FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1986010238> ?p ?o ?g. }

• W1986010238 endingPage "5145" @default.
• W1986010238 startingPage "5137" @default.
• W1986010238 abstract "Inflammatory cytokines tumor necrosis factor-α and interleukin-1 trigger the ceramide signaling pathway, initiated by neutral sphingomyelinase-elicited hydrolysis of cell membrane phospholipid sphingomyelin to ceramide, a new lipid second messenger. Here, we show that triggering the ceramide pathway by sphingomyelinase or C2- and C6-ceramide enhances collagenase-1 (matrix metalloproteinase-1; MMP-1) gene expression by fibroblasts. C2-ceramide activates three distinct mitogen-activated protein kinases (MAPKs) in dermal fibroblasts, i.e.extracellular signal-regulated kinase 1/2 (ERK1/2), stress-activated protein kinase/Jun N-terminal-kinase (SAPK/JNK), and p38. Stimulation of MMP-1 promoter activity by C2-ceramide is dependent on the presence of a functional AP-1 cis-element and is entirely inhibited by overexpression of MAPK inhibitor, dual specificity phosphatase CL100 (MAPK phosphatase-1). Activation of MMP-1 promoter by C2-ceramide is also effectively inhibited by kinase-deficient forms of ERK1/2 kinase (MEK1/2) activator Raf-1, ERK1 and ERK2, SAPK/JNK activator SEK1, or SAPKβ. In addition, ceramide-dependent induction of MMP-1 expression is potently prevented by PD 98059, a selective inhibitor of MEK1 activation, and by specific p38 inhibitor SB 203580. These results show that triggering the ceramide signaling pathway activates MMP-1 gene expression via three distinct MAPK pathways, i.e. ERK1/2, SAPK/JNK, and p38, and suggest that targeted modulation of the ceramide signaling pathway may offer a novel therapeutic approach for inhibiting collagenolytic activity, e.g. in inflammatory disorders. Inflammatory cytokines tumor necrosis factor-α and interleukin-1 trigger the ceramide signaling pathway, initiated by neutral sphingomyelinase-elicited hydrolysis of cell membrane phospholipid sphingomyelin to ceramide, a new lipid second messenger. Here, we show that triggering the ceramide pathway by sphingomyelinase or C2- and C6-ceramide enhances collagenase-1 (matrix metalloproteinase-1; MMP-1) gene expression by fibroblasts. C2-ceramide activates three distinct mitogen-activated protein kinases (MAPKs) in dermal fibroblasts, i.e.extracellular signal-regulated kinase 1/2 (ERK1/2), stress-activated protein kinase/Jun N-terminal-kinase (SAPK/JNK), and p38. Stimulation of MMP-1 promoter activity by C2-ceramide is dependent on the presence of a functional AP-1 cis-element and is entirely inhibited by overexpression of MAPK inhibitor, dual specificity phosphatase CL100 (MAPK phosphatase-1). Activation of MMP-1 promoter by C2-ceramide is also effectively inhibited by kinase-deficient forms of ERK1/2 kinase (MEK1/2) activator Raf-1, ERK1 and ERK2, SAPK/JNK activator SEK1, or SAPKβ. In addition, ceramide-dependent induction of MMP-1 expression is potently prevented by PD 98059, a selective inhibitor of MEK1 activation, and by specific p38 inhibitor SB 203580. These results show that triggering the ceramide signaling pathway activates MMP-1 gene expression via three distinct MAPK pathways, i.e. ERK1/2, SAPK/JNK, and p38, and suggest that targeted modulation of the ceramide signaling pathway may offer a novel therapeutic approach for inhibiting collagenolytic activity, e.g. in inflammatory disorders. Matrix metalloproteinases (MMPs) 1The abbreviations used are: MMP, matrix metalloproteinase; TNF, tumor necrosis factor; IL, interleukin; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; SAPK/JNK, stress-activated protein kinase/Jun N-terminal kinase; MEK, MAPK/ERK kinase; SEK1, SAPK/ERK kinase-1; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; CS, calf serum; kb, kilobase pair; TIMP, tissue inhibitor of metalloproteinases; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CAT, chloramphenicol acetyltransferase; RSV, Rous sarcoma virus; MOPS, 4-morpholinepropanesulfonic acid; mU, milliunit(s). are a family of zinc-dependent metalloendopeptidases collectively capable of degrading essentially all extracellular matrix components (1Birkedal-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: 197-250Google Scholar, 2Kähäri V.-M. Saarialho-Kere U. Exp. Dermatol. 1997; 6: 199-213Google Scholar). MMPs play an important role in tissue remodeling during fetal development, angiogenesis, and tissue repair, and they are also responsible for excessive breakdown of connective tissue in inflammatory disorders, e.g. rheumatoid arthritis, osteoarthritis, autoimmune blistering disorders of skin, dermal photoaging, and periodontitis (1Birkedal-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: 197-250Google Scholar, 2Kähäri V.-M. Saarialho-Kere U. Exp. Dermatol. 1997; 6: 199-213Google Scholar). In addition, degradation of basement membrane and extracellular matrix by MMPs is crucial for invasion and metastasis of tumor cells. To date, the MMP gene family consists of 16 members, which according to structure and substrate specificity can be divided into subgroups of collagenases, gelatinases, stromelysins, and membrane-type MMPs (2Kähäri V.-M. Saarialho-Kere U. Exp. Dermatol. 1997; 6: 199-213Google Scholar). Collagenase-1 (MMP-1) is the principal fibroblast-derived secreted neutral proteinase capable of degrading native fibrillar collagens of types I, II, III, and V, and it apparently plays an important role in the remodeling of collagenous connective tissues in various physiological and pathological situations. The expression of MMP-1 by fibroblasts is potently up-regulated by cytokines, growth factors, and tumor promoters (see Refs. 1Birkedal-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: 197-250Google Scholar, 2Kähäri V.-M. Saarialho-Kere U. Exp. Dermatol. 1997; 6: 199-213Google Scholar, 3Mauviel A. J. Cell. Biochem. 1993; 53: 288-295Google Scholar). Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine, which potently inhibits accumulation of connective tissue components. TNF-α stimulates degradation of extracellular matrix by inducing the expression of MMP-1 and stromelysin-1 (MMP-3) by fibroblasts (4Brenner D.A. O'Hara M. Angel P. Chojkier M. Karin M. Nature. 1989; 337: 661-663Google Scholar, 5MacNaul K.L. Chartrain N. Lark M. Tocci M.J. Hutchinson N.I. J. Biol. Chem. 1990; 265: 17238-17245Google Scholar, 6Westermarck J. Häkkinen L. Fiers W. Kähäri V.-M. J. Invest. Dermatol. 1995; 105: 197-202Google Scholar). In addition, TNF-α inhibits type I collagen gene expression by fibroblasts in culture (6Westermarck J. Häkkinen L. Fiers W. Kähäri V.-M. J. Invest. Dermatol. 1995; 105: 197-202Google Scholar, 7Mauviel A. Daireaux M. Rédini F. Galera P. Loyau G. Pujol J.-P. FEBS Lett. 1988; 236: 47-52Google Scholar, 8Solis-Herruzo J.A. Brenner D.A. Chojkier M. J. Biol. Chem. 1988; 263: 5841-5845Google Scholar, 9Kähäri V.-M. Chen Y.Q. Su M.W. Ramirez F. Uitto J. J. Clin. Invest. 1990; 86: 1489-1495Google Scholar) and in vivo (10Rapala K.T. Vähä-Kreula M.O. Heino J.J. Vuorio E.I. Laato M.K. Experientia. 1996; 52: 70-74Google Scholar), down-regulates elastin (11Kähäri V.-M. Chen Y.Q. Bashir M.M. Rosenbloom J. Uitto J. J. Biol. Chem. 1992; 267: 26134-26141Google Scholar) and decorin (12Mauviel A. Santra M. Chen Y.Q. Uitto J. Iozzo R.V. J. Biol. Chem. 1995; 270: 11692-11700Google Scholar) gene expression at the transcriptional level, and is able to abrogate the activation of type I collagen and elastin gene expression by transforming growth factor-β (9Kähäri V.-M. Chen Y.Q. Su M.W. Ramirez F. Uitto J. J. Clin. Invest. 1990; 86: 1489-1495Google Scholar, 11Kähäri V.-M. Chen Y.Q. Bashir M.M. Rosenbloom J. Uitto J. J. Biol. Chem. 1992; 267: 26134-26141Google Scholar). The effects of TNF-α on extracellular matrix formation partially overlap with those of interleukin-1 (IL-1), which also induces expression of MMP-1 and MMP-3 in fibroblasts (see Refs. 1Birkedal-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: 197-250Google Scholar, 2Kähäri V.-M. Saarialho-Kere U. Exp. Dermatol. 1997; 6: 199-213Google Scholar, 3Mauviel A. J. Cell. Biochem. 1993; 53: 288-295Google Scholarand 5MacNaul K.L. Chartrain N. Lark M. Tocci M.J. Hutchinson N.I. J. Biol. Chem. 1990; 265: 17238-17245Google Scholar). The cellular effects of TNF-α are mediated by two distinct cell surface receptors: TNF-RI (TNF-R55) and TNF-RII (TNF-R75), both of which are expressed by fibroblastic cells (see Ref. 13Vandenabeele P. Declerq W. Beyaert R. Fiers W. Trends Cell Biol. 1995; 5: 392-399Google Scholar). We have recently shown that the effects of TNF-α on the expression of MMP-1, MMP-3, and type I collagen in dermal fibroblasts are primarily mediated by TNF-R55 (6Westermarck J. Häkkinen L. Fiers W. Kähäri V.-M. J. Invest. Dermatol. 1995; 105: 197-202Google Scholar). It has been shown that binding of TNF-α to TNF-R55 activates neutral sphingomyelinase, a cell membrane-associated phospholipase, which hydrolyzes cell membrane structural phospholipid sphingomyelin to phosphocholine and ceramide, a novel lipid second messenger (see Refs. 14Spiegel S. Foster D. Kolesnick R. Curr. Opin. Cell Biol. 1996; 8: 159-167Google Scholar and 15Obeid L. Hannun Y.A. J. Cell. Biochem. 1995; 58: 191-198Google Scholar). The role of the ceramide pathway in TNF-α-induced apoptosis in various cells has been recently elucidated (15Obeid L. Hannun Y.A. J. Cell. Biochem. 1995; 58: 191-198Google Scholar). In addition, it has been shown that ceramides activate the expression of cyclooxygenase, stimulate synthesis of prostaglandin E2 (16Ballou L.R. Chao C.P. Holness M.A. Barker S.C. Raghow R. J. Biol. Chem. 1992; 267: 20044-20050Google Scholar), and enhance production of IL-6 by cultured fibroblasts (17Laulederkind S.J. Bielawska A. Raghow R. Hannun Y.A. Ballou L.H. J. Exp. Med. 1995; 182: 599-604Google Scholar), indicating a role for this signaling pathway in mediating the inflammatory effects of TNF-α on fibroblasts. However, the role of the ceramide pathway as a mediator of the effects of TNF-α and IL-1 on the synthesis and degradation of extracellular matrix is not known. In this study, we show for the first time that triggering the ceramide pathway with neutral sphingomyelinase or cell-permeable ceramides in human skin fibroblasts results in marked stimulation of MMP-1 expression and that this effect is dependent on the presence of a functional AP-1 cis-element in the MMP-1 promoter region as well as on the activity of extracellular signal-regulated kinase 1/2 (ERK1/2), stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK), and p38 mitogen-activated protein kinases (MAPKs). These results show that the effects of TNF-α and IL-1 on MMP-1 gene expression can be mimicked by activating the ceramide pathway in dermal fibroblasts, suggesting that targeted modulation of this pathway may offer a novel approach for therapeutic inhibition of matrix degradation, e.g. in inflammatory disorders. Neutral sphingomyelinase (fromStaphylococcus aureus) and cycloheximide were obtained from Sigma. C2- and C6-ceramide, C2-dihydroceramide, and PD 98059 were obtained from Calbiochem. SB 203580 was provided by SmithKline Beecham (King of Prussia, PA). Human recombinant interleukin-1β was obtained from Boehringer Mannheim (Mannheim, Germany). Human TNF-R55-specific TNF-α (double mutant R32W/S86T) (18Barbara J.A.J. Smith W.B. Gamble J.R. Van Ostade X. Vandenabeele P. Tavernier J. Fiers W. Vadas M.A. Lopez A.F. EMBO J. 1994; 13: 843-850Google Scholar) were kindly provided by Dr. Walter Fiers (University of Gent, Belgium). Normal human skin fibroblast cultures were established from punch biopsy obtained from a voluntary healthy male donor (age 23) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mmglutamine, 100 IU/ml penicillin G, and 100 μg/ml streptomycin. Murine NIH-3T3 fibroblasts were obtained from ATCC (Rockville, MD) and cultured in similar medium supplemented with 10% calf serum (CS). For experiments, fibroblast cultures were maintained in culture medium supplemented with 0.5% FCS for 18 h. Thereafter, sphingomyelinase, ceramides, TNF-R55-specific human TNF-α, or IL-1β was added in concentrations and combinations indicated, and incubations were continued for 24 h. In experiments involving MAPK inhibitors or cycloheximide, these were added to the cultures 1 h prior to the addition of ceramides. To estimate the viability of cells after a 24-h incubation with ceramides and sphingomyelinase, cells were washed with PBS and stained with 0.4% trypan blue in phosphate-buffered saline (PBS) for 10 min. Cells were then washed with PBS and fixed with 10% formaldehyde, and the number of stained cells was counted. Total cellular RNA was isolated from cell cultures using the single step method (19Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Google Scholar). Aliquots of total RNA were fractionated on 0.8% agarose gels containing 2.2 mformaldehyde, transferred to Zeta Probe filters (Bio-Rad) 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 cDNAs labeled with [α-32P]dCTP using random priming. The filters were then washed, the final stringency being 0.1 × SSC, 0.1% SDS at 60 °C (20Westermarck J. Lohi J. Keski-Oja J. Kähäri V.-M. Cell Growth Differ. 1994; 5: 1205-1213Google Scholar). The following cDNAs were used for hybridizations: a 2.0-kb human cDNA for collagenase-1 (MMP-1) (21Goldberg G.I. Wilhelm S.M. Kronberger A. Bauer E.A. Grant G.A. Eisen A.Z. J. Biol. Chem. 1986; 261: 6600-6605Google Scholar); a 1.5-kb human cDNA for stromelysin-1 (MMP-3) (22Saus J. Quinones S. Otani Y. Nagase H. Harris Jr., E.D. Kurkinen M. J. Biol. Chem. 1988; 263: 6742-6745Google Scholar); a 0.7-kb human cDNA for TIMP-1 (23Carmichael D.F. Sommer A. Thompson R.G. Anderson D.C. Smith C.G. Welgus H.G. Stricklin G.P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2407-2411Google Scholar); a 1.3-kb rat cDNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (24Fort P. Marty L. Piechaczyk M. El Sabrouty S. Dani C. Jeanteur P. Blanchard J.M. Nucleic Acids Res. 1985; 13: 1431-1442Google Scholar); a human 0.4-kb cDNA for c-jun (25Angel P. Allegretto E.A. Okino S.T. Hattori K. Boyle W.J. Hunter T. Karin M. Nature. 1988; 332: 166-171Google Scholar); a human 1.2-kb cDNA for junB(26Schütte J. Viallet J. Nau M. Segal S. Fedorko J. Minna J. Cell. 1989; 89: 987-997Google Scholar); and a human 3.1-kb genomic fragment for c-fos(obtained from Amersham Corp.). The [32P]cDNA-mRNA hybrids were visualized by autoradiography, and the mRNA levels were quantitated by scanning densitometry of the autoradiographs using MCID software (Imaging Research Inc., St. Catharines, Ontario, Canada). MMP-1 and MMP-3 mRNA levels were corrected for the levels of GAPDH mRNA in the same samples. The cells were maintained in serum-free DMEM for 18 h, after which sphingomyelinase (100 mU/ml) or C2-ceramide (10 μm) was added either alone or in combination with TNF-R55-specific TNF-α (20 ng/ml) or IL-1β (5 units/ml), and the incubations were continued for 24 h. Equal aliquots of the conditioned media, relative to cell number (27Kueng W. Silber E. Eppenberger U. Anal. Biochem. 1989; 182: 16-19Google Scholar) were analyzed for the amount of MMP-1 by Western blotting, as described previously (6Westermarck J. Häkkinen L. Fiers W. Kähäri V.-M. J. Invest. Dermatol. 1995; 105: 197-202Google Scholar) using a polyclonal rabbit antiserum against human MMP-1 (kindly provided by Dr. Henning Birkedal-Hansen, NIDR, National Institutes of Health, Bethesda, MD), in a 1:2000 dilution, and the enhanced chemiluminescence detection system (Amersham). The levels of immunoreactive MMP-1 were quantitated by densitometric scanning of the x-ray films. Confluent NIH-3T3 fibroblast cultures were transiently transfected either with 4 μg of the construct p2278CLCAT, which contains 2.278 kb of the human MMP-1 promoter linked to the CAT reporter gene, or with a similar construct with a mutated AP-1 element (28Pierce R.A. Sandefur S. Doyle G.A.R. Welgus H.G. J. Clin. Invest. 1996; 97: 1890-1899Google Scholar) (both kindly provided by Dr. William C. Parks (Washington University, St. Louis, MO)). In co-transfection experiments, the cells were transiently transfected with the MMP-1 promoter/CAT construct pCLCAT3 (2 μg), which contains 3.8 kb of the 5′-flanking region of human MMP-1 gene linked to the CAT gene (29Frisch S.M. Reich R. Collier I.E. Genrich L.T. Martin G. Goldberg G.I. Oncogene. 1990; 5: 75-83Google Scholar) (kindly provided by Dr. Steven Frisch (La Jolla Cancer Research Foundation, La Jolla, CA)), together with 10 μg of the following expression plasmids: RSV/AS-c-jun (30Mauviel A. Chung K.-Y. Agarwal A. Tamai K. Uitto J. J. Biol. Chem. 1996; 271: 10917-10923Google Scholar), a c-junantisense expression construct (kindly provided by Dr. Alain Mauviel (Thomas Jefferson University, Philadelphia, PA)); SG5-CL100 (31Alessi D.R. Smythe C. Keyse S.M. Oncogene. 1993; 8: 2015-2020Google Scholar) for MAPK inhibitor; dual specificity phosphatase CL100 (MAPK phosphatase-1) (32Keyse S.M. Emslie E.A. Nature. 1992; 359: 644-647Google Scholar) (kindly provided by Dr. Steven Keyse (Ninewells Hospital, Dundee, Scotland)); RSV-Raf-C4 (33Bruder T. Heidecker G. Rapp U.R. Genes Dev. 1993; 6: 545-556Google Scholar) for kinase-deficient Raf-1 and SAPKβKK→RR (34Ludwig S. Engel K. Hoffmeyer A. Sithanandam G. Neufeld B. Palm D. Gaestel M. Rapp U.R. Mol. Cell. Biol. 1996; 16: 6687-6697Google Scholar) for kinase-deficient SAPKβ (both kindly provided by Dr. Ulf Rapp (University of Würzburg, Germany)); pEBG-SEK1-K→R (35Sánchez I. Hughes R.T. Mayer B.J. Yee K. Woodgett J.R. Avruch J. Kyriakis J.M. Zion L.I. Nature. 1994; 372: 794-798Google Scholar), specific for kinase-deficient SEK1 (kindly provided by Dr. John Kyriakis (Harvard University, Boston, MA)); and CEp4LK71RERK1 or CEp4LK52RERK2 (36Frost J.A. Geppert T.D. Cobb M.H. Feramisco J.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3844-3848Google Scholar), specific for kinase-deficient ERK1 and ERK2, respectively (kindly provided by Dr. Melanie Cobb, Southwestern Medical Center, Dallas, TX). Control cultures were co-transfected in parallel with the respective empty expression vectors. Transfections were performed with the calcium phosphate/DNA co-precipitation method, followed by a 2-min glycerol shock, as described previously (20Westermarck J. Lohi J. Keski-Oja J. Kähäri V.-M. Cell Growth Differ. 1994; 5: 1205-1213Google Scholar). The cultures were then maintained in DMEM and 1% CS for 16 h, C2-ceramide was added, and the incubations were continued for 24 h. Cells were harvested and lysed by three cycles of freezing and thawing. As an index of promoter activity, CAT activity was measured from aliquots of cell extracts in reactions containing 0.25 m Tris-HCl buffer (pH 8.0), 5 μg of n-butyryl-coenzyme A (Sigma), and 0.0625 μCi of [14C]chloramphenicol (Amersham), in a total volume of 125 μl. The butyrylated chloramphenicol products were extracted with 300 μl of xylene after overnight incubation at 37 °C. The CAT activity was quantitated by scintillation counting after two back-extractions of the xylene phase with 100 μl of 0.25 m Tris-HCl. The transfection efficiency was monitored by co-transfecting cells with 4 μg of RSV/β-galactosidase construct and correcting the CAT activities for β-galactosidase activity (20Westermarck J. Lohi J. Keski-Oja J. Kähäri V.-M. Cell Growth Differ. 1994; 5: 1205-1213Google Scholar). For assay of ERK1/2 and SAPK/JNK activity, confluent cultures of human skin fibroblasts were incubated for 18 h in DMEM containing 0.5% FCS. Thereafter, ceramide was added, and the incubations were continued for different periods of time. Cells (2 × 106/sample) were lysed in 400 μl of lysis buffer (PBS, pH 7.4; 1% Nonidet P-40; 0.5% sodium deoxycholate; 1 mm Na3VO4; 0.1% SDS; 1 mm EDTA; 1 mm EGTA; 20 mmNaF; 1 mm PMSF; and 1 μg/ml aprotinin, leupeptin, and pepstatin). For immunoprecipitation of ERK1/2, cell lysates were centrifuged (3000 × g for 15 min), and the supernatant was incubated with an antibody generated against ERK2 (p42 MAPK; Transduction Laboratories, Lexington, KY), coupled to protein A-Sepharose (Sigma). This antibody also cross-reacts with ERK1. Immunoprecipitates were washed three times in lysis buffer and three times in kinase assay buffer (10 mm Tris, pH 7.4, 150 mm NaCl, 10 mm MgCl2, 0.5 mm dithiothreitol). The kinase reaction was carried out by adding to the immunoprecipitate 20 μl of kinase assay buffer, including 25 μm ATP, 2.5 μCi of [γ-32P]ATP (Amersham), and 1 μg/μl myelin basic protein (Sigma) as substrate. The reaction was carried out for 15 min at 37 °C and stopped by adding 3 × Laemmli sample buffer. The samples were resolved on 12.5% SDS-polyacrylamide gel electrophoresis, and myelin basic protein phosphorylation was quantified with a phosphor imager (Bio-Rad). To measure SAPK/JNK activity, the cells were lysed in 400 μl of lysis buffer as described above. The cell lysates were centrifuged (3000 × g for 15 min), and SAPK/JNK was immunoprecipitated by incubating the supernatant with an antibody generated against SAPK/JNK (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) coupled to protein A-Sepharose. Immunoprecipitates were washed three times in lysis buffer, three times in LiCl wash buffer (500 mm LiCl, 100 mm Tris, pH 7.6, 0.1% Triton X-100, 1 mmdithiothreitol), and three times in kinase assay buffer (20 mm MOPS, pH 7.2, 2 mm EGTA, 10 mmMgCl2, 0.1% Triton X-100, 1 mmdithiothreitol). The kinase reaction was carried out by adding to the immunoprecipitate 20 μl of kinase assay buffer, including 25 μm ATP, 2.5 μCi of [γ-32P]ATP (Amersham), and 6 μg of bacterially expressed glutathioneS-transferase-c-Jun (a kind gift from Eleanor Coffey, Åbo Akademi, Turku, Finland) as substrate. The reaction was carried out for 20 min at 37 °C and stopped by the addition of 3 × Laemmli sample buffer. The samples were resolved on 12.5% SDS-polyacrylamide gel electrophoresis, and c-Jun phosphorylation was quantified as above. To determine the activation of p38 MAPK, confluent cultures of human skin fibroblasts were treated with C2-ceramide for different periods of time and lysed in 100 μl of Laemmli sample buffer. The samples were then sonicated, fractionated on a 10% SDS-polyacrylamide gel, and transferred to nitrocellulose membrane (Amersham). The levels of activated, phosphorylated p38 in the samples were determined by Western blotting performed as described above using a phosphospecific p38 antibody (New England Biolabs, Beverly, MA) in a 1:400 dilution. The levels of activated p38 were quantitated by densitometric scanning of the x-ray films. To elucidate the role of ceramide pathway in the regulation of fibroblast collagenase (collagenase-1, MMP-1) gene expression, we first activated this signaling pathway by treatment of human skin fibroblasts with S. aureus neutral sphingomyelinase for 24 h and assayed MMP-1 mRNA levels with Northern blot hybridizations. As shown in Fig. 1 A, sphingomyelinase treatment of cells with 1 mU/ml resulted in a marked enhancement (7-fold) in MMP-1 mRNA expression, and an even more potent increase (14-fold) was noted with a concentration of 100 mU/ml after correction of the MMP-1 mRNA abundance for the level of GAPDH mRNA in the same samples (Fig. 1 A). Next, we triggered the ceramide pathway in dermal fibroblasts by treatment with cell-permeable ceramide analogs C6- and C2-ceramide. Exposure of cells to C6-ceramide resulted in a dose-dependent enhancement of MMP-1 mRNA abundance (6- and 19-fold) with concentrations of 10 and 100 μm, respectively (Fig. 1 B). The mRNA abundance for TIMP-1 was slightly (25%) reduced with the highest C6-ceramide concentration when corrected for the levels of GAPDH mRNA (Fig. 1 B). In a parallel experiment, fibroblasts were treated with different concentrations of C2-ceramide, which stimulated the expression of MMP-1 mRNA even more potently (22- and 385-fold) with the two highest concentrations, 10 and 100 μm, respectively (Fig. 1 C). In addition, the level of stromelysin-1 (MMP-3) mRNA was markedly enhanced with C2-ceramide (100 μm). Treatment of cells with an inactive ceramide analog, C2-dihydroceramide (10 μm) had no effect on MMP-1, MMP-3, or TIMP-1 mRNAs (not shown). The viability of fibroblasts was only affected by 100 μmC2-ceramide, whereas lower concentrations of C2-ceramide as well as all concentrations of C6-ceramide or sphingomyelinase used did not affect the viability of cells as estimated by trypan blue exclusion (not shown). To examine the effect of sphingomyelinase and C2-ceramide on the production of MMP-1 by dermal fibroblasts, we assayed the amount of immunoreactive MMP-1 in the conditioned media of cells treated with sphingomyelinase (100 mU/ml) and C2-ceramide (10 μm) using Western blot analysis. As shown in Fig. 2 A, both sphingomyelinase and C2-ceramide stimulated production of MMP-1 by fibroblasts by 5-fold. We have recently shown that the effect of TNF-α on MMP-1 expression in dermal fibroblasts is primarily mediated via TNF-R55 (6Westermarck J. Häkkinen L. Fiers W. Kähäri V.-M. J. Invest. Dermatol. 1995; 105: 197-202Google Scholar). In this context, we examined whether the activation of the ceramide pathway by sphingomyelinase augments the maximal enhancement of MMP-1 production obtained with 20 ng/ml TNF-R55-specific TNF-α (6Westermarck J. Häkkinen L. Fiers W. Kähäri V.-M. J. Invest. Dermatol. 1995; 105: 197-202Google Scholar). In this experiment, TNF-R55-specific TNF-α enhanced production of immunoreactive MMP-1 into the culture media of dermal fibroblasts 45-fold, and simultaneous treatment with sphingomyelinase (100 mU/ml) potentiated this stimulation of MMP-1 production 1.8-fold, the final stimulation being 81-fold, as compared with the untreated control cultures (Fig. 2 B). The cellular signaling of IL-1 also involves activation of neutral sphingomyelinase and subsequent triggering of the ceramide pathway (14Spiegel S. Foster D. Kolesnick R. Curr. Opin. Cell Biol. 1996; 8: 159-167Google Scholar,15Obeid L. Hannun Y.A. J. Cell. Biochem. 1995; 58: 191-198Google Scholar). Therefore, we also examined the effect of sphingomyelinase on IL-1β-elicited induction of MMP-1 production. As expected, IL-1β (5 units/ml) enhanced (18-fold) the production of immunoreactive MMP-1 by dermal fibroblasts (Fig. 2 C), and sphingomyelinase augmented this maximal IL-1β-elicited induction of MMP-1 production by 2.7-fold, the final enhancement being 49-fold over the control cells (Fig. 2 C). Stimulation of MMP-1 gene transcription by various stimuli involves induction of dimeric AP-1 trans-activating factor complex (Jun plus Fos), which binds to the correspondingcis-element in the MMP-1 promoter (see Refs. 1Birkedal-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: 197-250Google Scholar, 2Kähäri V.-M. Saarialho-Kere U. Exp. Dermatol. 1997; 6: 199-213Google Scholar, 3Mauviel A. J. Cell. Biochem. 1993; 53: 288-295Google Scholar). We therefore examined the effect of C2-ceramide on the expression of members of Jun and Fos families in dermal fibroblasts. Treatment of cells with C2-ceramide (100 μm) resulted in rapid stimulation of c-jun mRNA expression, first noted at 1 h of incubation with further increase up to 6 h (Fig. 3 A). The levels of junB mRNA were also enhanced by C2-ceramide, the peak induction noted at 2 and 6 h (Fig. 3 A). In addition, a rapid induction of c-fos mRNA was detected maximally after 1- and 2-h incubations (Fig. 3 A). Interestingly, the levels of c-jun, junB, and c-fos mRNAs were still enhanced after 24 h (Fig. 3 A). In the same experiment, the expression of MMP-1 mRNA was first induced after a 6-h exposure to C2-ceramide (Fig. 3 A). In parallel experiments, C6-ceramide (100 μm) and sphingomyelinase (100 mU/ml) also induced expression of MMP-1 mRNA with similar kinetics (not shown). As shown in Fig. 3 B, enhancement of MMP-1 expression by C2-ceramide was abrogated by co-treatment of cells with cycloheximide (10 μg/ml). Similarly, activation of MMP-1 expression by sphingomyelinase was inhibited by cycloheximide (not shown). These observations show that ceramide-elicited activation of MMP-1 gene expression is dependent on synthesis of new regulatory proteins. To examine whether ceramide treatment of fibroblasts results in transcriptional activation of MMP-1 promoter, we transiently transfected NIH-3T3 fibroblasts with an MMP-1 promoter/CAT construct p2278CLCAT, in which a 2.278-kb human MMP-1 promoter segment is linked to the CAT reporter gene. Treatment of transiently transfected NIH-3T3 cells with C2-ceramide (50 μm) potently (7.7-fold) enhanced the activity of this MMP-1 promoter construct (Fig. 3 C). To elucidate the role of AP-1 in ceramide-elicited activation of the MMP-1 promoter, we transfected parallel cultures with an MMP-1 promoter/CAT construct containing the same promoter segment except with a mutation rendering the AP-1 binding site at −72 to −65 incapable of binding AP-1. As shown in Fig. 3 C, loss of the functional AP-1 cis-element reduced the basal activity of p2278CLCAT by 95%. Interestingly, the C2-ceramide-elicited enhancement of the MMP-1 promoter construct lacking the AP-1 element was clearly lower (2.4-fold), as compared with the wild type MMP-1 promoter, the final promoter activity being 12% of the untreated wild type (Fig. 3 C). To elucidate the role of c-Jun in ceramide-mediated activation of the MMP-1 promoter, we transiently co-" @default.
• W1986010238 created "2016-06-24" @default.
• W1986010238 creator A5008528112 @default.
• W1986010238 creator A5031503332 @default.
• W1986010238 creator A5035567894 @default.
• W1986010238 creator A5041488438 @default.
• W1986010238 creator A5058045826 @default.
• W1986010238 creator A5066095948 @default.
• W1986010238 creator A5090125749 @default.
• W1986010238 date "1998-02-01" @default.
• W1986010238 modified "2023-10-14" @default.
• W1986010238 title "Enhancement of Fibroblast Collagenase (Matrix Metalloproteinase-1)Gene Expression by Ceramide Is Mediated by Extracellular Signal-regulated and Stress-activated Protein Kinase Pathways" @default.
• W1986010238 cites W1491598928 @default.
• W1986010238 cites W1521095043 @default.
• W1986010238 cites W1523015364 @default.
• W1986010238 cites W1534320734 @default.
• W1986010238 cites W1555579413 @default.
• W1986010238 cites W1562575195 @default.
• W1986010238 cites W1571224400 @default.
• W1986010238 cites W1577422435 @default.
• W1986010238 cites W1577946620 @default.
• W1986010238 cites W1894541755 @default.
• W1986010238 cites W1964201687 @default.
• W1986010238 cites W1968590447 @default.
• W1986010238 cites W1969158591 @default.
• W1986010238 cites W1969785844 @default.
• W1986010238 cites W1970161839 @default.
• W1986010238 cites W1971915141 @default.
• W1986010238 cites W1972402566 @default.
• W1986010238 cites W1973645860 @default.
• W1986010238 cites W1982390204 @default.
• W1986010238 cites W1982396402 @default.
• W1986010238 cites W1984113136 @default.
• W1986010238 cites W1984361597 @default.
• W1986010238 cites W1999017992 @default.
• W1986010238 cites W2003533394 @default.
• W1986010238 cites W2007986629 @default.
• W1986010238 cites W2011245371 @default.
• W1986010238 cites W2015379482 @default.
• W1986010238 cites W2016894764 @default.
• W1986010238 cites W2017517432 @default.
• W1986010238 cites W2022589671 @default.
• W1986010238 cites W2022914311 @default.
• W1986010238 cites W2023189063 @default.
• W1986010238 cites W2036985660 @default.
• W1986010238 cites W2038682684 @default.
• W1986010238 cites W2042289154 @default.
• W1986010238 cites W2046280437 @default.
• W1986010238 cites W2046413765 @default.
• W1986010238 cites W2051819610 @default.
• W1986010238 cites W2057094603 @default.
• W1986010238 cites W205984844 @default.
• W1986010238 cites W2064439294 @default.
• W1986010238 cites W2075104267 @default.
• W1986010238 cites W2077156149 @default.
• W1986010238 cites W2078261279 @default.
• W1986010238 cites W2084098251 @default.
• W1986010238 cites W2089372467 @default.
• W1986010238 cites W2093676869 @default.
• W1986010238 cites W2094336459 @default.
• W1986010238 cites W2120033507 @default.
• W1986010238 cites W2171569176 @default.
• W1986010238 cites W2181679132 @default.
• W1986010238 cites W4294216491 @default.
• W1986010238 doi "https://doi.org/10.1074/jbc.273.9.5137" @default.
• W1986010238 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9478967" @default.
• W1986010238 hasPublicationYear "1998" @default.
• W1986010238 type Work @default.
• W1986010238 sameAs 1986010238 @default.
• W1986010238 citedByCount "198" @default.
• W1986010238 countsByYear W19860102382012 @default.
• W1986010238 countsByYear W19860102382013 @default.
• W1986010238 countsByYear W19860102382014 @default.
• W1986010238 countsByYear W19860102382015 @default.
• W1986010238 countsByYear W19860102382016 @default.
• W1986010238 countsByYear W19860102382017 @default.
• W1986010238 countsByYear W19860102382018 @default.
• W1986010238 countsByYear W19860102382019 @default.
• W1986010238 countsByYear W19860102382020 @default.
• W1986010238 countsByYear W19860102382021 @default.
• W1986010238 countsByYear W19860102382022 @default.
• W1986010238 countsByYear W19860102382023 @default.
• W1986010238 crossrefType "journal-article" @default.
• W1986010238 hasAuthorship W1986010238A5008528112 @default.
• W1986010238 hasAuthorship W1986010238A5031503332 @default.
• W1986010238 hasAuthorship W1986010238A5035567894 @default.
• W1986010238 hasAuthorship W1986010238A5041488438 @default.
• W1986010238 hasAuthorship W1986010238A5058045826 @default.
• W1986010238 hasAuthorship W1986010238A5066095948 @default.
• W1986010238 hasAuthorship W1986010238A5090125749 @default.
• W1986010238 hasBestOaLocation W19860102381 @default.
• W1986010238 hasConcept C104317684 @default.
• W1986010238 hasConcept C109523444 @default.
• W1986010238 hasConcept C150194340 @default.
• W1986010238 hasConcept C153911025 @default.
• W1986010238 hasConcept C181199279 @default.
• W1986010238 hasConcept C184235292 @default.
• W1986010238 hasConcept C185592680 @default.

• next