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• W2007609332 abstract "Growth hormone (GH) exerts sexually dimorphic effects on liver gene transcription through its sex-dependent temporal pattern of pituitary hormone secretion. CYP2C12 encodes a female-specific rat liver P450 steroid hydroxylase whose expression is activated by continuous GH stimulation of hepatocytes. Presently, we investigated the role of liver-enriched and GH-regulated transcription factors in the activation of CYP2C12 gene expression in GH-stimulated liver cells. Transcription of a CYP2C12 promoter-luciferase reporter gene in transfected HepG2 cells was activated 15–40-fold by the liver-enriched hepatocyte nuclear factor (HNF) 3α, HNF3β, and HNF6. Synergistic interactions leading to an ∼300-fold activation of the promoter by HNF3β in combination with HNF6 were observed. 5′-Deletion analysis localized the HNF6 response to a single 5′-proximal 96-nucleotide segment. By contrast, the stimulatory effects of HNF3α and HNF3β were attributable to five distinct regions within the 1.6-kilobase CYP2C12 proximal promoter. GH activation of the signal transducer and transcriptional activator STAT5b, which proceeds efficiently in male but not female rat liver, inhibited CYP2C12 promoter activation by HNF3β and HNF6, despite the absence of a classical STAT5-binding site. The female-specific pattern of CYP2C12 expression is thus proposed to reflect the positive synergistic action in female liver of liver-enriched and GH-regulated transcription factors, such as HNF3β and HNF6, coupled with a dominant inhibitory effect of GH-activated STAT5b that is manifest in males. Growth hormone (GH) exerts sexually dimorphic effects on liver gene transcription through its sex-dependent temporal pattern of pituitary hormone secretion. CYP2C12 encodes a female-specific rat liver P450 steroid hydroxylase whose expression is activated by continuous GH stimulation of hepatocytes. Presently, we investigated the role of liver-enriched and GH-regulated transcription factors in the activation of CYP2C12 gene expression in GH-stimulated liver cells. Transcription of a CYP2C12 promoter-luciferase reporter gene in transfected HepG2 cells was activated 15–40-fold by the liver-enriched hepatocyte nuclear factor (HNF) 3α, HNF3β, and HNF6. Synergistic interactions leading to an ∼300-fold activation of the promoter by HNF3β in combination with HNF6 were observed. 5′-Deletion analysis localized the HNF6 response to a single 5′-proximal 96-nucleotide segment. By contrast, the stimulatory effects of HNF3α and HNF3β were attributable to five distinct regions within the 1.6-kilobase CYP2C12 proximal promoter. GH activation of the signal transducer and transcriptional activator STAT5b, which proceeds efficiently in male but not female rat liver, inhibited CYP2C12 promoter activation by HNF3β and HNF6, despite the absence of a classical STAT5-binding site. The female-specific pattern of CYP2C12 expression is thus proposed to reflect the positive synergistic action in female liver of liver-enriched and GH-regulated transcription factors, such as HNF3β and HNF6, coupled with a dominant inhibitory effect of GH-activated STAT5b that is manifest in males. growth hormone Janus kinase 2 signal transducer and activator of transcription cytochrome P450 gene 2C12 GH-activated nuclear factor hepatocyte nuclear factor insulin response element-A binding protein CAAT/enhancer-binding protein nucleotide(s) fetal bovine serum luciferase polymerase chain reaction electrophoretic mobility shift assay oligonucleotide GH1 signals to hepatocytes and other target cells via its plasma membrane-bound receptor, which is a member of the cytokine/growth factor receptor superfamily (1Moutoussamy S. Kelly P.A. Finidori J. Eur. J. Biochem. 1998; 255: 1-11Crossref PubMed Scopus (104) Google Scholar). Binding of GH to the GH receptor induces receptor dimerization and activation of Janus kinase 2 (JAK2), a tyrosine kinase that interacts with the cytoplasmic domain of the GH receptor and phosphorylates both itself and the cytoplasmic domain of the GH receptor on multiple tyrosine residues. These phosphorylated tyrosines, in turn, serve as docking sites for downstream signaling molecules that contain Src homology 2 domains, including STAT transcription factors and insulin receptor substrates 1 and 2 (1Moutoussamy S. Kelly P.A. Finidori J. Eur. J. Biochem. 1998; 255: 1-11Crossref PubMed Scopus (104) Google Scholar, 2Argetsinger L.S. Carter-Su C. Physiol. Rev. 1996; 76: 1089-1107Crossref PubMed Scopus (247) Google Scholar). These primary signaling molecules activate secondary messengers, such as diacylglycerol, calcium, and nitric oxide, and enzymes, such as mitogen-activated protein kinase, protein kinase C, phospholipase A2, and phosphatidylinositol 3′-kinase. These GH-stimulated signaling pathways regulate a variety of intracellular events, including gene transcription, metabolite transport, and enzymatic activity, and contribute to the overall regulation by GH of whole body growth and metabolism (3Davey H.W. Wilkins R.J. Waxman D.J. Am. J. Hum. Genet. 1999; 65: 959-965Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The cytochrome P450 gene CYP2C12 encodes a steroid-disulfate 15β-hydroxylase that is induced in the livers of female rats at puberty. At this same developmental stage, a related P450 gene,CYP2C11, which encodes a steroid 16α- and 2α-hydroxylase, is induced in the livers of male rats (4MacGeoch C. Morgan E.T. Halpert J. Gustafsson J.A. J. Biol. Chem. 1984; 259: 15433-15439Abstract Full Text PDF PubMed Google Scholar, 5Waxman D.J. J. Biol. Chem. 1984; 259: 15481-15490Abstract Full Text PDF PubMed Google Scholar). The differential expression of these two CYP genes in male and female rats is established by the sexually dimorphic patterns of pituitary GH secretion (6Waxman D.J. Chang T.K.H. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 2nd Ed. Plenum Press, New York1995: 391-417Crossref Google Scholar, 7Mode A. Tollet P. Strom A. Legraverend C. Liddle C. Gustafsson J.A. Adv. Enzyme Regul. 1992; 32: 255-263Crossref PubMed Scopus (34) Google Scholar). In male rats, GH is secreted intermittently to give regular plasma GH peaks of large amplitude (∼200 ng/ml) each 3–4 h, followed by trough periods of no detectable GH (8Tannenbaum G.S. Martin J.B. Endocrinology. 1976; 98: 562-570Crossref PubMed Scopus (652) Google Scholar). In contrast, the female rat GH secretory pattern is characterized by more frequent plasma peaks of smaller amplitude, resulting in a near continuous presence of GH in blood at an average level of 40–60 ng/ml (9Jansson J.-O. Ekberg S. Isaksson O. Endocr. Rev. 1985; 6: 128-150Crossref PubMed Scopus (661) Google Scholar). The cellular mechanisms by which the temporal plasma profile of GH regulates CYP2C11 and CYP2C12 gene expression in rat liver are only partially understood. We and others have demonstrated that expression of these two genes is regulated by GH at the level of transcription initiation (10Sundseth S.S. Alberta J.A. Waxman D.J. J. Biol. Chem. 1992; 267: 3907-3914Abstract Full Text PDF PubMed Google Scholar, 11Legraverend C. Mode A. Westin S. Strom A. Eguchi H. Zaphiropoulos P.G. Gustafsson J.-A. Mol. Endocrinol. 1992; 6 (J. A.): 259-266Crossref PubMed Scopus (117) Google Scholar). We have further established that a liver-expressed, latent cytoplasmic transcription factor, designated STAT5b, undergoes repeated tyrosine phosphorylation and nuclear translocation in direct response to the male pattern of GH secretion, leading us to propose that STAT5b is a transcriptional activator of male-specific, GH pulse-activated genes such asCYP2C11 (12Waxman D.J. Ram P.A. Park S.H. Choi H.K. J. Biol. Chem. 1995; 270: 13262-13270Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 13Choi H.K. Waxman D.J. Endocrinology. 2000; 141: 3245-3255Crossref PubMed Scopus (98) Google Scholar). Indeed, targeted disruption of theStat5b gene in male mice leads to GH pulse resistance (14Davey H.W. Park S.H. Grattan D.R. McLachlan M.J. Waxman D.J. J. Biol. Chem. 1999; 274: 35331-35336Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) associated with a selective loss of the male-specific pattern of liver gene expression (15Udy G.B. Towers R.P. Snell R.G. Wilkins R.J. Park S.H. Ram P.A. Waxman D.J. Davey H.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7239-7244Crossref PubMed Scopus (836) Google Scholar, 16Park S.H. Liu X. Hennighausen L. Davey H.W. Waxman D.J. J. Biol. Chem. 1999; 274: 7421-7430Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Further support for this proposal is provided by the correlation between STAT5b nuclear translocation and gender-specific P450 expression in mouse liver (17Sueyoshi T. Yokomori N. Korach K.S. Negishi M. Mol. Pharmacol. 1999; 56: 473-477Crossref PubMed Scopus (60) Google Scholar) and by the presence of bona fide STAT5-binding sites in the 5′-flanking region of several male-specific, GH-dependent liver P450 genes (18Subramanian A. Wang J. Gil G. Nucleic Acids Res. 1998; 26: 2173-2178Crossref PubMed Scopus (40) Google Scholar). 2S. H. Park and D. J. Waxman, unpublished data. Several liver-expressed nuclear factors have been proposed to contribute to the female-specific expression of CYP2C12. A continuous GH-activated nuclearfactor, termed GHNF, was demonstrated to be specifically induced or activated by the female plasma pattern of GH secretion and to bind to multiple sites along the female-specific CYP2C12promoter (19Waxman D.J. Zhao S. Choi H.K. J. Biol. Chem. 1996; 271: 29978-29987Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Thus far, however, GHNF has resisted purification and has been only partially characterized. A distinct liver-enriched transcription factor, termed HNF6 (20Lemaigre F.P. Durviaux S.M. Truong O. Lannoy V.J. Hsuan J.J. Rousseau G.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9460-9464Crossref PubMed Scopus (131) Google Scholar, 21Samadani U. Costa R.H. Mol. Cell. Biol. 1996; 16: 6273-6284Crossref PubMed Scopus (131) Google Scholar), whose expression in liver is stimulated by GH, can bind to and enhance transcription from theCYP2C12 promoter ∼3-fold (22Lahuna O. Fernandez L. Karlsson H. Maiter D. Lemaigre F.P. Rousseau G.G. Gustafsson J. Mode A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12309-12313Crossref PubMed Scopus (92) Google Scholar). However, HNF6 expression displays only a modest sex difference in rat liver (female/male = 2:1) and by itself cannot account for the striking female-specific expression of CYP2C12. The insulin response element-A binding protein (IRE-ABP), a member of the SRY family of transcriptional regulators, was hypothesized to be a transcriptional repressor of CYP2C12 (23Buggs C. Nasrin N. Mode A. Tollet P. Zhao H.F. Gustafsson J.A. Alexander-Bridges M. Mol. Endocrinol. 1998; 12: 1294-1309PubMed Google Scholar). Indeed, IRE-ABP inhibits activation of CYP2C12 by the liver transcription factor C/EBPα, and this effect was correlated with the overlapping binding sites for C/EBPα and IRE-ABP in the CYP2C12 promoter (23Buggs C. Nasrin N. Mode A. Tollet P. Zhao H.F. Gustafsson J.A. Alexander-Bridges M. Mol. Endocrinol. 1998; 12: 1294-1309PubMed Google Scholar). Moreover, one of these sites overlaps with the GHNF-binding site ofCYP2C12 found at nt −231 to −185 (19Waxman D.J. Zhao S. Choi H.K. J. Biol. Chem. 1996; 271: 29978-29987Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). However, IRE-ABP has not been shown to be expressed in female liver, and there is no evidence for the regulation of this factor by GH. STAT5b, which is proposed to be a transcriptional activator of male-specific, GH pulse-induced genes (12Waxman D.J. Ram P.A. Park S.H. Choi H.K. J. Biol. Chem. 1995; 270: 13262-13270Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar), was recently proposed to additionally act as a transcriptional repressor of female dominant mouseCyp genes, based on our finding that a subset of such genes is up-regulated in livers of STAT5b-deficient male mice (16Park S.H. Liu X. Hennighausen L. Davey H.W. Waxman D.J. J. Biol. Chem. 1999; 274: 7421-7430Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). This latter proposal is consistent with our finding that the induction of CYP2C12 gene expression by the female plasma pattern of GH secretion is associated with a dramatic down-regulation of the STAT5b signaling pathway (12Waxman D.J. Ram P.A. Park S.H. Choi H.K. J. Biol. Chem. 1995; 270: 13262-13270Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 24Gebert C.A. Park S.H. Waxman D.J. Mol. Endocrinol. 1999; 13: 213-227Crossref PubMed Scopus (63) Google Scholar). This study was carried out to identify both liver-enriched and GH-regulated nuclear factors that may contribute to regulated expression of CYP2C12. We demonstrate that the liver-enriched transcription factors HNF3α and HNF3β (25Lai E. Prezioso V.R. Smith E. Litvin O. Costa R.H. Darnell Jr., J.E. Genes Dev. 1990; 4: 1427-1436Crossref PubMed Scopus (407) Google Scholar, 26Lai E. Prezioso V.R. Tao W.F. Chen W.S. Darnell Jr., J.E. Genes Dev. 1991; 5: 416-427Crossref PubMed Scopus (436) Google Scholar) and HNF6 (20Lemaigre F.P. Durviaux S.M. Truong O. Lannoy V.J. Hsuan J.J. Rousseau G.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9460-9464Crossref PubMed Scopus (131) Google Scholar, 21Samadani U. Costa R.H. Mol. Cell. Biol. 1996; 16: 6273-6284Crossref PubMed Scopus (131) Google Scholar) bind to and strongly activate the CYP2C12promoter. We show that these latter two factors act in concert, via distinct sets of binding sites, to synergistically enhanceCYP2C12 gene activation. Additionally, we demonstrate that, when activated by GH, STAT5b and the closely related STAT5a down-regulate HNF3- and HNF6-induced CYP2C12 gene transcription. Our findings led us to propose a model whereby STAT5 proteins activated in liver in vivo by the male pulsatile GH pattern contribute to the sex specificity of CYP2C12expression by actively suppressing HNF3- and HNF6-dependentCYP2C12 transcription in adult male rats. The human hepatoma cell line HepG2, obtained from the American Type Culture Collection repository, was maintained in minimal essential medium containing 10% fetal bovine serum (FBS), 50 units/ml penicillin, and 50 units/ml streptomycin. An SV40-transformed African green monkey kidney cell line, COS-1, was maintained in Dulbecco's modified Eagle's medium containing 10% FBS and 50 units/ml penicillin/streptomycin. Mammalian expression plasmids for rat HNF3α and rat HNF3β, cloned into pNEO-SV40, were provided by Dr. Eseng Lai (Sloan-Kettering Cancer Center, New York) (25Lai E. Prezioso V.R. Smith E. Litvin O. Costa R.H. Darnell Jr., J.E. Genes Dev. 1990; 4: 1427-1436Crossref PubMed Scopus (407) Google Scholar, 26Lai E. Prezioso V.R. Tao W.F. Chen W.S. Darnell Jr., J.E. Genes Dev. 1991; 5: 416-427Crossref PubMed Scopus (436) Google Scholar). Rat HNF4 expression plasmid pLEN4S was provided by Dr. Frances Sladek (University of California, Riverside, CA) (27Sladek F.M. Zhong W.M. Lai E. Darnell Jr., J.E. Genes Dev. 1990; 4: 2353-2365Crossref PubMed Scopus (859) Google Scholar). Rat HNF6 expression plasmid pECE-HNF6 was obtained from Dr. Guy Rousseau (University of Louvain Medical School, Brussels, Belgium) (20Lemaigre F.P. Durviaux S.M. Truong O. Lannoy V.J. Hsuan J.J. Rousseau G.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9460-9464Crossref PubMed Scopus (131) Google Scholar). Rat GH receptor cDNA, cloned into expression plasmid pcDNAI, was provided by Dr. Nils Billestrup (Hagedorn Research Institute, Gentofte, Denmark). Mouse STAT1, STAT3, STAT5a, and STAT5b cDNAs, cloned into expression plasmid pME18S, were obtained from Dr. Alice Mui (DNAX Research Institute of Molecular and Cellular Biology, Inc., Palo Alto, CA). cDNAs encoding tyrosine-to-phenylalanine mutated forms of mouse STAT5a and STAT5b (designated STAT5a-Y694F and STAT5b-Y699F, respectively) and cDNAs encoding serine-to-alanine mutated STAT5 forms (designated STAT5a-S725A and STAT5b-S730A, respectively), all cloned into pcDNAI, were provided by Dr. Hallgeir Rui (Uniformed Services University of the Health Sciences, Bethesda, MD) (28Yamashita H. Xu J. Erwin R.A. Farrar W.L. Kirken R.A. Rui H. J. Biol. Chem. 1998; 273: 30218-30224Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). cDNAs encoding constitutively active forms of mouse STAT5a and STAT5b (designated STAT5a1*6 and STAT5b1*6, respectively) were provided by Dr. Toshiro Kitamura (University of Tokyo) (29Onishi M. Nosaka T. Misawa K. Mui A.L. Gorman D. McMahon M. Miyajima A. Kitamura T. Mol. Cell. Biol. 1998; 18: 3871-3879Crossref PubMed Scopus (348) Google Scholar) and were subcloned into expression plasmid pCI (Promega). The STAT5-activated luciferase reporter plasmid 4X-pT109-LUC was provided by Dr. Mary Vore (University of Kentucky, Lexington, KY) (30Ganguly T.C. O'Brien M.L. Karpen S.J. Hyde J.F. Suchy F.J. Vore M. J. Clin. Invest. 1997; 99: 2906-2914Crossref PubMed Scopus (88) Google Scholar). A2C12 promoter-firefly luciferase reporter plasmid containing 1632 base pairs of 5′-upstream sequence, designated2C12(−1632/+10)-LUC, was constructed as follows. A 1642-base pair DNA fragment corresponding to nt −1632 to +10 of the rat CYP2C12 gene (nucleotide numbering relative to the2C12 transcription start site) (RATP4515B1; GenBankTM/EBI Data Bank) was synthesized by PCR using the cloned 2C12 promoter as template (plasmid p42) (10Sundseth S.S. Alberta J.A. Waxman D.J. J. Biol. Chem. 1992; 267: 3907-3914Abstract Full Text PDF PubMed Google Scholar) and synthetic oligonucleotides corresponding to nt −1632 to −1614 (ON-477, upstream primer containing a 5′-end MluI restriction site) and nt +10 to −9 (ON-366, downstream primer containing a 5′-end BglII restriction site). PCRs were carried out at 94 °C for 1 min, 56 °C for 1.5 min, and 72 °C for 2 min for 30 cycles in a Stratagene RoboCycler. The resultant DNA fragment was double-digested by BglII and MluI and then ligated to BglII/MluI-digested pGL3-basic (Promega), which encodes a modified firefly luciferase reporter. The sequence was confirmed by DNA sequence analysis. 5′-Deletions of the 2C12(−1632/+10)-LUC plasmid were prepared by PCR amplification of various lengths of the 2C12promoter using upstream oligonucleotides (each containing a 5′-endMluI site) corresponding to the following sequences upstream of the transcription start site: 5′-primer start positions beginning at nt −1398, −1319, −1182, −970, −904, −856, −745, −611, −516, −240, and −96 (see Fig. 2). Primer ON-366 was used as the fixed downstream primer. PCRs using plasmid p42 as template (10Sundseth S.S. Alberta J.A. Waxman D.J. J. Biol. Chem. 1992; 267: 3907-3914Abstract Full Text PDF PubMed Google Scholar) were carried out for 30 cycles consisting of 94 °C for 1 min, 40 to 56 °C for 1.5 min, and 72 °C for 2 min. The resultant PCR products were then cloned into pGL3-basic as described above, and the sequences were confirmed by DNA sequencing. For HepG2 transient transfections, cells were plated in minimal essential medium containing 10% FBS in triplicate in 24-well tissue culture plates (35% confluency; ∼90,000 cells/177 mm2/well) and transfected 24 h later using FuGENETM6 transfection reagent as described by the manufacturer (Roche Molecular Biochemicals). For COS-1 transient transfections, similar conditions were used, except that the cells were plated at ∼50% confluency and transfected 5 h later. All transfections were carried out using 185 ng of 2C12promoter-LUC reporter plasmid together with 200 ng of liver-enriched transcription factor expression plasmid or 200 ng of a control expression plasmid (pCI), unless indicated otherwise. When present, 95 ng of GH receptor expression plasmid, 200 ng of STAT expression plasmid, and 90 ng of the STAT-activated reporter plasmid 4X-pT109-LUC (30Ganguly T.C. O'Brien M.L. Karpen S.J. Hyde J.F. Suchy F.J. Vore M. J. Clin. Invest. 1997; 99: 2906-2914Crossref PubMed Scopus (88) Google Scholar) were included. In addition, 50 ng of the Renillaluciferase reporter plasmid pRL-TK (Promega) was included in each sample as an internal standard for transfection efficiency. Firefly andRenilla luciferase activities were determined 20–24 h after the initiation of transfection using the Dual-LuciferaseTMreporter assay system (Promega) and a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). Firefly luciferase values were normalized on the basis of the Renillaluciferase values. In experiments in which HepG2 cells were treated with GH or prolactin (500 ng/ml, unless specified otherwise), cells were plated in minimal essential medium + 10% FBS. The cells were washed 6 h later to remove the FBS and then incubated overnight (∼18 h) in minimal essential medium without serum. Cells were then transfected as described above and were simultaneously treated with either GH or prolactin for a total of 24 h. Normalized luciferase activities were determined as described above. Rat GH and rat prolactin were hormonally pure preparations (SIAFP grade) obtained from Dr. A. Parlow and the National Hormone and Pituitary Program, NIDDK, National Institutes of Health. Total cell extracts were prepared as follows. HepG2 cells were washed once with ice-cold phosphate-buffered saline and then scraped in lysis buffer containing 20 mmHEPES (pH 7.9), 1% Triton X-100, 20% glycerol, 20 mm NaF, 1 mm EDTA, 1 mm EGTA, 1 mmNa3VO4, 1 mmNa2P2O7, 1 mmdithiothreitol, 0.5 mm phenylmethanesulfonyl fluoride, 1 μg/ml pepstatin, and 1 μg/ml leupeptin. Crude extracts were aspirated 10 times through a 27-gauge needle, adjusted to 150 mm NaCl, and centrifuged at 13,000 × g for 30 min at 4 °C. Supernatants were stored in liquid N2until analysis. Protein concentrations were determined using the Bio-Rad Dc detergent protein assay kit. Rat liver nuclear extracts were prepared as described previously (12Waxman D.J. Ram P.A. Park S.H. Choi H.K. J. Biol. Chem. 1995; 270: 13262-13270Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). The CYP2C12 double-stranded oligonucleotides shown in Table I were synthesized by Bio-Synthesis, Inc. (Lewisville, TX) and used as EMSA probes. Other EMSA probes used in this study include the following: 1) an HNF3 consensus sequence derived from nt −109 to −85 of the transthyretin promoter and mutated to eliminate HNF6 binding (21Samadani U. Costa R.H. Mol. Cell. Biol. 1996; 16: 6273-6284Crossref PubMed Scopus (131) Google Scholar) (5′-TGA-CTA-AAC-AAA-CAT-TCA-GAA-TCG-3′; probe ON-523, with mutated residues underlined); 2) an HNF3β-specific sequence derived from the transthyretin-2 enhancer site (31Samadani U. Qian X. Costa R.H. Gene Expr. 1996; 6: 23-33PubMed Google Scholar) (5′-GGC-CCC-TGT-TCA-AAC-ATG-TCC-TAAT-3′; probe ON-569, with the core HNF3β sequence underlined); and 3) HNF6 consensus sequence derived from nt −109 to −85 of the transthyretin promoter and mutated to eliminate HNF3 binding (21Samadani U. Costa R.H. Mol. Cell. Biol. 1996; 16: 6273-6284Crossref PubMed Scopus (131) Google Scholar) (5′-TGA-CTA-AAT-CAA-TAT-CGA-GAA-TCA-G-3′; probe ON-527, with mutated residues underlined). The sense strand oligonucleotide of each probe was labeled with [γ-32P]ATP (PerkinElmer Life Sciences) using T4 polynucleotide kinase (Promega), annealed to the antisense strand, and then purified. Total HepG2 or COS-1 cell extract (10 μg of protein in 5 μl) or rat liver nuclear extract (3 μg of protein in 5 μl) (12Waxman D.J. Ram P.A. Park S.H. Choi H.K. J. Biol. Chem. 1995; 270: 13262-13270Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) was preincubated for 10 min at room temperature with 9 μl of gel mobility shift buffer (12.5 mm Tris-HCl (pH 7.5) containing 2 μg of poly(dI-dC) (Roche Molecular Biochemicals), 5% glycerol, 1.25 mm MgCl2, 625 μm EDTA, and 625 μm dithiothreitol). Double-stranded,32P-labeled oligonucleotide probe (10 fmol, 1 μl) was then added, and incubation was continued for 20 min at room temperature, followed by a 10-min incubation on ice. After addition of 2 μl of loading dye (30% glycerol, 0.25% bromphenol blue, and 0.25% xylene cyanol), samples were electrophoresed at room temperature through a pre-electrophoresed nondenaturing polyacrylamide gel (5.5% acrylamide and 0.07% bisacrylamide; National Diagnostics, Inc., Atlanta, GA) in 0.5× buffer containing 44.5 mm Trizma (Tris base), 44.5 mm boric acid, and 5 mm EDTA. Unlabeled probe competitions were carried out by including up to a 100-fold molar excess of unlabeled DNA probe in gel mobility shift buffer.Figure 2Deletion analysis of 2C12promoter: functional characterization of HNF3- and HNF6-binding sites. A, shown is a schematic representation of the 5′-deleted 2C12 promoter-LUC constructs. B–D, each 5′-deleted 2C12 promoter-LUC construct was transiently cotransfected into HepG2 cells with the control expression plasmid (pCI) or with HNF3α (B), HNF3β (C), or HNF6 (D) expression plasmid (200 ng of total pCI or HNF plasmid/well) in the presence of the internal control plasmid pRL-TK. Normalized firefly luciferase activity values were determined. Thebar values represent mean ± S.E. of three independent series of transfections. Numbers to the right of each bar indicate the -fold increase in reporter gene activity stimulated by HNF3α, HNF3β, or HNF6 compared with the pCI control plasmid shown alongside each bar.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table ISynthetic oligonucleotides derived from the HNF3 DNA-binding sites of the CYP2C12 promoterOligonucleotide (location) 1-aNucleotide position, numbered relative to the CYP2C12 transcription start site.Sequence 1-bThe HNF3-binding sites present in oligos A–D are written in boldface uppercase letters. Underlined sequences on either the sense or the anti-sense strand are compared with the HNF3 consensus recognition sequence (37) with matches of 8/9 (oligo A, each site of oligo B, and oligo D) and either 7/9 or 9/9 (sites of oligo C).A (−587/−563)5′-cctataaaa TGTTTACTA aaaacac-3′3′-ggatattttACAAATGAT ttttgtg-5′B (−355/−326)5′-gaaAAGAAAATAaggaaATGCAAATAttag-3′3′-ctt TTCTTTTAT tcctt TACGTTTAT aatc-5′C (−277/−241)5′-TCATAAATAAATAatttaaaattaACTCAAATAtgtt-3′3′- AGTATTTATTTAT taaattttaat TGAGTTTAT acaa-5′D (−96/−73)5′-gagAGATAAACAgtggccagatgg-3′3′-ctc TCTATTTGT caccggtctacc-5′HNF3 (consensus sequence)T(A/G)TT(T/G)(G/A)(C/T)T(T/C)1-a Nucleotide position, numbered relative to the CYP2C12 transcription start site.1-b The HNF3-binding sites present in oligos A–D are written in boldface uppercase letters. Underlined sequences on either the sense or the anti-sense strand are compared with the HNF3 consensus recognition sequence (37Overdier D.G. Porcella A. Costa R.H. Mol. Cell. Biol. 1994; 14: 2755-2766Crossref PubMed Scopus (330) Google Scholar) with matches of 8/9 (oligo A, each site of oligo B, and oligo D) and either 7/9 or 9/9 (sites of oligo C). Open table in a new tab GH-dependent expression ofCYP2C12 mRNA requires ongoing protein synthesis (32Tollet P. Enberg B. Mode A. Mol. Endocrinol. 1990; 4: 1934-1942Crossref PubMed Scopus (115) Google Scholar), suggesting that CYP2C12 transcription may be regulated by a transcription factor that is itself regulated by GH at the level of gene expression. Because genes coding for several liver-enriched transcription factors are reported to be inducible by GH (e.g. Refs. 33Clarkson R.W. Chen C.M. Harrison S. Wells C. Muscat G.E. Waters M.J. Mol. Endocrinol. 1995; 9: 108-120Crossref PubMed Scopus (83) Google Scholar and 34Lahuna O. Rastegar M. Maiter D. Thissen J.P. Lemaigre F.P. Rousseau G.G. Mol. Endocrinol. 2000; 14: 285-294Crossref PubMed Scopus (96) Google Scholar), we examined the role of liver-enriched nuclear factors in CYP2C12 transactivation as determined by transient transfection into the human hepatoblastoma cell line HepG2. The 5′-flanking sequence of CYP2C12 (nt −1632 to +10 relative to the transcription start site) was found to have significant basal expression activity when fused to a firefly luciferase reporter gene. This basal activity was increased significantly when the cells were cotransfected with expression plasmids encoding the liver factor HNF3β (39.1 ± 2.0-fold stimulation) or HNF6 (14.6 ± 2.6-fold stimulation) (Fig.1). HNF3α, a liver-enriched transcription factor that is closely related to HNF3β (25Lai E. Prezioso V.R. Smith E. Litvin O. Costa R.H. Darnell Jr., J.E. Genes Dev. 1990; 4: 1427-1436Crossref PubMed Scopus (407) Google Scholar, 26Lai E. Prezioso V.R. Tao W.F. Chen W.S. Darnell Jr., J.E. Genes Dev. 1991; 5: 416-427Crossref PubMed Scopus (436) Google Scholar), also transactivated the 2C12 promoter (17.5 ± 2.8-fold increase in luciferase reporter activity) (Fig. 1). By contrast, cotransfection of the liver factor HNF4 (27Sladek F.M. Zhong W.M. Lai E. Darnell Jr., J.E. Genes Dev. 1990; 4: 2353-2365Crossref PubMed Scopus (859) Google Scholar) did not significantly increase reporter gene activity (Fig. 1), in agreement with an earlier report (35Strom A. Westin S. Eguchi H. Gustafsson J.A. Mode A. J. Biol. Chem. 1995; 270: 11276-11281Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Control experiments verified that HNF4 was expressed in the transfected HepG2 cells and that it specifically bound its cognate DNA response element (36Xanthopoulos K.G. Prezioso V.R. Chen W.S. Sladek F.M. Cortese R. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3807-3811Crossref PubMed Scopus (142) Google Scholar) in EMSAs (data not shown). 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