JunD Regulates Transcription of the Tissue Inhibitor of Metalloproteinases-1 and Interleukin-6 Genes in Activated Hepatic Stellate Cells
2001; Elsevier BV; Volume: 276; Issue: 26 Linguagem: Inglês
10.1074/jbc.m101840200
ISSN1083-351X
AutoresDavid E. Smart, Karen J. Vincent, Michael J.P. Arthur, Oliver Eickelberg, Marc Castellazzi, Jelena Mann, Derek A. Mann,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoActivation of hepatic stellate cells (HSCs) to a myofibroblast-like phenotype is the pivotal event in hepatic wound healing and fibrosis. Rat HSCs activated in vitro express JunD, Fra2, and FosB as the predominant AP-1 DNA-binding proteins, and all three associate with an AP-1 sequence that is essential for activity of the tissue inhibitor of metalloproteinases-1 (TIMP-1) promoter. In this study, we used expression vectors for wild-type, dominant-negative, and forced homodimeric (Jun/eb1 chimeric factors) forms of JunD and other Fos and Jun proteins to determine the requirement for JunD in the transcriptional regulation of the TIMP-1 and interleukin-6 (IL-6) genes. JunD activity was required for TIMP-1 gene promoter activity, whereas overexpression of Fra2 or FosB caused a repression of promoter activity. The ability of homodimeric JunD/eb1 to elevate TIMP-1 promoter activity supports a role for JunD homodimers as the major AP-1-dependent transactivators of the TIMP-1 gene. IL-6 promoter activity was induced upon activation of HSCs and also required JunD activity; however, expression of JunD/eb1 homodimers resulted in transcriptional repression. Mutagenesis of the IL-6 promoter showed that an AP-1 DNA-binding site previously reported to be an activator of transcription in fibroblasts functions as a suppressor of promoter activity in HSCs. We conclude that JunD activates IL-6 gene transcription as a heterodimer and operates at an alternative DNA-binding site in the promoter. The relevance of these findings to events occurring in the injured liver was addressed by showing that AP-1 DNA-binding complexes are induced during HSC activation and contain JunD as the predominant Jun family protein. JunD is therefore an important transcriptional regulator of genes responsive to Jun homo- and heterodimers in activated HSCs. Activation of hepatic stellate cells (HSCs) to a myofibroblast-like phenotype is the pivotal event in hepatic wound healing and fibrosis. Rat HSCs activated in vitro express JunD, Fra2, and FosB as the predominant AP-1 DNA-binding proteins, and all three associate with an AP-1 sequence that is essential for activity of the tissue inhibitor of metalloproteinases-1 (TIMP-1) promoter. In this study, we used expression vectors for wild-type, dominant-negative, and forced homodimeric (Jun/eb1 chimeric factors) forms of JunD and other Fos and Jun proteins to determine the requirement for JunD in the transcriptional regulation of the TIMP-1 and interleukin-6 (IL-6) genes. JunD activity was required for TIMP-1 gene promoter activity, whereas overexpression of Fra2 or FosB caused a repression of promoter activity. The ability of homodimeric JunD/eb1 to elevate TIMP-1 promoter activity supports a role for JunD homodimers as the major AP-1-dependent transactivators of the TIMP-1 gene. IL-6 promoter activity was induced upon activation of HSCs and also required JunD activity; however, expression of JunD/eb1 homodimers resulted in transcriptional repression. Mutagenesis of the IL-6 promoter showed that an AP-1 DNA-binding site previously reported to be an activator of transcription in fibroblasts functions as a suppressor of promoter activity in HSCs. We conclude that JunD activates IL-6 gene transcription as a heterodimer and operates at an alternative DNA-binding site in the promoter. The relevance of these findings to events occurring in the injured liver was addressed by showing that AP-1 DNA-binding complexes are induced during HSC activation and contain JunD as the predominant Jun family protein. JunD is therefore an important transcriptional regulator of genes responsive to Jun homo- and heterodimers in activated HSCs. hepatic stellate cell tissue inhibitor of metalloproteinases nuclear factor-κB interleukin intercellular adhesion molecule-1 electrophoretic mobility shift assay chloramphenicol acetyltransferase base pair(s) Rous sarcoma virus phosphate-buffered saline polymerase chain reaction cAMP-responsive element Hepatic stellate cells (HSCs)1 represent up to 15% of the resident cells of the liver and play a pivotal role in the cellular pathology underlying hepatic fibrosis (1Friedman S.L. J. Biol. Chem. 2000; 275: 2247-2250Abstract Full Text Full Text PDF PubMed Scopus (1895) Google Scholar). In response to liver injury of any etiology, the normally quiescent HSC undergoes a progressive process of trans-differentiation into a proliferating myofibroblast-like activated HSC (1Friedman S.L. J. Biol. Chem. 2000; 275: 2247-2250Abstract Full Text Full Text PDF PubMed Scopus (1895) Google Scholar). Through increased secretion of extracellular matrix proteins and the tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2, activated HSCs are responsible for deposition and accumulation of the majority of the excess extracellular matrix in the fibrotic liver (2Friedman S.L. N. Engl. J. Med. 1993; 328: 1828-1835Crossref PubMed Scopus (0) Google Scholar). Furthermore, activated HSCs can contribute to the fibrogenic process through their ability to secrete and respond to a wide range of cytokines and growth factors (3Arthur M.J. Mann D.A. Iredale J.P. J. Gastroenterol. Hepatol. 1998; 13 suppl.: S33-S38Crossref PubMed Scopus (132) Google Scholar). Details of the molecular events that regulate HSC activation are beginning to be unraveled, as is the potential for specific members of the AP-1, NF-κB, and Kruppel-like transcription factor families to control key profibrogenic features of the activated HSCs (1Friedman S.L. J. Biol. Chem. 2000; 275: 2247-2250Abstract Full Text Full Text PDF PubMed Scopus (1895) Google Scholar, 4Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar, 5Hellerbrand C. Jobin C. Limuro Y. Licato L. Sartor R.B. Brenner D.A. Hepatology. 1998; 27: 1285-1295Crossref PubMed Scopus (175) Google Scholar, 6Elsharkawy A.M. Wright M.C. Hay R.T. Arthur M.J.P. Hughes T. Bahr M.J. Degitz K. Mann D.A. Hepatology. 1999; 30: 761-769Crossref PubMed Scopus (122) Google Scholar). Putative AP-1 and NF-κB sites are found in the promoters of many genes that are induced upon HSC activation and contribute to the fibrotic process, including TIMP-1 (AP-1), IL-6 (AP-1 and NF-κB), and ICAM-1 (NF-κB) (4Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar, 5Hellerbrand C. Jobin C. Limuro Y. Licato L. Sartor R.B. Brenner D.A. Hepatology. 1998; 27: 1285-1295Crossref PubMed Scopus (175) Google Scholar, 7Eickelberg O. Pansky A. Mussmann R. Bihl M. Tamm M. Hildebrand P. Perruchoud A.P. Roth M. J. Biol. Chem. 1999; 274: 12933-12938Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Since in vivo activation of HSCs can be closely mimicked by culturing HSCs isolated from normal rat liver on plastic and in the presence of serum, it has been possible to investigate the transcriptional control of potential profibrotic genes during HSC activation (1Friedman S.L. J. Biol. Chem. 2000; 275: 2247-2250Abstract Full Text Full Text PDF PubMed Scopus (1895) Google Scholar). Investigators including ourselves have previously demonstrated that basal and cytokine/growth factor-inducible transcription of these genes is dependent on interaction of specific AP-1 and NF-κB (Rel) protein dimers with their putative promoter-binding sites (4Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar, 5Hellerbrand C. Jobin C. Limuro Y. Licato L. Sartor R.B. Brenner D.A. Hepatology. 1998; 27: 1285-1295Crossref PubMed Scopus (175) Google Scholar, 6Elsharkawy A.M. Wright M.C. Hay R.T. Arthur M.J.P. Hughes T. Bahr M.J. Degitz K. Mann D.A. Hepatology. 1999; 30: 761-769Crossref PubMed Scopus (122) Google Scholar). These observations indicate that these inducible transcription factors are likely to play a key role in the activation and/or persistence of myofibroblast-like HSCs. Recent studies have identified target genes of NF-κB (IL-6 and ICAM-1) and have also indicated that NF-κB may protect activated HSCs against apoptosis (5Hellerbrand C. Jobin C. Limuro Y. Licato L. Sartor R.B. Brenner D.A. Hepatology. 1998; 27: 1285-1295Crossref PubMed Scopus (175) Google Scholar, 6Elsharkawy A.M. Wright M.C. Hay R.T. Arthur M.J.P. Hughes T. Bahr M.J. Degitz K. Mann D.A. Hepatology. 1999; 30: 761-769Crossref PubMed Scopus (122) Google Scholar, 8Lang A. Schoonhoven R. Tuvia S. Brenner D.A. Rippe R.A. J. Hepatol. ( Amst. ). 2000; 33: 49-58Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Less attention has been directed at studying the role played by AP-1 in HSC activation. Although in vitrostudies have shown that activated HSCs express inducible AP-1 DNA-binding activity (4Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar, 9Armendariz-Borunda J. Simkevitch C.P. Roy N. Raghow R. Kang A.H. Sayer J.M. Biochem. J. 1994; 304: 817-824Crossref PubMed Scopus (72) Google Scholar, 10Tao J. Mallat A. Gallois C. Belmadani S. Mery P.-F. Nhieu J.T.-V. Pavoine C. Lotersztajn S. J. Biol. Chem. 1999; 274: 23761-23769Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), there is little direct evidence that AP-1 plays a key role in the transcriptional regulation of the activated HSC phenotype. Chen and Davis (11Chen A. Davis B.H. J. Biol. Chem. 1999; 271: 25994-25998Abstract Full Text Full Text PDF Scopus (46) Google Scholar, 12Chen A. Davis B.H. Mol. Cell. Biol. 2000; 20: 2818-2826Crossref PubMed Scopus (83) Google Scholar) recently reported that acetaldehyde- and UV-induced transcription of the αI(I) collagen gene is mediated via AP-1-dependent activation of BTEB, a GC box-binding transcription factor that regulates αI(I) collagen gene transcription. We have previously shown that an AP-1-binding site in the human TIMP-1 gene promoter is required for high level transcription in activated HSCs (4Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar). In this study, we have addressed the role of the AP-1 transcription factor JunD in the control of TIMP-1 and IL-6 gene transcription in activated HSCs. The jun family proto-oncogenes (c-jun,junB, and junD) are critical components of the AP-1 transcription factor (13Karin M. Liu Z. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2324) Google Scholar, 14Ziff E.B. Genetics. 1990; 6: 69-72Scopus (189) Google Scholar). The Jun proteins are bZip transcription factors that can form either AP-1 homodimers (Jun/Jun) or AP-1 heterodimers. Jun heterodimers are created through interaction of Jun proteins with members of the related bZip protein family, notably those of the fos proto-oncogene family (c-fos,fosB, fra1, and fra2) or the ATF family (ATF2, ATF3, and ATF4) (13Karin M. Liu Z. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2324) Google Scholar, 14Ziff E.B. Genetics. 1990; 6: 69-72Scopus (189) Google Scholar, 15Hai T. Curren T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3720-3724Crossref PubMed Scopus (1119) Google Scholar). An evolutionarily conserved non-canonical AP-1 site (TGAGTAA) in the human TIMP-1 promoter is required for induction of transcription during culture activation of primary rat HSCs and binds Jun/Jun and Jun/Fos dimers (4Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar). Western blot and electrophoretic mobility shift assay (EMSA) studies revealed that JunD is the predominant Jun family protein expressed in culture-activated rat HSCs, with little or no detectable expression of c-Jun and JunB after the first 48 h of culture. This observation indicated a role for JunD not only in the transcriptional activation of TIMP-1, but also in other AP-1-dependent regulatory processes of activated HSCs. In this study, we demonstrate that JunD is required for high level activity of both the TIMP-1 and IL-6 promoters in activated HSCs. We also show that expression of different combinations of AP-1 proteins leads to differential effects on transcription and that the repressive or stimulatory effects induced by Jun/Jun and Jun/Fos dimers are dependent on the target promoter. HSCs were isolated from the livers of normal male Sprague-Dawley rats (400 ± 50 g) by sequential perfusion with Pronase and collagenase as previously described (16Iredale J.P. Benyon R.C. Arthur M.J.P. Ferris W.F. Alcolado R. Winwood P.J. Clark N. Murphy G. Hepatology. 1996; 24: 176-184Crossref PubMed Google Scholar). Induction of acute liver damage in rats was achieved by intraperitoneal injection of a 1:1 ratio of CCl4 (0.2 ml/100 g of body weight) and olive oil as previously described (16Iredale J.P. Benyon R.C. Arthur M.J.P. Ferris W.F. Alcolado R. Winwood P.J. Clark N. Murphy G. Hepatology. 1996; 24: 176-184Crossref PubMed Google Scholar). Control rats were administered an intraperitoneal injection of olive oil alone. HSCs were separated from the cell suspension over an 11.5% Optiprep gradient (Nycomed Pharma AS, Oslo, Sweden), followed by elution. HSCs were seeded onto plastic, cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 16% fetal calf serum (Life Technologies, Inc.), and maintained at 37 °C in an atmosphere of 5% CO2. All plasmid DNA was prepared using a commercial DNA extraction and isolation kit (Maxiprep, QIAGEN). A chloramphenicol acetyltransferase (CAT) reporter plasmid (pTIMP1) containing a 162-bp minimal human TIMP-1 promoter cloned into theHindIII and PstI sites of pBLCAT3 was used to determine TIMP-1 promoter function (4Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar, 17Trim J.E. Samra S.K. Arthur M.J.P. Wright M.C. McAuley M. Beri R. Mann D.A. J. Biol. Chem. 2000; 275: 6657-6663Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). IL-6 promoter function was studied using the luciferase reporter vector pIL6-Luc651, containing nucleotides −651 to +1 of the human IL-6 gene (7Eickelberg O. Pansky A. Mussmann R. Bihl M. Tamm M. Hildebrand P. Perruchoud A.P. Roth M. J. Biol. Chem. 1999; 274: 12933-12938Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Construction of pIL6-Luc651 and derivatives carrying site-directed mutations in the AP-1 (−283 to −276), NF-IL6 (−154 to −146), and NF-κB (−72 to −63) sequences has been described elsewhere (7Eickelberg O. Pansky A. Mussmann R. Bihl M. Tamm M. Hildebrand P. Perruchoud A.P. Roth M. J. Biol. Chem. 1999; 274: 12933-12938Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The controlRenilla luciferase vector pRL-TK was purchased from Promega(Southampton, United Kingdom). Expression vectors for mouse Jun (pCMV2-c-Jun, pCMV2-JunB, and pCMV2-JunD) and Fos (pCMV2-c-Fos, pCMV2-FosB, pCMV2-Fra1, and pCMV2-Fra2) were a kind gift of Dr. Paul Dobner and are as described by Harrison et al. (18Harrison R.J. McNeil G.P. Dobner P.R. Mol. Endocrinol. 1995; 9: 981-993Crossref PubMed Google Scholar). Expression of the chimeric Jun/eb1 proteins was provided by transfection of pDP7c-Jun/eb1, pDP7JunD/eb1, and pDP7JunB/eb1, in which expression is driven by the RSV long terminal repeat. Construction of the pDP7 vectors has been described by Vandel et al. (19Vandel L. Montreau N. Vial E. Pfarr C.M. Binetruy B. Castellazzi M. Mol. Cell. Biol. 1996; 16: 1881-1888Crossref PubMed Scopus (56) Google Scholar). An expression vector (RSVβ-JunD) for dominant-negative JunD lacking amino acids 1–162 was obtained from Dr. Ernst Lengyel (20Reid S. Jager C. Jeffers M. Vande Woude G.F. Graeff H. Schmitt M. Lengyel E. J. Biol. Chem. 1999; 274: 16377-16386Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). AP-1 DNA binding was determined by EMSA as previously described (4Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar) using a 32P end-labeled double-stranded oligonucleotide probe containing a consensus AP-1 site: sense oligonucleotide, 5′-TATAAAGCATGAGTCAGACACCTCT-3′; and antisense oligonucleotide, 5′-AGAGGTGTCTGACTCATGCTTTATA-3′. Nuclear extracts were prepared from HSCs by a protocol modified from that described by Dignamet al. (21Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9164) Google Scholar). Harvested cells were washed twice in ice-cold phosphate-buffered saline (PBS) prior to lysis in Buffer A (21Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9164) Google Scholar) supplemented with 0.2% Nonidet P-40, 0.5 mm4-(2-aminoethyl)benzenesulfonyl fluoride, 0.2 mm EDTA, and 15 μg/ml aprotinin. Lysates were centrifuged for 10 s at 13,000 rpm to collect crude nuclear pellets. Supernatants were discarded, and pellets were washed twice in lysis buffer prior to resuspension in Buffer C (21Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9164) Google Scholar) supplemented with 0.5 mm4-(2-aminoethyl)benzenesulfonyl fluoride, 0.2 mm EDTA, and 15 μg/ml aprotinin. After a 10-min incubation on ice with occasional vortexing, the extracts were cleared of insoluble nuclear material by centrifugation at 13,000 rpm for 30 s. Cleared nuclear extracts were transferred to fresh Eppendorf tubes, and their protein content was determined using the Bradford DC assay kit (Bio-Rad). EMSA reactions were assembled on ice and consisted of an initial 10-min incubation of 4 μl of Buffer C containing 5 μg of nuclear protein extract and 12 μl of water containing 2 μg of poly(dI·dC). 4 μl of water containing 0.4 ng of radiolabeled double-stranded AP-1 probe was then added to the reaction and, after mixing, was incubated for a further 20 min. For supershift assays, reactions were incubated for a further 16 h in the presence of 1 μg of anti-Jun antiserum (Santa Cruz Biotechnology, Inc.). EMSA and supershift reaction mixtures were then resolved by electrophoresis on an 8% nondenaturing polyacrylamide gel (37:5:1). Whole cell protein extracts were prepared by lysis of PBS-washed cultures in 60 mm Tris-HCl (pH 6.8), 2% (w/v) SDS, 10% (v/v) glycerol, and 5% (v/v) 2-mercaptoethanol. Equal quantities (10 μg) of whole cell extract were then fractionated by electrophoresis through a 12.5% SDS-polyacrylamide gel. Gels were run at a 20-mA constant current for 1.5 h prior to transfer onto nitrocellulose as previously described (4Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar, 6Elsharkawy A.M. Wright M.C. Hay R.T. Arthur M.J.P. Hughes T. Bahr M.J. Degitz K. Mann D.A. Hepatology. 1999; 30: 761-769Crossref PubMed Scopus (122) Google Scholar). Following blockade of nonspecific protein binding, nitrocellulose blots were incubated for 2 h with primary antibodies (diluted in PBS/Tween 20 (0.05%)) containing 5% Marvel. Rabbit polyclonal antibody recognizing JunD (Santa Cruz Biotechnology, Inc.) was used at a 1:100 dilution. Blots were then washed twice in PBS/Tween 20 prior to incubation for 1 h with sheep anti-rabbit horseradish peroxidase antibody (1:2000) and after extensive washing in PBS/Tween 20 before being processed to distilled water for detection of antigen using the ECL system (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). 3.2 μg of RNA extracted from freshly isolated and 7-day culture-activated rat HSCs was used to generate first-strand cDNA using a random hexamer primer (oligo(dN)6). PCR amplification of rat IL-6 and β-actin cDNAs was carried out using specific oligonucleotide primers selected within the coding regions of the rat genes. IL-6 primers used were 5′-CCACCCACAACAGACCAGTAT-3′ (sense) and 5′-TCCAGAAGACCAGAGCAGATT-3′ (antisense) and were designed to amplify sequences located between nucleotides 180 and 421 of the rat IL-6 cDNA. Primers used for detection of β-actin were 5′-AGAGGGAAATCGTGCGTGACA-3′ (sense) and 5′-ACATCTGCTGGAAGGTGGACA-3′ (antisense) and were designed to produce a 350-bp product. PCRs were composed of 1 μl of cDNA template, 100 ng each of sense and antisense oligonucleotide primers, 2.5 μl of optimized TaqPCR buffer (Promega), 0.4 mm dNTP mixture, and 2 units ofTaq polymerase in a total reaction volume of 25 μl. Following an initial 5-min incubation at 94 °C, PCRs were performed using a 1-min annealing step (at 51.5 °C for IL-6 and 57.0 °C for β-actin), followed by a 2-min elongation step at 72.0 °C and a 30-s denaturation step at 94 °C. A total number of 28 and 30 PCR cycles were carried out for detection of β-actin and IL-6, respectively, followed by a final elongation reaction for 10 min at 72.0 °C. PCR products were separated by electrophoresis at 80 V for 60 min through a 1% agarose gel and were detected by ethidium bromide staining. Expected sizes of specific PCR products (241 bp for IL-6 and 350 bp for β-actin) were verified by reference to a 1-kilobase DNA ladder, and sequence identity of the IL-6 product was confirmed by DNA sequence analysis. HSCs were transfected by the non-liposomal Effectene protocol (QIAGEN) according to the manufacturer's instructions. CAT assays were performed as previously described (4Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar, 6Elsharkawy A.M. Wright M.C. Hay R.T. Arthur M.J.P. Hughes T. Bahr M.J. Degitz K. Mann D.A. Hepatology. 1999; 30: 761-769Crossref PubMed Scopus (122) Google Scholar, 17Trim J.E. Samra S.K. Arthur M.J.P. Wright M.C. McAuley M. Beri R. Mann D.A. J. Biol. Chem. 2000; 275: 6657-6663Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) and normalized for differences in transfection efficiency either by the Hirts assay or by measurement of the activity of a cotransfected Renillaluciferase vector. Luciferase assays were performed using a dual luciferase kit (Promega) according to the manufacturer's instructions. IL-6 promoter-driven expression of firefly luciferase was normalized by reference to the level of activity of a cotransfectedRenilla luciferase vector. TIMP-1 promoter function in activated HSCs is dependent on an intact AP-1 site that binds JunD (4Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar). To determine the influence of JunD on the activity of the TIMP-1 promoter, rat HSCs were culture-activated for a minimum of 7 days prior to cotransfection with a human TIMP-1-CAT reporter (pTIMP1) and expression vectors for c-Jun, JunB, and JunD. Overexpression of JunD in activated rat HSCs resulted in a 2.5-fold enhancement of TIMP-1 promoter activity that was reproducible in replicate experiments (Fig.1 A). In contrast, overexpression of c-Jun or JunB resulted in a 2-fold or greater inhibition of TIMP-1 promoter activity. Overexpression of JunD failed to enhance the activity of a TIMP-1 promoter lacking an AP-1 site and did not alter the low activity of the TIMP-1 promoter in freshly isolated HSCs (data not shown). We next determined if the endogenous JunD activity expressed in activated rat HSCs is required for TIMP-1 gene transcription. Activated HSCs were cotransfected with pTIMP1 and a vector (RSVβ-JunD) producing expression of a mutant JunD protein lacking a functional transactivation domain (20Reid S. Jager C. Jeffers M. Vande Woude G.F. Graeff H. Schmitt M. Lengyel E. J. Biol. Chem. 1999; 274: 16377-16386Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). As shown in Fig. 1 B, RSVβ-JunD expression resulted in a profound inhibition of TIMP-1 promoter activity, with levels of transcription that were only marginally higher than those observed with pBLCAT3, which lacks a promoter. We have previously reported that culture-activated HSCs express JunD together with Fra2 and FosB (4Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar). To assess the role of Fos family proteins in the transcriptional control of the TIMP-1 gene, we cotransfected activated HSCs with pTIMP1 and expression vectors for c-Fos, FosB, Fra1, and Fra2 (Fig.2). Overexpression c-Fos or Fra1 caused a moderate reduction of promoter activity that lacked statistical significance; by contrast, overexpression of FosB or Fra2 resulted in a significant 2-fold repression of transcription. As activation of rat HSCs is accompanied by induction of FosB and Fra2 expression (4Bahr M.J. Vincent K.J. Arthur M.J.P. Fowler A.V. Smart D.E. Wright M.C. Clark I.M. Benyon R.C. Iredale J.P. Mann D.A. Hepatology. 1999; 29: 839-848Crossref PubMed Scopus (78) Google Scholar), it is possible that changes in the activity of these Fos proteins serve to fine-tune TIMP-1 transcription by forming AP-1 dimers that are less active than JunD homodimers. It was therefore of interest to establish if JunD homodimers are able to influence TIMP-1 promoter activity. Activated HSCs were cotransfected with pTIMP1 and a vector (JunD/eb1) that drives expression of a JunD protein in which the JunD dimerization domain is replaced with the dimerization domain from the Epstein-Barr virus EB1 transcription factor (19Vandel L. Montreau N. Vial E. Pfarr C.M. Binetruy B. Castellazzi M. Mol. Cell. Biol. 1996; 16: 1881-1888Crossref PubMed Scopus (56) Google Scholar). This mutant JunD protein is able to form transcriptionally active homodimers, but cannot form dimers with endogenous wild-type JunD, Fra2, or FosB. Expression of JunD/eb1 substantially enhanced TIMP-1 promoter function, generating a 4-fold higher level of CAT activity relative to cells transfected with a control empty expression vector (Fig.3 A). Hence, JunD/eb1 is a powerful positive regulator of TIMP-1 promoter function, and the data suggest that JunD homodimers are stronger AP-1 transactivators than JunD/Fra2 or JunD/FosB heterodimers. As Fra2 can also negatively regulate c-Jun activity (22Suzuki T. Okuno H. Yoshida T. Endo T. Nishina H. Iba H. Nucleic Acids Res. 1991; 19: 5537-5542Crossref PubMed Scopus (194) Google Scholar, 23Sonobe M.H. Yoshida T. Murakami M. Kameda T. Iba H. Oncogene. 1995; 10: 689-696PubMed Google Scholar) and can form heterodimers with JunB that act as transcriptional repressors in keratinocytes (24Rutberg S.E. Saez E. Lo S. Jang S.-I. Markova N. Spiegelmann B.M. Yuspa S.H. Oncogene. 1997; 15: 1337-1346Crossref PubMed Scopus (66) Google Scholar), it was conceivable that the negative influence of c-Jun and JunB on TIMP-1 promoter function in HSCs may arise from formation of repressive Jun/Fra2 heterodimers. We therefore determined the ability of c-Jun/eb1 and JunB/eb1 dimers to attenuate TIMP-1 promoter activity (Fig.3 B). In contrast to wild-type c-Jun, overexpression of the c-Jun/eb1 homodimer enhanced TIMP-1 promoter activity by 2-fold; however, overexpression of a JunB/eb1 dimer resulted in only a weak and statistically insignificant elevation of transcription.Figure 3Effects of expression of Jun/eb1 homodimers on TIMP-1 promoter activity in activated rat HSCs. 7-Day culture-activated rat HSCs were cotransfected with 1 μg of pTIMP1 and 3 μg of empty vector pDP7 (Control) or a pDP7-derived vector carrying a junD/eb1 fusion gene ( A) or 3 μg of empty vector pDP7 (Control) or a pDP7-derived vector carrying c-jun/eb1 or junB/eb1fusion genes (B). In both experiments, sister cultures were also cotransfected with 1 μg of the promoterless plasmid pBLCAT3 and 3 μg of pDP7 as a reference. CAT activities were determined 48 h after transfection. Results are expressed as the mean % CAT conversion with respect to control (pTIMP + pDP7) ± S.E. for three independent transfection experiments. Statistical analysis was performed by Student's t test. *, **, and ***,p < 0.05, 0.01, and 0.005, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It has previously been established that activated rat and human HSCs express IL-6 and that induction of IL-6 protein expression in response to stimulation of serum-starved HSCs can be suppressed by inhibition of NF-κB (5Hellerbrand C. Jobin C. Limuro Y. Licato L. Sartor R.B. Brenner D.A. Hepatology. 1998; 27: 1285-1295Crossref PubMed Scopus (175) Google Scholar, 25Tiggelman A.M. Boers W. Linthorst C. Brand H.S. Sala M. Chamuleau R.A. J. Hepatol. ( Amst. ). 1995; 23: 295-306Abstract Full Text PDF PubMed Scopus (73) Google Scholar). To determine if IL-6 mRNA expression is induced during HSC activation, we used reverse transcriptase-PCR to detect IL-6 mRNA in freshly isolated (quiescent) and culture-activated rat HSCs. The presence of IL-6 mRNA was detected in this assay by amplification of a 241-bp cDNA fragment (Fig. 4), which was later verified as a fragmen
Referência(s)