Transcriptional Regulation of Decorin Gene Expression
1995; Elsevier BV; Volume: 270; Issue: 19 Linguagem: Inglês
10.1074/jbc.270.19.11692
ISSN1083-351X
AutoresAlain Mauviel, Manor an jan Santra, Yue Qiu Chen, Jouni Uitto, Renato V. Iozzo,
Tópico(s)Cell Adhesion Molecules Research
ResumoDecorin, a leucine-rich proteoglycan with ubiquitous tissue distribution, may play essential biological roles during inflammation and cancer growth through its ability to bind extracellular matrix constituents and growth factors. In this study, we demonstrate that decorin gene expression is greatly enhanced after normal diploid fibroblasts reach confluency and cease to proliferate. Elevation of decorin mRNA steady state levels was maintained for up to 16 days posteonfluency. In vitro transcription analyses indicated enhanced transcriptional activity in quiescent fibroblasts when compared to cells harvested in their logarithmic phase of growth. This phenotypic trait was reversed by the exogenous addition of tumor necrosis factor-α (TNF-α). Furthermore, transforming growth faetor-β (TGF-β) down-regulated deeorin gene expression in an additive manner with TNF-α. Transient cell transfection assays using plasmid constructs harboring the decorin promoter linked to the chloramphenicol acetyltransferase reporter gene demonstrated a dose-dependent transcriptional repression by TNF-α. These findings were further corroborated by in vitro transcription experiments using nuclear extracts from control and TNF-α-treated quiescent fibroblasts. In contrast, the decorin promoter constructs failed to respond to TGF-β, thus suggesting either post-transcriptional regulation by this growth factor or lack of TGF-β-responsive elements. Further experiments with 5′ deletion constructs showed two TNF-α response elements, one residing within the 5-untranslated region (exon Ib), the other one between residues –188 and –140 of the decorin promoter. Collectively, our results indicate that TNF-α, through its ability to transcriptionally inhibit decorin gene expression in growth-arrested cells, may be a key modulator of the biological functions of this proteoglycan. Decorin, a leucine-rich proteoglycan with ubiquitous tissue distribution, may play essential biological roles during inflammation and cancer growth through its ability to bind extracellular matrix constituents and growth factors. In this study, we demonstrate that decorin gene expression is greatly enhanced after normal diploid fibroblasts reach confluency and cease to proliferate. Elevation of decorin mRNA steady state levels was maintained for up to 16 days posteonfluency. In vitro transcription analyses indicated enhanced transcriptional activity in quiescent fibroblasts when compared to cells harvested in their logarithmic phase of growth. This phenotypic trait was reversed by the exogenous addition of tumor necrosis factor-α (TNF-α). Furthermore, transforming growth faetor-β (TGF-β) down-regulated deeorin gene expression in an additive manner with TNF-α. Transient cell transfection assays using plasmid constructs harboring the decorin promoter linked to the chloramphenicol acetyltransferase reporter gene demonstrated a dose-dependent transcriptional repression by TNF-α. These findings were further corroborated by in vitro transcription experiments using nuclear extracts from control and TNF-α-treated quiescent fibroblasts. In contrast, the decorin promoter constructs failed to respond to TGF-β, thus suggesting either post-transcriptional regulation by this growth factor or lack of TGF-β-responsive elements. Further experiments with 5′ deletion constructs showed two TNF-α response elements, one residing within the 5-untranslated region (exon Ib), the other one between residues –188 and –140 of the decorin promoter. Collectively, our results indicate that TNF-α, through its ability to transcriptionally inhibit decorin gene expression in growth-arrested cells, may be a key modulator of the biological functions of this proteoglycan. Decorin, also known as DS-PGII or PG-40, is a leucine-rich proteoglycan with ubiquitous tissue distribution and may play important biological roles through its ability to bind other extracellular matrix proteins and certain growth factors (Gallagher, 1989Gallagher J.T. Curr. Opin. Cell Biol. 1989; 1: 1201-1218Crossref PubMed Scopus (211) Google Scholar; Kresse et al., 1993Kresse H. Hausser H. Schönherr E. Experientia. 1993; 49: 403-416Crossref PubMed Scopus (124) Google Scholar; Iozzo and Cohen, 1993Iozzo R.V. Cohen I. Experientia. 1993; 49: 447-455Crossref PubMed Scopus (104) Google Scholar). Specifically, decorin is involved in the regulation of fundamental biological functions, such as matrix assembly, cell attachment, migration, and proliferation. These functions are thought to be mediated by its ability to bind collagen types I, II (Vogel et al., 1984Vogel K.G. Paulsson M. Heinegârd D. Biochem. J. 1984; 223: 587-597Crossref PubMed Scopus (701) Google Scholar, and VI (Bidanset et al., 1992Bidanset D.J. LeBaron R. Rosenberg L. Murphy-Ullrich J.E. Hook M. J. Cell Biol. 1992; 118: 1523-1531Crossref PubMed Scopus (90) Google Scholar, fibronectin (Winnemöller et al., 1991Winnemöller M. Schmidt G. Kresse H. Eur. J. Cell Biol. 1991; 54: 10-17PubMed Google Scholar; Schmidt et al., 1991Schmidt G. Hausser H. Kresse H. Biochem. J. 1991; 280: 411-414Crossref PubMed Scopus (86) Google Scholar, and throm-bospondin (Winnemöller et al., 1992Winnemöller M. Schön P. Vischer P. Kresse H. Eur. J. Cell Biol. 1992; 59: 47-55PubMed Google Scholar. These binding properties of decorin, which are mediated by the protein core of the molecule, imply an important role for the protein component of decorin in the structural organization and assembly of the extracellular matrix (Gallagher, 1989Gallagher J.T. Curr. Opin. Cell Biol. 1989; 1: 1201-1218Crossref PubMed Scopus (211) Google Scholar; Kresse et al., 1993Kresse H. Hausser H. Schönherr E. Experientia. 1993; 49: 403-416Crossref PubMed Scopus (124) Google Scholar. Furthermore, decorin may be involved in the control of cell proliferation and matrix assembly through its ability to bind and neutralize transforming growth faetor-β (TGF-β) 1The abbreviations used are: TGF-β, transforming growth factor-β; TK, thymidine kinase; TNF-α, tumor necrosis factor-α; TNFRE, TNF response element; CAT, chloramphenicol acetyltransferase; IL, interleukin; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; kb, kilobase(s); bp, base pair(s).1The abbreviations used are: TGF-β, transforming growth factor-β; TK, thymidine kinase; TNF-α, tumor necrosis factor-α; TNFRE, TNF response element; CAT, chloramphenicol acetyltransferase; IL, interleukin; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; kb, kilobase(s); bp, base pair(s). (Yamaguchi et al., 1990Yamaguchi Y. Mann D.M. Ruoslahti E. Nature. 1990; 346: 281-284Crossref PubMed Scopus (1285) Google Scholar. This affinity of decorin for TGF-β could lead to the removal of this potent growth factor from the cellular microenvironment, thereby neutralizing its biological activity. This property has been demonstrated in an experimental animal model of glomerulonephritis in which infusion of recombinant decorin prevents the fibrosis of renal glomeruli induced by TGF-β (Border et al., 1992Border W.A. Noble N.A. Yamamoto T. Harper J.R. Yamaguchi Y. Pierschbacher M.D. Ruoslahti E. Nature. 1992; 360: 361-364Crossref PubMed Scopus (924) Google Scholar. Also, decorin may play a role in the development of cancer, as evidenced by the altered decorin gene expression in the stroma of human colon carcinomas (Iozzo, 1985Iozzo R.V. J. Biol. Chem. 1985; 260: 7464-7473Abstract Full Text PDF PubMed Google Scholar; Adany et al., 1990Adany R. Heimer R. Caterson B. Sorrell J.M. Iozzo R.V. J. Biol. Chem. 1990; 265: 11389-11396Abstract Full Text PDF PubMed Google Scholar, suggesting that decorin could mediate signaling between neoplastic cells and the adjacent stromal elements (Iozzo and Cohen, 1993Iozzo R.V. Cohen I. Experientia. 1993; 49: 447-455Crossref PubMed Scopus (104) Google Scholar).Our recent cloning of the decorin gene in both human (Danielson et al., 1993Danielson K.G. Fazzio A. Cohen I. Cannizzaro L.A. Eichstetter I. Iozzo R.V. Genomics. 1993; 15: 146-160Crossref PubMed Scopus (89) Google Scholar and murine (Scholzen et al., 1994Scholzen T. Solursh M. Susuki S. Reiter R. Morgan J.L. Buchberg A.M. Siracusa L.D. Iozzo R.V. J. Biol. Chem. 1994; 269: 28270-28281Abstract Full Text PDF PubMed Google Scholar and the development of various decorin promoter/CAT reporter gene constructs (Santra et al., 1994Santra M. Danielson K.G. Iozzo R.V. J. Biol. Chem. 1994; 269: 579-587Abstract Full Text PDF PubMed Google Scholar have allowed us to investigate the molecular mechanisms regulating decorin gene expression during growth and quiescence of normal diploid human skin cells. Our results indicate a novel transcriptional activation of the decorin gene associated with confluency-induced quiescence of human dermal fibroblasts or when HeLa cells are rendered quiescent by serum deprivation. We also provide evidence for a transcriptional repression of decorin gene expression by TNF-α, through down-regulation of the decorin promoter activity. These results raise the possibility of a delicately balanced regulation of the transcriptional activity of the decorin gene during important phases of the cell cycle.MATERIALS AND METHODSCell CulturesHuman dermal fibroblast cultures, established from tissue specimens obtained from adult individuals during surgical procedures or from neonatal foreskins, were utilized in passages 3–8. The cell cultures were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 units/ml penicillin, and 50 μg/ml streptomycin.Cytokines/Growth FactorsHuman recombinant TGF-β2 was a generous gift from Dr. David Olsen, Celtrix Laboratories, Santa Clara, CA. Human recombinant TNF-α was purchased from Boehringer Mannheim.Northern AnalysesTotal RNA was isolated using standard procedures (Chirgwin et al., 1979Chirgwin J.M. Przybyla A.E. MacDonald R.J. Rutter W.J. Biochemistry. 1979; 18: 5294-5299Crossref PubMed Scopus (16619) Google Scholar; Chomczynski and Sacchi, 1987Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62983) Google Scholar) and analyzed by Northern hybridization with 32P-labeled cDNA probes (Sambrook et al., 1989Sambrook J. Frisch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar. The [32PIcDNA-mRNA hybrids were visualized by autoradiography, and the steady state levels of mRNA were quantitated by scanning densitometry using a He-Ne laser scanner at 633 nm (LKB Produkter, Bromma, Sweden).cDNAs and Plasmid ConstructsThe following cDNAs were used for Northern hybridizations to detect specific mRNA transcripts: a 1.8-kb human decorin cDNA (kindly provided by Dr. Tom Krusius, University of Helsinki, Finland) (Krusius and Ruoslahti, 1986Krusius T. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7683-7687Crossref PubMed Scopus (412) Google Scholar) or a 411-bp polymerase chain reaction fragment developed in our laboratory and described previously (Santra et al., 1994Santra M. Danielson K.G. Iozzo R.V. J. Biol. Chem. 1994; 269: 579-587Abstract Full Text PDF PubMed Google Scholar; a l.-kb biglycan cDNA, kindly provided by Dr. Larry W. Fisher, NIH (Fisher et al., 1989Fisher L.W. Termine J.D. Young M.F. J. Biol. Chem. 1989; 264: 4571-4576Abstract Full Text PDF PubMed Google Scholar; and a 1.3-kb rat glyceraldehyde-3-phosphate dehydrogenase used as a control (Fort et al., 1985Fort P. Marty L. Piechaczyk M. El Sabrouty S. Dani C. Jeanteur P. Blanchard J.-M. Nucleic Acids Res. 1985; 13: 1431-1442Crossref PubMed Scopus (1970) Google Scholar.To study the transcriptional regulation of decorin gene expression, transient transfection experiments were performed with various 5′ deletion constructs derived from pUCDEC-10/CAT, a plasmid containing ∼1 kb of decorin promoter linked to the CAT reporter gene cloned into pUCCAT vector. Preparation of these deletion constructs is described in detail elsewhere (Santra et al., 1994Santra M. Danielson K.G. Iozzo R.V. J. Biol. Chem. 1994; 269: 579-587Abstract Full Text PDF PubMed Google Scholar. Also, a synthetic oligonucleotide spanning the region from –188 to –140 of the decorin promoter, which is fundamental for TNF-α response (see text) was cloned as a Hindlll/Sall fragment upstream of the thymidine kinase (TK) promoter linked to the CAT gene, generating the plasmid pDEC-188–140TK/CAT used in transfection experiments to investigate the function of the elements comprised within this region of the decorin promoter. The plasmid pTK/CAT was used as a control.Transient Transfection and CAT AssaysTransient transfections of human foreskin fibroblasts were performed by the calcium phosphate/DNA co-precipitation method, as described previously (Santra et al., 1994Santra M. Danielson K.G. Iozzo R.V. J. Biol. Chem. 1994; 269: 579-587Abstract Full Text PDF PubMed Google Scholar. Briefly, the cells were transfected with 10 or 20 μg of DNA mixed with 5 μg of the pRSV-β-galactosidase plasmid DNA in order to monitor transfection efficiencies. After glycerol shock, the cells were placed in DMEM containing 1% FCS, 4 h prior to the addition of growth factors and cytokines. In experiments without growth factors and cytokines, the cells were placed in DMEM containing 10% FCS. After an additional 40 h of incubation, the cells were rinsed twice with phosphate-buffered saline, harvested by scraping, and lysed by three cycles of freeze-thawing in 100 ml of 0.25 M Tris-HCl, pH 7.8. The β-galactosid-ase activities were measured according to standard protocols (Sambrook et al., 1989Sambrook J. Frisch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar. Aliquots corresponding to identical β-galacto-sidase activity were used for each CAT assay with [14C]chloramphenicol as substrate (Gorman et al., 1982Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5288) Google Scholar using thin layer chromatography. Following autoradiography, the plates were cut and counted by liquid scintillation to quantify the acetylated [14C]chloramphenicol.In Vitro Transcription AssaysA polymerase chain reaction-generated 1008-bp fragment containing exon Ib, and ∼1 kb of the 5′-flanking sequence was subcloned into pBluescript-KS+ as a SmaI/SacI insert (Santra et al., 1994Santra M. Danielson K.G. Iozzo R.V. J. Biol. Chem. 1994; 269: 579-587Abstract Full Text PDF PubMed Google Scholar. Further deletions were prepared by restriction enzyme digestion. Detailed procedures for preparation and characteristics of the generated constructs are provided in the legend to Fig. 2 and in the text under “Results.” Plasmid DNA constructs were purified by affinity chromatography on DNA purification columns (QIAGEN Inc., Chatsworth, CA), according to the manufacturer's protocol and digested with EcoRI in order to linearize the plasmids at the 3′ end of exon lb. Fibroblast nuclear extracts were prepared according to previously described methods (Stein et al., 1989Stein B. Rahmsdorf H.J. Steffen A. Liftin M. Herrlich P. Mol. Cell. Biol. 1989; 9: 5169-5181Crossref PubMed Scopus (452) Google Scholar; Andrews and Faller, 1991Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 192499Crossref PubMed Scopus (2209) Google Scholar). In vitro transcription products were generated from this linearized DNA (1 μg) in reactions containing either fibroblast nuclear extracts or HeLa cell nuclear protein extracts (Promega, in vitro transcription grade), according to the method of the manufacturer, and analyzed on a denaturing 8% Polyacrylamide gel, as described previously (Santra et al., 1994Santra M. Danielson K.G. Iozzo R.V. J. Biol. Chem. 1994; 269: 579-587Abstract Full Text PDF PubMed Google Scholar.Gel Mobility Shift AssaysFor gel retardation assays, nuclear extracts were prepared according to either the method of Andrews and Faller, 1991Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 192499Crossref PubMed Scopus (2209) Google Scholar or to that of Stein et al., 1989Stein B. Rahmsdorf H.J. Steffen A. Liftin M. Herrlich P. Mol. Cell. Biol. 1989; 9: 5169-5181Crossref PubMed Scopus (452) Google Scholar. For DNA binding assays, two double-stranded oligomers containing the potential TNFREs identified in our study were generated: a 54-bp fragment containing the region –188 to –140 of the decorin promoter: 5′-AGCTTAGTGATGGT-CAATTGAGTCATTTGTGTGCAAAATATTGTGCAAGGCCCG-3′ and a 22-bp fragment corresponding to a sequence present in the exon lb of the decorin gene, sharing high homology with the osteocalcin gene TNFRE (underlined): 5′-TTGCCTGGATGAGCCAGGGGAC-31. The end-labeled oligomer (∼7 × 104 cpm) was incubated with 3–12 μg of protein extracts for 30 min on ice in 20 μl of binding reaction buffer (12 mM HEPES, pH 7.9, 4 mM Tris, pH 7.9, 60 mM KCl, 1 IHM EDTA, 12% glycerol), in the presence of 2–4 μg of poly(dI-dC), as described previously (Dignam et al., 1983Dignam J.D. Martin P.L. Shastry B.S. Roeder R.G. Methods Enzymol. 1983; 101: 582-598Crossref PubMed Scopus (744) Google Scholar. For competition experiments, a 20- to 500-fold molar excess of DNA was added to the binding reaction. Details of the competition assays are provided in the legends to the figures and the corresponding text under “Results.” DNA-protein complexes were separated from unbound oligomers on 4 or 5% acrylamide gels in 0.4 × TBE. The gels were fixed for 30 min in 30% methanol, 10% acetic acid, dried, and exposed to x-ray films at –70 °C.RESULTSGrowth Arrest Induces Decorin Gene ExpressionModulation of cellular growth is often associated with the expression of key regulatory genes. To investigate whether the growth phase of normal diploid cells had any effect on decorin gene expression, three experimental approaches were taken. First, RNA was isolated from fibroblast cultures at different time points before and after reaching confluency and hybridized under high stringency with a decorin cDNA probe. Significant expression of decorin was noted in exponentially growing fibroblasts (Fig. 1, lanes 1–3). However, decorin expression was dramatically enhanced (∼8-fold above level in proliferating fibroblast cultures) 8 days after fibroblast cultures reached confluency and had stopped proliferating (lane 4), and this enhancement (∼4fold) persisted for up to 16 days postconfluency (lane 5).FIG. 1Effect of growth state on decorin mRNA levels in human dermal fibroblasts in culture. Total RNA was extracted from fibroblast cultures growing in logarithmic phase for 1, 2, and 3 days (lanes 1–3, respectively) or 8 and 16 days after reaching confluency (lanes 4 and 5, respectively). RNA (10 μg/lane) was analyzed by Northern hybridization with a decorin cDNA under stringent conditions, allowing the detection of closely migrating transcripts of 1.6 and 1.9 kb, which encode human decorin (upperpanel). Ethidium bromide staining of ribosomal RNA (rRNA) prior to transfer in shown in the lower panel.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Secondly, to understand the mechanisms leading to increased decorin expression in growth-arrested cells and to determine whether the elevation of decorin mRNA levels observed in confluent fibroblast cultures was due to transcriptional activation of the decorin gene, nuclear proteins were extracted from both proliferating and 5-day postconfluent fibroblast cultures. The nuclear extracts were examined for their ability to drive the transcription of the decorin gene using various 5′ deletion constructs of the decorin promoter (Fig. 2A). Fig. 2B depicts an autoradiogram of a denaturing 89< Polyacrylamide gel electrophoresis of in vitro transcription products generated from a 983-bp decorin promoter construct with nuclear extracts from both proliferating and confluent fibroblasts. The transcription reactions generated an ∼ 100-bp RNA whose synthesis was elevated when transcription was driven by the nuclear extracts from confluent fibroblasts (Fig. 2B, lane 4 versus lane 3). Transcription products generated from quiescent HeLa cell nuclear extracts are shown in lane 2 (B).To identify the promoter region responsible for enhanced decorin expression in nonproliferating fibroblasts, we performed parallel in vitro transcription reactions using DNA constructs containing three 5′ deletions of the decorin promoter (Fig. 2C). The amounts of transcription products generated with nuclear extracts from either proliferating (C, lanes 1–3) or confluent (lanes 6–8) fibroblast cultures were not dependent on the deletion segments of the decorin gene 5′-flanking region. Increased transcription in nuclear extracts from confluent fibroblast cultures was specific for the decorin promoter because neither SV40 promoter-driven nor cytomegalovirus promoter-driven transcription of the decorin gene was affected by the growth state of fibroblasts (lanes 4, 5, 9, and 11, respectively). Taken together, these results indicate that up-regulation of decorin gene expression during quiescence is transcriptionally regulated and involves a region comprised between position –140 and the transcription start site.Thirdly, to further ascertain that the elevation of decorin mRNA levels in confluent fibroblasts was due to increased decorin promoter activity, we performed transient cell transfection experiments with pUCDEC-140/CAT, which contains 140 bp of decorin promoter linked to the CAT reporter gene, together with pRSV-j3-galactosidase construct, utilizing either proliferating or confluent fibroblast cultures. The CAT activity, reflecting decorin promoter activity, was significantly elevated in confluent cultures of fibroblasts (Fig. 3, lane 2) as compared to that in proliferating fibroblast cultures (Fig. 3, lane 1), after normalization of the CAT activity by β-galactosidase activity in the same cell extracts, so as to correct for differences in transfection efficiency. Interestingly, induction of decorin promoter activity was also observed in HeLa cells rendered quiescent by serum deprivation (Fig. 3, lane 4 versus lane 3). These findings indicate that elevated decorin gene expression correlates with growth arrest.FIG. 3Enhancement of decorin promoter activity upon cell growth arrest. Fibroblast cultures, either proliferating or confluent (lanes 1 and 2, respectively) and HeLa cell cultures, either exponentially growing in DMEM containing 10% fetal calf serum or rendered quiescent by serum-starvation (lanes 3 and 4, respectively) were co-transfected with a minimal decorin promoter (-140 relative to the transcriptional start site +D/CAT construct, together with pRSV-β-galactosidase by the calcium phosphate/DNA coprecipitation procedure, as described under “Materials and Methods.” After transfection, the cultures were either incubated for 72 h in DMEM containing 10% FCS in order to maintain them in their logarithmic phase of growth (lanes 1 and 3) or allowed to grow in DMEM containing 10% FCS for 24 h, replaced with serum-free medium for another 48 h in order to force them into a quiescent state. The cell extracts were assayed for CAT activity using 1HC [chloramphenicol as a substrate by thin layer chromatography after normalization for β-galactosidase activity. The autoradiogram of a representative experiment is shown. C, [14C]chloramphenicol; AC, acetylated [14C]chloramphenicol.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Collectively, these data indicate that the elevated decorin mRNA levels present in confluent fibroblast cultures result from transcriptional activation of the decorin gene. Furthermore, it appears that the proximal 140-bp segment of the decorin promoter is sufficient to allow transcriptional activation of the gene upon quiescence.Elevated Decorin Gene Expression in Confluent Fibroblast Cultures Is Reduced by TNF-αIn a set of experiments, we investigated whether the growth state of fibroblasts could influence the response of the decorin gene to cytokine modulation. For this purpose, fibroblast cultures, either proliferating (Fig. 4, lanes 1 and 2) or 3 days (lanes 3 and 4) or 6 days (lanes 5 and 6) following confluency, were cultured for 24 h in the absence (-) or in the presence (+) of TNF-α (10 ng/ml). Total RNA was extracted and subjected to Northern analysis. The results confirmed the previous finding of an up-regulation of decorin gene expression after fibroblasts reach confluency (lanes 3 and 5 versus lane 1). Interestingly, TNF-α was a potent inhibitor of decorin gene expression in confluent fibroblast cultures (lanes 4 and 6 versus lanes 3 and 5, respectively), whereas it had no effect on proliferating cells (lane 2 versus lane 1). This selective effect of TNF-α was specific for decorin, as demonstrated by unaltered glyceraldehyde-3-phosphate dehydrogenase mRNA levels in the same experiments. Quantitation of the autoradiograms by scanning densitometry and correction of the values against glyceraldehyde-3-phosphate dehydrogenase mRNA levels in the same RNA preparations revealed that TNF-α inhibited decorin gene expression in confluent fibroblast cultures by approximately 60%, at both 3 and 6 days after confluency, whereas it had no effect on the decorin transcript levels when cells were still proliferating.FIG. 4Effect of TNF-α on decorin mRNA levels in human dermal fibroblasts in culture. Fibroblast cultures, in logarithmic growth phase (lanes 1 and 2), 3 days (lanes 3 and 4), and 6 days (lanes 5 and 6) after reaching confluency, were incubated in DMEM containing 10%, fetal calf serum, without (–) or with (+) TNF-α (10 ng/ml) for 24 h. Total RNA was extracted and analyzed by Northern hybridization with a decorin cDNA. A glyceraldehyde-3-phosphate dehydrogenase IGAPDH) cDNA was used as a control probe to ensure the specificity of the TNF-α effect on decorin gene expression.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To investigate whether the inhibitory effect of TNF-α on decorin mRNA levels occurred at the transcriptional level, we performed in vitro transcription assays utilizing nuclear extracts from either proliferating or confluent fibroblast cultures incubated with or without TNF-α (Fig. 5). In vitro transcription using the 662-bp decorin promoter template together with nuclear extracts from proliferating fibroblast cultures generated small amounts of transcripts (Fig. 5, lane 2). Consistent with the results observed at the mRNA level (see Fig. 4), TNF-α treatment did not alter the transcriptional activity of proliferating fibroblasts (Fig. 5, lane 3 versus lane 2). In contrast, as expected from the data of the experiments presented in Fig. 3, nuclear extracts from confluent fibroblast cultures exhibited elevated transcriptional activity as compared to that from proliferating cultures (lanes 4 and 6). This elevated activity was dramatically reduced when the cell cultures were treated for 24 h with TNF-α (lanes 5 and 7 versus lanes 4 and 6), indicating transcriptional repression of decorin gene expression.FIG. 5Effect of TNF-α on decorin promoter transcription, as measured by in vitro transcription assay. Experimental conditions were the same as those described in Fig. 4. The arrow indicates the exon lb transcripts generated by in vitro transcription from the –661-bp decorin promoter construct utilizing nuclear extracts from proliferating (lanes 2 and 3), 3 days (lanes 4 and 5), and 6 days (lanes 6 and 7) postconfluency fibroblast cultures, treated with (+) or without (-) TNF-α (10 ng/ml). Lane I shows radioactive DNA molecular mass markers whose sizes (bp) are indicated on the left of the panel.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TGF-β and TNF-α Separately and Additively Inhibit Decorin Gene ExpressionTGF-β has been previously shown to inhibit fibroblast decorin gene expression (Kähäri et al., 1991Kähäri V.-M. Larjava H. Uitto J. J. Biol. Chem. 1991; 266: 10608-10615Abstract Full Text PDF PubMed Google Scholar; Vogel and Hernandez, 1992Vogel K.G. Hernandez D.J. Eur. J. Cell Biol. 1992; 59: 304-313PubMed Google Scholar) and to antagonize the effect of TNF-α on the expression on various extracellular matrix-related genes (Kähäri et al., 1990Kähäri V.-M. Chen Y.Q. Su M.W. Ramirez F. Uitto J. J. Clin. Invest. 1990; 86: 1489-1495Crossref PubMed Scopus (167) Google Scholar; Armendariz-Borunda et al., 1992Armendariz-Borunda J. Katayama K. Seyer J.M. J. Biol. Chem. 1992; 267: 14316-14321Abstract Full Text PDF PubMed Google Scholar; Mauviel and Uitto, 1993Mauviel A. Uitto J. Wounds. 1993; 5: 137-152Google Scholar; Mauviel et al., 1993aMauviel A. Chen Y.Q. Dong W. Evans C.H. Uitto J. Curr. Biol. 1993; 3: 822-831Abstract Full Text PDF PubMed Scopus (50) Google Scholar. We therefore examined whether TGF-β and TNF-α utilize the same mechanism(s) to inhibit decorin gene expression. To this end, confluent fibroblast cultures, which exhibited maximal levels of decorin mRNA transcripts, were incubated for 24 h with TNF-α (10 ng/ml) or TGF-β (5 ng/ml), either alone or in combination. Total RNA was then extracted and analyzed for decorin gene expression. The two cytokines, tested separately, inhibited decorin
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