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• W2037992255 abstract "The purpose of this study was to clarify the mechanism(s) responsible for the growth hormone (GH)-induced expression of the CYP2C12 gene. To identify a functional GH-responsive element (GHRE) in vivo, we performed the direct injection of promoter-luciferase chimeric genes into female rat livers. The results showed that the luciferase activity was decreased to approximately 20% by the deletion of the sequence between nucleotides −4213 and −4161. Within this region, two copies of a possible GHRE were present. The sequence of the GHRE was overlapped with that of an interferon-γ-activated sequence, known to be recognized by the signal transducer and activator of transcription (STAT) proteins. In fact, a supershift assay showed that STAT5 was capable of binding to the core sequence of the GHRE. Furthermore, a luciferase assay with reporter plasmids, Δ−4161/−3781, mutated hepatocyte nuclear factor-4 (HNF-4), and mutated HNF-6, revealed that the GH-stimulated expression of the CYP2C12 gene was regulated cooperatively by STAT5, HNF-4, HNF-6, and the factor(s) that binds to the elements, 2C12-I (−4095 to −4074) and 2C12-II (−4072 to −4045). The cooperative regulation by STAT5 and the liver-enriched transcription factors account for the GH-dependent and the liver-specific expression of the CYP2C12 gene in female rats. The purpose of this study was to clarify the mechanism(s) responsible for the growth hormone (GH)-induced expression of the CYP2C12 gene. To identify a functional GH-responsive element (GHRE) in vivo, we performed the direct injection of promoter-luciferase chimeric genes into female rat livers. The results showed that the luciferase activity was decreased to approximately 20% by the deletion of the sequence between nucleotides −4213 and −4161. Within this region, two copies of a possible GHRE were present. The sequence of the GHRE was overlapped with that of an interferon-γ-activated sequence, known to be recognized by the signal transducer and activator of transcription (STAT) proteins. In fact, a supershift assay showed that STAT5 was capable of binding to the core sequence of the GHRE. Furthermore, a luciferase assay with reporter plasmids, Δ−4161/−3781, mutated hepatocyte nuclear factor-4 (HNF-4), and mutated HNF-6, revealed that the GH-stimulated expression of the CYP2C12 gene was regulated cooperatively by STAT5, HNF-4, HNF-6, and the factor(s) that binds to the elements, 2C12-I (−4095 to −4074) and 2C12-II (−4072 to −4045). The cooperative regulation by STAT5 and the liver-enriched transcription factors account for the GH-dependent and the liver-specific expression of the CYP2C12 gene in female rats. growth hormone GH nuclear factor GH-responsive element cyclic AMP-response element CRE-binding protein CREB-binding protein CCAAT/enhancer-binding protein hepatocyte nuclear factor signal transducer and activator of transcription tyrosine aminotransferase thymidine kinase transthyretin base pair(s) CYP2C12 is known to be one of the steroid hydroxylases and is constitutively expressed in female rats but not in male rats (1Kamataki T. Maeda K. Yamazoe Y. Nagai T. Kato R. Arch. Biochem. Biophys. 1983; 225: 758-770Crossref PubMed Scopus (230) Google Scholar, 2MacGeoch C. Morgan E.T. Halpert J. Gustafsson J.-Å. J. Biol. Chem. 1984; 259: 15433-15439Abstract Full Text PDF PubMed Google Scholar, 3Kamataki T. Maeda K. Shimada M. Kitani K. Nagai T. Kato R. J. Pharmcol. Exp. Ther. 1985; 233: 222-228PubMed Google Scholar, 4Zaphiropoulos P.G. Mode A. Ström A. Möller C. Fernandez C. Gustafsson J.-Å. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4214-4217Crossref PubMed Scopus (56) Google Scholar). The sex-specific expression of the CYP2C12 gene is regulated by the plasma growth hormone (GH)1 pattern (5Kamataki T. Shimada M. Maeda K. Kato R. Biochem. Biophys. Res. Commun. 1985; 130: 1247-1253Crossref PubMed Scopus (64) Google Scholar, 6MacGeoch C. Morgan E.T. Gustafsson J.-Å. Endocrinology. 1985; 117: 2085-2092Crossref PubMed Scopus (87) Google Scholar, 7Kato R. Yamazoe Y. Shimada M. Murayama N. Kamataki T. J. Biochem. ( Tokyo ). 1986; 100: 895-902Crossref PubMed Scopus (98) Google Scholar, 8Yamazoe Y. Shimada M. Kamataki T. Kato R. Jpn. J. Pharmacol. 1986; 42: 371-382Crossref PubMed Scopus (55) Google Scholar, 9MacGeoch C. Morgan E.T. Cordell B. Gustafsson J.-Å. Biochem. Biophys. Res. Commun. 1987; 143: 782-788Crossref PubMed Scopus (24) Google Scholar, 10Legraverend C. Mode A. Westin S. Ström A. Eguchi H. Zaphiropoulos P.G. Gustafsson J.-Å. Mol. Endocrinol. 1992; 6: 259-266Crossref PubMed Scopus (117) Google Scholar, 11Mode A. J. Reprod. Fertil. 1993; 46 (suppl.): 77-86Google Scholar, 12Sundseth S.S. Alberta J.A. Waxman D.J. J. Biol. Chem. 1992; 267: 3907-3914Abstract Full Text PDF PubMed Google Scholar). Although the pattern of GH secretion in male rats is characterized by the peaks of large amplitude every 3–4 h with undetectable levels in interpulse periods, the pattern of GH secretion in female rats shows more frequent oscillation of small amplitude (13Tannenbaum G.S. Martin J.B. Endocrinology. 1976; 98: 562-570Crossref PubMed Scopus (652) Google Scholar, 14Edén S. Endocrinology. 1979; 105: 555-560Crossref PubMed Scopus (476) Google Scholar). Thus, the transcription of the CYP2C12 gene is thought to be activated by the constant level of GH seen in female rats (9MacGeoch C. Morgan E.T. Cordell B. Gustafsson J.-Å. Biochem. Biophys. Res. Commun. 1987; 143: 782-788Crossref PubMed Scopus (24) Google Scholar, 10Legraverend C. Mode A. Westin S. Ström A. Eguchi H. Zaphiropoulos P.G. Gustafsson J.-Å. Mol. Endocrinol. 1992; 6: 259-266Crossref PubMed Scopus (117) Google Scholar, 12Sundseth S.S. Alberta J.A. Waxman D.J. J. Biol. Chem. 1992; 267: 3907-3914Abstract Full Text PDF PubMed Google Scholar). The effects of GH are mediated by a GH receptor, which belongs to a cytokine/hematopoietin receptor superfamily (15Argetsinger L.S. Carter-Su C. Physiol. Rev. 1996; 76: 1089-1107Crossref PubMed Scopus (247) Google Scholar). On the basis of a proposed pathway for the induction of gene expression by GH, the binding of GH to the GH receptor promotes the association of the GH receptor with Janus kinase 2 and the tyrosyl phosphorylation of the Janus kinase 2. (16Argetsinger L.S. Campbell G.S. Yang X. Witthuhn B.A. Silvennoinen O. Ihle J.N. Carter-Su C. Cell. 1993; 74: 237-244Abstract Full Text PDF PubMed Scopus (827) Google Scholar). Subsequently, the activated Janus kinase 2 phosphorylates the tyrosine residues of the STAT protein. After the formation of the homodimer of the STAT protein or the heterodimer of the STAT protein with other factor(s) in the cytoplasm, the complex translocates to the nucleus and then binds to target sequences (17Darnell J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5062) Google Scholar). Among the STAT family, STAT1, STAT3, and STAT5 have been identified as GH-stimulated proteins (15Argetsinger L.S. Carter-Su C. Physiol. Rev. 1996; 76: 1089-1107Crossref PubMed Scopus (247) Google Scholar, 18Ram P.A. Park S.-H. Choi H.K. Waxman D.J. J. Biol. Chem. 1996; 271: 5929-5940Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Particularly, STAT5 (19Wood T.J.J. Sliva D. Lobie P.E. Pircher T.J. Gouilleux F. Wakao H. Gustafsson J.-Å. Groner B. Norstedt G. Haldosén L.-A. J. Biol. Chem. 1995; 270: 9448-9453Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) has been reported to participate in the GH-related expression of some genes including c-fos (20Gronowski A.M. Zhong Z. Wen Z. Thomas M.J. Darnell J.E. Rotwein P. Mol. Endocrinol. 1995; 9: 171-177Crossref PubMed Google Scholar), serine protease inhibitor 2.1 (21Sliva D. Wood T.J.J. Schindler C. Lobie P.E. Norstedt G. J. Biol. Chem. 1994; 269: 26208-26214Abstract Full Text PDF PubMed Google Scholar), and the acid-labile subunit gene (22Ooi G.T. Hurst K.R. Poy M.N. Rechler M.M. Boisclair Y.R. Mol. Endocrinol. 1998; 12: 675-687Crossref PubMed Google Scholar). Pulsatile plasma GH secretion seen in male rats but not in female rats has been shown to activate STAT5 in the liver (23Waxman 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. 1997; 11: 400-414Crossref PubMed Scopus (111) Google Scholar). Based on these lines of evidence, STAT5 has been regarded as the male-specific regulator for the expression of genes including the sex-limited protein gene (25Varin-Blank N. Dondi E. Tosi M. Hernandez C. Boucontet L. Gotoh H. Shiroishi T. Moriwaki K. Meo T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8750-8755Crossref PubMed Scopus (16) Google Scholar) andCYP3A10 (26Subramanian A. Teixeira J. Wang J. Gil G. Mol. Cell. Biol. 1995; 15: 4672-4682Crossref PubMed Google Scholar). To date, the mechanism responsible for the GH-dependent activation of the CYP2C12 gene in females has been studiedin vitro. HNF-6 has been reported to be involved in the GH-dependent transcription of the CYP2C12 gene (27Lahuna O. Fernandez L. Karlsson H. Maiter D. Lemaigre F.P. Rousseau G.G. Gustafsson J.-Å. Mode A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12309-12313Crossref PubMed Scopus (92) Google Scholar). Additionally, it was found that the female-enriched GH-dependent complex, termed GHNF, bound to five distinct regions within the CYP2C12 promoter region between nucleotides −1560 and +60 (28Waxman 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, it has not been clarified as yet whether or not the above factors play a key role in the in vivo expression of the CYP2C12 gene. In addition to the uncertainty of the in vivo role of these factors, a functional GH-responsive element(s) has not yet been clarified in the regulatory region of the CYP2C12 gene. To identify a functional factor(s) responsible for the modulation of the GH-dependent transcription of the CYP2C12gene, we attempted to search the cultured cells to show the inducibility of CYP2C12 by GH. However, no suitable cultured cells were found. Recently, it has been reported that the direct injection of plasmid DNA into the liver is a useful method to assay the in vivo activity of the CYP2B and CYP2Cpromoters (29Park Y. Li H. Kemper B. J. Biol. Chem. 1996; 271: 23725-23728Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Thus, we employed the direct injection method to search for a critical factor(s) responsible for the GH-dependent expression and to determine the relative contribution of each transcription factor to the GH-dependent expression of theCYP2C12 gene. In the present study, we provide evidence that the GH-dependent and liver-specific expression of theCYP2C12 gene in female rats results from a cooperative regulation with STAT5, HNF-4, HNF-6, and factors that bind to elements 2C12-I and 2C12-II in the upstream region of the CYP2C12gene. Adult female Harlan Sprague Dawley rats (8 weeks old; Sankyo Experimental Animals, Tokyo, Japan) were used. When necessary, female rats were hypophysectomized at 6 weeks of age. Recombinant human methionylated GH (Somatonorm, Kabi Vitrum, Stockholm, Sweden) was kindly supplied by Sumitomo Pharmaceutical Co. (Osaka, Japan). Human GH was administered by a continuous infusion (0.94 IU/kg·day), which mimics female-type GH secretion, with an osmotic minipump (model 2001, Alza, Palo Alto, CA) or by a pulsatile injection (0.94 IU/kg·day), which mimics male-type GH secretion (10Legraverend C. Mode A. Westin S. Ström A. Eguchi H. Zaphiropoulos P.G. Gustafsson J.-Å. Mol. Endocrinol. 1992; 6: 259-266Crossref PubMed Scopus (117) Google Scholar), for 6 days. To isolate the 5′-flanking region of the CYP2C12 gene (30Zaphilopous P.G. Westin S. Ström A. Mode A. Gustafsson J.-Å. DNA Cell Biol. 1990; 9: 49-56Crossref PubMed Scopus (26) Google Scholar), a gene library prepared from the rat genomic DNA, which had been cleaved by Sau3A I and cloned into λFIXII vector (Stratagene, La Jolla, CA), was screened with the fragment of a CYP2C12promoter as a probe. Consequently, a positive clone containing the 9–10-kilobase fragment of the CYP2C12 gene was obtained. The clone was digested with XhoI and HindIII to obtain a 5′-flanking region between nucleotides −5132 and +113 from the transcription start site (30Zaphilopous P.G. Westin S. Ström A. Mode A. Gustafsson J.-Å. DNA Cell Biol. 1990; 9: 49-56Crossref PubMed Scopus (26) Google Scholar). The resultant fragment was subcloned to the XhoI/HindIII site of pUC19 (pUC5.2). Luc5132 reporter plasmid was constructed as follows. AHindIII site was introduced downstream of the nucleotide +16 of pUC5.2 by site-directed mutagenesis (pUC5.2(+16)). pUC5.2(+16) was then cleaved with XhoI and HindIII. The fragment corresponding to nucleotides from −5132 to +16 was ligated into the unique XhoI–HindIII site of basic vector 2 (Toyoinki, Tokyo, Japan). Reporter plasmids, Luc4213, Luc4118, and Luc1944, were constructed by the digestion of Luc5132 with restriction enzymes, HincII, BstXI, and BamHI, respectively. Luc4200, Luc4187, and Luc4161 were generated by polymerase chain reaction using S-4200, S-4187, or S-4161 as a 5′-primer and AS-3614 as a 3′-primer. Synthesized fragments were digested with BglII at position −3780 in theCYP2C12 gene. Resultant fragments were inserted into theSmaI/BglII site of Luc3780. To construct Luc4200ΔGHRE2 without the region between nucleotides −4174 and −4162, the region corresponding to nucleotides from −4200 to −4175 was inserted into the KpnI site of Luc4161. To confirm the contribution of GHRE to GH-dependent transcriptional activity, reporter plasmids, 1×GHRE-LucTK, 2×GHRE-LucTK, 4×GHRE-LucTK, and GHRE-Luc1944 were constructed. TK promoter was inserted into the BglII/HindIII site of basic vector 2 (LucTK). 1×GHRE, 2×GHRE, or 4×GHRE was then introduced into the XhoI site of LucTK. The copy number and the direction of the oligonucleotides inserted into the reporter plasmids were confirmed by a sequence analysis (31Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52769) Google Scholar). To construct Luc4200Δ(−4161/−3781) or Luc4200Δ (−4161/−4118), the region corresponding to nucleotides from −4200 to −4162 was inserted into theKpnI/NheI site of Luc3780 or Luc4118. To construct Luc4200Δ(−3776/−3138), Luc4200Δ(−3137/−1944), Luc4200Δ(−1939/−536), Luc4200Δ(−535/−81), Luc4200Δ(−4122/−4036), or Luc4200Δ(−4030/−3781), Luc4200 was digested with BglII/EcoRV,EcoRV/BamHI, BamHI/StuI,StuI/MscI, BstXI/HgaI, orHgaI/BglII, respectively. Resultant fragments were blunt-ended and self-ligated. To construct GHRE-Luc3780, the region corresponding to nucleotides from −4200 to −4162 was inserted into the KpnI/NheI site of Luc3780. To construct GHRE-2C12-I-Luc3780 and GHRE-2C12-II-Luc3780, the regions corresponding to nucleotides from −4095 to −4074 (2C12-I) and from −4072 to −4045 (2C12-II) were cloned into the GHRE-Luc3780. To construct GHRE-2C12-I/II-Luc3780, the region corresponding to nucleotides from −4072 to −4045 (2C12-II) was cloned into the GHRE-2C12-I-Luc3780. Luc4200mtC/EBP, Luc4200mtHNF-4, and Luc4200mtHNF-6 were generated by site-directed mutagenesis. The oligonucleotide primers used for the synthesis of DNA fragment or site-directed mutagenesis are as follows: S-4200, 5′-AAATTTCCTAGAAGTGAAATTG-3′; S-4187, 5′-GTGAAATTGTGGTAAATTCC-3′; S-4161, 5′-TCATTGCCAGAGGAGACA-3′; AS-3614, 5′-TGATGAGTGAGAACATTCTA-3′; −4200/−4175, 5′-CAAATTTCCTAGAAGTGAAATTGTGGTGGTAC-3′ (sense strand) and 3′-CATGGTTTAAAGGATCTTCACTTTAACACCAC-5′ (antisense strand); −4200/−4162, 5′-CAAATTTCCTAGAAGTGAAATTGTGGTAAATTCCTAGAACG-3′ (sense strand) and 3′-CATGGTTTAAAGGATCTTCACTTTAACACCATTTAAGGATCTTGCGATC-5′ (antisense strand); GHRE, 5′-TCGAGTTTCCTAGAAGCTCGAGTTTCCTAGAAGC-3′ (sense strand) and 3′-CAAAGGATCTTCGAGCTCAAAGGATCTTCGAGCT-5′ (antisense strand); mutated 2C12-C/EBP (−228/−205) (32Tollet P. Lahuna O. Ahlgren R. Mode A. Gustafsson J.-Å. Mol. Endocrinol. 1995; 9: 1771-1781Crossref PubMed Scopus (1) Google Scholar), 5′-TGAGTGTAGATATCGGTGTTACAT-3′ and 3′-ACTCACATCTATAGCCACAATGTA-5′; mutated 2C12-HNF-4 (−122/−96) (33Ström A. Westin S. Eguchi H. Gustafsson J.-Å. Mode A. J. Biol. Chem. 1995; 270: 11276-11281Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), 5′-ATCATTGAGTCTGTCTTCATTTGAAAG-3′ and 3′-TAGTAACTCAGACAGAAGTAAACTTTC-5′; mutated 2C12-HNF-6 (−52/−30) (27Lahuna O. Fernandez L. Karlsson H. Maiter D. Lemaigre F.P. Rousseau G.G. Gustafsson J.-Å. Mode A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12309-12313Crossref PubMed Scopus (92) Google Scholar), 5′-GCAAAAGATGGTTTTTTATGGTG-3′ and 3′-CGTTTTCTACCAAAAAATACCAC-5′. Direct DNA injection was performed with minor modifications of the method previously described (34Malone R.W. Hickman M.A. Lehmann-Bruinsma K. Sih T.R. Walzem R. Carlson D.M. Powell J.S. J. Biol. Chem. 1994; 269: 29903-29907Abstract Full Text PDF PubMed Google Scholar, 29Park Y. Li H. Kemper B. J. Biol. Chem. 1996; 271: 23725-23728Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The CYP2C12-Luc chimeric genes (600 μg of plasmid DNA diluted with 1.5 ml of Dulbecco's modified Eagle's medium (Nisssui Pharmaceutical, Tokyo, Japan)) were injected into the peripheral site of single liver lobes. Rats were sacrificed 7 days later to obtain the injected liver weighing from 0.1 to 0.2 g. Human GH was administered 1 day after the DNA injection by a continuous infusion for 6 days (30Zaphilopous P.G. Westin S. Ström A. Mode A. Gustafsson J.-Å. DNA Cell Biol. 1990; 9: 49-56Crossref PubMed Scopus (26) Google Scholar). When a reporter plasmid with TK promoter was injected into the livers of hypophysectomized rats, GH was administered 5 days prior to the DNA injection. Rats were sacrificed 1 day after the DNA injection, when the highest luciferase activity was seen. Three ml of LCβ/PGC-51 lysis buffer (Toyoinki, Tokyo, Japan) per 1 g of liver tissue was added to prepare liver cell extracts. After homogenization with a Dounce homogenizer, insoluble materials were removed by centrifugation. The supernatants were kept frozen at −80 °C until use. Luciferase activity was measured using a Berthold Lumat LB 9501 luminometer with photon detection integrated over 10-s intervals. Statistical significance was judged by a Mann-Whitney U test. Nuclear extracts were prepared from the livers from hypophysectomized rats and from the hypophysectomizd rats treated with GH by the constant infusion as described elsewhere (35Gorski K. Carneiro M. Schibler U. Cell. 1986; 47: 767-776Abstract Full Text PDF PubMed Scopus (973) Google Scholar) except for 1% aprotinin (Sigma) in an initial homogenization buffer. Subsequently, the extracts were snap-frozen in 0.01-ml aliquots at 5–10 mg of protein/ml and immediately frozen in liquid nitrogen for storage. The gel shift assay was performed with 20 μl of a reaction mixture containing 25 mm Hepes (pH 7.8), 100 mm KCl, 5 mm MgCl2, 0.3 mm EDTA, 0.5 mm dithiothreitol, 2.5% glycerol, 2% Ficoll 400, 10 μg of liver nuclear extracts, and a32P-labeled probe (10 fmol). The mixture was incubated at 0 °C for 90 min. Resulting DNA-protein complex was subjected to a 6% polyacrylamide gel in a buffer containing 25 mm Tris borate and 0.5 mm EDTA. For a competition assay, liver nuclear extracts were preincubated at 0 °C for 30 min with a 20-fold molar excess of an unlabeled oligonucleotide. CRE and mutated CRE used for a competition assay were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). For a supershift assay, liver nuclear extracts were preincubated at 0 °C for 30 min with 1 μl of antibodies to STAT1α, STAT3, STAT5, STAT5a, CREB1, or CREB2. These antibodies to STAT proteins were purchased from Santa Cruz Biotechnology. After a labeled probe was added, the mixture was incubated at 0 °C for 90 min. The oligonucleotide primers used as a probe are as follows: GHRE1, 5′-AAAATTTCCTAGAAGTG-3′ and 3′-TTAAAGGATCTTCACTT-5′; GHRE2, 5′-GAAATTGTGGTAAATTCCTAGAACTC-3′ and 3′-TTAACACCATTTAAGGATCTTGAGTA-5′; Ly6E interferon γ-activated sequence core site (36Khan K.D. Shuai K. Lindwall G. Maher S.E. Darnell J.E. Bothwell A.L.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6806-6810Crossref PubMed Scopus (149) Google Scholar), 5′-GATCATATTCCTGTAAGTGAT-3′ and 3′-TATAAGGACATTCACTACTAG-5′; M67sis-inducible element from c-fos (37Wagner B.J. Hayes T.E. Hoban C.J. Cochran B.H. EMBO J. 1990; 9: 4477-4484Crossref PubMed Scopus (554) Google Scholar), 5′-GATCCATTTCCCGTAAATCAT-3′ and 3′-GTAAAGGGCATTTAGTACTAG-5′; rat α2-macroglobulin acute-phase response element (38Wegenka U.M. Buschmann J. Lütticken C. Heinrich P.C. Horn F. Mol. Cell. Biol. 1993; 13: 276-288Crossref PubMed Scopus (490) Google Scholar), 5′-GATCGGAATTCCCAGAAGGAT-3′ and 3′-CCTTAAGGGTCTTCCTACTAG-5′; rat β-casein gene STAT5/mammary gland factor response element (39Wakao H. Gouilleux F. Groner B. EMBO J. 1994; 13: 2182-2191Crossref PubMed Scopus (717) Google Scholar), 5′-GATCGGACTTCTTGGAATTAAGGGA-3 and 3′-CCTGAAGAACCTTAATTCCCTCTAG-5′; mutated β-casein, 5′-GATCGGACTTAGTTTAATTAAGGGA-3′ and 3′-CCTGAATCAAATTAATTCCCTCTAG-5′; mouse α-globin GATA-1 binding site (40Tsai S.-F. Martin D.I.K. Zon L.I. D'Andrea A.D. Wong G.G. Orkin S.H. Nature. 1989; 339: 446-451Crossref PubMed Scopus (667) Google Scholar), 5′-TCCGGCAACTGATAAGGATTCCCT-3′ and 3′-AGGCCGTTGACTATTCCTAAGGGA-5′; TTR (41Costa R.H. Grayson D.R. Darnell J.E. Mol. Cell. Biol. 1989; 9: 1415-1425Crossref PubMed Scopus (428) Google Scholar), 5′-TCTGATTATTGACTTAGTCAAG-3′ and 3′-AGACTAATAACTGAATCAGTTC -5′; TAT (42Nitsch D. Schütz G. Mol. Cell. Biol. 1993; 13: 4494-4504Crossref PubMed Scopus (38) Google Scholar), 5′-GACGTTTCTCAATATTTGCTCTGGCAG-3′ and 3′-TGCAAAGAGTTATAAACGAGACCGTCT-5′. Southwestern blot analysis was performed according to the method of Miskimins et al.(43Miskimins W.K. Roberts M.P. McClelland A. Ruddle F.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6741-6744Crossref PubMed Scopus (201) Google Scholar). To examine whether or not the Luc5132 reporter gene was activated in vivo by GH secreted in a female-type pattern, we injected the DNA of Luc5132 into the liver of female rats (Fig. 1 A). As expected, the luciferase activity was detected in the liver transfected with Luc5132. Hypophysectomy of female rats resulted in the abolishment of the luciferase activity. However, the activity was restored to the level seen in intact rats by the continuous infusion of GH to the livers of hypophysectomized female rats. These results indicate that the direct DNA injection is a useful method to clarify the mechanism responsible for the GH-dependent transcriptional regulation of the CYP2C12 gene. To identify possible regulatory element(s) involved in the GH-induced expression of the CYP2C12 gene, the 5′-deletion mutants of the gene were constructed as shown in Fig. 1 B. When the mutants were injected into livers of female rats, the maximal activity of luciferase was seen in the liver that received Luc4213. The activity was decreased to approximately 20% by the deletion of the nucleotide sequences between −4213 and −4161. Further deletion between −4118 and −1944 also resulted in a significant decrease to a basal activity. These results suggest that at least two regions are necessary for the GH-dependent expression of the CYP2C12 gene in female rats. The sequence of the 5′-flanking region of the CYP2C12 gene was found to contain 9-bp palindrome sequences (5′-TTCCTAGAA-3′), designated as GHRE1 and GHRE2, in a region between nucleotides −4213 and −4161 (Fig. 2 A). Compared with elements reported so far, the sequence of the GHREs overlapped with those of the interferon γ-activated sequence and acute-phase response element (5′-TTCCNNNAA-3′), known to be the binding sites of STAT proteins. As can be seen in Fig. 2 B, the deletion of either the GHRE1 or the GHRE2 caused an 80–90% decrease in the luciferase activity. This result indicates that both GHREs contribute to the GH-dependent activation of the CYP2C12gene in female rats. Furthermore, a gel shift assay using the GHRE1 or the GHRE2 as a probe showed that a factor(s) in nuclear extracts from female livers bound to each GHRE (Fig. 2 C). Interestingly, the binding of the constitutive factor(s) to the GHREs disappeared with the hypophysectomy of female rats. The binding of the factor(s) to the GHREs was, however, restored by the continuous infusion of GH, which mimics female-type GH secretion, to hypophysectomized female rats. To further confirm the contribution of the GHREs to the GH-dependent expression of the CYP2C12 gene, GHRE-Luc1944, which contains 10 copies of the GHRE, was injected into the liver of female rats (Fig. 3 A). The results indicate that the luciferase activity in the liver transfected with GHRE-Luc1944 is much higher than that in the liver transfected with Luc1944. In the livers from hypophysectomized female rats, the activation of the GHRE-Luc1944 was not seen. The continuous infusion of GH to hypophysectomized rats elevated the luciferase activity of GHRE-Luc1944 by approximately 10-fold. To further characterize GHRE, 1×GHRE-, 2×GHRE-, and 4×GHRE-LucTK were used for a luciferase assay. The luciferase activity was significantly increased with the copy number of GHRE (Fig. 3 B). Additionally, the luciferase activity with 4×GHRE-LucTK was elevated by the infusion of GH to the livers of hypophysectomized rats (Fig. 3 C).FIG. 3GH-dependent activation of theCYP2C12 or TK promoter through the GHRE . A, GHRE-Luc1944 was constructed as described under “Materials and Methods.” The reporter plasmid was injected into the livers of nontreated females (NT), hypophysectomized females (Hypox), and hypophysectomized females infused with human GH for 6 days (Hypox + GH). B, effects of the copy number of GHRE on the transcriptional activity of TK promoter.C, GH-dependent activation of 4×GHRE-LucTK. 4×GHRE-LucTK was injected into the livers of hypophysectomized females (Hypox) or hypophysectomized females infused with human GH for 5 days (Hypox + GH). All values represent the mean ± S.D. from independent experiments shown in parentheses. The data are expressed as the ratio of the luciferase activity of each deletion mutant to the basal activity obtained with GHRE-Luc1944 or 4×GHRE-LucTK in female rats. * and **, significantly different between two groups at p < 0.05 and p < 0.01, respectively.View Large Image Figure ViewerDownload (PPT) Searching the sequence similar to the GHRE, we found that the sequence of the GHRE overlapped with those of STAT-binding sites (Fig. 4). Particularly, the sequence of the GHRE was completely identical to the STAT5 binding sequence found in the acid-labile subunit gene (22Ooi G.T. Hurst K.R. Poy M.N. Rechler M.M. Boisclair Y.R. Mol. Endocrinol. 1998; 12: 675-687Crossref PubMed Google Scholar) and interleukin-2 receptor α gene (44Lécine P. Algarté M. Rameil P. Beadling C. Bucher P. Nabholz M. Imbert J. Mol. Cell. Biol. 1996; 16: 6829-6840Crossref PubMed Google Scholar). The STAT3 binding sites of the α2-macrogloblin gene (38Wegenka U.M. Buschmann J. Lütticken C. Heinrich P.C. Horn F. Mol. Cell. Biol. 1993; 13: 276-288Crossref PubMed Scopus (490) Google Scholar) and the STAT5 binding sites of the β-casein gene (39Wakao H. Gouilleux F. Groner B. EMBO J. 1994; 13: 2182-2191Crossref PubMed Scopus (717) Google Scholar) possessed only one base change as compared with the GHRE sequence. To confirm the binding of STAT protein to the GHRE2, we performed a competition assay with STAT-binding sequences seen in the rat β-casein, rat α2-macroglobulin, and human Ly6E and M67 genes (Fig. 5 A). We found that the binding of the GH-stimulated factor(s) to the GHRE2 disappeared with the presence of a 20-fold molar excess of the STAT-binding sequences of the β-casein and α2-macroglobulin genes. The competitor of Ly6E and M67 partially inhibited the formation of the complex of the GH-stimulated factor(s) with the GHRE2. Unlike the competitors, the β-casein, α2-macroglobulin, Ly6E, andM67 genes, mutated β-casein, and nonspecific competitor, GATA-1, did not affect the binding of the complex to the GHRE2. To identify a STAT protein to bind to the GHRE, a supershift assay using antibodies to STAT1α, STAT3, STAT5, and STAT5a was performed (Fig. 5,B and C). The results showed that a supershifted band appeared in the presence of antibodies against STAT5 and STAT5a. Thus, it appeared that STAT5a is one of the modulators for the expression of CYP2C12 in the livers of female rats.FIG. 5Binding of STAT5 to the GHRE . A, effects of competitors on the binding of STAT proteins to GHRE2. A 32P-labeled double strand GHRE2 was incubated with 10 μg of nuclear extracts prepared from the liver of female rats in the presence of a 20-fold molar excess amount of a competitor at 0 °C for 90 min. mt, mutated; α 2 -MG, α2-macroglobulin. B and C, supershift assay using antibodies to STAT1α, STAT3, STAT5, and STAT5a. A32P-labeled double strand GHRE2 was incubated with nuclear extracts prepared from the liver of female rats in the presence of antibodies to STAT1α, STAT3, STAT5, or STAT5a as described under “Materials and Methods.”View Large Image Figure ViewerDownload (PPT) The liver-specific expression of CYP2C12 may not be accounted for solely by STAT5, since this protein is also expressed in extrahepatic tissues. Thus, we postulated that liver-specific factors other than STAT5 were required for the expression of CYP2C12 in the livers of female rats. To identify the regulatory regions necessary for the liver-specific expression of theCYP2C12 gene, we performed a luciferase assay with reporter plasmids, Luc4200Δ(−4161/−3781), Luc4200" @default.
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• W2037992255 title "Cooperative Regulation of CYP2C12 Gene Expression by STAT5 and Liver-specific Factors in Female Rats" @default.
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