Sterol Regulatory Element-binding Proteins Activate Insulin Gene Promoter Directly and Indirectly through Synergy with BETA2/E47
2005; Elsevier BV; Volume: 280; Issue: 41 Linguagem: Inglês
10.1074/jbc.m506718200
ISSN1083-351X
AutoresMichiyo Amemiya-Kudo, Junko Oka, Tomohiro Ide, Takashi Matsuzaka, Hirohito Sone, Tomohiro Yoshikawa, Naoya Yahagi, Shun Ishibashi, Jun-ichi Osuga, Nobuhiro Yamada, Toshio Murase, Hitoshi Shimano,
Tópico(s)Diet, Metabolism, and Disease
ResumoInsulin gene expression is regulated by pancreatic β cell-specific factors, PDX-1 and BETA2/E47. Here we have demonstrated that the insulin promoter is a novel target for SREBPs established as lipid-synthetic transcription factors. Promoter analyses of rat insulin I gene in non-β cells revealed that nuclear SREBP-1c activates the insulin promoter through three novel SREBP-binding sites (SREs), two of which overlap with E-boxes, binding sites for BETA2/E47. SREBP-1c activation of the insulin promoter was markedly enhanced by co-expression of BETA2/E47. This synergistic activation by SREBP-1c/BETA2/E47 was not mediated through SREs but through the E-boxes on which BETA2/E47 physically interacts with SREBP-1c, suggesting a novel function of SREBP as a co-activator. These two cis-DNA regions, E1 and E2, with an appropriate distance separating them, were mandatory for the synergism, which implicates formation of SREBP-1c·BETA2·E47 complex in a DNA looping structure for efficient recruitment of CREB-binding protein/p300. However, in the presence of PDX1, the synergistic action of SREBP-1c with BETA2/E47 was canceled. SREBP-1c-mediated activation of the insulin promoter and expression became overt in β cell lines and isolated islets when endogenous PDX-1 expression was low. This cryptic SREBP-1c action might play a compensatory role in insulin expression in diabetes with β cell lipotoxicity. Insulin gene expression is regulated by pancreatic β cell-specific factors, PDX-1 and BETA2/E47. Here we have demonstrated that the insulin promoter is a novel target for SREBPs established as lipid-synthetic transcription factors. Promoter analyses of rat insulin I gene in non-β cells revealed that nuclear SREBP-1c activates the insulin promoter through three novel SREBP-binding sites (SREs), two of which overlap with E-boxes, binding sites for BETA2/E47. SREBP-1c activation of the insulin promoter was markedly enhanced by co-expression of BETA2/E47. This synergistic activation by SREBP-1c/BETA2/E47 was not mediated through SREs but through the E-boxes on which BETA2/E47 physically interacts with SREBP-1c, suggesting a novel function of SREBP as a co-activator. These two cis-DNA regions, E1 and E2, with an appropriate distance separating them, were mandatory for the synergism, which implicates formation of SREBP-1c·BETA2·E47 complex in a DNA looping structure for efficient recruitment of CREB-binding protein/p300. However, in the presence of PDX1, the synergistic action of SREBP-1c with BETA2/E47 was canceled. SREBP-1c-mediated activation of the insulin promoter and expression became overt in β cell lines and isolated islets when endogenous PDX-1 expression was low. This cryptic SREBP-1c action might play a compensatory role in insulin expression in diabetes with β cell lipotoxicity. Insulin gene expression is restricted to pancreatic β cells and is induced by glucose (1Permutt M.A. Kipnis D.M. J. Biol. Chem. 1972; 247: 1194-1199Abstract Full Text PDF PubMed Google Scholar). Extensive studies on the insulin promoter have unveiled β cell-specific transcription factors responsible for glucose-inducible transcription, such as PDX-1, BETA2/E47, and MafA, and have located their respective binding sites, A3, E1, and C1 (2Melloul D. Ben-Neriah Y. Cerasi E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3865-3869Crossref PubMed Scopus (159) Google Scholar, 3Naya F.J. Stellrecht C.M. Tsai M.J. Genes Dev. 1995; 9: 1009-1019Crossref PubMed Scopus (515) Google Scholar, 4Olson L.K. Qian J. Poitout V. Mol. 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Natl. Acad. Sci. U. S. A. 1996; 93: 15057-15062Crossref PubMed Scopus (151) Google Scholar) in addition to its important role in the development and differentiation of pancreatic islets. BETA2, a β cell-specific basic helix-loop-helix (bHLH) 2The abbreviations used are: bHLH, basic helix-loop-helix; CREB, cAMP-response element-binding protein; CBP, CREB-binding protein; HEK, human embryonic kidney; Luc, luciferase; SREBP, sterol regulatory element-binding protein; CMV, cytomegalovirus; GST, glutathione S-transferase; ChIP, chromatin immunoprecipitation; β-gal, β-galactosidase; TA, transactivation domain; HIT, hamster insulinoma tumor cells. 2The abbreviations used are: bHLH, basic helix-loop-helix; CREB, cAMP-response element-binding protein; CBP, CREB-binding protein; HEK, human embryonic kidney; Luc, luciferase; SREBP, sterol regulatory element-binding protein; CMV, cytomegalovirus; GST, glutathione S-transferase; ChIP, chromatin immunoprecipitation; β-gal, β-galactosidase; TA, transactivation domain; HIT, hamster insulinoma tumor cells. protein is also an important regulator of the insulin gene (3Naya F.J. Stellrecht C.M. Tsai M.J. 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BETA2/E47 binds to E2-box and PDX-1 binds to A3/4. PDX-1, BETA2/E47, and HMGI(Y) physically interact (12Ohneda K. Mirmira R.G. Wang J. Johnson J.D. German M.S. Mol. Cell. Biol. 2000; 20: 900-911Crossref PubMed Scopus (166) Google Scholar) and show synergistic activation of insulin gene expression by facilitating recruitment of coactivator CBP/p300 (13Qiu Y. Sharma A. Stein R. Mol. Cell. Biol. 1998; 18: 2957-2964Crossref PubMed Scopus (120) Google Scholar, 14Sharma A. Moore M. Marcora E. Lee J.E. Qiu Y. Samaras S. Stein R. Mol. Cell. Biol. 1999; 19: 704-713Crossref PubMed Scopus (81) Google Scholar). Sterol regulatory element-binding proteins (SREBPs) are members of the basic helix-loop-helix leucine zipper family of transcription factors that regulate fatty acid and cholesterol synthesis (reviewed in Refs. 15Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2916) Google Scholar and 16Brown M.S. Ye J. Rawson R.B. Goldstein J.L. Cell. 2000; 100: 391-398Abstract Full Text Full Text PDF PubMed Scopus (1135) Google Scholar). SREBPs are initially bound to the rough endoplasmic reticulum membrane. By virtue of the SREBP cleavage-activating protein/Insig system, SREBP is cleaved in a sterol-dependent manner to liberate the amino-terminal portion containing a basic helix-loop-helix leucine zipper domain (nuclear SREBP), which enters the nucleus where it can bind to specific sterol response elements (SREs) in the promoters of target genes. Three isoforms of SREBP, -1a, -1c, and -2, are known. Although SREBP-2 plays a crucial role in the regulation of cholesterol synthesis, SREBP-1c controls the gene expression of lipogenic enzymes (reviewed in Refs. 17Shimano H. Vitam. Horm. 2002; 65: 167-194Crossref PubMed Google Scholar and 18Horton J.D. Biochem. Soc. Trans. 2002; 30: 1091-1095Crossref PubMed Scopus (275) Google Scholar). Nuclear SREBP-2 has a high affinity with classic sterol regulatory elements, which are usually found in the promoters of cholesterogenic genes and the LDL receptor gene. Nuclear SREBP-1 also has broad binding capacity to SRE-like sequences, including E-boxes that are occasionally found in the promoters of lipogenic genes (19Amemiya-Kudo M. Shimano H. Hasty A.H. Yahagi N. Yoshikawa T. Matsuzaka T. Okazaki H. Tamura Y. Iizuka Y. Ohashi K. Osuga J. Harada K. Gotoda T. Sato R. Kimura S. Ishibashi S. Yamada N. J. Lipid Res. 2002; 43: 1220-1235Abstract Full Text Full Text PDF PubMed Google Scholar). SREBP-1c has been well established as controlling the nutritional regulation of lipogenic genes in the liver (20Shimano H. Yahagi N. Amemiya-Kudo M. Hasty A.H. Osuga J. Tamura Y. Shionoiri F. Iizuka Y. Ohashi K. Harada K. Gotoda T. Ishibashi S. Yamada N. J. Biol. Chem. 1999; 274: 35832-35839Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar). However, the role of SREBP-1c in pancreatic β cells is yet to be elucidated. Because an excess of fatty acids or triglycerides could impair the functions of β cells, a potential pathophysiologic consequence of diabetes, often referred to as lipotoxicity, estimation of SREBP-1c in β cell function is an important issue to be addressed. It has been reported that insulin-resistant obese animals have a high expression of SREBP-1c in β cells (21Kakuma T. Lee Y. Higa M. Wang Z. Pan W. Shimomura I. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8536-8541Crossref PubMed Scopus (228) Google Scholar). A β cell line in which nuclear SREBP-1c is overexpressed showed impaired insulin secretion (22Wang H. Maechler P. Antinozzi P.A. Herrero L. Hagenfeldt-Johansson K.A. Bjorklund A. Wollheim C.B. J. Biol. Chem. 2003; 278: 16622-16629Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 23Andreolas C. da Silva Xavier G. Diraison F. Zhao C. Varadi A. Lopez-Casillas F. Ferre P. 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In the current study, we performed promoter analysis on rat insulin promoter I to investigate the potential effects of SREBPs on insulin gene expression in both non-β and β cell lines. Standard molecular biology techniques were used. We obtained cholesterol and 25-hydroxycholesterol from Sigma, Redivue [α-32P]dCTP (6000 Ci/mmol) from Amersham Biosciences, and restriction enzymes from Takara Bio Inc. Plasmid DNAs for transfection were prepared with EndoFree Plasmid Midi kits (Qiagen). Expression Plasmids—Expression vectors encoding rat PDX-1, rat BETA2, and rat E47 were generated by PCR amplification and insertion of the cDNAs into the cytomegalovirus (CMV)-driven vector (pCMV7) (27Andersson S. Davis D.L. Dahlback H. Jornvall H. Russell D.W. J. Biol. Chem. 1989; 264: 8222-8229Abstract Full Text PDF PubMed Google Scholar). The following primers were used: PDX-1-(1–284), 5′-primer 5′-ATGAACAGTGAGGAGCAGT-3′, 3′-primer 5′-TCACCGGGGTTCCTGCGG-3′; BETA2-(1–357), 5′-primer 5′-ATGACCAAATCATACAGCGAGAG-3′, 3′-primer 5′-CTAATCGTGAAAGATGGCAT-3′; E47-(1–647), 5′-primer 5′-ATGAACCAGTCTCAGAGAATG-3′, 3′-primer 5′-TCACAGGTGCCCGGCTGGGTTGT-3′; BETA2ΔAD2-(1–299), 5′-CTAAAAGGCATAATTTTTTTCAAACTC-3′; BETA2ΔAD1AD2-(1–170), 5′-CTACTGTACAAAGGAGACGAG-3′; BETA2ΔbHLH-(169–357), 5′-GTACAGACACTCTG-3′; E47ΔAD1-(178–647), 5′-CCTGGTCTTCCTTCCTCG-3′; E47ΔAD1AD2-(510–647), 5′-GAAGACAAGAAGGACCTG-3′; E47ΔbHLH-(1–537), 5′-TCACAGGTCCTTCTCCTC-3′. CMV expression plasmids encoding human nuclear SREBPs (pCMV-SREBP-1a, -1c, and -2) (28Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Investig. 1996; 98: 1575-1584Crossref PubMed Scopus (693) Google Scholar), Tyr→Arg mutated versions (pCMV-SREBP-1aM, -1cM, and -2M) (19Amemiya-Kudo M. Shimano H. Hasty A.H. Yahagi N. Yoshikawa T. Matsuzaka T. Okazaki H. Tamura Y. Iizuka Y. Ohashi K. Osuga J. Harada K. Gotoda T. Sato R. Kimura S. Ishibashi S. Yamada N. J. Lipid Res. 2002; 43: 1220-1235Abstract Full Text Full Text PDF PubMed Google Scholar) and pCMV-ΔTA-SREBP-1c, which lacks the amino-terminal transactivation domain (1–90) of SREBP-1c (20Shimano H. Yahagi N. Amemiya-Kudo M. Hasty A.H. Osuga J. Tamura Y. Shionoiri F. Iizuka Y. Ohashi K. Harada K. Gotoda T. Ishibashi S. Yamada N. J. Biol. Chem. 1999; 274: 35832-35839Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar), were previously described. Reporter Plasmids—The reporter plasmid Ins715-Luc contains a fragment of the rat insulin I gene promoter from –715 to +31 bp cloned into the SmaI site of pGL2 basic vector (Promega) containing the coding sequences of firefly luciferase cDNA. Other constructs were produced by PCR using this construct as a DNA template, and the PCR products were inserted into pGL2 basic vector. The primers used for PCR were as follows: 5′-primer Ins715-Luc, 5′-TCTCAACTCCTTGAAAATAGCTACCT-3′; Ins646-Luc, 5′-GCTGTGCTACTGAGGCCTGATG-3′; Ins265-Luc, 5′-GGTACCTGATTGTGCTGTGAACTGCT-3′; Ins118-Luc, 5′-GGTACCTCTCGCCATCTGCCTACC-3′; Ins104-Luc, 5′-GGTACCTACCCCTCCTAGAGCCCTT-3′; SRE2 (m)-Luc, 5′-GGTACCTGTGAACTGCTTCATAAAGCCATCTGGCCC-3′; E2 (m)-Luc, 5′-GGTACCTGTGAACTGCTTCATCAGGCCATCAAGCCCCTTGTTAAT-3′; del SRE2-Luc, 5′-GCCATCTGGCCCCTTGTTAAT-3′, 3′-primer 5′-AAGCTTGTAGCTGGTCACTTAGGGTT-3′. Restriction sites KpnI and HindIII were added to each 5′-primer and 3′-primer, respectively. The site-directed mutagenesis constructs, SRE1(m)-Luc and E1(m)-Luc were produced by PCR with the following primers: SRE1 (m)-5′, 5′-GCTAGCCATCTGCCTACCTACCC-3′; SRE1 (m)-3′, 5′-GCTAGCGGGGCTGAAGCTGTAATT-3′;E1(m)-5′,5′-GGTACCAGGCCTACCTACCCCTC-3′; E1 (m)-3′, 5′-AGGCCTATGGCGAGAGGGGCTGAA-3′. The constructs lacking the intervening sequences between E1 and E2 regions were produced as follows: The fragment was amplified by 5′-primer, 5′-CCCTCTCGCCATCTG-3′ and 3′-primer, 5′-AAGCTTGTAGCTGGTCACTTAGGGTT-3′ using Ins265-Luc as a template and was inserted to the pGL2 basic vector. Restriction sites NheI and HindIII were added to each primer. 50-bp- and 86-bp-deleted fragments were amplified by 5′-primer 5′-GGTACCTGATTGTGCTGTGAACTGCT-3′ and 3′-primer, 5′-GGGTAATTAGATTATTAA-3′ (del 86) or 3′-primer, 5′-GCGCTCATTGGACGTCA-3′ (del 50). These fragments were inserted into the above vector. Restriction sites KpnI and NheI were added to each primer. Rat insulin II gene promoter InsII251-Luc was previously described (29Kajihara M. Sone H. Amemiya M. Katoh Y. Isogai M. Shimano H. Yamada N. Takahashi S. Biochem. Biophys. Res. Commun. 2003; 312: 831-842Crossref PubMed Scopus (54) Google Scholar). Transfection and Luciferase Assays—HEK293 cells (3.5 × 104 cells/well), HepG2 cells (4.2 × 104 cells/well), and HIT cells (3.5 × 105 cells/well) were plated on 12-well plates. Each expression plasmid (0–0.5 μg), luciferase reporter plasmid (0.5 μg), and pSV-β-gal (0.5 μg) were co-transfected using SuperFect transfection reagent (Qiagen) for HEK293 and HepG2 cells and using JetPEI™-Man (Polyplus-transfection) for HIT cells. The total amount of DNA in each transfection was adjusted to 1.5–2.0 μg/well. The luciferase activity in transfectants was measured by MicroLumat Plus (Berthold) and normalized to the β-galactosidase activity as measured by standard kit (Promega). Gel Mobility Shift Assays—Gel shift assays were performed as previously described (30Amemiya-Kudo M. Shimano H. Yoshikawa T. Yahagi N. Hasty A.H. Okazaki H. Tamura Y. Shionoiri F. Iizuka Y. Ohashi K. Osuga J. Harada K. Gotoda T. Sato R. Kimura S. Ishibashi S. Yamada N. J. Biol. Chem. 2000; 275: 31078-31085Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Recombinant SREBPs (SREBP-1c and -2) (Fig. 1E) and nuclear extracts from HEK293 cells transfected with pCMV-SREBP-1c, pCMV-SREBP-1cM, or pCMV-ΔTA-SREBP-1c-Myc (Fig. 6B) were used. The DNA probes prepared by annealing both strands were as follows: SRE1/E1-box, 5′-TCAGCCCCTCTCGCCATCTGCCTACCTA-3′; SRE2, 5′-GAACTGCTTCATCAGGCCATC-3′; E2-box, 5′-GGCCCATCTGGCCCCTTGTTAAC-3′; and SRE2/E2-box, 5′-GGACTGCTTCATCAGGCCATCTGGCCCCTTGTTAATAATCTAATTACCCTAGGTCTAAC-3′.FIGURE 6Effects of TA deletion (ΔTA-SREBP-1) and Tyr→Arg mutation (SREBP-1cM) on the synergistic activation by SREBP-1c/BETA2/E47. A, schematic representation of wild type of SREBP-1c, ΔTA-SREBP-1 in which the amino-terminal transactivation domain of SREBP-1c was deleted, and SREBP-1cM in which arginine (R) was substituted for tyrosine (Y) residue conserved in the basic region of DNA binding sites of SREBP. Basic, DNA binding site; HLH, helix-loop-helix structure. B, left, inhibitory effects of ΔTA-SREBP-1 and SREBP-1cM on the activation of Ins265-Luc by SREBP-1c. Ins265-luc, pSV-β-gal, and pCMV-SREBP-1c (0.125 μg) were transfected with the indicated amounts of ΔTA-SREBP-1 or SREBP-1cM (0–0.5 μg) in HepG2 cells. Right, electrophoretic mobility shift analysis (EMSA) of DNA-binding activities of SREBP-1c, SREBP-1cM, and ΔTA-SREBP-1. Each of the SREBP plasmids was transfected into HEK293 cells, and nuclear proteins were extracted. The SRE2+E2-box region was used as a probe. Supershift assay was performed using antibodies against SREBP-1 or Myc. C, effects of ΔTA-SREBP-1 or SREBP-1cM on the activation of Ins265-Luc by BETA2+E47 or SREBP-1c+BETA2+E47. The indicated amounts of ΔTA-SREBP-1 or SREBP-1cM (0–0.5 μg) were transfected with Ins265-luc and pSV-β-gal in HepG2 cells. The relative Luc activity (mean ± S.E.) was expressed as a fold change to that of the control vector (pCMV7).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The 32P-labeled probe and proteins were incubated in 15 μl of binding buffer (10 mm Tris-HCl, pH 7.6, 50 mm KCl, 0.05 mm EDTA, 2.5 mm MgCl2, 8.5% glycerol, 1 mm dithiothreitol, 0.5 mg/ml poly(dI-dC), 0.1% Triton X-100, and 1 mg/ml nonfat milk) for 30 min on ice. Myc or SREBP antibodies were preincubated with proteins for 60 min before adding the labeled probes. The DNA·protein complex was resolved on a 4.6% polyacrylamide gel. Gels were dried and exposed to the Bioimaging Analyzer System station software (Fuji Photo Film). In Vitro Protein-Protein Interaction Assay—[35S]Methionine-labeled proteins, luciferase, SREBP-1c, ΔTA-SREBP1c, BETA2, and E47 were prepared using the in vitro translation transcription system (Promega). Labeled protein (3 μl) was mixed with 1 μl of Myc antibody bound to 5 μl of protein G-Sepharose beads (Amersham Biosciences) in 200 μlof interaction buffer (20 mm Hepes, pH 7.5, 150 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40, 10 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride) and incubated for 1 h at 4 °C. The beads were washed three times with interaction buffer. The bound proteins were eluted with 30 μl of sample buffer (1% SDS, 100 mm dithiothreitol, 50 mm Tris-HCl, pH 7.5, 0.1% Bromothymol Blue) and heated 95 °C for 5 min. The bound proteins (15 μl) were subjected to SDS-PAGE and autoradiography. GST Binding Assay—GST fusion proteins, GST alone, GST-SREBP-1c (nuclear form-(24–460)), GST-SREBP-1c (bHLH-(286–364)) (31Najima Y. Yahagi N. Takeuchi Y. Matsuzaka T. Sekiya M. Nakagawa Y. Amemiya-Kudo M. Okazaki H. Okazaki S. Tamura Y. Iizuka Y. Ohashi K. Harada K. Gotoda T. Nagai R. Kadowaki T. Ishibashi S. Yamada N. Osuga J. Shimano H. J. Biol. Chem. 2005; 280: 27523-27532Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) were expressed in Escherichia coli and purified according to the manufacturer's recommendations (Promega). [35S]Methionine-labeled E47 (bHLH)-(498-634)) was prepared using the in vitro translation transcription system (Promega). GST precipitation assays were modified as previously reported (12Ohneda K. Mirmira R.G. Wang J. Johnson J.D. German M.S. Mol. Cell. Biol. 2000; 20: 900-911Crossref PubMed Scopus (166) Google Scholar, 32Yamamoto T. Shimano H. Nakagawa Y. Ide T. Yahagi N. Matsuzaka T. Nakakuki M. Takahashi A. Suzuki H. Sone H. Toyoshima H. Sato R. Yamada N. J. Biol. Chem. 2004; 279: 12027-12035Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Preparation of Nuclear Extracts—Nuclear extracts from HEK293, HIT, and INS1 cells were performed as described by Hua et al. (33Hua X. Sakai J. Brown M.S. Goldstein J.L. J. Biol. Chem. 1996; 271: 10379-10384Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Two hours prior to cell collection, ALLN (N-acetyl-Leu-Leu-norleucinalaldehyde, calpain inhibitor I) (Calbiochem) (25 μg/μl) was added to the cells. After collection, the cells were washed with phosphate-buffered saline and suspended in buffer A (10 mm Hepes, pH 7.9, 10 mm KCl, 1.5 mm MgCl2,1 mm EDTA, 1 mm EGTA). Pellets were passed through a 26-gauge needle eight times and centrifuged at 1,600 × g for 5 min. The supernatant was centrifuged at 55,000 rpm for 20 min, and the membrane-containing pellets were suspended in membrane buffer (50 mm Tris-HCl, pH 8.0, 2 mm CaCl2, 80 mm NaCl, 1% Triton X-100). The pellet containing the nuclei was suspended in buffer C (20 mm Hepes, pH 7.9, 25% glycerol, 0.4 m NaCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA), rotated at 4 °C for 30 min, and centrifuged at 10,000 × g for 20 min. The protein from the nuclear extract is in the supernatant. All buffers used contain a mixture of protease inhibitors (25 μg/μl ALLN, 10 μg/μl leupeptin, 2.1 μg/μl aprotinin, 5 μg/μl pepstatin, and 5 μg/μl phenylmethylsulfonyl fluoride). Isolation of Rat Islet and Mouse Pancreas—Pancreatic islets of Langerhans were isolated from 18-h-fasting male Sprague-Dawley rats (8 weeks old) and C57Bl6 mice (12 weeks old) by perfusion of the pancreatic duct and in situ collagenase digestion (34Shapiro A.M. Hao E. Rajotte R.V. Kneteman N.M. Cell Transplant. 1996; 5: 631-638Crossref PubMed Scopus (38) Google Scholar). The islets were subsequently purified by Ficoll density gradient and by hand picking. Immediately after isolation, the islets were cultured overnight in RPMI 1640 medium containing 5.5 mm glucose with or without T0901317 (3 μm). Total RNA was prepared using TRIzol reagent (Invitrogen). Immunoblot Analysis—Aliquots of proteins were subjected to 10% SDS-PAGE transferred to nitrocellulose membrane (Hybond-ECL, Amersham Biosciences) and incubated with rabbit anti-mouse SREBP-1 or goat anti-PDX-1 (N-18) antibodies (Santa Cruz Biotechnology). Secondary antibodies were horseradish peroxidase-conjugated donkey anti-rabbit or donkey anti-goat (Santa Cruz Biotechnology), and detection of immunoreactive bands was performed using the ECL kit (Amersham Biosciences). Chromatin Immunoprecipitation (ChIP) Assay—ChIP assays were performed as previously published with some modification (35Chakrabarti S.K. James J.C. Mirmira R.G. J. Biol. Chem. 2002; 277: 13286-13293Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 36Ide T. Shimano H. Yahagi N. Matsuzaka T. Nakakuki M. Yamamoto T. Nakagawa Y. Takahashi A. Suzuki H. Sone H. Toyoshima H. Fukamizu A. Yamada N. Nat. Cell Biol. 2004; 6: 351-357Crossref PubMed Scopus (274) Google Scholar). A total of 3.0 × 107 INS-1 cells (from two confluent 10-cm plates) was cross-linked with formaldehyde (final concentration 1%) after washing with phosphate-buffered saline and incubation for 15 min at room temperature. The reaction was stopped by adding glycine at a final concentration of 0.125 m. After 5 min, the cells were scraped and washed two times with cold phosphate-buffered saline with protease inhibitors (10 μg/ml leupeptin, 2 μg/ml aprotinin, 12.5 μg/ml ALLN, 2.5 μg/μl pepstatin A, and 1 mm phenylmethylsulfonyl fluoride). The pellets were resuspended in 0.5 ml of lysis buffer (1% SDS, 10 mm EDTA, 50 mm Tris-HCl, pH 8.1, plus protease inhibitors) and subjected to sonication at setting 10 for six 10-s pulses plus setting 30 for five 10-s pulses to share the chromatin to 1000-bp fragments. The samples were diluted 4-fold with immunoprecipitation dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mm EDTA, 167 mm NaCl, 16.7 mm Tris-HCl, pH 8.0, plus protease inhibitors). To reduce the nonspecific binding, the samples were incubated with 60 μl of sonicated salmon sperm DNA/protein A-Sepharose slurry (Amersham Biosciences) on a rotating platform at 4 °C for 2 h. After centrifugation, the supernatant was incubated with 10 μl of rabbit anti-mouse SREBP-1 antibody and rabbit normal IgG as a negative control and rotated overnight at 4 °C. 0.9 × 107 INS-1 cells for each antibody were used. Immune complexes were collected by adding 20 μl of salmon sperm DNA/protein G-Sepharose slurry (Amersham Biosciences) for 4 h with rotation. Samples were subsequently washed using 1.0 ml of wash buffer A (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 150 mm NaCl, 20 mm Tris-HCl, pH 8.1), wash buffer B (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 500 mm NaCl, 20 mm Tris-HCl, pH 8.1), wash buffer C (0.25 m LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mm EDTA, 10 mm Tris-HCl, pH 8.1), and wash buffer D (1 mm EDTA, 10 mm Tris-HCl, pH 8.0), and eluted by 30-min incubations with 0.5 ml of elution buffer (1% SDS, 50 mm NaHCO3, 10 mm dithiothreitol). NaCl was added to the elution at a final concentration of 0.3 m, and the samples were incubated at 65 °C for 6 h to reverse the formaldehyde-induced cross-linking. DNA and protein were ethanol-precipitated overnight at –20 °C and dissolved in 100 μl of Tris EDTA buffer (pH 7.4). Digestion was performed by the addition 25 μl of proteinase K buffer (50 mm Tris-HCl, pH 7.5, 25 mm EDTA, 1.25% SDS) with 2 μg/μl proteinase K (2 μl), and samples were placed at 55 °C for 1 h. Chromatin DNA was extracted with phenol/CHCl3 followed by ethanol precipitation. Samples were dissolved in 100 μl of Tris EDTA buffer (pH 7.4). 3-μl aliquots were used for PCR analysis. To amplify the rat insulin I promoter region containing SRE1 and SRE2, the following primer sets were used: 5′-TGCTTCATCAGGCCATCTGG-3′ for sense and 5′-GGTAGGCAGATGGCGAGAGGG-3′ for antisense. Primers sets for rat fatty acid synthase promoter regions containing SRE were used as a positive control: 5′-GACGCTCATTGGCCTGG-3′ for sense and 5′-TCTGGAGGCAGACGACAAG-3′ for antisense. The PCR conditions were 5 min at 94 °C and 30 cycles of 30 s at 94 °C, 30 s at 55 °C, and 1 min at 72 °C. Following amplification, PCR products were electrophoresed in a 3% agarose gel and visualized by ethidium bromide staining. Statistics—Statistical significance was assessed with the Student's t test for unpaired data. Identification of Insulin Gene Promoters as SREBP Targets—As schematized in Fig. 1A, pancreatic β cell-specific cis-elements for the insulin promoter included E-boxes (E1 and E2) as binding sites for bHLH protein heterodimers BETA2/E47, and A-boxes (A1 and A3/4) for a homeobox PDX-1. In the process of searching for glucose/insulin-responsive elements in promoters of nutritionally regulated genes in relation to SREBP regulation, we found that the rat insulin I gene promoter possesses three potential binding sites for nuclear SREBPs putatively designated SRE1, SRE2, and SRE3 (Fig. 1A). Intriguingly, SRE1 and SRE2 were located adjacent to the E1- and E2-boxes with partial overlapping sequences, respectively. The cluster of A1, E1, and SRE1 was tentatively designated as the E1 region and the distal cluster of A3/4, E2, and SRE2 as the E2 region (Fig. 1A). SRE3 was located upstream and in a reverse orientation. These new potential SREBP binding elements are highly conserved in rat II insulin and human insulin gene promoters (37Melloul D. Marshak S. Cerasi E. Diabetologia. 2002; 45: 309-326Crossref PubMed Scopus (269) Google Scholar). Luciferase assays in non-β HEK293 and HepG2 cells (data not shown) demonstrated that overexpression of the active form (nuclear) of SREBP-1a markedly and dose-dependently induced the activity of the rat insulin I promoter containing these regions (Ins715-Luc) (Fig. 1B). Strikingly, the activation of Ins715-Luc by nuclear SREBP-1a was even higher than by co-expression of PDX-1 and BETA2, both of which are crucial for β cell-specific and robust expression of the insulin gene (12Ohneda K. Mirmira R.G. Wang J. Johnson J.D. German M.S. Mol. Cell. Biol. 2000; 20: 900-911Crossref PubMed Scopus (166) Google Scholar, 38Glick E. Leshkowitz D. Walker M.D. J. Biol. Chem. 2000; 275: 2199-2204Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Among the nuclear SREBP isoforms (referred to as SREBPs, hereafter), SREBP-1a had the highest activity (Fig. 1C). SREBP-1 has been shown to bind both SRE and some E-boxes, low stringent consensus sequences for bHLH proteins
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