SOX6 Attenuates Glucose-stimulated Insulin Secretionby Repressing PDX1 Transcriptional Actvity and IsDown-regulated in Hyperinsulinemic Obese Mice*□S

Received for publication, May 17, 2005, and in revised form, August 29, 2005 Published, JBC Papers in Press, September 7, 2005, DOI 10.1074/jbc.M505392200

Haruhisa Iguchi‡§1, 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§**2, Tokuo T. Yamamoto‡‡, Tatsuhiko Kodama‡, and Juro Sakai‡§3

From the ‡Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, University ofTokyo, Tokyo 153-8904, Japan, §Yanagisawa Orphan Receptor Project, Exploratory Research for Advanced Technology, JapanScience and Technology Agency, Tokyo 135-0064, Japan, the ¶Department of Cellular Function, Division of Cellular and MolecularPathology, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan, the �Department ofMolecular Biology and Biochemistry, University of California, Irvine, California 92717-3900, **Howard Hughes Medical Instituteand Department of Molecular Genetics, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9050, and‡‡Center for Advanced Genome Research, Institute of Development, Aging and Cancer, Tohoku University, Sendai 981-8555, Japan

In obesity-related insulin resistance, pancreatic islets compen-sate for insulin resistance by increasing secretory capacity. Here, wereport the identification of sex-determining regionY-box 6 (SOX6),a member of the high mobility group box superfamily of transcrip-tion factors, as a co-repressor for pancreatic-duodenal homeoboxfactor-1 (PDX1). SOX6 mRNA levels were profoundly reduced byboth a long term high fat feeding protocol in normal mice and ingenetically obese ob/ob mice on a normal chow diet. Interestingly,we show that SOX6 is expressed in adult pancreatic insulin-produc-ing �-cells and that overexpression of SOX6 decreased glucose-stimulated insulin secretion, which was accompanied by decreasedATP/ADP ratio, Ca2� mobilization, proinsulin content, and insulingene expression. In a complementary fashion, depletion of SOX6 bysmall interfering RNAs augmented glucose-stimulated insulinsecretion in insulinoma mouse MIN6 and rat INS-1E cells. Theseeffects can be explained by ourmechanistic studies that show SOX6acts to suppress PDX1 stimulation of the insulin II promoterthrough a direct protein/protein interaction. Furthermore, SOX6retroviral expression decreased acetylation of histones H3 and H4in chromatin from the promoter for the insulin II gene, suggestingthat SOX6 may decrease PDX1 stimulation through changes inchromatin structure at specific promoters. These results suggestthat perturbations in transcriptional regulation that are coordi-nated through SOX6 and PDX1 in �-cells may contribute to the�-cell adaptation in obesity-related insulin resistance.

Insulin resistance is tissue insensitivity to the regulatory effects ofinsulin and is the leading cause of type 2 diabetes (1, 2). Most affectedindividuals with insulin resistance do not directly develop diabetes butrather adapt to chronic insulin resistance by expanding pancreatic�-cell mass and/or insulin secretory capacity. To provide the requiredamount of insulin to maintain normal glucose levels, �-cell massincreases by islet neogenesis, �-cell replication, and �-cell hypertrophy.Pancreatic�-cells eventually fail to compensate for the increased insulindemand created by insulin resistance, leading to type 2 diabetes (1–6).Pancreatic-duodenal homeobox factor-1 (PDX1),4 a homeodomain

transcription factor, and the insulin/insulin-like growth factor signalingpathway are critical for �-cell replication and the compensatoryresponse to insulin resistance (7). PDX1 is expressed in �-cells of theislets of Langerhans and is involved in regulating the expression of anumber of key �-cell genes. It plays a pivotal role in the development ofthe pancreas and islet cell ontogeny (8). In a mouse model, inactivationof both pdx1 alleles results in pancreas agenesis, whereas heterozygouspdx1�/� mice or animals carrying a�-cell-specificmutation of the geneexhibit glucose intolerance (9–11).Mutations in the human PDX1 geneare associated with maturity onset diabetes of the young (MODY4) andpredispose to late onset type II diabetes (12–14). Although these resultsshow that PDX1 plays a key role in the development of the pancreas andglucose-stimulated insulin secretion (GSIS) from �-cells, its functionalrole in the �-cell adaptation seen in chronic insulin resistance is poorlyunderstood.PDX1 is a 284-amino acid protein consisting of 1) an NH2-terminal

transactivation domain of 144 amino acids, 2) a homeodomain of 60amino acids, and 3) a COOH-terminal domain of 80 amino acids. PDX1binds through its homeodomain to target sequences called A-boxes(A/T-rich elements) of the insulin gene promoter (15). TheNH2-termi-nal activation domain of PDX1 recruits the coactivator p300 and stim-ulates insulin gene expression synergistically with E12 and E47, whichbind to E-boxes that are also located in the insulin gene promoter (16–

* This work was supported in part by research grants from the Ministry of Education,Science, and Culture of Japan, Special Coordination Funds for Promoting Science andTechnology from the Ministry of Education, Culture, Sports, Science, and Technologyof the Japanese Government, Astellas Foundation for Research on Metabolic Disor-ders, Research Fund of Mitsukoshi Health and Welfare Foundation, and ExploratoryResearch for Advanced Technology/Japan Science and Technology Agency (Yanagi-sawa orphan receptor project). The costs of publication of this article were defrayed inpart by the payment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) contains supple-mental Fig. 1 and Table 1.

1 Present address: Genomics Science Laboratories, Sumitomo Pharmaceuticals Co. Ltd.,Takarazuka 665-0051, Japan.

2 An Investigator of the Howard Hughes Medical Institute.3 To whom correspondence should be addressed: Laboratory for Systems Biology and

Medicine, Research Center for Advanced Science and Technology, University ofTokyo, Tokyo 153-8904, Japan. Tel.: 81-3-5452-5472; Fax: 81-3-5452-5429; E-mail:jmsakai-tky@umin.ac.jp.

4 The abbreviations used are: PDX1, pancreatic-duodenal homeobox factor-1; �-KIC,�-ketoisocaproate; ChIP, chromatin immunoprecipitation; CMV, cytomegalovirus;GSIS, glucose-stimulated insulin secretion; GST, glutathione S-transferase; HFD, highfat diet; NCD, normal chow diet; SOX, sex-determining region Y-box; QRT-PCR, quan-titative real-time polymerase chain reaction; HMG, high mobility group; siRNA, smallinterfering RNA; KRBH, Krebs-Ringer bicarbonate HEPES buffer; ALP, alkalinephosphatase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 45, pp. 37669 –37680, November 11, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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19). Interestingly, p300 is recruited to the insulin gene promoter onlywhen cells are cultured in high glucose media (20).To identify additional factors that may contribute to the �-cell adap-

tation in insulin resistance, we have been characterizing genes that areselectively regulated in the islets of mice fed a high fat diet (HFD) usingmicroarray analysis. Through the evaluation of transcriptional changesby microarray and quantitative real time PCR analyses, we found thatone of the sex-determining region Y-box (SOX) transcription factors,SOX6, is markedly down-regulated in the islets of HFD-fed mice andnormal chow fed ob/obmice. Functional analyses with pancreatic�-cellline MIN6 cells revealed that SOX6 reduces GSIS by inhibiting PDX1transcriptional activity, and our evidence indicates this occurs througha direct interaction between SOX6 and PDX1 proteins. We furthershow that overexpression of SOX6 results in decreased expression ofgenes involved in mitochondrial metabolism, including the NADHdehydrogenase complex of the mitochondrial respiratory chain, ATPsynthase, and a subunit of cytochrome c oxidase. Taken together, thecurrent data suggest that SOX6 is a key protein in the regulation of GSISand that, together with PDX1, it contributes to the adaptive compensa-tion of �-cells during the progression of obesity-related insulinresistance.

EXPERIMENTAL PROCEDURES

Materials—The luciferase reporter assay system and pGL3-basic(Promega) were used as the source of the luciferase gene in all con-structs and for luciferase assay components. The RNeasy kit was pur-chased from Qiagen. Acetyl-histone H3 and H4 immunoprecipitationassay kits were purchased from Upstate Biotechnology, Inc. Otherreagents were obtained from sources as described previously (21–24).Antibodies were obtained from the following sources: a goat polyclonalanti-PDX1 (sc-14664) and anti-SOX6 (sc-17332), rabbit polyclonalanti-SOX5 (sc-20091) and anti-SOX9 (sc-20095), and peroxidase-con-jugated affinity-purified donkey anti-rabbit and anti-goat IgG fromSanta Cruz Biotechnology (Santa Cruz, CA); rabbit polyclonal anti-SOX6 (ab-12054) (directed against amino acids 349–354 of humanSOX6) and anti-�-actin (ab-8226) from Abcam Ltd.; rabbit polyclonalanti-PDX1 (KR059) from TransGenic Inc. (Hyogo, Japan); Alexa Fluor488 anti-guinea pig and anti-rabbit IgG and Zenon Alexa Fluor 594anti-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 ChemicomInternational, Inc.; and control rabbit IgG (I-1000) from VectorLaboratories.

Animals, Diets, and Pancreatic Islet Preparation—10-Week maleC57BL/6J mice and ob/obmice were purchased from Charles River andhoused in a temperature- and humidity-controlled (26.5 °C and 35%)facilitywith a 12-h light/dark cycle (09:00 to 21:00 h).Micewere fedwitha normal chow diet (NCD) (CE-2; CLEA,Osaka, Japan) or a high fat diet(HFD) (24) ad libitum for 9 weeks and then sacrificed for islet prepara-tions at 11:00. Mouse pancreatic islets were isolated by a standard col-lagenase digestion method as described previously (22, 25, 26).

Quantitative Real Time PCR (QRT-PCR) and Affymetrix Oligonu-cleotide Microarray—The methods for microarray and QRT-PCR havebeen described (23, 24).We used Affymetrix GenechipMOE430-A and-B arrays that contain probe sets for �30,000 mouse genes. All primersequences used in this article are available by request.

Immunohistochemistry—For light microscopy of paraffin-embeddedsections,mouse pancreatic tissueswere fixedwith 10% (w/v) formalin atroom temperature for 20 h. The samples were dehydrated with an alco-hol series and embedded in paraffin. Antigen retrieval was performed by

heating the sections in an autoclave at 121 °C for 15 min. The sectionswere incubated with anti-SOX6 antibody (ab-12054) (1:2000 dilution)for 16 h at 4 °C. Bound antibody was detected with the Simple StainMAX-PO (Multi) reagent (Nichirei), an amino acid polymer coatedwith goat anti-rabbit IgG (Fab�) and peroxidase using 3,3�-diaminoben-zidine (Dojindo, Kumamoto, Japan) as a substrate, and a hematoxylincounterstain was applied. For double immunofluorescence of adultmouse pancreas, fixed frozen tissues were permeabilized with 0.2% Tri-ton 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 label-ing kit and an anti-insulin or an anti-PDX1 antibody (KR059) (1:2000dilution). For the detection of PDX1 and insulin, Alexa Fluor 488 anti-rabbit IgG (1:2000 dilution) and Alexa Fluor 488 anti-guinea pig IgGwere used as a secondary antibody, respectively. Control experimentswere carried out by omitting the primary antibody. Immunofluores-cence was captured with a confocal laser scanning microscope (Fluo-view FV500, Olympus, Japan).

Expression Plasmids—Retroviral expression vectors encoding mouseSOX6 and other SOX genes were generated by PCR and insertion of thecDNAs into the pMX, a cytomegalovirus (CMV) promoter-driven ret-roviral expression vector (provided by Dr. Toshio Kitamura at Univer-sity of Tokyo) (27). pCMV-PDX1, a pcDNA3-based plasmid encodingmouse PDX1, was obtained from Dr. Kazuya Yamagata at Osaka Uni-versity (28), and to create pcDNA3-based plasmids encoding mutantPDX1, the deletion sequences of PDX1 (amino acids 1–205, 1–144, and145–284) were amplified by PCR and ligated into pcDNA3 (Invitrogen).To create pcDNA3-based plasmids encoding full-length and mutantSOX6, the full-length and deletion sequences of SOX6 (amino acids181–827, 263–827, 617–827, 697–827, and 617–696) were generatedby PCR amplification and ligated into pcDNA3. pCMV-�HMG-SOX6encodes 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 digestionof pCMV-SOX6 with ApaI to remove the ApaI-ApaI 0.8-kbp fragmentcontaining sequences for the HMG domain, and the plasmid was sub-sequently religated. A GAL4-PDX1 fusion construct, pBIND-PDX1,and a GAL4-E47 fusion construct, pBIND-E47, were constructed byinserting each cDNA fragment into a polylinker site of pBIND plasmid(Promega), which contains the DNA binding domain of the yeast GAL4protein.

Reporter Plasmids—pINS(�872)-luc is the rat insulin II gene pro-moter-luciferase reporter construct that spans�872 to�176 relative tothe translation initiation site. pINS(�552)-luc and pINS(�413)-luc are5�-deletion mutants of pINS(�872)-luc, and each contains a deletionwith the 5�-end denoted in parentheses and the same 3�-end point at�176. pINS(�413mut)-luc is identical to pINS(�413)-luc except thatthe potential SOX binding site (nucleotides �248 to �242) is deleted.pINS(�370mut)-luc was constructed in an identical manner topINS(�413mut)-luc, but starting from position �370. The 5�-flankingregion of the rat insulin II gene (�872 to �176) (29) was amplified byPCR using a forward primer starting from �872 (5�-TATAGGTAC-CCCCAACCACTCCAA-3�) and a reverse primer OLi(�176R) (5�-TATACCCGGGGGTTACTGAATCC-3�) and cloned into pGL3-ba-sic. pINS(�552)-luc and pINS(�413)-luc were constructed in a similarmanner to pINS(�872)-luc using the respective forward primers start-ing from the positions �552 (5�-TATAGGTACCTGTGAAACAA-CAGTTCAAGGG-3�) and �413 (5�-TATAGGTACCTTCATCAG-GCCACCCAGGAG-3�) and coupled with a common reverse primerOLi(�176R).

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p(�E5� �E2� �E3)4-luc is a luciferase reporter plasmid driven by apromoter consisting of four tandem copies of the E47-responsive ele-ment (5�-ACACCTGCAGCAGCTGGCAGGAAGCAGGTCATGT-GGCA-3�) from the mouse IgH promoter (30). It was constructed byannealing the oligonucleotides for the top and bottom strands and sub-sequent ligation into theMluI and BglII sites of pGL3-basic. All plasmidconstructs were verified by restriction endonucleasemapping andDNAsequencing. pG5luc is a luciferase reporter construct driven by a pro-moter consisting of five copies of GAL4 binding sites plus the adenovi-rus E1B TATA box (Promega).

Cell Culture and Retroviral Infection—MIN6 cells (a line of mousepancreatic �-cells) (31) and INS-1E cells (a clone of parental rat �-cellline INS-1E cells (32) selected for insulin content and adequate prolif-eration (33)) were kind gifts fromDr. Jun-Ichi Miyazaki (Osaka Univer-sity) and Dr. Pierre Maechler (University Medical Center at Switzer-land), respectively. MIN6 cells were grown in Dulbecco’s modifiedEagle’s medium containing 25 mM glucose, 5.5 �M �-mercaptoethanol,100 units/ml penicillin, and 100 �g/ml of streptomycin sulfate, supple-mented with 15% fetal bovine serum at 37 °C in 5% CO2. INS-1E cellswere cultured in RPMI1640 containing 11.6mMglucose, 10mMHEPES,pH 7.4, 1 mM sodium pyruvate, 50 �M �-mercaptoethanol, 2 mM gluta-mine, 100 units/ml penicillin, and 100 �g/ml streptomycin sulfate, sup-plemented with 5% fetal bovine serum at 37 °C in 5% CO2. Retroviralinfection to MIN6 cells was performed as previously described (23)using pMX plasmids (27, 34). Human embryonic kidney 293 cells andBHK21 cells (a line of hamster kidney cells) were obtained from the CellResource Center for Biomedical Research at Tohoku University (Sen-dai, Japan) andmaintained inDulbecco’smodified Eagle’s medium con-taining 100 units/ml penicillin and 100 �g/ml of streptomycin sulfate,supplemented with 10% fetal bovine serum at 37 °C in 5% CO2.

Transient Transfection Assays—MIN6, HEK293, or BHK21 cellswere plated on day 0 at a density of 5 � 104 cells/24-well plates. On day1, cells were transfected with luciferase reporter plasmid, expressionplasmids, and pCMV� (Stratagene), a �-galactosidase reference gene,using Lipofectamine PLUS reagent (Invitrogen) as previously described(23, 35–37). The total amount of DNA in each transfectionwas adjustedto 0.2–0.7 �g/well. On day 2, the cells were harvested and assayed forfirefly luciferase activity and normalized to �-galactosidase activityusing kits from Promega and BD Biosciences, respectively.

siRNA Experiments—The duplexes of each small interfering RNA(siRNA), targeting SOX6 mRNA (target sequences of 5�-CGACCA-CACCAUCACCUCAdTdT-3� and 5�-UGAGGUGAUGGUGUG-GUCGdTdT-3�) and negative control (siCONTROL nontargetingsiRNA 2) were purchased from Dharmacon Inc. (Lafayette, CO). PDX1siRNA (identification number 155849, target sequences of 5�-GGUCU-GAGCCUUGUCUUUAdTdT-3� and 5�-UAAAGACAAGGCUCA-GACCdTdT-3�) was purchased from Ambion (Austin, TX). ThesiRNAswere transfected by using Lipofectamine PLUS as described (35,36, 38). Cells were harvested for RNA as well as for protein. SOX6expression was confirmed by QRT-PCR and immunoblot analysis.

Insulin Secretion, Content, and Adenine Nucleotide Determinationsand an Intracellular Ca2� Assay—The secretory responses to glucoseand other secretagogues were tested in MIN6 cells and INS-1E cellsbetween passages 16–35 and 54–95, respectively (31, 39). Before theexperiments,MIN6 or INS-1E cells were washed twice with phosphate-buffered saline and preincubated for 30 min at 37 °C in glucose-freeKrebs-Ringer bicarbonate HEPES buffer (KRBH) of the following com-position: 129mMNaCl, 4.7mMKCl, 5.0mMNaHCO3, 1.2mMKH2PO4,

1.2mMMgSO4, 2.0mMCaCl2, and 10mMHEPES, pH7.4. Bovine serumalbumin (0.1%) was added as an insulin carrier. Next, cells were washed

once with glucose-free KRBH and then incubated for 1 h in KRBH andstimuli as indicated. Incubationwas stopped by putting the plates on ice,and the supernatants were collected for insulin secretion. Cellular insu-lin was extracted with acid-ethanol (0.4 M HCl in 74% ethanol) over-night at 4 °C as described previously (22). Insulin secretion and contentwere determined by a rat insulin enzyme-linked immunosorbent assaykit (Shibayagi Co., Shibukawa, Japan). ATP and ADP content in MIN6cells were determined using theATP assay system (Toyobo-Net, Tokyo,Japan) as previously described (22, 40). To determine the intracellularCa2� levels, MIN6 cells were loaded with 2 �M fura-2/AM (Dojindo) inKRBH containing 10 mM glucose at room temperature for 1 h asdescribed (26). The loading solution was removed and then applied to aFunctional Drug Screening System 6000 (Hamamatsu Photonics,Shizuoka, Japan). Intracellular Ca2� concentrationwasmeasured by theratio of emission fluorescence of 510 nm by excitation at 340 and380 nm.

Chromatin Immunoprecipitation (ChIP) Assay—A commerciallyavailable assay kit (Upstate Biotechnologies, Charlottesville, VA) wasused for ChIP studies according to the manufacturer’s protocol.Approximately 2� 106MIN6 cells were cross-linked for 15min at 37 °Cwith formaldehyde (1% final concentration) in Dulbecco’s modifiedEagle’s medium, subsequently washed twice with phosphate-bufferedsaline containing proteinase inhibitors. Cells were scraped, centrifuged,and resuspended in 0.5 ml of lysis buffer (50 mM Tris-HCl at pH 8.1, 10mMEDTA, 1% SDS). The cells were then sonicated on ice 10 times using30-s pulses using a Sonifier cell disrupter model Micro-150 (GENEQInc., Montreal, Canada), and then the debris was removed by centrifu-gation. Supernatants were collected and used for immunoprecipitation.To reduce the nonspecific binding, samples were preincubated withprotein A-Sepharose and sonicated salmon sperm DNA for 1 h at 4 °C.After centrifugation, the supernatant was incubated with specific anti-bodies (anti-PDX1 (sc-14664), anti-SOX6 (sc-17332), anti-acetyl-his-tone H3 and H4), or control rabbit IgG overnight at 4 °C followed byincubation with Protein A-Sepharose for 1 h. After centrifugation andwashing, the immunocomplexes were eluted twice with 250 �l of elu-tion buffer (1% SDS and 0.1 M NaHCO3) for 15 min at room tempera-ture. The cross-linking was then reversed by adding 20 �l of 5 M NaCland 1 �l of 10 mg/ml RNase A and by incubating at 65 °C for 6 h. Aftertreatingwith 1.5�l of proteinaseK (10�g/�l), theDNAswere extractedwith phenol/chloroform, subsequently ethanol-precipitated using 20�g of glycogen carrier, and dissolved in 50 �l of distilled water. Theamount ofDNA recovered from immunoprecipitateswith specific antibodiesorcontrol IgGwasquantifiedbyQRT-PCR,whichwasperformedintriplicate.Data were represented as -fold change over DNA input. The primers used toamplify the insulin II promoter sequences were 5�-GGAACTGTGAAA-CAGTCCAAGG-3� and 5�-CCCCTGGACTTTGCTGTTTG-3�.

GST Pull-down Assay—Glutathione S-transferase (GST) fusion con-structs containing full-length PDX1 (amino acids 1–284) and full-length SOX6 (amino acids 1–827) were created in the bacterial expres-sion vector pGEX-4T-2 (Amersham Biosciences), expressed in BL21bacteria, and purified as previously described (21, 23). pcDNA3 con-structs, a TNT quick coupled transcription/translation system (Pro-mega), and L-[35S]methionine (1000 Ci/mmol; Amersham Biosciences)were used for synthesizing 35S-labeled in vitro proteins. Purified GST orGST fusion proteins were incubated with 35S-labeled proteins for 2 h at4 °C and washed five times before SDS-PAGE was carried out.

Immunoblot Analysis—Aliquots of proteins were subjected to SDS-PAGE followed by immunoblot analysis using anti-SOX5, anti-SOX6(ab-12054), anti-SOX9, and anti-SOX15 (1:1000 dilution); anti-PDX1(sc-14664) (1:500 dilution); or anti-�-actin antibodies (1:5000 dilution).

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Immunoblots were visualized using the ECL-Plus® system (AmershamBiosciences) as previously described (36, 38, 41) andmeasured by Lumi-VisionPRO (TAITEC, Osaka, Japan).

Statistical Analysis—For unpaired comparisons of two groups, Stu-dent’s t test was used. For multiple comparisons, one-way analysis ofvariance, followed by Tukey’s honestly significant difference test wasused.

RESULTS

Identification of SOX6 as a Down-regulated Transcription Factor inHyperinsulinemic Animals—In an attempt to uncover the mechanismunderlying hyperinsulinemia and the �-cell adaptation phenotype inobesity-induced insulin resistance, we examined the gene expressionprofile in pancreatic islets of hyperinsulinemic obese mice that were fedan HFD. Using the Affymetrix mouse gene chip MOE430 and QRT-PCR of 59 transcription factors from the data base of the Beta CellBiology Consortium (on theWorldWideWeb at www.cbil.upenn.edu/EPConDB/index.shtml), we identified 17 transcription factors whoseexpression levels were altered either more than 2 or less than 0.5 inresponse to HFD (supplemental Table 1) and subsequently examinedtheir properties in insulinoma �-cell lineMIN6 cells (supplemental Fig.1). Through these processes, we identified the unique SOX transcrip-tion factor, SOX6, as a specifically down-regulated gene in these HFD-

fed mice, which also has the ability to modulate GSIS (supplementalFig. 1). As shown in Fig. 1A, the islet expression of SOX6 mRNA wasseveralfold lower in both the HFD-induced obese mice and geneticallyobese ob/ob mice than in normal mice. This result suggested that thelevels of SOX6 mRNA might be negatively regulated by insulin or glu-cose. To test this hypothesis, we carried out fasting/refeeding experi-ments where insulin levels change to regulate blood glucose. As shownin Fig. 1B, the pancreatic levels of SOX6 RNA were essentiallyunchanged either by fasting or subsequent refeeding after 12 h of fast-ing, whereas expression of stearoyl-CoA desaturase 2, a well character-ized insulin-regulated gene, was down- and up-regulated by fasting andrefeeding, respectively. These results indicate that the expression ofSOX6may not be simply regulated by acute changes in insulin or bloodglucose levels but instead by the prolonged hyperglycemia, hyperinsu-linemia, and/or insulin resistance generated in the course of developingobesity.Next, we examined whether SOX6 protein was expressed in the pan-

creatic islet. Nuclear extracts were prepared from mouse pancreaticislets and insulinomaMIN6 cells and immunoblotted with an antibodyraised against SOX6. A strong signal was detected at the predictedmolecular weight in the nuclear extract from mouse islets and MIN6cells (Fig. 1C). This signal was diminished by the application of siRNAfor SOX6 (see Fig. 2D), indicating that this antibody specifically recog-

FIGURE 1. SOX6 gene expression in pancreaticislets from diet-induced obese and geneticallyobese ob/ob mice. A, relative amounts of SOX6mRNA in pancreatic islets from C57BL/6J malemice on either an HFD or NCD or those from maleob/ob mice on NCD. Male C57BL/6J mice (8 weeksof age) and ob/ob mice were fed with either HFD orNCD for 9 weeks as described under “ExperimentalProcedures.” B, relative amounts of mRNAs forSOX6 and stearoyl-CoA desaturase 2 in islets frommice subjected to fasting and refeeding. C57BL/6Jmale mice, 8 weeks of age, were divided into threegroups: fed, fasted, and refed. The fed group wasfed ad libitum, the fasted group was fasted for 24 h,and the refed group was fasted for 24 h and thenrefed a normal chow diet for 1 h prior to study. Aand B, total RNA from pancreatic islets were pre-pared and subjected to QRT-PCR as described.Mouse 36B4 (NM_007475) mRNA was used for theinvariant control for QRT-PCR. Values representthe amount of mRNA relative to those in NCD mice(A) or in the fed condition experiment (B), which isarbitrarily defined as 1. Each bar and symbol repre-sent mean � S.E. of three independent experi-ments performed in triplicate. *, p � 0.01 com-pared with control. SCD2, stearoyl-CoA desaturase2. C, immunoblot analysis of nuclear extracts pre-pared from mouse pancreatic islets and MIN6 cells.Nuclear extracts were prepared as previouslydescribed (36, 41), and aliquots (20 �g) were sub-jected to SDS-PAGE and immunoblot analysis withanti-SOX6 antibody (top). The filter was exposedfor 30 s. The position of SOX6 is indicated by anarrow. D, immunohistochemistry of paraffin-em-bedded section from control C57BL/6J (left) andob/ob (right) mice using anti-SOX6 antibody. E,double immunofluorescence of fixed frozen sec-tions of adult mouse pancreas for SOX6 and insu-lin. F, double immunofluorescence of fixed frozensections of adult mouse pancreas for SOX6 andPDX1.

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nized SOX6 protein. Immunostaining of the pancreas with the SOX6antibody showed that SOX6 is localized in both the nucleus and cytosolof islets of Langerhans in a similar pattern as reported for SOX13 (42).Consistent with the decrease in SOX6mRNA level shown in ob/obmicein Fig. 1A, the number of cells expressing SOX6 protein was alsoreduced in ob/ob mice (Fig. 1D). To examine whether SOX6 isexpressed in �-cells, double immunofluorescence for SOX6 and insulinwas carried out, and this analysis showed that SOX6 was expressed inthe majority of �-cells (Fig. 1E). Double staining with glucagon showedthat SOX6was also expressed in�-cells (data not shown). Since PDX1 isalso expressed in the majority of � cells, we examined whether SOX6co-localizedwith PDX1 in�-cells by double immunofluorescence stain-ing. Endogenous SOX6 and PDX1 were diffusely distributed in thenuclei of pancreatic �-cells and co-localized (Fig. 1F).

SOX6 Negatively Regulates Glucose-stimulated Insulin Secretion—We next examined the effects of SOX6 expression on GSIS. MIN6 cellswere transduced with a recombinant retrovirus encoding SOX6, andinsulin secretion in response to 5.6 or 16.7 mM glucose was examined asdescribed under “Experimental Procedures.” As a control experiment, arecombinant retrovirus encoding alkaline phosphatase (ALP) wastransduced into companion dishes of MIN6 cells. As shown in Fig. 2A,retrovirus-mediated induction of SOX6 resulted in 60% inhibition ofGSIS evoked by 16.7 mM glucose as compared with the control ALP-transduced cells. Control experiments for these studies showed thatboth SOX6 and ALP RNAs were expressed in the transduced cells (datanot shown).

The SOX gene family encodes a group of transcription factorsdefined by the conserved HMG DNA-binding domain and consists ofmore than 20 individual members (43). SOX proteins are classified intoeight subgroups, A–H. SOX6 and its highly related proteins, SOX5 andSOX13, belong to groupD, which is unique, because all three contain anadditional specifically positioned leucine zipper motif. To evaluate theeffects of these and other SOX proteins on GSIS, we selected SOX5, -9,-13, and -15 plus several other randomly selected SOX proteins andexpressed each through retrovirus transduction in MIN6 cells. ThemRNA expression of each SOX gene was confirmed by either QRT-PCR or immunoblotting or both. As shown in Fig. 2B, similar to SOX6,expression of the highly related SOX5 and SOX13 also attenuatedGSIS,whereas expression of the other more distally related SOX proteins(SOX9 and SOX15) had minimal effects. The expression of SOX5,SOX9, and SOX15was confirmed by immunoblotting (Fig. 2B, inset). Inexperiments not shown, we also determined that similar expression ofsix other more distantly related SOX proteins had minimal effects onGSIS. These studies demonstrate that SOX6 and its closest relatives allcan potentially inhibit GSIS. However, unlike SOX6, the levels of theSOX5 and SOX13 transcripts are not down-regulated by the HFD or inob/ob obese mice (data not shown), suggesting that SOX6 is the majorSOX protein involved in modulating GSIS in obesity-induced pancre-atic islets.To complement the results obtained by retroviral expression, we next

employed an RNA interference approach to knock down the expressionof the endogenous SOX6 inmouse insulinomaMIN6 cells and rat insu-

FIGURE 2. Glucose-stimulated insulin secretionin insulinoma cell lines with SOX6 either retro-virally overexpressed (A and B) or down-regu-lated by the application of specific siRNA (C–E).A and B, MIN6 cells were transduced with eitherindicated SOX genes or control ALP retroviruses.Three days after the infection, insulin release fromtransduced cells for 1 h in response to either 5.6 or16.7 mM glucose (A) or 16.7 mM glucose (B) wasassessed as described under “Experimental Proce-dures.” Inset, an immunoblot for SOX5, SOX9, andSOX15 from cells transduced with control retrovi-rus (lanes 1, 3, and 5) or retrovirus for SOX5 (lane 2),SOX9 (lane 4), and SOX15 (lane 6). C–E, siRNA-me-diated knockdown of SOX6. MIN6 and INS-1E cellswere transfected with either control (si-cont) orSOX6-specific siRNA (si-SOX6). Two days aftertransfection, cells were applied for insulin secre-tion assay. In parallel, the cells were harvested forisolation of total RNA and whole cell extracts. C,quantification of SOX6 mRNA levels by QRT-PCR intransfected cells. To correct for variations in inputRNA, data ware normalized to the quantity ofmouse or rat 36B4 (NM_022402). D, immunoblotfor SOX6 and �-actin proteins from whole cellextracts of transfected cells. E, insulin release for1 h in response to either 5.6 or 16.7 mM glucose.Each bar and symbol represent mean � S.E. of trip-licate experiments. *, p � 0.05; **, p � 0.01 com-pared with ALP (A and B) or control siRNA (E). n.s.,not significant.

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linoma INS-1E cells. Transfection of an siRNA duplex, correspondingto nucleotides 2249–2267 of themouse and rat SOX6mRNAs, resultedin a reduction of SOX6 mRNA levels by 50% in both MIN6 andINS-1E cells (Fig. 2C), whereas it had almost no effect on the levels ofSOX5 or SOX13 (data not shown). Consistent with the decrease inmRNA level, immunodetectable SOX6 was reduced in both MIN6 andINS-1E cells treated with SOX6-specific siRNA (Fig. 2D). Under theseconditions, insulin secretion in response to 16.7 mM glucose was signif-icantly enhanced by the SOX6 siRNA (1.4-fold, p � 0.05) in both celllines as compared with companion dishes where a control siRNA wastransfected (Fig. 2E). In contrast, the increase of insulin secretion bySOX6 siRNA was not observed in 5.6 mM glucose. Together with theretroviral expression findings, these data indicate that SOX6 negativelyregulates GSIS in insulin-secreting cells.

SOX6 Expression Reduces ATP Production and Insulin GeneTranscripts—To further evaluate the mechanism for the reduced GSISmediated by SOX6,we analyzed the insulin secretory responses to�-ke-toisocaproate (�-KIC), tolbutamide, and KCl in MIN6 cells transducedwith the SOX6-expressing retrovirus. These secretagogues wereselected for their actions at different and specific stages in the insulinsecretion pathway; after glucose, which is the primary event, �-KIC actsat mitochondrial metabolism, and KCl and tolbutamide act at mem-brane depolarization (44). Whereas SOX6 expression had almost noeffect on the depolarization-induced insulin secretion by 0.3 mM tolbu-tamide or 20mMKCl, it profoundly reduced insulin secretion by 10mM

�-KIC at glucose concentrations of 5.6 and 16.7 mM (Fig. 3A). Thisresult suggests that mitochondrial ATP production from �-KIC isinhibited by the expression of SOX6. Consistent with the reduced insu-lin secretion from �-KIC by SOX6, the ATP content and ATP/ADPratio in the presence of 16.7mMglucosewere significantly decreased (by25–30%) in SOX6-expressing cells as compared with control ALP-transduced cells (Fig. 3B). Furthermore, glucose-induced intracellularCa2� ([Ca2�]i) transients (as determined by changes in the 340/380-nmfluorescence ratio), the eventual trigger for the exocytosis of insulin-containing vesicle (Fig. 3C), and total cellular insulin content (80% ofALP control cells) were significantly decreased in SOX6-expressingcells (Fig. 3D).

SOX6Negatively Regulates Transcripts of Genes for Insulin asWell asATP Production in Mitochondria—To further examine the molecularmechanism for the reduction in ATP generation and cellular insulincontent, we examined gene expression in MIN6 cells transduced withSOX6. Consistent with reduced levels of ATP production, genes for theNADH dehydrogenase complex subunit (complex I), the cytochromebc1 complex subunit (complex III), cytochrome c oxidase complex sub-unit (complex IV), and the ATP synthase subunit (complex V) werereduced by 40–60% comparedwith control ALP-expressingMIN6 cells(TABLE ONE). Importantly, consistent with the reduction of total cel-lular insulin content, the insulin I and II gene transcript levels in SOX6cells were markedly decreased, as quantified by QRT-PCR (to 60% ofALP control cells) (Fig. 3E).

SOX6 Acts as a Co-repressor for PDX1 on the Insulin Promoter—Based on the reduced insulin I and II genemRNA in SOX6-overexpress-ing cells, we examined the effects of SOX6 expression on insulin genepromoter activity using the promoter region of the insulin II gene. ADNA fragment extending from nucleotide �872 to �176 of the ratinsulin II gene was subcloned into the promoterless luciferase reportergene, pGL3-basic, to create the promoter reporter constructpINS(�872)-luc. This promoter reporter construct was transientlytransfected into MIN6 cells along with increasing amounts of pCMV-

SOX6. As shown in Fig. 4A, the normalized luciferase activities inMIN6cells were decreased in proportion to the amounts of co-transfectedpCMV-SOX6.

FIGURE 3. Secretagogue-stimulated insulin secretions (A), glucose-stimulated ATP/ADP ratio (B), [Ca2�]i transient (C), proinsulin content (D), and insulin gene tran-scripts (E) in MIN6 cells overexpressing SOX6. A–E, 3 days after retroviral transductionwith either SOX6 or control ALP to MIN6 cells, each assay was performed. A, insulinsecretion from transduced MIN6 cells in response to 10 mM �-KIC, 0.3 mM tolbutamide(Tolb), or 20 mM KCl was assessed in 1-h static incubations under 5.6 and 16.7 mM glucoseconditions. B, transduced MIN6 cells were preincubated in KRBH containing 1 mM glu-cose at 37 °C for 30 min and incubated in the presence of 5.6 or 16.7 mM glucose at 37 °Cfor 1 h. ATP and ADP content was measured as described, and the ATP/ADP ratio wascalculated. C, transduced MIN6 cells were subjected to a Ca2� influx assay as describedunder “Experimental Procedures.” The arrows indicate the time glucose (40 mM finalconcentration) (left) or tolbutamide (0.1 mM final concentration) (right) was applied. D,proinsulin content in transduced MIN6 cells was measured after extraction with acidethanol as described. E, mRNA levels for insulin I and II genes in transduced MIN6 cellswere determined by QRT-PCR. Mouse 36B4 mRNA was used for the invariant control forQRT-PCR. Each bar and symbol represents mean � S.E. of triplicate experiments. *, p �0.01 compared with control. n.s., not significant.

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There are three potential SOX elements in the rat insulin II gene (Fig.4B). To analyze whether SOX6-mediated suppression of the insulinpromoter is mediated by the direct binding of these SOX-like elementsby SOX6, we constructed a series of deletion mutants, each lacking oneof the three SOX-like elements (Fig. 4B). Co-transfection of thesemutants with SOX6 revealed that deletion of any of these SOX elementsdid not affect the suppressive effects of SOX6. These results suggest thatthe suppression of the insulin promoter by SOX6 is either dependent onSOX6 binding to nonconsensus sites or independent of SOX6 bindingto DNA. Consistent with previously reported observations (45, 46),deletion of nucleotides �413 to �370 of the insulin II promoter abol-ished all of the promoter activity (Fig. 4B).

We next examined the effects of SOX6 on the insulin II promoteractivation by either PDX1, E47 (an E2A gene product), or both. Asshown in Fig. 4C and consistent with a previous report (17), PDX1together with E47 synergistically activated the insulin II promoter, andincreasing amounts of the SOX6 expression plasmid resulted in a dose-dependent inhibition. Fig. 4D shows that SOX6 had almost no effect onthe E47-mediated transactivation of a highly E47-responsive IgH

enhancer reporter, p(�E5 � �E2 � �E3)4-luc, suggesting that SOX6does not specifically repress E47 function.To evaluate whether SOX6 might inhibit activation mediated by

PDX1, we fused the coding sequence of PDX1 and E47 to the GAL4DNA-binding domain, and the transactivation potential of the resultingGAL4 fusions was examined in the absence and presence of SOX6. Asshown in Fig. 4E, SOX6 strongly suppressed GAL4-PDX1 transactiva-tion, whereas it had almost no effects on GAL4-E47.These results suggest that SOX6 and PDX1 proteins may directly

interact together to inhibit the insulin II promoter. To evaluate thispossibility, we carried out GST pull-down assays using a GST-SOX6fusion protein and in vitro-translated full-length PDX1, and to mapthe interaction domain we also evaluated a series of deletionmutants. As shown in Fig. 5A, i and ii, the full-length protein and themutant lacking the COOH terminus (residues 206–284) were co-precipitated with GST-SOX6, whereas the mutant lacking the home-odomain and the COOH terminus had weak binding to GST-SOX6(Fig. 5A, iii), and deletion of the NH2-terminal 144 amino acidsabolished the binding to GST-SOX6 (Fig. 5A, iv). These data indicate

TABLE ONE

The relative amounts of mRNAs for mitochondrial oxidative phosphorylation complexes in SOX6-transduced MIN6 cellsTotal RNA fromMIN6 cells transducedwith retrovirus encodingALPor SOX6was subjected toQRT-PCRas described under “Experimental Procedures.”Mouse36B4 mRNA was used for the invariant control for QRT-PCR. Values are depicted relative to ALP-transduced MIN6 cells, which are arbitrarily defined as 1.Relative amounts and S.E. of each experiment are shown in the columns 3 and 4. OXPHOS, oxidative phosphorylation complex; Expt., experiment.

Gene Accession no.SOX6/ALP

Expt. 1 Expt. 2

Complex I of mitochondrial OXPHOSNADH dehydrogenase (ubiquinone) 1,

subcomplex unknown, 1NM_025523 0.45 � 0.014 0.62 � 0.015

NADH dehydrogenase (ubiquinone) 1 �subcomplex, 3

NM_025348 0.64 � 0.007 0.59 � 0.023

NADH dehydrogenase (ubiquinone) 1 �subcomplex, 1, 7 kDa

AW_060701 0.62 � 0.014 0.63 � 0.014

NADH dehydrogenase (ubiquinone) 1 �subcomplex, 2

NM_026612 0.64 � 0.030 0.67 � 0.010

NADH dehydrogenase (ubiquinone) 1 �subcomplex 4

NM_026610 0.59 � 0.007 0.63 � 0.010

NADH dehydrogenase (ubiquinone) 1 �subcomplex, 6, 17 kDa

XM_131359 0.62 � 0.013 0.52 � 0.028

NADH dehydrogenase (ubiquinone) Fe-Sprotein 2

NM_153064 0.57 � 0.013 0.44 � 0.014

NADH dehydrogenase (ubiquinone) Fe-Sprotein 7

NM_029272 0.50 � 0.006 0.48 � 0.011

NADH dehydrogenase (ubiquinone) Fe-Sprotein 8

NM_144870 0.59 � 0.014 0.53 � 0.010

Complex III of mitochondrial OXPHOSCytochrome c1 NM_025567 0.46 � 0.007 0.46 � 0.022Ubiquinol-cytochrome c reductase (6.4 kDa)

subunitNM_025650 0.62 � 0.024 0.64 � 0.013

Complex IV of mitochondrial OXPHOSCytochrome c oxidase, subunit Vb NM_009942 0.66 � 0.004 0.66 � 0.023cytochrome c oxidase, subunit Vib NM_025628 0.55 � 0.005 0.64 � 0.013

Complex V of mitochondrial OXPHOSATP synthase, H�-transporting, mitochondrial

F0 complex, subunit c (subunit 9), isoform 1NM_007506 0.66 � 0.011 0.49 � 0.008

ATP synthase, H�-transporting, mitochondrialF0 complex, subunit f, isoform 2

NM_020582 0.44 � 0.008 0.65 � 0.010

ATP synthase, H� transporting, mitochondrialF1 complex, � subunit, isoform 1

NM_025983 0.66 � 0.013 0.47 � 0.009

ATP synthase, H�-transporting, mitochondrialF1 complex, O subunit

NM_138597 0.45 � 0.004 0.43 � 0.015

ATP synthase, H�-transporting, mitochondrialF1F0 complex, subunit e

NM_007507 0.61 � 0.007 0.53 � 0.014

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that the NH2-terminal 144 amino acids of PDX1 were critical for theinteraction with SOX6.As a complement to the GST-SOX6 pull-down experiment, we con-

structed a GST-PDX1 fusion protein and evaluated it for interactionwith full-length SOX6 or a series of deletion mutants of SOX6. Asshown in Fig. 5B, it is clear that the minimum PDX1 binding site ofSOX6 is a region containing the functionally important HMG box ofSOX6. Together with the GST-SOX6 pull-down assay findings, thesedata indicate that the HMG box of SOX6 interacts physically with theNH2-terminal 144 amino acids of PDX1, as schematically depicted inFig. 5C.In Fig. 5D, top, we show that elimination of the HMG domain from

SOX6 resulted in a protein that was unable to attenuate GSIS. Theprotein expression ofmutant SOX6 lacking theHMGdomain (�HMG-SOX6) was confirmed by immunoblotting with an anti-SOX6 antibody(Fig. 5D, bottom). Furthermore, the deletion of the HMG domain ofSOX6 abolished the SOX6 suppressive effect on the insulin II genepromoter (Fig. 5E), whereas expression of the NH2 terminus (residues1–144) of PDX1 reversed the SOX6-mediated inhibition (Fig. 5F).Taken together with the GST interaction studies, these data demon-strate that the SOX6 HMG domain suppresses the insulin II gene pro-moter by interacting physically with the NH2 terminus of PDX1, thekey transactivator protein for positive regulation of insulin genetranscription.

siRNA-mediated Knockdown of PDX1 Decreased SOX6Occupancy ofthe Insulin II Promoter—To further determine the mechanismunderlying insulin II promoter inhibition by SOX6, we examined the

association of PDX1 and SOX6 with the insulin II promoter in SOX6or control ALP retrovirus and PDX1-specific siRNA-treated MIN6cells by ChIP assays. We used QRT-PCR to evaluate the results of theChIP, and the specific primers were designed as schematicallydepicted in Fig. 6.When SOX6 was expressed by retroviral transduction, the insulin

promoter sequence was present at a higher level in the SOX6 immuno-precipitate (Fig. 6A, top, compare lanes 2 and 4), suggesting an increasedassociation of SOX6 with the promoter under these conditions. In con-trast, SOX6 expression did not alter the DNA binding of PDX1 to theinsulin II promoter (Fig. 6A, top, compare lanes 6 and 8), indicating thatSOX6 does not simply interfere with the DNA binding of PDX1. Theimmunoblot in the bottom panel shows that SOX6 protein levels wereelevated (6.5-fold, as evaluated by LumiVisionPRO) in the SOX6 retro-viral transduced cells (Pre) and that SOX6 was efficiently collected bythe immunoprecipitation procedure with anti-SOX6 antibody (Post) inproportion to the overall levels (Fig. 6A, bottom; compare the gelslabeled Pre for direct immunoblot with those labeled Post).

It has been previously demonstrated that PDX1 specifically binds theproximal insulin promoter of MIN6 cells by a ChIP assay (47). 48 hfollowing PDX1-siRNA treatment, we observed an 80% reduction inPDX1 occupancy at the insulin II promoter in MIN6 cells (Fig. 6B, top,compare lanes 2 and 4). Interestingly, a decrease in PDX1 at the insulinII promoter in MIN6 cells was also accompanied by a 60% reduction inthe binding of SOX6 to the proximal insulin promoter (Fig. 6B, top,compare lanes 6 and 8). Taken together with the GST pull-down assays,

FIGURE 4. Transcriptional inhibition of the insulin II gene promoter (A and B) and specific inhibition of PDX1 transcriptional activity (C–E) by transfected SOX6. A, MIN6 cellswere transfected with 0.1 �g of the insulin II promoter luciferase reporter plasmid pINS(�872)-luc and 0.01 �g of pCMV� together with the indicated amount of pCMV-SOX6 asdescribed under “Experimental Procedures.” B, deletion mutants of the insulin II promoter luciferase reporter plasmid, in which different putative SOX elements were deleted, wereconstructed as described. MIN6 cells were transfected with 0.1 �g of pINS(�872)-luc or its mutants together with 0.01 �g of pCMV� in the presence or absence of 0.1 �g ofpCMV-SOX6. C, BHK21 cells were transfected with 0.1 �g of pINS(�872)-luc and 0.01 �g of pCMV� together with the indicated amounts of the following plasmids: pCMV-E47 (lanes2 and 5– 8), pCMV-PDX1 (lanes 3 and 5– 8), and pCMV-SOX6 (lanes 4 and 6 – 8). D, HEK293 cells were transfected with 0.1 �g of E47-responsive IgH enhancer reporter plasmid p(�E5� �E2 � �E3)4-luc (30) and 0.05 �g of pCMV� together with the indicated amounts of pCMV-E47 (lanes 3– 6) and pCMV-SOX6 (lanes 2 and 4 – 6). E, MIN6 cells were transfected with0.1 �g of pG5luc, the multimerized GAL4-responsive promoter luciferase reporter construct, and 0.02 �g of pCMV� together with 0.02 �g of GAL4 fusion constructs: pBIND-PDX1(lanes 3–5) and pBIND-E47 (lanes 6 and 7) together with the indicated amounts of pCMV-SOX6 (lanes 2 and 4 –7). A–E, luciferase activity was measured and normalized to �-galac-tosidase activity as described. Each bar and symbol represent mean � S.E. of triplicate experiments. *, p � 0.01 compared with control. RLU, relative luciferase units.

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these results indicate that endogenous SOX6 protein in eukaryotic cellsinteracts with DNA-bound PDX1 at the insulin promoter in vivo.

SOX6 Decreased Acetylation of Chromatin-associated Histones H3and H4—PDX1 transcriptional activity is reportedly regulated by therecruitment of p300, a coactivator protein that possesses intrinsic his-tone acetylase activity (20, 48), and similar to SOX6, p300 interactsdirectly with theNH2-terminal region of PDX1.We therefore evaluatedthe effects of SOX6 on the acetylation levels of histones H3 and H4 inchromatin at the insulin II promoter, using antibodies directed at theacetylated forms of either histone H3 or H4. Consistent with previouspapers (20, 48), cultivation of MIN6 cells with a high glucose concen-

tration (30 mM) leads to a significant increase in acetylation of histoneH4 at the insulin II promoter (Fig. 6D, compare lanes 2 and 4), whereasthere was no effect of glucose on the degree of H3 acetylation (Fig. 6C,compare lanes 2 and 4). In cells where SOX6was expressed by retroviraltransduction, the levels of both acetylated histones H3 and H4 at theinsulin II promoter were profoundly reduced (Fig. 6,C, compare lanes 2and 6, lanes 4 and 8, and D, compare lanes 4 and 8), indicating thatSOX6 leads to a reduction in acetylation of both H3 and H4 in thechromatin on the insulin II promoter. Because p300 has histone acety-lase activity, this suggests that SOX6may interfere with the recruitmentof p300 to PDX1 bound at the insulin II promoter.

FIGURE 5. Functional interaction between NH2-terminal domain of PDX1 and HMG domain within SOX6. A and B, in vitro interaction of SOX6 and PDX1. GST-fused full-lengthSOX6 (A) or GST-fused full-length PDX1 protein (B) immobilized to glutathione beads were incubated with in vitro translated [35S]PDX1 and its deletion mutants (A) or in vitrotranslated [35S]SOX6 and its deletion mutants (B), respectively, at room temperature for 1 h. Purified GST was used as a negative control for nonspecific binding. After washingextensively, the proteins bound to beads and 5% of the input proteins were resolved on SDS-PAGE and visualized by a FUJIX BAS2000 imaging system (Fuji Film, Tokyo, Japan). C,schematic diagram showing HMG domain within SOX6 interacts with NH2-terminal transactivation domain of PDX1. D, the effect of insulin secretion by retrovirally transducedmutant SOX6 lacking the HMG domain. Three days after infection with retroviruses for ALP, SOX6, or mutant SOX6 lacking HMG domain (�HMG) to MIN6 cells, insulin secretion for1 h in response to 5.6 and 16.7 mM glucose was measured (top). At the end of the GSIS experiment, medium was aspirated, these same cells used for 16.7 mM glucose concentrationwere lysed in SDS lysis buffer, and these cells were subjected to SDS-PAGE followed by immunoblotting with anti-SOX6 antibody (ab-12054), which also reacts with �HMG-SOX6(bottom). The migration of wild type and �HMG-SOX6 are noted. E and F, luciferase reporter gene assay. MIN6 cells were transfected with 0.1 �g of pINS(�872)-luc and 0.01 �g ofpCMV� together with 0.1 �g of either pCMV-SOX6 or pCMV-�HMG-SOX6 (E). MIN6 cells were transfected with 0.1 �g of pINS(�872)-luc and 0.01 �g of pCMV� together with theindicated amounts of pCMV-SOX6 (lanes 3– 6) and pCMV-PDX1 (1–144), a construct encoding an NH2-terminal domain (amino acids 1–144) of PDX1 (lanes 2 and 4 – 6). F, fireflyluciferase activity was measured and normalized to �-galactosidase activity. Each bar and symbol represents mean � S.E. of triplicate experiments. *, p � 0.01 compared with control.n.s., not significant.

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DISCUSSION

In the current study, we demonstrated that SOX6 protein isexpressed in adult pancreatic islets, where its expression is dramaticallydown-regulated both by HFD feeding and in genetically predisposedobese model (ob/ob) mice. In both cases, the mice are hyperinsulinemicwith insulin resistance, and they have an increase in pancreatic isletmass. In contrast, SOX6 mRNA levels were not altered by fasting andrefeeding experiments, suggesting that pancreatic expression of SOX6

is not regulated by acute changes of blood glucose or insulin levels.Based on the attenuation of GSIS by SOX6 and its down-regulation inhyperinsulinemicmice, we propose that changes in the ratio of SOX6 toPDX1 may be responsible for the compensatory islet hyperplasia thatoccurs in response to insulin resistance.The SOX family of transcription factors contains a DNA binding

HMG box, which is highly conserved across species. SOX proteins areinvolved in a number of developmental processes, including tissue spec-

FIGURE 6. ChIP assays for SOX6, PDX1, and acetylated histone association with the insulin II gene promoter. MIN6 cells infected with retrovirus encoding either SOX6 orcontrol ALP (A, C, and D) or MIN6 cells transfected with PDX1-specific or control siRNA (B) were harvested for ChIP as detailed under “Experimental Procedures.” Schematic diagramsof the insulin gene above the panels indicate the DNA fragments (and their positions in bp relative to the transcriptional start site at 1) that were amplified by real time PCR followingChIP. A, recovery of the insulin promoter fragment following ChIP using extracts from retrovirus-infected MIN6 cells and antibody (Ab) against SOX6 and PDX1. The bottom panelshows the presence of SOX6 in cell lysate before immunoprecipitation (Pre) and the immunoprecipitated (IP) samples with SOX6-Ab (Post) detected by immunoblot analysis withSOX6-Ab (ab-12054). B, recovery of the insulin promoter fragment following ChIP using SOX6 and PDX1 Ab and extracts from siRNA-transfected cells. The bottom panel shows thepresence of PDX1 in cell lysate before immunoprecipitation (Pre) and the immunoprecipitated samples with PDX1-Ab (Post) detected by immunoblot analysis with PDX1-Ab. C andD, recovery of the insulin promoter fragment following ChIP using anti-acetyl histone H3 and H4 antibodies and extracts from retrovirus-infected MIN6 cells. Retrovirus-infected MIN6cells were grown on media containing either 3 or 30 mM glucose for 16 h. ChIP assays were performed using anti-acetyl histones H3 and H4 antibodies for immunoprecipitation.Rabbit IgG was used as a negative control for immunoprecipitation. All data represent recovery, in percent, of each DNA fragment relative to total input DNA. Each bar and symbolrepresents mean � S.E. of triplicate experiments. *, p � 0.01 compared with control. n.s., not significant. The data represent the average of at least three independent experiments.

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ification and differentiation, embryonic patterning, and maturation.Numerous members of the SOX gene family are also expressed duringpancreas development, and they may have important and possiblyredundant functions in pancreas development (49). Although SOX6expression was not noticed in this previous study (49), our studies pro-vide strong evidence that it is indeed expressed in islets and regulated byHFD. SOX6 and highly related SOX5 are co-expressed during mousechondrogenesis and cooperatively activate the expression of the Col2agene, a chondrocyte differentiationmarker (50). Since SOX factors playimportant roles in key developmental processes, knockout mice tend todie in utero. Mice lacking both SOX5 and SOX6 have a severe general-ized chondrodysplasia and die in utero (51). Therefore, the roles for theSOX proteins in adult tissues including pancreatic islets are poorlyunderstood.Our data suggest that SOX6 modulates GSIS by regulating PDX1

transcriptional activities in pancreatic islets. Our studies also indicatethat a physical interaction between the HMG domain of SOX6 and theNH2-terminal activation domain of PDX1 is important for this repres-sion. This same NH2-terminal activation domain of PDX1 is known torecruit the histone acetylase coactivator p300, and the HMG domainhas been shown to be a common protein-protein interaction motif inother SOX proteins as well (52). Consistent with a model where SOX6interacts with DNA bound PDX1, our studies show that SOX6 expres-sion does not affect PDX1 DNA binding, and reduced PDX1 binding tothe insulin promoter by PDX1-specific siRNA results in a decrease inthe co-association of SOX6. Furthermore, SOX6 recruitment results inreduced histone acetylation at the insulin II promoter. Based on theseobservations, we hypothesize that the suppressive effect of SOX6 onPDX1 is mediated, in part, by decreasing the localized histone hyper-acetylation that normally would occur upon recruitment of p300 toDNA-bound PDX1. It is also possible that SOX6 interacts with PDX1 torecruit a repressor complex to the promoter of PDX1 target genes.Recently, Iype et al. (53) showed that decreased insulin transcription

was associated with decreased occupancy of the insulin promoter byPDX1 and p300. These investigators also showed that, whereas recruit-ment of RNA polymerase II to the insulin coding region was signifi-cantly reduced, there was no corresponding change in the recruitmentof RNA polymerase II to the proximal promoter. They suggested thatPDX1 directly regulates insulin transcription through formation of acomplex with transcriptional factors (E47, BETA2/NeuroD) and co-factors (p300) to lead to a physical and functional interaction with RNApolymerase II (53). Since SOX6 inhibits synergistic activation by PDX1and E47, our data suggest that SOX may disrupt this PDX1-mediatedincrease in recruitment of the polymerase II transcriptional machineryto the insulin gene.The decrease in insulin secretion by elevated SOX6 is likely to be

mediated in part by the decreased insulin gene expression. We alsoshowed that SOX6 overexpression resulted in a decrease in the ratio ofATP/ADP that was accompanied by a significant decrease in expressionfor several genes of oxidative metabolism (TABLE ONE). Because adecreased ATP/ADP ratio is also associated with decreased GSIS, ourresults are additionally consistent with a model where SOX6 repressesGSIS through decreasing expression of genes involved in ATP produc-tion. Whether these genes are direct targets of PDX1 or whether SOX6may decrease gene expression through other mechanisms requires fur-ther study. Interestingly, however, expression of a dominant negativeform of PDX1 through adenovirus transduction in vitro reduced theexpression of mitochondrial genes and also resulted in severe conse-quences in �-cell mitochondrial function (54).

Although SOXgenes of the same group tend to be co-expressed at the

same developmental stage and exhibit functional redundancy in devel-opment, it is intuitive that redundancymay not extend to key transcrip-tional regulatory decisions in adult tissues. Group D SOX proteins(SOX5, -6, and -13) are all detected in adult islets (49) (present work).SOX13 has been reported to be expressed in �-cells and functions asautoantigen in type 1 diabetes (42). Our data show that they are allcapable of suppressing GSIS when overexpressed; however, only SOX6gene expression was reduced by the HFD or in ob/ob mice. Since allthree are expressed in islets, it is not immediately clear why down-regulation of SOX6 alone by the HFD could result in an increase inGSIS. It is possible that the levels of endogenous SOX5 and SOX13proteins are relatively low, so a decrease in SOX6 alone would be suffi-cient to initiate a response, or additional proteins that are also aber-rantly expressed by the HFD augment the SOX6 effect specifically.Experiments are in progress to address these and related issues.It is conceivable that SOX genes in adult tissues are regulated in

response to the circulating nutrients or hormones, such as glucose,insulin, or other nutrients. Thus, SOX6 may contribute to the insulingene regulation in pathophysiological states where PDX1 function iscompromised as observed in insulin resistance and diabetes. Therefore,the modulation of PDX1 function by SOX6 may provide a promisingnew therapeutic target for treatment of type II diabetes.

Acknowledgments—We thank Dr. Kazuya Yamagata for plasmid constructs;Dr. Toshio Kitamura for a retroviral packaging cell line and pMXplasmid; Dr.Jun-Ichi Miyazaki for MIN6 cells; Dr. Pierre Maechler for INS-1E cells; andTakashi Aoyama and Kenji Oyachi for technical assistance.

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T. Yamamoto, Tatsuhiko Kodama and Juro SakaiTokuoMaejima, Aoi Uchida, Makoto Naito, Timothy F. Osborne, Masashi Yanagisawa,

Shuying Jiang, Go Hasegawa, Ryoichi X. Ioka, Kenta Magoori, Koichi Sumi, TakashiHiroto Ohguchi, Shinobu Takayasu, Noriaki Kojima, Satoshi Iwasaki, Riuko Ohashi, Haruhisa Iguchi, Yukio Ikeda, Masashi Okamura, Toshiya Tanaka, Yasuyo Urashima,Transcriptional Actvity and Is Down-regulated in Hyperinsulinemic Obese Mice

SOX6 Attenuates Glucose-stimulated Insulin Secretion by Repressing PDX1

doi: 10.1074/jbc.M505392200 originally published online September 7, 20052005, 280:37669-37680.J. Biol. Chem. 

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