT were confirmed by DNA sequencing (Norris Cancer Center, University of Southern California). The early passages of human dermal fibroblasts were seeded in 6-cm dishes. Transfection was conducted with 0.5-μg plasmid and LipofectAMINE-PLUS according to the manufacturer's instructions (Life Technologies, Inc.). After incubation for 3 h the plasmid complex was removed and replaced with 0.5% fetal bovine serum/DMEM, and then cytokines were added (TNF-α at 10 ng/ml and TGF-β 1 ng/ml). For the time course experiment the cells were harvested at 24, 48, and 60 h post-transfection. For promoter analysis experiments most of the transfection times were 62 h. The cells were washed by phosphate-buffered saline and harvested in 400-μl 0.25 mTris at pH 8.0 buffer. The cells were lysed via three rounds of quick freeze and thaw followed by heating for 10 min at 60 °C. The extracts were briefly centrifuged, and the supernatant was harvested for the CAT assay. The reaction was performed in a 125-μl system with 50 μl of lysate, 1 μl of [14-C]chloramphenicol (PerkinElmer Life Sciences, 1.9 MBq/ml), 5 μl of 10 mmbutyl-CoA (Roche Molecular Biochemicals), and 69 μl of the 0.25m Tris, pH 8.0. After incubating at 37 °C for 14 h, the lipid phase was extracted by chloroform. The products were resolved by silica gel TLC (Whatman) with 3% methanol and 97% chloroform. The acetylated products were detected by PhosphorImaging (Molecular Dynamics). To establish the profile of cytokine-exerted MMP-9 induction and activation, we first utilized organ culture of human skin. Normal human skin, discarded after reconstructive surgery, was cultured in serum-free DMEM and exposed to TNF-α and TGF-β either individually or combined (10 ng/ml for TNF-α and 1 ng/ml for TGF-β). The conditioned media were sampled at the indicated time points. The resultant conditioned media were assayed for type IV collagenase activity by gelatinolytic zymography. In the absence of exogenous cytokine, minimal 92-kDa gelatinolytic activity was present in the conditioned medium after culturing for 72 h (Fig.1 A). Treatment of the skin with TNF-α induced a small increase of the 92-kDa gelatinolytic activity. Remarkably, in the presence of TNF-α, the 92-kDa gelatinolytic activity progressively disappeared with a concomitant increase in the 82-kDa gelatinolytic activity. The 92- and 82-kDa gelatinolytic activities were confirmed as MMP-9 by Western blot (Fig.1 B). In contrast, exposure of the skin to TGF-β markedly induced the 92-kDa pro-MMP-9. Unlike the response to TNF-α, TGF-β failed to promote MMP-9 proteolytic activation to the 82-kDa form. When both cytokines were applied, a synergistic induction of pro-MMP-9 was detectable at 48 h. Furthermore, simultaneous exposure to the two cytokines led to substantial conversion of the pro-MMP-9 to the active form. After cultivation with the two cytokines for 96 h, most of the pro-MMP-9 was converted to the active 82-kDa active form. Clinical investigations have outlined a temporal pattern for pro-MMP-9 expression and activation in normal healing cutaneous wound sites. Persistent elevation of MMP-9 was found in association with chronic wounds (36Young P.K. Grinnell F. J. Invest. Dermatol. 1994; 103: 660-664Abstract Full Text PDF PubMed Scopus (85) Google Scholar, 37Tarlton J.F. Vickery C.J. Leaper D.J. Bailey A.J. Br. J. Dermatol. 1997; 137: 506-516Crossref PubMed Scopus (77) Google Scholar, 38Wysocki A.B. Kusakabe A.O. Chang S. Tuan T.L. Wound Repair Regen. 1999; 7: 154-165Crossref PubMed Scopus (97) Google Scholar). However, causal factors to induce and activate the proteinase in human tissue at wound sites are still obscure. Our findings reported here show a specific role for the inflammatory cytokines in the induction and activation of MMP-9 in human tissue. The next question was dissecting the nature of the TNF-α-induced activation of pro-MMP-9 in the organ-cultured human skin and whether activation is performed by a skin-associated factor or a secreted soluble factor. The skin explants were stimulated with or without TNF-α for 70 h, and the conditioned media were collected. The treated explants were washed and re-plated in fresh DMEM without additional cytokine. The previous conditioned medium and the re-plated culture were incubated at 37 °C and sampled at 0, 8, and 20 h (Fig. 2). As shown by zymography, the 82-kDa MMP-9 was generated by direct contact with the skin tissue but not by incubation with the conditioned medium. Next, we attempted to extract the pro-MMP-9 activator from the TNF-α-treated skin. The TNF-α-treated explant was extracted by nonionic detergent, Triton X-100. Purified pro-MMP-9 was added to the Triton-soluble and -insoluble fractions and incubated for 20 h (Fig.3). The results show that pro-MMP-9 is processed to the 82-kDa form in the Triton-insoluble fraction, which suggests that the unidentified pro-MMP-9 activator is tightly associated with the tissue structure.Figure 3Proteolytic activation of pro-MMP-9 in the cell-free skin extract. Adult human skin was stimulated with TNF-α (10 ng/ml) in DMEM for 70 h. The tissue was washed and minced following extraction using 2% Triton X-100 (see “Experimental Procedures”). The detergent-soluble and -insoluble fractions were separated by centrifugation. Purified pro-MMP-9 was added or not to these fractions and incubated at 37 °C for 20 h. MMP-9 activities were measured by gelatinolytic zymogram.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To understand the cellular mechanism of the cytokine-mediated induction and activation of pro-MMP-9 from intact skin, we studied the MMP profile in isolated dermal fibroblasts and epidermal keratinocytes. The dermal environment was simulated by embedding the fibroblasts in three-dimensional type I collagen lattices. Individual cytokines including TNF-α, TGF-β1, PDGF-AB, EGF, IL-8, and IL-6 were applied at 10 ng/ml in the serum-free culture medium. In another panel, a combination of TGF-β with other cytokines was applied to the cells. After culturing for 68 h, the conditioned media were analyzed for gelatinolytic activity (Fig.4 A). The results show that TNF-α alone could moderately induce the 92-kDa gelatinolytic activity, and the other cytokines in the list did not induce the 92-kDa gelatinolytic activity. Remarkably, the combin" @default.
• W2036733178 created "2016-06-24" @default.
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• W2036733178 creator A5041102310 @default.
• W2036733178 creator A5052901734 @default.
• W2036733178 creator A5054314117 @default.
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• W2036733178 date "2001-06-01" @default.
• W2036733178 modified "2023-09-30" @default.
• W2036733178 title "Transforming Growth Factor-β- and Tumor Necrosis Factor-α-mediated Induction and Proteolytic Activation of MMP-9 in Human Skin" @default.
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