Protein Kinase C-dependent, CCAAT/Enhancer-binding Protein β-mediated Expression of Insulin-like Growth Factor I Geneстатья из журнала
Аннотация: The possible involvement of the protein kinase C (PKC) pathway in transcriptional regulation of the human insulin-like growth factor-I (IGF-I) gene has been suggested. In this study, we sought to determine whether a PKC-dependent pathway is implicated in the transcriptional control, and if it is, how this occurs. Treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) caused an increase in the activity of the human IGF-I gene major promoter in HepG2 cells. A CCAAT/enhancer-binding protein (C/EBP) binding site located at +22 to +30 was bound by C/EBPβ in a TPA-dependent manner and was solely responsible for the TPA responsiveness. This increase in C/EBPβ activity occurs through transcriptional and posttranslational regulation, and the latter is mediated by activation of p90 ribosomal S6 kinase (RSK): co-expression of dominant negative RSK abolished the TPA-responsive and C/EBPβ-dependent transactivation. Also, TPA-responsive activation of GAL4-C/EBPβ chimera required the Ser residue known as the RSK target. In SK-N-MC cells, which display constitutive, high expression of IGF-I on use of the major promoter, a large amount of C/EBPβ binding was observed with the C/EBP site in the basal state. Treatment with PKC inhibitors substantially reduced the promoter activity and mRNA amounts of IGF-I, with the binding of C/EBPβ to the C/EBP site also being reduced. When the C/EBP site was disrupted, the basal promoter activity was reduced, but the reduction by the PKC inhibitor was no longer observed. These observations suggest that the increase of C/EBPβ binding to the C/EBP site, which is in part mediated via activation of RSK, can primarily explain the TPA responsiveness of the IGF-I gene promoter. The intrinsic PKC activity in SK-N-MC cells should play a major role in the constitutive, high expression of IGF-I and may therefore contribute in part to the maintenance of the tumor phenotype of the cells. The possible involvement of the protein kinase C (PKC) pathway in transcriptional regulation of the human insulin-like growth factor-I (IGF-I) gene has been suggested. In this study, we sought to determine whether a PKC-dependent pathway is implicated in the transcriptional control, and if it is, how this occurs. Treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) caused an increase in the activity of the human IGF-I gene major promoter in HepG2 cells. A CCAAT/enhancer-binding protein (C/EBP) binding site located at +22 to +30 was bound by C/EBPβ in a TPA-dependent manner and was solely responsible for the TPA responsiveness. This increase in C/EBPβ activity occurs through transcriptional and posttranslational regulation, and the latter is mediated by activation of p90 ribosomal S6 kinase (RSK): co-expression of dominant negative RSK abolished the TPA-responsive and C/EBPβ-dependent transactivation. Also, TPA-responsive activation of GAL4-C/EBPβ chimera required the Ser residue known as the RSK target. In SK-N-MC cells, which display constitutive, high expression of IGF-I on use of the major promoter, a large amount of C/EBPβ binding was observed with the C/EBP site in the basal state. Treatment with PKC inhibitors substantially reduced the promoter activity and mRNA amounts of IGF-I, with the binding of C/EBPβ to the C/EBP site also being reduced. When the C/EBP site was disrupted, the basal promoter activity was reduced, but the reduction by the PKC inhibitor was no longer observed. These observations suggest that the increase of C/EBPβ binding to the C/EBP site, which is in part mediated via activation of RSK, can primarily explain the TPA responsiveness of the IGF-I gene promoter. The intrinsic PKC activity in SK-N-MC cells should play a major role in the constitutive, high expression of IGF-I and may therefore contribute in part to the maintenance of the tumor phenotype of the cells. insulin-like growth factor-I protein kinase C 12-O-tetradecanoylphorbol 13-acetate CCAAT/enhancer-binding protein p90 ribosomal S6 kinase hemagglutinin dominant negative phosphate-buffered saline amino acids mitogen-activated protein kinases mitogen-activated protein kinase/extracellular signal-regulated kinase kinase type-I insulin-like growth factor receptor Insulin-like growth factor I (IGF-I),1 a 70-residue single-chain growth-promoting polypeptide, is produced in many organs and tissues and plays a major role in somatic growth, cell survival, tissue differentiation, and intermediary metabolism (1Jones J.I. Clemmons D.R. Endocr. Rev. 1995; 16: 3-34Crossref PubMed Google Scholar, 2Stewart C.E. Rotwein P. Physiol. Rev. 1996; 76: 1005-1026Crossref PubMed Scopus (698) Google Scholar, 3Baserga R. Hongo A. Rubini M. Prisco M. Valentinis B. Biochim. Biophys. Acta. 1997; 1332: F105-F126Crossref PubMed Scopus (488) Google Scholar). Although various tissue-dependent factors as well as endocrine hormones seem to regulate the IGF-I gene expression, their mechanisms, except for those involved in prostaglandin E2 or cAMP (4Thomas M.J. Umayahara Y. Shu H. Centrella M. Rotwein P. McCarthy T.L. J. Biol. Chem. 1996; 271: 21835-21841Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 5Ji Y. Umayahara C. Centrella M. Rotwein P. McCarthy T.L. J. Biol. Chem. 1997; 272: 31793-31800Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 6Umayahara Y. Ji J. Billiard C. Centrella M. McCarthy T.L. Rotwein P. J. Biol. Chem. 1999; 274: 10609-10617Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), are poorly understood. The protein kinase-C (PKC) pathway is among the few that have been suggested to be involved in IGF-I gene regulation. The result of a nuclear run-on assay indicated that treatment of human macrophage-like cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) increased the transcription rate of the IGF-I gene 4- to 5-fold (7Nagaoka I. Trapnell B.C. Crystal R.G. J. Clin. Invest. 1990; 85: 448-455Crossref PubMed Scopus (73) Google Scholar), suggesting that the human IGF-I gene regulatory sequences contain something that responds to PKC. Support for this also comes from our recent observations with the chicken IGF-I gene, i.e. that the gene promoter can be activated by TPA through an AP-1 binding site located in it (8Kajimoto Y. Kawamori R. Umayahara Y. Iwama N. Imano E. Morishima T. Yamasaki Y. Kamada T. Biochem. Biophys. Res. Commun. 1993; 190: 767-773Crossref PubMed Scopus (9) Google Scholar). However, it is unknown whether the mammalian IGF-I genes are activated by PKC, and if they are, how this occurs. In contrast to protein kinase A, which seems to be involved in parathyroid hormone or prostaglandin E2-induced IGF-I gene activation, PKC has been often discussed in correlation with tumorigenesis. Indeed, the best-known activator of PKC, TPA, is a strong tumor promoter (9Blobe G.C. Obeid L.M. Hannun Y.A. Cancer Metastasis. Rev. 1994; 13: 411-431Crossref PubMed Scopus (259) Google Scholar, 10Gomez D.E. Skilton G. Alonso D.F. Kazanietz M.G. Oncol. Rep. 1999; 6: 1363-1370PubMed Google Scholar). In vitro overexpression studies have suggested that individual PKC isozymes control cell proliferation and malignant transformation. For example, when PKCβI was overexpressed in rat fibroblasts, the cells were partially transformed and could form tumors in nude mice (11Housey G.M. Johnson M.D. Hsiao W.L. O'Brian C.A. Murphy J.P. Kirschmeier P. Weinstein I.B. Cell. 1988; 52: 343-354Abstract Full Text PDF PubMed Scopus (424) Google Scholar). Overexpression of PKCα also occasionally leads to transformation of fibroblasts (12Megidish T. Mazurek N. Nature. 1989; 342: 807-811Crossref PubMed Scopus (105) Google Scholar). Because the IGF system is known to play an essential role in inducing transformation (13Baserga R. Exp. Cell Res. 1999; 253: 1-6Crossref PubMed Scopus (267) Google Scholar) or maintaining the tumor phenotype in some cells, such as a human neuroblastoma cell line SK-N-MC (14Kiess W. Koepf G. Christiansen H. Blum W.F. Regul. Pept. 1997; 72: 19-29Crossref PubMed Scopus (32) Google Scholar), it is likely that PKC-dependent activation of the IGF-I gene, if it occurs in mammals, may be partially involved in the tumorigenesis. As a step toward elucidating the molecular basis of IGF-I gene regulation, we examined whether the human IGF-I gene promoter is a target of PKC regulation and sought to elucidate the physiological roles of the PKC pathway in the gene expression. Here we report that the major promoter of the gene, which is located within the 5′-flanking region and untranslated region of exon 1, is indeed a target of TPA stimulation and a CCAAT/enhancer binding protein (C/EBP) site within the promoter is responsible for the phenomenon. C/EBPβ, which is activated by PKC both at the level of transcription and of posttranslation, binds to the C/EBP site and thereby mediates the phenomenon. Interestingly, the posttranslational activation of C/EBPβ occurs primarily through activation of p90 ribosomal S6 kinase (RSK). Moreover, as support for the pathophysiological significance of these findings, we found that the constitutive IGF-I gene expression in the human neuroblastoma-derived SK-N-MC cells depends on the intrinsically activated PKC. Antibodies to C/EBPα (14AA), C/EBPβ (C-19), C/EBPδ (C-22), HNF-1α (C-19), and to c-Myb (M-19) were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Antibodies to c-Fos and to c-Jun were purchased from Oncogene Science (Uniondale, NY). Antibody to HA tag was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). HepG2 cells (Riken Cell Bank, Tsukuba, Japan, catalog no. RCB459) were maintained as previously described (15Umayahara Y. Kawamori R. Watada H. Imano E. Iwama N. Morishima T. Yamasaki Y. Kajimoto Y. Kamada T. J. Biol. Chem. 1994; 269: 16433-16442Abstract Full Text PDF PubMed Google Scholar). SK-N-MC cells (ATCC catalog no. HTB10) were maintained in Earle's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, non-essential amino acids, penicillin, and streptomycin (basal condition medium). 293T cells were maintained as previously described (16Fujitani Y. Kajimoto Y. Yasuda T. Matsuoka T.A. Kaneto H. Umayahara Y. Fujita N. Watada H. Miyazaki J.I. Yamasaki Y. Hori M. Mol. Cell. Biol. 1999; 19: 8281-8291Crossref PubMed Scopus (56) Google Scholar). Human IGF-I promoter-1-luciferase fusion genes were constructed as follows. A phage clone containing the 5′-flanking region and exon 1 of the human IGF-I gene was isolated from a human genomic library and used as a template to make the reporter gene plasmids. A series of PCR was performed using the phage DNA as a template to amplify promoter-1 DNA fragments, which were comprised of either 1600 bp or 300 bp of the human IGF-I gene 5′-flanking region and the 197 bp of the exon 1 untranslated region. The PCR primers used were 5′-GCGGTACCGCCTCTCAATGACACAATCTG-3′(for the 1600-bp fragment), 5′-GCGGTACCGAGTTTGCTGGAGAGGGTCT-3′(for the 300-bp fragment) and 5′-GGCAAGCTTGCGCAGGCTCTATCTGCT-3′ (for both). To make the plasmid pIGFI-1600 (Fig. 1), the PCR-amplified 1600-bp fragment was made blunt-ended using the DNA Blunting Kit (Takara, Kyoto, Japan), digested with HindIII, and ligated into SmaI/HindIII-digested pA3Luc (a kind gift from I. H. Maxwell, University of Colorado Health Science Center, Denver, CO) (17Maxwell I.H. Harrison G.S. Wood W.M. Maxwell F. BioTechniques. 1989; 7: 276-280PubMed Google Scholar). The plasmid pIGFI-600 (Fig. 1) was constructed by digesting the 1600-bp fragment with KpnI andHindIII and subcloning the resulting 600-bp fragment into the KpnI/HindIII-digested pA3Luc. The 300-bp fragment was digested with KpnI and HindIII and ligated into KpnI/HindIII-digested pA3Luc to construct the plasmid pIGFI-300 (Fig. 1). Site-directed mutagenesis was performed as described previously (8Kajimoto Y. Kawamori R. Umayahara Y. Iwama N. Imano E. Morishima T. Yamasaki Y. Kamada T. Biochem. Biophys. Res. Commun. 1993; 190: 767-773Crossref PubMed Scopus (9) Google Scholar). C/EBPβ expression vector pcDNA3C/EBPβ has been described previously (5Ji Y. Umayahara C. Centrella M. Rotwein P. McCarthy T.L. J. Biol. Chem. 1997; 272: 31793-31800Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Wild type and dominant negative (DN) type RSK expression vectors were gifts from Dr. J. Blenis (Boston, MA). The plasmid pMSV β-gal is an expression plasmid of the β-galactosidase gene driven by the murine sarcoma virus long terminal repeat (18Rosenthal N. Methods Enzymol. 1987; 152: 704-720Crossref PubMed Scopus (403) Google Scholar). The plasmid pRL-CMV was purchased from Promega Corp (Madison, WI). Transfection studies using HepG2 cells were performed as follows. One microgram of IGF-I promoter-1-lucferase fusion genes were cotransfected with 500 ng of pMSV β-gal to normalize for transfection efficiency. Cultures at 50% confluent density were rinsed in serum-free medium and exposed to plasmids in the presence of LipofectAMINETM for 5 h. After washing the plates two times with PBS, the solution was then replaced with serum-free medium (15Umayahara Y. Kawamori R. Watada H. Imano E. Iwama N. Morishima T. Yamasaki Y. Kajimoto Y. Kamada T. J. Biol. Chem. 1994; 269: 16433-16442Abstract Full Text PDF PubMed Google Scholar), and the cells were incubated for 24 h. Next, the cells were treated for 24 h with vehicle (Me2SO) or 10−7m TPA. After incubation, the medium was aspirated, the cultures were rinsed with PBS twice and lysed in cell lysis buffer (Promega), and luciferase activity was measured as described previously (15Umayahara Y. Kawamori R. Watada H. Imano E. Iwama N. Morishima T. Yamasaki Y. Kajimoto Y. Kamada T. J. Biol. Chem. 1994; 269: 16433-16442Abstract Full Text PDF PubMed Google Scholar). Transfection studies using SK-N-MC cells were performed as follows. One microgram of IGF-I promoter-1-lucferase fusion genes were cotransfected with 5 ng of pRL-CMV to normalize for transfection efficiency. Cultures at 50% confluent density were rinsed in serum-free medium and exposed to plasmids in the presence of LipofectAMINETM for 5 h. After washing the plates two times with PBS, the solution was replaced with culture medium containing 10% fetal bovine serum, and the cells were incubated for 24 h. Next, the cells were treated for 24 h with vehicle (Me2SO) or 10−7m GF109203X. After incubation, the medium was aspirated, the cultures were rinsed with PBS twice and lysed in cell lysis buffer, and dual-luciferase assay was performed according to the manufacturer’s instructions (Promega). Transfection studies using 293T cells were performed basically in the same way as the SK-N-MC cells described above. One microgram of IGF-I promoter-1-luciferase fusion genes were cotransfected with the indicated amount of C/EBPβ expression vector, 1 μg of wild type or dominant negative type RSK expression vector, when required, and 5 ng of pRL-CMV. After transfection, the cells were incubated for 24 h and dual-luciferase assay (Promega) was performed following the manufacturer’s directions. The Gal4 fusion constructs (Gal4C/EBPβ118 and Gal4C/EBPβ166) were generated by isolating (by PCR) and introducing appropriate DNA fragments of C/EBPβ into the EcoRI-BglII site of pFACMV plasmid (Stratagene), which contained the DNA-binding domain (positions 1–147) of Gal4. Site-directed mutagenesis was performed with the QuikChange site-directed mutagenesis kit (Stratagene) using two synthetic complementary oligonucleotides, 5′-CCGAGCAAGAAGCCGGCCGACTACGGTTACG-3′ and 5′-CGTAACCGTAGTCGGCCGGCTTCTTGCTCGG-3′ (mutated sequence is underlined), to generate Gal4C/EBPβ118mutAla105 and Gal4C/EBPβ166mut105Ala. The Gal4-responsive reporter plasmid pFR-Luc plasmid containing five copies of Gal4-binding element upstream of the basic promoter element (TATA box) linked to luciferase structural gene was purchased from Stratagene. By lipofection, 1 μg of each Gal4 fusion plasmid was cotransfected into the host cell with 1 μg of pFR-Luc and 5 ng of pRL-CMV. The cells were then incubated for 48 h, followed by a dual-luciferase assay (Promega) performed according to the manufacturer’s directions. HepG2 and SK-N-MC cell nuclear extracts were prepared by the method of Lee et al. (19Lee K.A. Bindereif A. Green M.R. Gene Anal. Tech. 1988; 5: 22-31Crossref PubMed Scopus (394) Google Scholar) with minor modifications. Cells were harvested with a cell scraper and gently pelleted, and the pellets were washed with phosphate-buffered saline. The cells were then lysed in hypotonic buffer (10 mm HEPES, pH 7.4, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol). Nuclei were pelleted and resuspended in hypertonic buffer containing 20 mm HEPES, pH 7.9, 0.42 m NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 25% glycerol, 0.5 mm dithiothreitol, and 0.5 mmphenylmethylsulfonyl fluoride. Soluble proteins released by a 30-min incubation at 4 °C were collected by centrifugation at 12,000 × g for 20 min, and the supernatant was collected. The protein concentration was measured using a modified Bradford assay (Bio-Rad). Gel mobility shift experiments followed previously published methods (4Thomas M.J. Umayahara Y. Shu H. Centrella M. Rotwein P. McCarthy T.L. J. Biol. Chem. 1996; 271: 21835-21841Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Radiolabeled double-stranded DNA probes were synthesized by annealing complementary end-labeled oligonucleotides. Nuclear protein extracts (5 μg) were preincubated for 20 min on ice with 2 mg of poly(dI-dC) with or without unlabeled specific or nonspecific DNA competitor or antibodies in 25 mm HEPES, pH 7.6, 60 mm KCl, 7.5% glycerol, 0.1 mm EDTA, 5 mm dithiothreitol, and 0.025% bovine serum albumin. After the addition of 5 × 104cpm of DNA probe for 30 min on ice, the samples were applied to 12% nondenaturing polyacrylamide gel that had been pre-electrophoresed for 30 min at 12.5 V/cm at 25 °C in 45 mm Tris, 45 mm boric acid, and 1 mm EDTA. Electrophoresis was conducted for 2.5 h under identical conditions. The dried gels were exposed to x-ray film at −80 °C with an intensifying screen. Deoxynuclease I (DNase) footprinting was performed as described elsewhere (6Umayahara Y. Ji J. Billiard C. Centrella M. McCarthy T.L. Rotwein P. J. Biol. Chem. 1999; 274: 10609-10617Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). End-labeled double-stranded DNA probes flanking the C/EBP site in human IGF-I promoter-1, which corresponds to −46 to +96 bp (relative to transcriptional start site) of the human IGF-I gene, were generated by polymerase chain reaction using one end-labeled oligonucleotide primer (5′-ATGCTCTGTCTCTAGTT-3′) and one unlabeled primer (5′-ACTGTAGACAGGAAACAGCT-3′). Nuclear protein (10 μg) was preincubated for 15 min with poly(dI-dC) in 25 mm HEPES, pH 7.6, 60 mm KCl, 7.5% glycerol, 0.1 mm EDTA, 5 mm dithiothreitol, and 0.05% bovine serum albumin, followed by the addition of labeled probe (5.0 × 105cpm/sample) and incubation for 60 min on ice. The reaction mixture was then treated with DNase I (final concentration 1.15 mg/ml, Worthington Biochemical Corp., Freehold, NJ) in 2.5 mmMgCl2 and 2.5 mm CaCl2 for 1 min at 25 C. Nuclease treatment was terminated by addition of 20 mm EDTA, 200 mm NaCl, 1% sodium dodecyl sulfate, and 10 mg of yeast transfer RNA followed by phenol-chloroform extraction and ethanol precipitation. Samples were analyzed after electrophoresis on 8% polyacrylamide, 8 m urea gel, and autoradiography for 16 h at −80 °C with an intensifying screen. Total RNA was extracted from HepG2 cells or SK-N-MC cells by homogenization in guanidine thiocyanate. Northern blots followed standard procedures using 10 μg of total RNA, and the buffer conditions were as described. The hybridization probes were 7 × 106 cpm of32P-labeled rat C/EBPβ cDNA probe and human IGF-I cDNA probe. Reverse transcription-PCR were performed using primers 5′-ATCAGCGTCTTCCAACCCAATTA-3′ and 5′-TGGCGCTGGGCACGGACAGA-3′ (for human IGF-I), and 5′-AAGGCCGGCTTCGCGGCGA-3′ and 5′-CCGGCCAGCCAGGTCCAGAC-3′ (for β-actin). 293T cell nuclear proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. After the membranes were blocked with 5% nonfat dry milk and 2% fetal bovine serum in 20 mm Tris-Cl, pH 7.6, and 137 mm NaCl for 1 h at 25 °C, they were incubated with an antibody to HA for 1 h at 25 °C. Subsequent steps were performed as described elsewhere (5Ji Y. Umayahara C. Centrella M. Rotwein P. McCarthy T.L. J. Biol. Chem. 1997; 272: 31793-31800Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). First we examined whether the major promoter (promoter-1) of the human IGF-I gene is a target of PKC activation. A series of gene transfer studies were performed with human hepatocellular carcinoma-derived HepG2 cells. Under the basal condition, HepG2 cells barely express the IGF-I gene according to reverse transcription-PCR results, but the IGF-I mRNA derived from the promoter 1 was induced when the cells were treated with 10−7m TPA for 4 h (data not shown). Each reporter gene plasmid contained various lengths of IGF-I gene 5′-flanking sequences and 197 bp of the exon 1 untranslated region linked to the firefly luciferase reporter because this portion of exon 1 untranslated region appeared to be important for basal promoter activity of promoter-1 in SK-N-MC cells (20Mittanck D.W. Kim S.W. Rotwein P. Mol. Cell. Endocrinol. 1997; 126: 153-163Crossref PubMed Scopus (54) Google Scholar). As shown in Fig. 1, despite differences in the basal promoter activities, the promoter activities of the 1600 bp (pIGFI-1600), 600 bp (pIGFI-600), and 300 bp (pIGFI-300) were activated to a similar extent after treatment with TPA, about 2.5-fold. This result showed that the human IGF-I gene promoter-1 could be activated by TPA treatment of the cells and that the major portion of the cis-active elements mediating this phenomenon is located within the 300 bp of the 5′-flanking sequence and/or the 197 bp of the untranslated region of exon 1. There was no region that perfectly matched the consensus AP-1 motif (T(G/T)AGTCA) within the region of the IGF-I gene which revealed the TPA responsiveness (−300 to ∼+197). However, a region of high similarity to the AP-1 consensus was seen within the exon 1 untranslated region (+23 to ∼+29; TTACTCA); indeed, the same sequence in the JE-1 gene was shown to be a target for TPA-responsive activation in MC3T3-E1 cells (21Koike M. Kuroki T. Nose K. Mol. Carcinog. 1993; 8: 105-111Crossref PubMed Scopus (9) Google Scholar). To find whether this portion is involved in the TPA responsiveness, we performed a mutation analysis. Because this portion was located within a region where multiple transcription initiation sites are clustered, it seemed possible that a mutation in this region could cause unpredictable, nonspecific damage to the promoter activity. To avoid this, we changed one sequence of the possible TPA-responsive region (A (+29) to G) so that the sequence became the same as the homologous regions of the chicken and rat IGF-I genes (22Kajimoto Y. Rotwein P. J. Biol. Chem. 1991; 266: 9724-9731Abstract Full Text PDF PubMed Google Scholar, 23Hall L.J. Kajimoto Y. Bichell D. Kim S.W. James P.L. Counts D. Nixon L.J. Tobin G. Rotwein P. DNA Cell Biol. 1992; 11: 301-313Crossref PubMed Scopus (121) Google Scholar). As shown in Fig. 1, when one nucleotide mutation was introduced into the portion (pIGFI-300M), TPA-induced promoter activation was completely abolished, showing that this portion does play an essential role in mediating TPA effects on the IGF-I gene promoter. The mutated promoter also caused a decrease in the basal promoter activity (Fig.1). Interestingly, the putative TPA-responsive region in the human IGF-I gene also contains the consensus for the C/EBP binding motif, CTTACTCAA. Indeed, Nolten et al. previously demonstrated in vitro that C/EBPα and C/EBPβ, when overexpressed, can bind to this region (24Nolten L.A. van Schaik F.M. Steenbergh P.H. Sussenbach J.S. Mol. Endocrinol. 1994; 8: 1636-1645Crossref PubMed Google Scholar). To characterize the factors involved in the TPA activation of the human IGF-I gene, we performed gel-mobility shift analyses. As shown in Fig.2, TPA enhanced specific protein bindings to the putative TPA-responsive region (lanes 1 and5). The unlabeled wild type competitor, but not the mutated competitor to which the same point mutation was introduced as in the reporter gene construct (A (+29) to G), inhibited the DNA-protein bindings (lanes 6–11). Also, when the mutation was introduced to the labeled probe, no binding was observed at all (lane 12). Thus, a certain factor or factors bind to the putative TPA-responsive region in the human IGF-I gene promoter in a TPA-responsive manner, and this mediates the TPA responsiveness of the gene transcription. As mentioned above, the putative TPA-responsive region reveals similarity to the AP-1 motif but also is a potential C/EBP binding site. To identify the factor that mediates the TPA responsiveness by binding to the region, we next performed a gel mobility supershift assay using specific antibodies against C/EBPα, C/EBPβ, C/EBPδ, c-Fos, c-Jun, HNF-1α, and c-Myc, respectively. Each antibody was added to a tube prior to the binding reaction. As shown in Fig.3, a supershifted band was observed together with a reduction of the gel-shift complex only when the C/EBPβ antibody was added (lane 9). Thus, the results clearly indicated that the TPA-responsive binding protein includes C/EBPβ, and the TPA-responsive region works as a C/EBP binding site. Next, we performed a DNase footprinting assay with end-labeled double-stranded DNA probes derived from human IGF-I promoter-1 and nuclear extracts from HepG2 cell or C/EBPβ overexpressing 293T cells (Fig. 4). The results indicated that overexpressed C/EBPβ protected the C/EBP site from nuclease digestion (lanes 4 and 5). With the nuclear extract of HepG2 cell, the same site was protected (lane 2), and this protection was enhanced by 4 h of incubation with TPA (lane 3), confirming that C/EBPβ binds to the C/EBP site in a TPA-dependent manner in HepG2 cells. Thus, C/EBPβ binds to the C/EBP site in IGF-I gene promoter-1 and mediates the TPA effects on the IGF-I gene promoter. To investigate whether C/EBPβ that binds to the C/EBP site can activate the IGF-I gene transcription, we overexpressed C/EBPβ in 293T cells and evaluated the effects on the IGF-I gene promoter activity. 293T cells were chosen because they lack intrinsic expression of C/EBPβ (data not shown). As shown in Fig.5, overexpression of C/EBPβ transactivates the IGF-I promoter-1 in a dose-dependent manner. When a point mutation was introduced to the C/EBP site, the transactivation effect of C/EBPβ disappeared, suggesting that C/EBPβ transactivates human IGF-I promoter-1 through the C/EBP site. Taken together, these results indicate that PKC activation induces human IGF-I gene transcription through enhancement of the C/EBPβ binding to promoter 1. Next we investigated the mechanism that underlies the PKC-dependent activation of C/EBPβ. First, the effects of TPA on C/EBPβ mRNA were evaluated in HepG2 cells. The results of Northern blot analysis (Fig. 6) revealed that TPA stimulation increases the C/EBPβ mRNA amount by ∼3-fold, suggesting that PKC can stimulate C/EBPβ gene transcription, and this may in part explain the PKC activation of the human IGF gene promoter. Recently, RSK was shown to stimulate C/EBPβ activity, and this facilitates TGFβ-induced hepatocyte proliferation (25Buck M. Poli V. van der Geer P. Chojkier M. Hunter T. Mol. Cell. 1999; 4: 1087-1092Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Because RSK is known to be a downstream target of PKC, we investigated the possibility of RSK also being involved in the PKC-dependent activation of C/EBPβ and IGF-I gene activation. For this purpose, we used a DN mutant of RSK. As shown in Fig. 7, the DN RSK mutant, when co-overexpressed in the 293T cells with C/EBPβ, significantly suppressed the transactivation potential of C/EBPβ in terms of the activation of the human IGF-I gene promoter. This effect was not observed when C/EBPβ was absent (Fig. 7). These results suggest that the transactivation potential of C/EBPβ depends on the RSK activity in 293T cells. To further clarify the mechanism underlying this RSK-dependent activation of C/EBPβ, we employed theSaccharomyces cerevisiae GAL4 fusion protein reporter system (Fig 8). It is known that the N-terminal region of C/EBPβ contains a transcription activation domain and a transrepression domain (Fig. 8b, (26Williams S.C. Baer M. Dillner A.J. Johnson P.F. EMBO J. 1995; 14: 3170-3183Crossref PubMed Scopus (200) Google Scholar)). It also includes a Ser residue at 105, which was previously shown to be critical for the C/EBPβ activation by TPA (27Trautwein C. Caelles C. van der Geer P. Hunter T. Karin M. Chojkier M. Nature. 1993; 364: 544-547Crossref PubMed Scopus (293) Google Scholar) and was also identified recently as the phosphorylated site by RSK (25Buck M. Poli V. van der Geer P. Chojkier M. Hunter T. Mol. Cell. 1999; 4: 1087-1092Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Accordingly, we prepared GAL4 fusion constructs containing either 118 or 166 amino acids (aa) of N-terminal region of C/EBPβ fused to the heterologous DNA-binding domain of the GAL4 transcription factor (Fig.8b). These chimeric GAL4-C/EBPβ fusion proteins were expressed in HepG2 cells, and effects on the GAL4 reporter were evaluated. HepG2 cells were used for this experiment because they show a very good response to TPA and have intrinsic C/EBPβ. As shown in Fig. 8a (lane 3), the Gal4C/EBPβ118 construct transactivated the GAL4 reporter in serum-free medium. On the other hand, the Gal4C/EBPβ166 construct did not activate the GAL4 reporter in the basal state (lane 5). This observation is consistent with former report b
Год издания: 2002
Авторы: Yutaka Umayahara, Yoshitaka Kajimoto, Yoshio Fujitani, Shin-ichi Gorogawa, Tetsuyuki Yasuda, Akio Kuroda, Kentaro Ohtoshi, S. Yoshida, Dan Kawamori, Yoshimitsu Yamasaki, Masatsugu Hori
Издательство: Elsevier BV
Источник: Journal of Biological Chemistry
Ключевые слова: Growth Hormone and Insulin-like Growth Factors, Metabolism, Diabetes, and Cancer, Protein Kinase Regulation and GTPase Signaling
Другие ссылки: Journal of Biological Chemistry (PDF)
Journal of Biological Chemistry (HTML)
PubMed (HTML)
Journal of Biological Chemistry (HTML)
PubMed (HTML)
Открытый доступ: hybrid
Том: 277
Выпуск: 18
Страницы: 15261–15270