SOX6 Attenuates Glucose-stimulated Insulin Secretion by Repressing PDX1 Transcriptional Actvity and Is Down-regulated in Hyperinsulinemic Obese Mice
2005; Elsevier BV; Volume: 280; Issue: 45 Linguagem: Inglês
10.1074/jbc.m505392200
ISSN1083-351X
AutoresHaruhisa Iguchi, Yukio Ikeda, Masashi Okamura, Toshiya Tanaka, Yasuyo Urashima, Hiroto Ohguchi, Shinobu Takayasu, Noriaki Kojima, Satoshi Iwasaki, Riuko Ohashi, Shuying Jiang, Go Hasegawa, Ryoichi X. Ioka, Kenta Magoori, Koichi Sumi, Takashi Maejima, Aoi Uchida, Makoto Naito, Timothy F. Osborne, Masashi Yanagisawa, Tokuo Yamamoto, Tatsuhiko Kodama, Juro Sakai,
Tópico(s)Diabetes and associated disorders
ResumoIn obesity-related insulin resistance, pancreatic islets compensate for insulin resistance by increasing secretory capacity. Here, we report the identification of sex-determining region Y-box 6 (SOX6), a member of the high mobility group box superfamily of transcription factors, as a co-repressor for pancreatic-duodenal homeobox factor-1 (PDX1). SOX6 mRNA levels were profoundly reduced by both a long term high fat feeding protocol in normal mice and in genetically obese ob/ob mice on a normal chow diet. Interestingly, we show that SOX6 is expressed in adult pancreatic insulin-producing β-cells and that overexpression of SOX6 decreased glucose-stimulated insulin secretion, which was accompanied by decreased ATP/ADP ratio, Ca2+ mobilization, proinsulin content, and insulin gene expression. In a complementary fashion, depletion of SOX6 by small interfering RNAs augmented glucose-stimulated insulin secretion in insulinoma mouse MIN6 and rat INS-1E cells. These effects can be explained by our mechanistic studies that show SOX6 acts to suppress PDX1 stimulation of the insulin II promoter through a direct protein/protein interaction. Furthermore, SOX6 retroviral expression decreased acetylation of histones H3 and H4 in chromatin from the promoter for the insulin II gene, suggesting that SOX6 may decrease PDX1 stimulation through changes in chromatin structure at specific promoters. These results suggest that perturbations in transcriptional regulation that are coordinated through SOX6 and PDX1 in β-cells may contribute to the β-cell adaptation in obesity-related insulin resistance. In obesity-related insulin resistance, pancreatic islets compensate for insulin resistance by increasing secretory capacity. Here, we report the identification of sex-determining region Y-box 6 (SOX6), a member of the high mobility group box superfamily of transcription factors, as a co-repressor for pancreatic-duodenal homeobox factor-1 (PDX1). SOX6 mRNA levels were profoundly reduced by both a long term high fat feeding protocol in normal mice and in genetically obese ob/ob mice on a normal chow diet. Interestingly, we show that SOX6 is expressed in adult pancreatic insulin-producing β-cells and that overexpression of SOX6 decreased glucose-stimulated insulin secretion, which was accompanied by decreased ATP/ADP ratio, Ca2+ mobilization, proinsulin content, and insulin gene expression. In a complementary fashion, depletion of SOX6 by small interfering RNAs augmented glucose-stimulated insulin secretion in insulinoma mouse MIN6 and rat INS-1E cells. These effects can be explained by our mechanistic studies that show SOX6 acts to suppress PDX1 stimulation of the insulin II promoter through a direct protein/protein interaction. Furthermore, SOX6 retroviral expression decreased acetylation of histones H3 and H4 in chromatin from the promoter for the insulin II gene, suggesting that SOX6 may decrease PDX1 stimulation through changes in chromatin structure at specific promoters. These results suggest that perturbations in transcriptional regulation that are coordinated through SOX6 and PDX1 in β-cells may contribute to the β-cell adaptation in obesity-related insulin resistance. Insulin resistance is tissue insensitivity to the regulatory effects of insulin and is the leading cause of type 2 diabetes (1Butler A.E. Janson J. Bonner-Weir S. Ritzel R. Rizza R.A. Butler P.C. Diabetes. 2003; 52: 102-110Crossref PubMed Scopus (3240) Google Scholar, 2Rhodes C.J. Science. 2005; 307: 380-384Crossref PubMed Scopus (755) Google Scholar). Most affected individuals with insulin resistance do not directly develop diabetes but rather adapt to chronic insulin resistance by expanding pancreatic β-cell mass and/or insulin secretory capacity. To provide the required amount of insulin to maintain normal glucose levels, β-cell mass increases by islet neogenesis, β-cell replication, and β-cell hypertrophy. Pancreatic β-cells eventually fail to compensate for the increased insulin demand created by insulin resistance, leading to type 2 diabetes (1Butler A.E. Janson J. Bonner-Weir S. Ritzel R. Rizza R.A. Butler P.C. Diabetes. 2003; 52: 102-110Crossref PubMed Scopus (3240) Google Scholar, 2Rhodes C.J. Science. 2005; 307: 380-384Crossref PubMed Scopus (755) Google Scholar, 3Liu Y.Q. Jetton T.L. Leahy J.L. J. Biol. Chem. 2002; 277: 39163-39168Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 4Laybutt D.R. Glandt M. Xu G. Ahn Y.B. Trivedi N. Bonner-Weir S. Weir G.C. J. Biol. 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Pancreatic-duodenal homeobox factor-1 (PDX1), 4The abbreviations used are: PDX1pancreatic-duodenal homeobox factor-1α-KICαketoisocaproateChIPchromatin immunoprecipitationCMVcytomegalovirusGSISglucose-stimulated insulin secretionGSTglutathione S-transferaseHFDhigh fat dietNCDnormal chow dietSOXsex-determining region Y-boxQRT-PCRquantitative real-time polymerase chain reactionHMGhigh mobility groupsiRNAsmall interfering RNAKRBHKrebs-Ringer bicarbonate HEPES bufferALPalkaline phosphatase 4The abbreviations used are: PDX1pancreatic-duodenal homeobox factor-1α-KICαketoisocaproateChIPchromatin immunoprecipitationCMVcytomegalovirusGSISglucose-stimulated insulin secretionGSTglutathione S-transferaseHFDhigh fat dietNCDnormal chow dietSOXsex-determining region Y-boxQRT-PCRquantitative real-time polymerase chain reactionHMGhigh mobility groupsiRNAsmall interfering RNAKRBHKrebs-Ringer bicarbonate HEPES bufferALPalkaline phosphatase a homeodomain transcription factor, and the insulin/insulin-like growth factor signaling pathway are critical for β-cell replication and the compensatory response to insulin resistance (7Kulkarni R.N. Jhala U.S. Winnay J.N. Krajewski S. Montminy M. Kahn C.R. J. Clin. Invest. 2004; 114: 828-836Crossref PubMed Scopus (224) Google Scholar). PDX1 is expressed in β-cells of the islets of Langerhans and is involved in regulating the expression of a number of key β-cell genes. It plays a pivotal role in the development of the pancreas and islet cell ontogeny (8Hui H. Perfetti R. Eur. J. Endocrinol. 2002; 146: 129-141Crossref PubMed Scopus (122) Google Scholar). In a mouse model, inactivation of both pdx1 alleles results in pancreas agenesis, whereas heterozygous pdx1+/- mice or animals carrying a β-cell-specific mutation of the gene exhibit glucose intolerance (9Brissova M. Shiota M. Nicholson W.E. Gannon M. Knobel S.M. Piston D.W. Wright C.V. Powers A.C. J. Biol. Chem. 2002; 277: 11225-11232Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 10Dutta S. Gannon M. Peers B. Wright C. Bonner-Weir S. Montminy M. Proc. Natl. Acad. Sci. U. S. 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Although these results show that PDX1 plays a key role in the development of the pancreas and glucose-stimulated insulin secretion (GSIS) from β-cells, its functional role in the β-cell adaptation seen in chronic insulin resistance is poorly understood. pancreatic-duodenal homeobox factor-1 αketoisocaproate chromatin immunoprecipitation cytomegalovirus glucose-stimulated insulin secretion glutathione S-transferase high fat diet normal chow diet sex-determining region Y-box quantitative real-time polymerase chain reaction high mobility group small interfering RNA Krebs-Ringer bicarbonate HEPES buffer alkaline phosphatase pancreatic-duodenal homeobox factor-1 αketoisocaproate chromatin immunoprecipitation cytomegalovirus glucose-stimulated insulin secretion glutathione S-transferase high fat diet normal chow diet sex-determining region Y-box quantitative real-time polymerase chain reaction high mobility group small interfering RNA Krebs-Ringer bicarbonate HEPES buffer alkaline phosphatase PDX1 is a 284-amino acid protein consisting of 1) an NH2-terminal transactivation domain of 144 amino acids, 2) a homeodomain of 60 amino acids, and 3) a COOH-terminal domain of 80 amino acids. PDX1 binds through its homeodomain to target sequences called A-boxes (A/T-rich elements) of the insulin gene promoter (15Moede T. Leibiger B. Pour H.G. Berggren P. Leibiger I.B. FEBS Lett. 1999; 461: 229-234Crossref PubMed Scopus (81) Google Scholar). The NH2-terminal activation domain of PDX1 recruits the coactivator p300 and stimulates insulin gene expression synergistically with E12 and E47, which bind to E-boxes that are also located in the insulin gene promoter (16Petersen H.V. Peshavaria M. Pedersen A.A. Philippe J. Stein R. Madsen O.D. Serup P. FEBS Lett. 1998; 431: 362-366Crossref PubMed Scopus (73) Google Scholar, 17Qiu Y. Guo M. Huang S. Stein R. Mol. Cell. Biol. 2002; 22: 412-420Crossref PubMed Scopus (154) Google Scholar, 18Peers B. Leonard J. Sharma S. Teitelman G. Montminy M.R. Mol. Endocrinol. 1994; 8: 1798-1806PubMed Google Scholar, 19Peshavaria M. Cissell M.A. Henderson E. Petersen H.V. Stein R. Mol. Endocrinol. 2000; 14: 1907-1917PubMed Google Scholar). Interestingly, p300 is recruited to the insulin gene promoter only when cells are cultured in high glucose media (20Mosley A.L. Corbett J.A. Ozcan S. Mol. Endocrinol. 2004; 18: 2279-2290Crossref PubMed Scopus (97) Google Scholar). To identify additional factors that may contribute to the β-cell adaptation in insulin resistance, we have been characterizing genes that are selectively regulated in the islets of mice fed a high fat diet (HFD) using microarray analysis. Through the evaluation of transcriptional changes by microarray and quantitative real time PCR analyses, we found that one of the sex-determining region Y-box (SOX) transcription factors, SOX6, is markedly down-regulated in the islets of HFD-fed mice and normal chow fed ob/ob mice. Functional analyses with pancreatic β-cell line MIN6 cells revealed that SOX6 reduces GSIS by inhibiting PDX1 transcriptional activity, and our evidence indicates this occurs through a direct interaction between SOX6 and PDX1 proteins. We further show that overexpression of SOX6 results in decreased expression of genes involved in mitochondrial metabolism, including the NADH dehydrogenase complex of the mitochondrial respiratory chain, ATP synthase, and a subunit of cytochrome c oxidase. Taken together, the current data suggest that SOX6 is a key protein in the regulation of GSIS and that, together with PDX1, it contributes to the adaptive compensation of β-cells during the progression of obesity-related insulin resistance. Materials—The luciferase reporter assay system and pGL3-basic (Promega) were used as the source of the luciferase gene in all constructs and for luciferase assay components. The RNeasy kit was purchased from Qiagen. Acetyl-histone H3 and H4 immunoprecipitation assay kits were purchased from Upstate Biotechnology, Inc. Other reagents were obtained from sources as described previously (21Ikeda Y. Yamamoto J. Okamura M. Fujino T. Takahashi S. Takeuchi K. Osborne T.F. Yamamoto T.T. Ito S. Sakai J. J. Biol. Chem. 2001; 276: 34259-34269Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 22Fujino T. Asaba H. Kang M.J. Ikeda Y. Sone H. Takada S. Kim D.H. Ioka R.X. Ono M. Tomoyori H. Okubo M. Murase T. Kamataki A. Yamamoto J. Magoori K. Takahashi S. Miyamoto Y. Oishi H. Nose M. Okazaki M. Usui S. Imaizumi K. Yanagisawa M. Sakai J. Yamamoto T.T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 229-234Crossref PubMed Scopus (332) Google Scholar, 23Yamamoto J. Ikeda Y. Iguchi H. Fujino T. Tanaka T. Asaba H. Iwasaki S. Ioka R.X. Kaneko I.W. Magoori K. Takahashi S. Mori T. Sakaue H. Kodama T. Yanagisawa M. Yamamoto T.T. Ito S. Sakai J. J. Biol. Chem. 2004; 279: 16954-16962Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 24Tanaka T. Yamamoto J. Iwasaki S. Asaba H. Hamura H. Ikeda Y. Watanabe M. Magoori K. Ioka R.X. Tachibana K. Watanabe Y. Uchiyama Y. Sumi K. Iguchi H. Ito S. Doi T. Hamakubo T. Naito M. Auwerx J. Yanagisawa M. Kodama T. Sakai J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15924-15929Crossref PubMed Scopus (730) Google Scholar). Antibodies were obtained from the following sources: a goat polyclonal anti-PDX1 (sc-14664) and anti-SOX6 (sc-17332), rabbit polyclonal anti-SOX5 (sc-20091) and anti-SOX9 (sc-20095), and peroxidase-conjugated affinity-purified donkey anti-rabbit and anti-goat IgG from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit polyclonal anti-SOX6 (ab-12054) (directed against amino acids 349-354 of human SOX6) and anti-β-actin (ab-8226) from Abcam Ltd.; rabbit polyclonal anti-PDX1 (KR059) from TransGenic Inc. (Hyogo, Japan); Alexa Fluor 488 anti-guinea pig and anti-rabbit IgG and Zenon Alexa Fluor 594 anti-rabbit IgG labeling kit from Molecular Probes, Inc. (Eugene, OR); guinea pig polyclonal antibody to pig insulin from Nichirei (Tokyo, Japan); chicken polyclonal anti-SOX15 (AB-9180) from Chemicom International, Inc.; and control rabbit IgG (I-1000) from Vector Laboratories. Animals, Diets, and Pancreatic Islet Preparation—10-Week male C57BL/6J mice and ob/ob mice were purchased from Charles River and housed in a temperature- and humidity-controlled (26.5 °C and 35%) facility with a 12-h light/dark cycle (09:00 to 21:00 h). Mice were fed with a normal chow diet (NCD) (CE-2; CLEA, Osaka, Japan) or a high fat diet (HFD) (24Tanaka T. Yamamoto J. Iwasaki S. Asaba H. Hamura H. Ikeda Y. Watanabe M. Magoori K. Ioka R.X. Tachibana K. Watanabe Y. Uchiyama Y. Sumi K. Iguchi H. Ito S. Doi T. Hamakubo T. Naito M. Auwerx J. Yanagisawa M. Kodama T. Sakai J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15924-15929Crossref PubMed Scopus (730) Google Scholar) ad libitum for 9 weeks and then sacrificed for islet preparations at 11:00. Mouse pancreatic islets were isolated by a standard collagenase digestion method as described previously (22Fujino T. Asaba H. Kang M.J. Ikeda Y. Sone H. Takada S. Kim D.H. Ioka R.X. Ono M. Tomoyori H. Okubo M. Murase T. Kamataki A. Yamamoto J. Magoori K. Takahashi S. Miyamoto Y. Oishi H. Nose M. Okazaki M. Usui S. Imaizumi K. Yanagisawa M. Sakai J. Yamamoto T.T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 229-234Crossref PubMed Scopus (332) Google Scholar, 25Okamoto H. Mol. Cell Biochem. 1981; 37: 43-61Crossref PubMed Scopus (126) Google Scholar, 26Ikeda Y. Iguchi H. Nakata M. Ioka R.X. Tanaka T. Iwasaki S. Magoori K. Takayasu S. Yamamoto T.T. Kodama T. Yada T. Sakurai T. Yanagisawa M. Sakai J. Biochem. Biophys. Res. Commun. 2005; 333: 778-786Crossref PubMed Scopus (28) Google Scholar). Quantitative Real Time PCR (QRT-PCR) and Affymetrix Oligonucleotide Microarray—The methods for microarray and QRT-PCR have been described (23Yamamoto J. Ikeda Y. Iguchi H. Fujino T. Tanaka T. Asaba H. Iwasaki S. Ioka R.X. Kaneko I.W. Magoori K. Takahashi S. Mori T. Sakaue H. Kodama T. Yanagisawa M. Yamamoto T.T. Ito S. Sakai J. J. Biol. Chem. 2004; 279: 16954-16962Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 24Tanaka T. Yamamoto J. Iwasaki S. Asaba H. Hamura H. Ikeda Y. Watanabe M. Magoori K. Ioka R.X. Tachibana K. Watanabe Y. Uchiyama Y. Sumi K. Iguchi H. Ito S. Doi T. Hamakubo T. Naito M. Auwerx J. Yanagisawa M. Kodama T. Sakai J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15924-15929Crossref PubMed Scopus (730) Google Scholar). We used Affymetrix Genechip MOE430-A and -B arrays that contain probe sets for >30,000 mouse genes. All primer sequences used in this article are available by request. Immunohistochemistry—For light microscopy of paraffin-embedded sections, mouse pancreatic tissues were fixed with 10% (w/v) formalin at room temperature for 20 h. The samples were dehydrated with an alcohol series and embedded in paraffin. Antigen retrieval was performed by heating the sections in an autoclave at 121 °C for 15 min. The sections were incubated with anti-SOX6 antibody (ab-12054) (1:2000 dilution) for 16 h at 4 °C. Bound antibody was detected with the Simple Stain MAX-PO (Multi) reagent (Nichirei), an amino acid polymer coated with goat anti-rabbit IgG (Fab′) and peroxidase using 3,3′-diaminobenzidine (Dojindo, Kumamoto, Japan) as a substrate, and a hematoxylin counterstain was applied. For double immunofluorescence of adult mouse pancreas, fixed frozen tissues were permeabilized with 0.2% Triton X-100 for 20 min at 4 °C and stained with an anti-SOX6 antibody (ab-12054) (1:2000 dilution) labeled with Zenon Alexa Fluor 594 labeling kit and an anti-insulin or an anti-PDX1 antibody (KR059) (1:2000 dilution). For the detection of PDX1 and insulin, Alexa Fluor 488 anti-rabbit IgG (1:2000 dilution) and Alexa Fluor 488 anti-guinea pig IgG were used as a secondary antibody, respectively. Control experiments were carried out by omitting the primary antibody. Immunofluorescence was captured with a confocal laser scanning microscope (Fluoview FV500, Olympus, Japan). Expression Plasmids—Retroviral expression vectors encoding mouse SOX6 and other SOX genes were generated by PCR and insertion of the cDNAs into the pMX, a cytomegalovirus (CMV) promoter-driven retroviral expression vector (provided by Dr. Toshio Kitamura at University of Tokyo) (27Kitamura T. Int. J. Hematol. 1998; 67: 351-359Crossref PubMed Google Scholar). pCMV-PDX1, a pcDNA3-based plasmid encoding mouse PDX1, was obtained from Dr. Kazuya Yamagata at Osaka University (28Okita K. Yang Q. Yamagata K. Hangenfeldt K.A. Miyagawa J. Kajimoto Y. Nakajima H. Namba M. Wollheim C.B. Hanafusa T. Matsuzawa Y. Biochem. Biophys. Res. Commun. 1999; 263: 566-569Crossref PubMed Scopus (58) Google Scholar), and to create pcDNA3-based plasmids encoding mutant PDX1, the deletion sequences of PDX1 (amino acids 1-205, 1-144, and 145-284) were amplified by PCR and ligated into pcDNA3 (Invitrogen). To create pcDNA3-based plasmids encoding full-length and mutant SOX6, the full-length and deletion sequences of SOX6 (amino acids 181-827, 263-827, 617-827, 697-827, and 617-696) were generated by PCR amplification and ligated into pcDNA3. pCMV-ΔHMG-SOX6 encodes an internal deletion mutant form of SOX6 in which a 265-amino acid region containing the high mobility group (HMG) domain (amino acids 563-827) was deleted. This was constructed by digestion of pCMV-SOX6 with ApaI to remove the ApaI-ApaI 0.8-kbp fragment containing sequences for the HMG domain, and the plasmid was subsequently religated. A GAL4-PDX1 fusion construct, pBIND-PDX1, and a GAL4-E47 fusion construct, pBIND-E47, were constructed by inserting each cDNA fragment into a polylinker site of pBIND plasmid (Promega), which contains the DNA binding domain of the yeast GAL4 protein. Reporter Plasmids—pINS(-872)-luc is the rat insulin II gene promoter-luciferase reporter construct that spans -872 to -176 relative to the translation initiation site. pINS(-552)-luc and pINS(-413)-luc are 5′-deletion mutants of pINS(-872)-luc, and each contains a deletion with the 5′-end denoted in parentheses and the same 3′-end point at -176. pINS(-413mut)-luc is identical to pINS(-413)-luc except that the potential SOX binding site (nucleotides -248 to -242) is deleted. pINS(-370mut)-luc was constructed in an identical manner to pINS(-413mut)-luc, but starting from position -370. The 5′-flanking region of the rat insulin II gene (-872 to -176) (29Hwung Y.P. Gu Y.Z. Tsai M.J. Mol. Cell. Biol. 1990; 10: 1784-1788Crossref PubMed Scopus (53) Google Scholar) was amplified by PCR using a forward primer starting from -872 (5′-TATAGGTACCCCCAACCACTCCAA-3′) and a reverse primer OLi(-176R) (5′-TATACCCGGGGGTTACTGAATCC-3′) and cloned into pGL3-basic. pINS(-552)-luc and pINS(-413)-luc were constructed in a similar manner to pINS(-872)-luc using the respective forward primers starting from the positions -552 (5′-TATAGGTACCTGTGAAACAACAGTTCAAGGG-3′) and -413 (5′-TATAGGTACCTTCATCAGGCCACCCAGGAG-3′) and coupled with a common reverse primer OLi(-176R). p(μE5 + μE2 + μE3)4-luc is a luciferase reporter plasmid driven by a promoter consisting of four tandem copies of the E47-responsive element (5′-ACACCTGCAGCAGCTGGCAGGAAGCAGGTCATGTGGCA-3′) from the mouse IgH promoter (30Libermann T.A. Baltimore D. Mol. Cell. Biol. 1993; 13: 5957-5969Crossref PubMed Google Scholar). It was constructed by annealing the oligonucleotides for the top and bottom strands and subsequent ligation into the MluI and BglII sites of pGL3-basic. All plasmid constructs were verified by restriction endonuclease mapping and DNA sequencing. pG5luc is a luciferase reporter construct driven by a promoter consisting of five copies of GAL4 binding sites plus the adenovirus E1B TATA box (Promega). Cell Culture and Retroviral Infection—MIN6 cells (a line of mouse pancreatic β-cells) (31Miyazaki J. Araki K. Yamato E. Ikegami H. Asano T. Shibasaki Y. Oka Y. Yamamura K. Endocrinology. 1990; 127: 126-132Crossref PubMed Scopus (1046) Google Scholar) and INS-1E cells (a clone of parental rat β-cell line INS-1E cells (32Asfari M. Janjic D. Meda P. Li G. Halban P.A. Wollheim C.B. Endocrinology. 1992; 130: 167-178Crossref PubMed Scopus (748) Google Scholar) selected for insulin content and adequate proliferation (33Janjic D. Maechler P. Sekine N. Bartley C. Annen A.S. Wolheim C.B. Biochem. Pharmacol. 1999; 57: 639-648Crossref PubMed Scopus (85) Google Scholar)) were kind gifts from Dr. Jun-Ichi Miyazaki (Osaka University) and Dr. Pierre Maechler (University Medical Center at Switzerland), respectively. MIN6 cells were grown in Dulbecco's modified Eagle's medium containing 25 mm glucose, 5.5 μm β-mercaptoethanol, 100 units/ml penicillin, and 100 μg/ml of streptomycin sulfate, supplemented with 15% fetal bovine serum at 37 °C in 5% CO2. INS-1E cells were cultured in RPMI1640 containing 11.6 mm glucose, 10 mm HEPES, pH 7.4, 1 mm sodium pyruvate, 50 μm β-mercaptoethanol, 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin sulfate, supplemented with 5% fetal bovine serum at 37 °C in 5% CO2. Retroviral infection to MIN6 cells was performed as previously described (23Yamamoto J. Ikeda Y. Iguchi H. Fujino T. Tanaka T. Asaba H. Iwasaki S. Ioka R.X. Kaneko I.W. Magoori K. Takahashi S. Mori T. Sakaue H. Kodama T. Yanagisawa M. Yamamoto T.T. Ito S. Sakai J. J. Biol. Chem. 2004; 279: 16954-16962Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) using pMX plasmids (27Kitamura T. Int. J. Hematol. 1998; 67: 351-359Crossref PubMed Google Scholar, 34Morita S. Kojima T. Kitamura T. Gene Ther. 2000; 7: 1063-1066Crossref PubMed Scopus (1348) Google Scholar). Human embryonic kidney 293 cells and BHK21 cells (a line of hamster kidney cells) were obtained from the Cell Resource Center for Biomedical Research at Tohoku University (Sendai, Japan) and maintained in Dulbecco's modified Eagle's medium containing 100 units/ml penicillin and 100 μg/ml of streptomycin sulfate, supplemented with 10% fetal bovine serum at 37 °C in 5% CO2. Transient Transfection Assays—MIN6, HEK293, or BHK21 cells were plated on day 0 at a density of 5 × 104 cells/24-well plates. On day 1, cells were transfected with luciferase reporter plasmid, expression plasmids, and pCMVβ (Stratagene), a β-galactosidase reference gene, using Lipofectamine PLUS reagent (Invitrogen) as previously described (23Yamamoto J. Ikeda Y. Iguchi H. Fujino T. Tanaka T. Asaba H. Iwasaki S. Ioka R.X. Kaneko I.W. Magoori K. Takahashi S. Mori T. Sakaue H. Kodama T. Yanagisawa M. Yamamoto T.T. Ito S. Sakai J. J. Biol. Chem. 2004; 279: 16954-16962Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 35Rawson R.B. Zelenski N.G. Nijhawan D. Ye J. Sakai J. Hasan M.T. Chang T.Y. Brown M.S. Goldstein J.L. Mol. Cell. 1997; 1: 47-57Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 36Sakai J. Duncan E.A. Rawson R.B. Hua X. Brown M.S. Goldstein J.L. Cell. 1996; 85: 1037-1046Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 37Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). The total amount of DNA in each transfection was adjusted to 0.2-0.7 μg/well. On day 2, the cells were harvested and assayed for firefly luciferase activity and normalized to β-galactosidase activity using kits from Promega and BD Biosciences, respectively. siRNA Experiments—The duplexes of each small interfering RNA (siRNA), targeting SOX6 mRNA (target sequences of 5′-CGACCACACCAUCACCUCAdTdT-3′ and 5′-UGAGGUGAUGGUGUGGUCGdTdT-3′) and negative control (siCONTROL nontargeting siRNA 2) were purchased from Dharmacon Inc. (Lafayette, CO). PDX1 siRNA (identification number 155849, target sequences of 5′-GGUCUGAGCCUUGUCUUUAdTdT-3′ and 5′-UAAAGACAAGGCUCAGACCdTdT-3′) was purchased from Ambion (Austin, TX). The siRNAs were transfected by using Lipofectamine PLUS as described (35Rawson R.B. Zelenski N.G. Nijhawan D. Ye J. Sakai J. Hasan M.T. Chang T.Y. Brown M.S. Goldstein J.L. Mol. 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Approximately 2 × 106 MIN6 cells were cross-linked for 15 min at 37 °C with formaldehyde (1% final concentration) in Dulbecco's modified Eagle's medium, subsequently washed twice with phosphate-b
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