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Transcriptional Regulation of Heme Oxygenase-1 Gene Expression by MAP Kinases of the JNK and p38 Pathways in Primary Cultures of Rat Hepatocytes

2003; Elsevier BV; Volume: 278; Issue: 20 Linguagem: Inglês

10.1074/jbc.m203929200

ISSN

1083-351X

Autores

Thomas Kietzmann, Anatoly Samoylenko, Stephan Immenschuh,

Tópico(s)

Aldose Reductase and Taurine

Resumo

Heme oxygenase-1 (HO-1) gene expression is induced by various oxidative stress stimuli including sodium arsenite. Since mitogen-activated protein kinases (MAPKs) are involved in stress signaling we investigated the role of arsenite and MAPKs for HO-1 gene regulation in primary rat hepatocytes. The Jun N-terminal kinase (JNK) inhibitor SP600125 decreased sodium arsenite-mediated induction of HO-1 mRNA expression. HO-1 protein and luciferase activity of reporter gene constructs with −754 bp of the HO-1 promoter were induced by overexpression of kinases of the JNK pathway and MKK3. By contrast, overexpression of Raf-1 and ERK2 did not affect expression whereas overexpression of p38α, β, and δ decreased and p38γ increased HO-1 expression. Electrophoretic mobility shift assays (EMSA) revealed that a CRE/AP-1 element (−668/−654) bound c-Jun, a target of the JNK pathway. Deletion or mutation of the CRE/AP-1 obliterated the JNK- and c-Jun-dependent up-regulation of luciferase activity. EMSA also showed that an E-box (−47/−42) was bound by a putative p38 target c-Max. Mutation of the E-box strongly reduced MKK3, p38 isoform-, and c-Max-dependent effects on luciferase activity. Thus, the HO-1 CRE/AP-1 element mediatesHO-1 gene induction via activation of JNK/c-Jun whereas p38 isoforms act through a different mechanism via the E-box. Heme oxygenase-1 (HO-1) gene expression is induced by various oxidative stress stimuli including sodium arsenite. Since mitogen-activated protein kinases (MAPKs) are involved in stress signaling we investigated the role of arsenite and MAPKs for HO-1 gene regulation in primary rat hepatocytes. The Jun N-terminal kinase (JNK) inhibitor SP600125 decreased sodium arsenite-mediated induction of HO-1 mRNA expression. HO-1 protein and luciferase activity of reporter gene constructs with −754 bp of the HO-1 promoter were induced by overexpression of kinases of the JNK pathway and MKK3. By contrast, overexpression of Raf-1 and ERK2 did not affect expression whereas overexpression of p38α, β, and δ decreased and p38γ increased HO-1 expression. Electrophoretic mobility shift assays (EMSA) revealed that a CRE/AP-1 element (−668/−654) bound c-Jun, a target of the JNK pathway. Deletion or mutation of the CRE/AP-1 obliterated the JNK- and c-Jun-dependent up-regulation of luciferase activity. EMSA also showed that an E-box (−47/−42) was bound by a putative p38 target c-Max. Mutation of the E-box strongly reduced MKK3, p38 isoform-, and c-Max-dependent effects on luciferase activity. Thus, the HO-1 CRE/AP-1 element mediatesHO-1 gene induction via activation of JNK/c-Jun whereas p38 isoforms act through a different mechanism via the E-box. heme oxygenase basic helix-loop-helix leucine zipper dominant-negative H-Ras electrophoretic mobility shift assay extracellular signal-regulated kinase firefly luciferase c-Jun N-terminal kinase luciferase mitogen-activated protein MAP kinase activated form of p38 hemagglutinin MAPK/ERK kinase MEK kinase MAP kinase kinase cAMP response element CRE-binding protein The microsomal enzyme heme oxygenase (HO,1 EC 1.14.99.3) catalyzes the first and rate-limiting step of heme degradation producing carbon monoxide (CO), Fe2+, and biliverdin, which is converted into bilirubin by biliverdin reductase (1Tenhunen R. Marver H.S. Schmid R. Proc. Natl. Acad. Sci. U. S. A. 1968; 61: 748-755Crossref PubMed Scopus (1501) Google Scholar). 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Holbrook N.J. Liu Y. Mol. Cell. Biol. 1998; 18: 5178-5188Crossref PubMed Scopus (139) Google Scholar) and recruitment of the Grb2-Sos complex which can activate the p21 small GTPases of the Rho family Ras and Rac. Ras, in turn, activates either the ERK pathway or via action on PAK the JNK or p38 pathway. Rac activation can lead again to PAK activation and subsequently to the activation of JNK and p38 (26Takai Y. Sasaki T. Matozaki T. Physiol. Rev. 2001; 81: 153-208Crossref PubMed Scopus (2048) Google Scholar). In liver cells the only detailed report so far regarding the role of MAPKs in HO-1 gene regulation had demonstrated that in the LMH chicken hepatoma cell line sodium arsenite-dependent induction of HO-1 gene expression was mediated by the ERK and p38 pathways (21Elbirt K.K. Whitmarsh A.J. Davis R.J. Bonkovsky H.L. J. Biol. Chem. 1998; 273: 8922-8931Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Despite the extensive characterization of the MAPK signal transduction pathways and the nearly universal induction of HO-1 expression by stress-related stimuli, conflicting data have been reported as to the involvement of MAPKs pathways in HO-1 gene activation in various tumor cell lines (20Masuya Y. Hioki K. Tokunaga R. Taketani S. J. Biochem. (Tokyo). 1998; 124: 628-633Crossref PubMed Scopus (57) Google Scholar, 21Elbirt K.K. Whitmarsh A.J. Davis R.J. Bonkovsky H.L. J. Biol. Chem. 1998; 273: 8922-8931Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 27Alam J. Wicks C. Stewart D. Gong P. Touchard C. Otterbein S. Choi A.M. Burow M.E. Tou J. J. Biol. Chem. 2000; 275: 27694-27702Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 28Yu R. Chen C. Mo Y.Y. Hebbar V. Owuor E.D. Tan T.H. Kong A.-N.T. J. Biol. Chem. 2000; 275: 39907-39913Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 29Chen K. Maines M.D. Cell Mol. Biol. Noisy. le. grand. 2000; 46: 609-617PubMed Google Scholar). Moreover, little is known about HO-1gene expression in response to the activation by arsenite and MAPKs in primary cultured cells. Thus, it was the goal of the present study to investigate the regulation of rat HO-1 gene expression by MAPKs using primary rat hepatocyte cultures as a model system. In our study basal levels of HO-1 mRNA expression as well as sodium arsenite-induced HO-1 mRNA levels were down-regulated in the presence of JNK inhibitor SP600125. It was demonstrated that the Ras pathway via JNK induced HO-1 gene expression while the Raf pathway via ERK was not involved in the regulation of HO-1gene expression. The promoter sequence −668/−654 of the ratHO-1 gene was shown to be responsible for HO-1 activation by JNK and Jun. The E-box element (−47/−42) was involved in the inhibition of HO-1 gene expression via an MKK3-independent mechanism by p38 and Max. All biochemicals and enzymes were of analytical grade and were purchased from commercial suppliers. Cells from male Wistar rats (200–250 g) were used for culture experiments. Hepatocytes were isolated by collagenase perfusion (30Immenschuh S. Iwahara S. Satoh H. Nell C. Katz N. Muller E.U. Biochemistry. 1995; 34: 13407-13411Crossref PubMed Scopus (67) Google Scholar). About 1 × 106 cells were cultured at 37 °C on 6-cm Falcon culture dishes under air/CO2 (19:5) in medium 199 with Earle's salts containing bovine serum albumin (2 g/liter), NaHCO3 (20 mm), Hepes (10 mm), streptomycin sulfate (117 mg/liter), penicillin (60 mg/liter), insulin (1 nm), and dexamethasone (10 nm). Fetal calf serum (5%) was present in the initial 5 h after which cultures were incubated in serum-free medium for another 18 h. Then, medium was changed again, and the cells were further cultured in serum-free medium for 24 h. Hepatocytes were treated with 5 μm sodium arsenite 8 h before harvesting. The specific inhibitors of MEK PD98059 (New England BioLabs) (5 μm) and U0126 (Cell Signaling) (10 μm), of JNK SP600125 (Biomol) (25 μm) and of p38 SB203580 (Calbiochem) (20 μm) were added to the culture medium also 8 h before harvesting. The luciferase reporter gene constructs pHO-754 and pHO-754ΔA were previously described (5Immenschuh S. Hinke V. Ohlmann A. Gifhorn K.S. Katz N. Jungermann K. Kietzmann T. Biochem. J. 1998; 334: 141-146Crossref PubMed Scopus (102) Google Scholar). The luciferase reporter gene construct pHO-347 was generated by deletion of the −754/−347 fragment of HO-754 by ApaI and KpnI followed by blunting of the remaining vector with Klenow enzyme and ligation. The luciferase construct pHO-754Em was generated with the QuickChangeTM XL site-directed mutagenesis kit (Stratagene) using the oligonucleotide 5′-GGCTCAGCTGGGCGGCCACctctagACTCGAGTAC-3′. The luciferase constructs p(CRE/AP-1)3-SV40Luc and p(CRE/AP-1)6-SV40Luc were generated by ligation of the oligonucleotide 5′-GATCCTGACTTCAGTCTGAATTCCTGACTTCAGTCTGACTTCAGTC-3′ containing 3 CRE/AP-1 elements into the BglII site of pGl3 prom (pSV40Luc) (Promega). Expression vectors for MAP kinase signaling pathway components and transcription factors have been described: ERK2 (31Whitmarsh A.J. Yang S.H. Su M.S. Sharrocks A.D. Davis R.J. Mol. Cell. Biol. 1997; 17: 2360-2371Crossref PubMed Scopus (438) Google Scholar), MEKK1 (32Xu S. Robbins D.J. Christerson L.B. English J.M. Vanderbilt C.A. Cobb M.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5291-5295Crossref PubMed Scopus (122) Google Scholar), JNK1 (33Derijard B. Hibi M. Wu I.H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2953) Google Scholar), MKK3, dominant-negative MKK3, dominant-negative MKK4 (34Raingeaud J. Whitmarsh A.J. Barrett T. Derijard B. Davis R.J. Mol. Cell. Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1140) Google Scholar), p38 isoforms, and dominant-negative p38 isoforms (16Han J. Lee J.D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2407) Google Scholar, 35Han J. Lee J.D. Jiang Y. Li Z. Feng L. Ulevitch R.J. J. Biol. Chem. 1996; 271: 2886-2891Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar, 36Jiang Y. Chen C. Li Z. Guo W. Gegner J.A. Lin S. Han J. J. Biol. Chem. 1996; 271: 17920-17926Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar, 37Jiang Y. Gram H. Zhao M. New L. Gu J. Feng L. Di Padova F. Ulevitch R.J. Han J. J. Biol. Chem. 1997; 272: 30122-30128Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar), H-Ras (38Feig L.A. Cooper G.M. Mol. Cell. Biol. 1988; 8: 3235-3243Crossref PubMed Scopus (679) Google Scholar), dominant-negative H-Ras (dnRas) (38Feig L.A. Cooper G.M. Mol. Cell. Biol. 1988; 8: 3235-3243Crossref PubMed Scopus (679) Google Scholar), Raf-1 (39Dent P. Reardon D.B. Morrison D.K. Sturgill T.W. Mol. Cell. Biol. 1995; 15: 4125-4135Crossref PubMed Scopus (124) Google Scholar), c-Jun (40Schonthal A. Buscher M. Angel P. Rahmsdorf H.J. Ponta H. Hattori K. Chiu R. Karin M. Herrlich P. Oncogene. 1989; 4: 629-636PubMed Google Scholar), c-Myc (41Kretzner L. Blackwood E.M. Eisenman R.N. Nature. 1992; 359: 426-429Crossref PubMed Scopus (380) Google Scholar). Isolation of total RNA and Northern analysis were performed as described (42Kietzmann T. Roth U. Freimann S. Jungermann K. Biochem. J. 1997; 321: 17-20Crossref PubMed Scopus (49) Google Scholar). Digoxigenin (DIG)-labeled antisense RNAs served as hybridization probes; they were generated as described (43Kietzmann T. Hirsch E.K. Kahl G.F. Jungermann K. Mol. Pharmacol. 1999; 56: 46-53Crossref PubMed Scopus (25) Google Scholar) by in vitro transcription from pBS-HO-1 (800-bp cDNA fragment) using T3 RNA polymerase or from pBS-β actin (550-bp cDNA fragment) using T7 RNA polymerase and RNA labeling mixture containing 3.5 mm 11-DIG-UTP, 6.5 mm UTP, 10 mm GTP, 10 mm CTP, 10 mm ATP. Hybridizations and detections were carried out essentially as described before (42Kietzmann T. Roth U. Freimann S. Jungermann K. Biochem. J. 1997; 321: 17-20Crossref PubMed Scopus (49) Google Scholar). Blots were quantified with a videodensitometer (Biotech Fischer, Reiskirchen). Western blot analysis was carried out as described (5Immenschuh S. Hinke V. Ohlmann A. Gifhorn K.S. Katz N. Jungermann K. Kietzmann T. Biochem. J. 1998; 334: 141-146Crossref PubMed Scopus (102) Google Scholar). In brief, total protein from primary cultured hepatocytes was prepared and the protein content was determined using the Bradford method. 50 μg of protein were loaded onto a 10% SDS-polyacrylamide gel and after electrophoresis blotted onto nitrocellulose membranes. The primary antibodies against rat HO-1 (Stressgen), rat HO-2 (Stressgen), phospho-c-Jun (Ser63) (Cell Signaling), phospho-HSP27 (Ser82) (Cell Signaling), phospho-p38 (Cell Signaling), H-Ras (Santa Cruz), and c-Fos (Santa Cruz) were rabbit and used at 1:1000 dilution. The primary monoclonal mouse antibody against β-actin (Sigma) was used at 1:5000 dilution, the primary mouse antibody against phospho-p44/42 MAPK (Thr-202/Tyr-204) (Cell Signaling) was used at 1:2000 dilution, the primary mouse antibody against c-Jun (Santa Cruz Biotechnology), the primary monoclonal mouse antibody against HA tag (Cell Signaling) and primary monoclonal mouse antibody against FLAG M2 (Sigma) were used at 1:1000 dilution. The secondary antibodies were goat anti-rabbit IgG (Bio-Rad) and goat anti-mouse IgG (Bio-Rad) and used at 1:2000 dilution. The ECL Western blotting system (Amersham Biosciences) was used for detection. HO-1 was visible as a 32-kDa band, HO-2 as a 36-kDa band, β-actin as a 42-kDa band, H-Ras as a 21-kDa band, c-Fos as a 55-kDa band, c-Jun as a 39-kDa band, phospho-HSP27 as a 27-kDa band, phospho-ERK was seen as a double band of 42 and 44 kDa. HA-MEKK1 was seen as 186-kDa band, HA-ERK as a 42-kDa band, FLAG-Raf as a 68-kDa band, FLAG-MKK3 as a 40-kDa band, FLAG-JNK1 as a 56-kDa band, and FLAG-p38 as a 38-kDa band. Freshly isolated rat hepatocytes (about 1 × 106 cells per dish) were transfected as described (5Immenschuh S. Hinke V. Ohlmann A. Gifhorn K.S. Katz N. Jungermann K. Kietzmann T. Biochem. J. 1998; 334: 141-146Crossref PubMed Scopus (102) Google Scholar). In brief, rat hepatocyte cultures (about 1 × 106 cells per dish) were transiently transfected with 2.5 μg of plasmid DNA containing 500 ng of theRenilla luciferase construct pRL-SV40 (Promega) to control transfection efficiency and 2 μg of the appropriate HO-1promoter Firefly luciferase (FL) construct. In every culture experiment two cultures were transfected per point. The DNA was transfected as a calcium phosphate precipitate (5Immenschuh S. Hinke V. Ohlmann A. Gifhorn K.S. Katz N. Jungermann K. Kietzmann T. Biochem. J. 1998; 334: 141-146Crossref PubMed Scopus (102) Google Scholar) for 5 h. After removal of the medium cells were cultured under standard conditions without serum. 24 h later cells were treated with fresh medium and were cultured for another 12 h. 12 h before harvesting the cells were treated with PD98059 (5 μm), U0126 (10 μm), SP600125 (25 μm), SB203580 (20 μm), or LY294002 (Cell Signaling) (10 μm), as indicated. The Luc activity of 20 μl of cell lysate was recorded in a luminometer (Berthold) using the dual luciferase assay kit (Promega). Nuclear extracts were prepared essentially as described (44Kietzmann T. Roth U. Jungermann K. Blood. 1999; 94: 4177-4185Crossref PubMed Google Scholar). The sequences of the HO-1 oligonucleotides used for the EMSA are 5′-TGTGTCAGAGCCATGTGTCCTGACTTCAGTCT-3′ (spanning the CRE/AP-1 (−664/−657), Fig. 4) and 5′-GGCTCAGCTGGGCGGCCACCACGTGACTCGAGTAC-3′ (spanning the E-box (−47/−42), Fig. 6). Equal amounts of complementary oligonucleotides were annealed and labeled by 5′ end-labeling with [γ-32P]ATP (Amersham Biosciences) and T4 polynucleotide kinase (MBI). They were purified with the Nucleotide Removal kit (Qiagen). Binding reactions were carried out in a total volume of 20 μl containing 50 mm KCl, 1 mmMgCl2, 1 mm EDTA, 5% glycerol, 10 μg of nuclear extract, 250 ng of poly(d(I-C)), and 5 mm DTE. For competition analyses a 50-fold molar excess of unlabeled AP1 consensus oligonucleotide (Promega) was added. After preincubation for 5 min at room temperature, 1 μl of the labeled probe (104 cpm) was added, and the incubation was continued for an additional 10 min. For supershift analysis 1 μl of the ATF/CREB-1 (24H4B), c-JunC (epitope corresponding to DNA binding domain), c-JunN (epitope mapping within the N-terminal domain), c-Fos (K-25), Myc (C33), Max (C17) or SP-1 (PEP2-G) antibody (all obtained from Santa Cruz Biotechnology) as well as a rabbit preimmune serum was added to the EMSA reaction, which was then incubated at 4 °C for 2 h. The electrophoresis was then performed with a 5% non-denaturing polyacrylamide gel in TBE buffer (89 mm Tris, 89 mm boric acid, 5 mmEDTA) at 200 V. After electrophoresis the gels were dried and exposed to a phosphorimager screen.Figure 6Overexpression of c-Jun induced luciferase expression controlled by either 754 bp of the HO-1promoter or by 6 and 3 copies of the HO-1 CRE/AP1 element.A, hepatocytes were transiently cotransfected with either c-Jun, c-Fos, or both expression plasmids and Luc gene constructs driven by the rat HO-1 promoter (pHO-754 and pHO-754ΔA) or by the SV40 promoter regulated by 6 or 3 copies of the HO-1 CRE/AP1 elements (p(CRE/AP-1)6-SV40Luc and p(CRE/AP-1)3-SV40Luc). In control experiments Luc constructs were cotransfected with pCMV plasmid and pSV40Luc construct was cotransfected with c-Jun, c-Fos, or both expression vectors. In each experiment the fold induction of Luc activity was determined relative to the pHO-754, pHO-754ΔA, p(CRE/AP-1)6-SV40Luc, p(CRE/AP-1)3-SV40Luc, or pSV40Luc controls, which were set equal to 1. In pHO-754ΔA the wild-type HO-1 sequence is shown on the upper strand, deleted bases are indicated by −, and mutated bases are shown in lowercase letters and are indicated by *. The values represent means ± S.E. of three independent experiments. Statistics, Student's t test for paired values: *, significant difference pHO-754+c-Fos versus pHO-754 control, pHO-754+c-Jun+c-Fos versus pHO-754 control, p(CRE/AP-1)6-SV40Luc+c-Jun versusp(CRE/AP-1)6-SV40Luc control, p(CRE/AP-1)6-SV40Luc+c-Jun+c-Fos versusp(CRE/AP-1)6-SV40Luc control, p(CRE/AP-1)3-SV40Luc+c-Jun versusp(CRE/AP-1)3-SV40Luc control, p ≤ 0.05.B, overexpression of Jun and Fos proteins was detected by Western blotting (see "Experimental Procedures").View Large Image Figure ViewerDownload (PPT) To investigate whether the sodium arsenite-dependent HO-1 induction occurs via the activation of various MAPK signaling pathways primary hepatocytes were treated with sodium arsenite in the presence of specific inhibitors of the ERK, JNK, or p38 pathways. 5 μm sodium arsenite induced HO-1 mRNA expression in primary rat hepatocytes after 8 h of treatment by about 8-fold (Fig. 1, A andB), in line with a previous study (45Jacobs J.M. Nichols C.E. Andrew A.S. Marek D.E. Wood S.G. Sinclair P.R. Wrighton S.A. Kostrubsky V.E. Sinclair J.F. Toxicol. Appl. Pharmacol. 1999; 157: 51-59Crossref PubMed Scopus (51) Google Scholar). The inhibitors of the ERK pathway PD98059 and U0126 as well as the p38 pathway inhibitor SB203580 did not change either basal or arsenite-enhanced HO-1 mRNA levels. In contrast, the JNK inhibitor SP600125 attenuated the sodium arsenite-dependent HO-1 mRNA induction. Interestingly, combination of the JNK and p38 inhibitors led to a more pronounced inhibition of the arsenite-dependent HO-1 mRNA induction pointing to the involvement of p38, which was overridden by the JNK pathway. The effectiveness of the kinase inhibitors at the concentrations used was demonstrated by Western blot analysis with antibodies against phospho-ERK (for ERK pathway), phospho-Jun63 (for JNK pathway), and phospho-HSP27 (for p38 pathway) (Fig. 1C). These results indicated that kinases of the JNK and p38 pathways were involved in the regulation of HO-1 expression by sodium arsenite. Since the JNK and p38 pathways can be activated by MEKK1, the question of whether overexpression of MEKK1 activates these pathways in hepatocytes was examined. Indeed, MEKK1 overexpression led to the activation of the JNK and p38 pathway as demonstrated by the phosphorylation of cJun and p38 (Fig. 1D). To further investigate the regulatory role of MAP kinases forHO-1 gene expression primary cultured rat hepatocytes were transfected with expression vectors for human H-Ras, Raf-1, MEKK1, MKK3, ERK2, JNK1, and p38. Overexpression of these proteins was confirmed by Western blot analysis. Hepatocytes transfected with H-Ras, MEKK1, JNK1, or MKK3 expression vectors showed enhanced HO-1 protein levels. By contrast, transfection with Raf-1 as well as ERK2 had no effect on the HO-1 expression while transfection with p38 led to a significant decrease of HO-1 protein levels (Fig.2). The observed pattern appeared to be specific for HO-1 since neither construct changed expression of HO-2 or actin. To examine the molecular mechanism of HO-1 gene regulation by MAPKs luciferase reporter gene constructs from the ratHO-1 promoter (46Muller R.M. Taguchi H. Shibahara S. J. Biol. Chem. 1987; 262: 6795-6802Abstract Full Text PDF PubMed Google Scholar) (pHO-754, pHO-750ΔA (5Immenschuh S. Hinke V. Ohlmann A. Gifhorn K.S. Katz N. Jungermann K. Kietzmann T. Biochem. J. 1998; 334: 141-146Crossref PubMed Scopus (102) Google Scholar), and pHO-347) were cotransfected with expression vectors encoding various components of the MAPK signaling pathways (Fig. 3). Luciferase expression of pHO-754 was up-regulated by constitutive active H-Ras, MEKK1, and MKK3 but not by Raf-1 or dominant-negative Ras (dnRas). After cotransfection of both H-Ras and dnRas expression vectors the stimulatory effect of H-Ras was attenuated by dnRas (Fig.3). Cotransfection of MEKK1 and dominant-negative mutants of MKK3 and MKK4 down-regulated HO-1 promoter activity. The up-regulation of Luc activity of pHO-754ΔA and pHO-347, both of which lack the previously described HO-1 CRE/AP-1 element (5Immenschuh S. Hinke V. Ohlmann A. Gifhorn K.S. Katz N. Jungermann K. Kietzmann T. Biochem. J. 1998; 334: 141-146Crossref PubMed Scopus (102) Google Scholar), by overexpressed MEKK1 was reduced compared with pHO-754 (Fig.3A). Furthermore, the dominant-negative MKK3 still abolished Luc activity whereas the dominant-negative MKK4 did not. The same pattern was seen with pHO-347. This indicated that the JNK pathway acted primarily through the CRE/AP-1 element, and the p38 pathway via an element inside the first −347 bases of the HO-1promoter. However, the remaining induction of pHO-347 by MEKK1 was not attenuated by the inhibitors of either ERK, JNK, p38, or PI3K/PKB because neither PD98059, U0126, SP600125, SB203580, nor the phosphatidylinositol 3-kinase inhibitor LY294002 down-regulated MEKK1-dependent enhancement of Luc activity. By contrast, the inhibitor of the p38 pathway SB203580 led to an induction of Luc activity. Because MEKK1 activates the JNK pathway, which is known to have the transcription factor AP-1 as a nuclear target (47Tibbles L.A. Woodgett J.R. Cell Mol. Life Sci. 1999; 55: 1230-1254Crossref PubMed Scopus (550) Google Scholar, 48Vuong H. Patterson T. Shapiro P. Kalvakolanu D.V. Wu R. Ma W.Y. Dong Z. Kleeberger S.R. Reddy S.P. J. Biol. Chem. 2001; 275: 32250-32259Abstract Full Text Full Text PDF Scopus (43) Google Scholar) the regulation of HO-1 gene promoter constructs was investigated in the presence of overexpressed JNK1, and for comparison also ERK2. JNK1 enhanced Luc activity of pHO-754, which was abolished in the presence of the JNK inhibitor SP600125. A JNK-dependent induction was not observed with pHO-754ΔA or pHO-347. By contrast, ERK2 did not affect Luc expression of the transfected HO-1 reporter gene constructs (Fig. 4). Taken together, these results indicate that activation of the JNK pathway may induce HO-1 gene expression through the HO-1 CRE/AP-1 site. Since the transcription factor Jun is a substrate of JNK it was investigated whether c-Jun can bind directly to the CRE/AP-1 element of the HO-1 promoter. EMSA studies with nuclear extracts of cultured rat hepatocytes and an oligonucleotide containing the HO-1 CRE/AP-1 site formed a strong protein-DNA complex. The intensity of this complex was reduced by an unlabeled AP-1 consensus oligonucleotide added in a 50-fold molar excess to the binding reaction (Fig.5). To investigate the presence of AP-1 and members of the ATF/CREB family in this complex antibodies against c-Jun, c-Fos, and ATF/CREB were included in the binding reactions. In the presence of the ATF/CREB antibody a slightly supershifted complex was observed (Fig. 5). In addition, the binding of the protein complex to the CRE/AP-1 nucleotide was enhanced. Addition of antibodies against c-Fos, as well as against the GC-box binding factor SP-1 or a rabbit preimmune serum, did not result in a supershift or inhibition of complex formation. The Jun antibody, which was raised against the highly conserved DNA binding domain of c-Jun (c-JunC) abolished DNA-protein complex formation. Addition of the c-JunN antibody generated against the N-terminal domain formed a strong supershifted complex, indicating that the CRE/AP-1 site was bound mostly by c-Jun. To verify the functional relevance of c-Jun binding to the HO-1 CRE/AP-1 element the pHO-754 and pHO-754ΔA luciferase constructs were cotransfected with expression vectors for c-Jun and c-Fos. Overexpression of both c-Jun and c-Fos was confirmed by Western blot ana

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