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Protein-tyrosine Phosphatase 1B as New Activator for Hepatic Lipogenesis via Sterol Regulatory Element-binding Protein-1 Gene Expression

2003; Elsevier BV; Volume: 278; Issue: 44 Linguagem: Inglês

10.1074/jbc.m306880200

ISSN

1083-351X

Autores

Shinya Shimizu, Satoshi Ugi, Hiroshi Maegawa, Katsuya Egawa, Yoshihiko Nishio, Takeshi Yoshizaki, Kun Shi, Yoshio Nagai, Katsutaro Morino, Ken-ichi Nemoto, Takaaki Nakamura, Michael Bryer‐Ash, Atsunori Kashiwagi,

Tópico(s)

RNA modifications and cancer

Resumo

Like hyperglycemia, postprandial (diet-induced) hypertriglyceridemia is thought to play crucial roles in the pathogenesis of insulin resistant/metabolic syndrome. Sterol regulatory element-binding protein-1 (SREBP-1) is a key transcription factor to induce postprandial hypertriglyceridemia. We found that insulin-resistant rats fed a diet high in fructose showed an increased proteintyrosine phosphatase 1B (PTP1B) content with strong expression of SREBP-1 mRNA in the liver. To clarify the association of PTP1B with SREBP-1 gene expression, we overexpressed PTP1B in rat hepatocytes, which led to increased mRNA content and promoter activity of SREBP-1a and -1c, resulting in the increased mRNA expression of fatty-acid synthase, one of the SREBP-1-responsive lipogenic genes. Because PTP1B overexpression increased phosphatase 2A (PP2A) activity, we inhibited PP2A activity by expression of its selective inhibitor, SV40 small t antigen and found that this normalized the PTP1B-enhanced SREBP-1a and -1c mRNA expressions through activation of the Sp1 site. These results indicate that PTP1B may regulate gene expression of SREBP-1 via enhancement of PP2A activity, thus mediating hepatic lipogenesis and postprandial hypertriglyceridemia. We demonstrate here a unique serial activation of the PTP1B-PP2A axis as a novel mechanism for the regulation of gene expression in the biosynthesis of triglyceride. Like hyperglycemia, postprandial (diet-induced) hypertriglyceridemia is thought to play crucial roles in the pathogenesis of insulin resistant/metabolic syndrome. Sterol regulatory element-binding protein-1 (SREBP-1) is a key transcription factor to induce postprandial hypertriglyceridemia. We found that insulin-resistant rats fed a diet high in fructose showed an increased proteintyrosine phosphatase 1B (PTP1B) content with strong expression of SREBP-1 mRNA in the liver. To clarify the association of PTP1B with SREBP-1 gene expression, we overexpressed PTP1B in rat hepatocytes, which led to increased mRNA content and promoter activity of SREBP-1a and -1c, resulting in the increased mRNA expression of fatty-acid synthase, one of the SREBP-1-responsive lipogenic genes. Because PTP1B overexpression increased phosphatase 2A (PP2A) activity, we inhibited PP2A activity by expression of its selective inhibitor, SV40 small t antigen and found that this normalized the PTP1B-enhanced SREBP-1a and -1c mRNA expressions through activation of the Sp1 site. These results indicate that PTP1B may regulate gene expression of SREBP-1 via enhancement of PP2A activity, thus mediating hepatic lipogenesis and postprandial hypertriglyceridemia. We demonstrate here a unique serial activation of the PTP1B-PP2A axis as a novel mechanism for the regulation of gene expression in the biosynthesis of triglyceride. The insulin resistance/metabolic syndrome is characterized by the variable coexistence of hyperinsulinemia, hyperlipidemia, obesity, and hypertension. Many individuals with obesity and insulin resistance suffer from fatty liver (steatohepatosis). Like hyperglycemia, postprandial (diet-induced) hypertriglyceridemia is thought to play a crucial role in the pathogenesis of the insulin resistance/metabolic syndrome. Several lines of evidence indicate that fatty liver in insulinresistant states is caused by activation of the sterol regulatory element-binding protein (SREBP)-1c, 1The abbreviations used are: SREBP-1, sterol regulatory element-binding protein-1; C/S, Cys215/Ser215; EMSA, electrophoretic mobility shift assay; Erk, extracellular signal-regulated kinase; IRS, insulin receptor substrate; PP2A, protein phosphatase 2A; PTP1B, proteintyrosine phosphatase 1B; ST, SV40 small t antigen; WT, wild type. which is elevated in response to high insulin levels (1Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Investig. 2002; 109: 1125-1131Crossref PubMed Scopus (3838) Google Scholar, 2Shimano H. Prog. Lipid Res. 2001; 40: 439-452Crossref PubMed Scopus (587) Google Scholar). Although insulin resistance is present in peripheral tissues, compensatory hyperinsulinemia is postulated to persistently activate SREBP-1c transcription and cleavage, which increases lipogenic gene expression, enhances fatty acid synthesis, and accelerates triglyceride accumulation in the liver in obese insulin-resistant mice (3Shimomura I. Bashmakov Y. Horton J.D. J. Biol. Chem. 1999; 274: 30028-30032Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar, 4Shimomura I. Matsuda M. Hammer R.E. Bashmakov Y. Brown M.S. Goldstein J.L. Mol. Cell. 2000; 6: 77-86Abstract Full Text Full Text PDF PubMed Scopus (708) Google Scholar, 5Tobe K. Suzuki R. Aoyama M. Yamauchi T. Kamon J. Kubota N. Terauchi Y. Matsui J. Akanuma Y. Kimura S. Tanaka J. Abe M. Ohsumi J. Nagai R. Kadowaki T. J. Biol. Chem. 2001; 276: 38337-38340Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). However, the reason that expression of hepatic SREBP-1c mRNA remains high in insulin-resistant states is little understood. We have reported that high fructose feeding causes insulin resistance with increased hepatic SREBP-1 mRNA content (6Nagai Y. Nishio Y. Nakamura T. Maegawa H. Kikkawa R. Kashiwagi A. Am. J. Physiol.. 2002; 282: E1180-E1190Crossref PubMed Scopus (174) Google Scholar), and we recently found that, as reported (7Taghibiglou C. Rashid-Kolvear F. Van Iderstine S.C. Le-Tien H. Fantus I.G. Lewis G.F. Adeli K. J. Biol. Chem. 2002; 277: 793-803Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar), protein-tyrosine phosphatase 1B (PTP1B) abundance is also increased in the liver. PTP1B is a major regulator of insulin sensitivity and body fat content, and deletion of its gene leads to augmented insulin sensitivity and resistance to obesity induced by high fat feeding (8Cheng A. Dube N. Gu F. Tremblay M.L. Eur. J. Biochem. 2002; 269: 1050-1059Crossref PubMed Scopus (175) Google Scholar, 9Johnson T.O. Ermolieff J. Jirousek M.R. Nat. Rev. Drug. Discov. 2002; 1: 696-709Crossref PubMed Scopus (567) Google Scholar, 10Elchebly M. Payette P. Michaliszyn E. Cromlish W. Collins S. Loy A.L. Normandin D. Cheng A. Himms-Hagen J. Chan C.C. Ramachandran C. Gresser M.J. Tremblay M.L. Kennedy B.P. Science. 1999; 283: 1544-1548Crossref PubMed Scopus (1926) Google Scholar, 11Klaman L.D. Boss O. Peroni O.D. Kim J.K. Martino J.L. Zabolotny J.M. Moghal N. Lubkin M. Kim Y.B. Sharpe A.H. Stricker-Krongrad A. Shulman G.I. Neel B.G. Kahn B.B. Mol. Cell. Biol. 2000; 20: 5479-5489Crossref PubMed Scopus (1130) Google Scholar). Moreover, at least one genetic polymorphism of PTP1B is associated with several features of the insulin resistance/metabolic syndrome (12Di Paola R. Frittitta L. Miscio G. Bozzali M. Baratta R. Centra M. Spampinato D. Santagati M.G. Ercolino T. Cisternino C. Soccio T. Mastroianno S. Tassi V. Almgren P. Pizzuti A. Vigneri R. Trischitta V. Am. J. Hum. Genet. 2002; 70: 806-812Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). We reported that overexpression of PTP1B in both liver and muscle cells led to insulin resistance (13Egawa K. Maegawa H. Shimizu S. Morino K. Nishio Y. Bryer-Ash M. Cheung A.T. Kolls J.K. Kikkawa R. Kashiwagi A. J. Biol. Chem. 2001; 276: 10207-10211Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). In high fructose-fed rats, overexpression of PTP1B may induce hepatic insulin resistance. However, high mRNA expression of SREBP-1 still continues in these rats. Although the magnitude of hyperinsulinemia is relatively less than that in fatless mice (3Shimomura I. Bashmakov Y. Horton J.D. J. Biol. Chem. 1999; 274: 30028-30032Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar), it is not known why overexpression of PTP1B fails to suppress insulin-induced SREBP-1 mRNA expression in high fructose-fed rats. Alternatively, it is possible that there may be another mechanism for the continued strong expression of hepatic SREBP-1c mRNA in insulinresistant states. The coexistent strong hepatic expression of PTP1B and SREBP-1 in high fructose-fed rats led us to postulate that PTP1B may regulate SREBP-1 gene expression. To clarify this issue, in the present study we examined the effect of PTP1B overexpression on SREBP-1 gene expression in rat hepatocytes. We found that PTP1B may enhance SREBP-1 gene expression by up-regulating Sp1 transcriptional activity via an increase in protein phosphatase 2A (PP2A) activity, as a distinct mechanism of insulin resistance. Materials—Porcine insulin was kindly provided by Eli Lilly, Inc. Polyclonal anti-PTP1B antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY), and monoclonal PTP1B antibody was from Oncogene Research Products (San Diego). Anti-IRS-1 antibody and anti-IRS-2 antibody were purchased from Upstate Biotechnology, Inc. Anti-phospho-Akt antibody and anti-phospho-MAP (mitogen-activated protein) kinase antibody were from New England Biolabs (Beverly, MA). Horseradish peroxidase-conjugated phosphotyrosine antibody (RC20H) and insulin receptor antibody were from Transduction Labs (Lexington, KY). Horseradish peroxidase-linked anti-rabbit and anti-mouse antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). William's E medium was obtained from Sigma. Dulbecco's modified Eagle's medium and fetal calf serum were obtained from Invitrogen. All radioisotopes were obtained from PerkinElmer Life Sciences. XAR-5 film was obtained from Eastman Kodak Co. All other reagents and chemicals were purchased from Sigma. Preparation of Recombinant Adenovirus—The recombinant adenovirus containing the cDNA encoding the rat PTP1B wild-type (WT) and the Cys215/Ser215 mutant (C/S) were prepared as described (13Egawa K. Maegawa H. Shimizu S. Morino K. Nishio Y. Bryer-Ash M. Cheung A.T. Kolls J.K. Kikkawa R. Kashiwagi A. J. Biol. Chem. 2001; 276: 10207-10211Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 14Wang J. Cheung A.T. Kolls J.K. Starks W.W. Martinez-Hernandez A. Dietzen D. Bryer-Ash M. Diabetes Obes. Metab. 2001; 3: 367-380Crossref PubMed Scopus (13) Google Scholar). Plasmid encoding small t antigen (pCMV5-small t) was a gift from Dr. Marc C. Mumby (University of Texas Southwestern Medical Center, Dallas) (15Sontag E. Fedorov S. Kamibayashi C. Robbins D. Cobb M. Mumby M. Cell. 1993; 75: 887-897Abstract Full Text PDF PubMed Scopus (461) Google Scholar). PP2A exists primarily as a heterotrimer composed of a catalytic subunit (C), a scaffolding subunit (A), and one of a variety of regulatory subunits (B). The C subunit is always associated with the A subunit. A number of variable B subunits can bind to this dimeric core, and these B subunits affect enzymatic activity and substrate specificity of PP2A. Small t antigen is known to specifically inhibit PP2A by binding to the A regulatory subunit, interfering with the ability of PP2A to bind to B subunits that determine its cellular substrates (15Sontag E. Fedorov S. Kamibayashi C. Robbins D. Cobb M. Mumby M. Cell. 1993; 75: 887-897Abstract Full Text PDF PubMed Scopus (461) Google Scholar). Adenoviral vector encoding small t antigen was generated using the Adeno-X Expression System (Clontech, Palo Alto, CA). Cells were infected at 5–10 multiplicities of infection for 1 h unless otherwise indicated and incubated for 56 h at 37 °Cin5%CO2 and the appropriate medium with 2% heat-inactivated serum followed by incubation in the starvation medium required for the assay (13Egawa K. Maegawa H. Shimizu S. Morino K. Nishio Y. Bryer-Ash M. Cheung A.T. Kolls J.K. Kikkawa R. Kashiwagi A. J. Biol. Chem. 2001; 276: 10207-10211Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). High Fructose Feeding—Six-week-old male Sprague-Dawley rats (Japan SLC, Shizuoka, Japan) were housed in an environmentally controlled room with a 12-h light/dark cycle. The animals were divided into a normal diet (control) and a diet high in fructose. The rats of each group were fed for 8 weeks. The normal diet (Oriental Yeast, Tokyo, Japan) consisted of 58% carbohydrate (no fructose), 12% fat, and 30% protein (energy percent of diet). The diet high in fructose (oriental yeast) contained 67% carbohydrate (98% of which was fructose), 13% fat, and 20% protein as described (6Nagai Y. Nishio Y. Nakamura T. Maegawa H. Kikkawa R. Kashiwagi A. Am. J. Physiol.. 2002; 282: E1180-E1190Crossref PubMed Scopus (174) Google Scholar). The day before the experiment, food was withdrawn from all animals at 8 p.m. Rats were re-fed in the dark from 6 to 8 a.m., and then the diet was removed again. After 4 h of food deprivation, rats were anesthetized, and the livers were removed, immediately frozen in liquid nitrogen, and stored at –80 °C until use. RNA and protein were extracted from the frozen samples. Plasma glucose, triglyceride, and insulin levels were determined as described (6Nagai Y. Nishio Y. Nakamura T. Maegawa H. Kikkawa R. Kashiwagi A. Am. J. Physiol.. 2002; 282: E1180-E1190Crossref PubMed Scopus (174) Google Scholar). Liver SREBP-1 mRNA content was determined by Northern blotting and PTP1B protein content was determined by Western blotting as described (6Nagai Y. Nishio Y. Nakamura T. Maegawa H. Kikkawa R. Kashiwagi A. Am. J. Physiol.. 2002; 282: E1180-E1190Crossref PubMed Scopus (174) Google Scholar, 13Egawa K. Maegawa H. Shimizu S. Morino K. Nishio Y. Bryer-Ash M. Cheung A.T. Kolls J.K. Kikkawa R. Kashiwagi A. J. Biol. Chem. 2001; 276: 10207-10211Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). All experiments were approved by Shiga University of Medical Science Animal Care Committees. Cell Culture—Hepatocytes were isolated from nonfasting 200-g male Sprague-Dawley rats by the collagenase method with minor modifications (6Nagai Y. Nishio Y. Nakamura T. Maegawa H. Kikkawa R. Kashiwagi A. Am. J. Physiol.. 2002; 282: E1180-E1190Crossref PubMed Scopus (174) Google Scholar). Cells were plated on 100-mm rat collagen dishes (Asahi Techno Glass, Chiba, Japan) and cultured in William's E medium supplemented with 10% fetal calf serum, 1 nm insulin, 100 nm triiodothyronine, 100 nm dexamethasone, 100 units/ml penicillin, and 100 μg/ml streptomycin (6Nagai Y. Nishio Y. Nakamura T. Maegawa H. Kikkawa R. Kashiwagi A. Am. J. Physiol.. 2002; 282: E1180-E1190Crossref PubMed Scopus (174) Google Scholar). Fao hepatoma cells, kindly provided by Dr. C. R. Kahn (Joslin Diabetes Center, Boston), were cultured in Dulbecco's modified Eagle's medium. Western Blotting—Each liver was homogenized in a solubilizing buffer containing 20 mm Tris, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, and 50 mm NaF (pH 7.5) for 30 min at 4 °C. The tissue lysates were centrifuged to remove insoluble materials. Each sample (20 μg protein/lane) was denatured by boiling in Laemmli sample buffer containing 100 mm dithiothreitol and resolved by SDS-PAGE. The gels were transferred to nitrocellulose by electroblotting in Towbin buffer containing 0.02% SDS and 20% methanol. Membranes were blocked and probed with specified antibodies. Blots were then incubated with horseradish peroxidase-linked second antibody followed by chemiluminescence detection as described (13Egawa K. Maegawa H. Shimizu S. Morino K. Nishio Y. Bryer-Ash M. Cheung A.T. Kolls J.K. Kikkawa R. Kashiwagi A. J. Biol. Chem. 2001; 276: 10207-10211Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Northern Blot Analysis—Total RNA was isolated with Trizol reagent (Invitrogen), and 20-μg RNA samples were run on a 1% agarose gel containing formaldehyde and transferred to a nylon membrane (Nytran N, Schleicher & Schuell). The cDNA probes were generated by reverse transcriptase-PCR amplification from total RNA isolated from the rat liver as reported (6Nagai Y. Nishio Y. Nakamura T. Maegawa H. Kikkawa R. Kashiwagi A. Am. J. Physiol.. 2002; 282: E1180-E1190Crossref PubMed Scopus (174) Google Scholar). Each cDNA probe labeled with [α-32P]dCTP (PerkinElmer Life Sciences) with a labeling kit (Takara, Shiga, Japan) was hybridized to UV cross-linked blots overnight at 68 °C in the hybridization buffer Perfecthyb (Toyobo, Tokyo) and washed at 68 °C for 40 min with 1× SSC, 0.1% SDS. The blots were exposed to Kodak Biomax MR film at –80 °C. The signal was quantified with a densitometer, and loading differences were normalized to the signal generated with a probe for 18 S ribosomal RNA. RNase Protection Assay—SREBP-1 mRNA is produced from a single gene through the use of alternative transcription start sites that produce alternate forms of exon 1, designated 1a and 1c (1Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Investig. 2002; 109: 1125-1131Crossref PubMed Scopus (3838) Google Scholar). Thus, the amount of SREBP-1a and SREBP-1c mRNA was assessed by RNase protection assay according to the method of Shimomura et al. (16Shimomura I. Bashmakov Y. Ikemoto S. Horton J.D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13656-13661Crossref PubMed Scopus (628) Google Scholar). Aliquots of total RNA (10 μg) from each sample were incubated with the SREBP-1 cRNA probe or with a cRNA probe for the mRNA of β-actin. After digestion with RNase A/T1, protected fragments corresponding to SREBP-1a and SREBP-1c were quantified. Measurement of Luciferase Reporter Gene Activity—The 5′-flanking region of rat Srebp-1c was amplified by PCR using rat genomic DNA (17Deng X. Cagen L.M. Wilcox H.G. Park E.A. Raghow R. Elam M.B. Biochem. Biophys. Res. Commun. 2002; 290: 256-262Crossref PubMed Scopus (95) Google Scholar). The resultant PCR product was inserted upstream of the luciferase gene in the pGL3-basic reporter vector (Promega, Madison, WI) to form pGL3-SREBP-1c (0.4 kb). A fragment composed of –0.9 kb of a human SREBP-1a gene promoter was made by PCR using primer (18Miserez A.R. Cao G. Probst L.C. Hobbs H.H. Genomics. 1997; 40: 31-40Crossref PubMed Scopus (90) Google Scholar) and inserted upstream of the luciferase gene in the pGL3-basic reporter vector to form pGL3-SREBP-1a (0.9 kb). For the luciferase reporter gene assay, transient transfection into Fao hepatoma cells was performed with SuperFECT in accordance with the manufacturer's instructions. Expression plasmids for WT, mutated PTP1B, small t antigen, or control vector without insert were cotransfected with the reporter plasmid and pRL-TK. Luciferase activity was assayed using the Dual-Luciferase Reporter Assay System as described (19Sekine O. Nishio Y. Egawa K. Nakamura T. Maegawa H. Kashiwagi A. J. Biol. Chem. 2002; 277: 36631-36639Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). PP2A Phosphatase Activity—Phosphatase activity was measured using para-nitrophenyl phosphate as a substrate as described (20Ugi S. Imamura T. Ricketts W. Olefsky J.M. Mol. Cell. Biol. 2002; 22: 2375-2387Crossref PubMed Scopus (84) Google Scholar). Briefly, starved rat hepatocytes were stimulated with 100 ng/ml insulin for 30 min at 37 °C and lysed. Clarified supernatants were incubated with anti-PP2A antibody and protein G-agarose for 2 h at 4 °C. The immunoprecipitates were washed and incubated with 900 μg/ml para-nitrophenyl phosphate for 30 min at 30 °C. The amount of para-nitrophenol produced was determined by measuring the absorbance at 405 nm. Electrophoretic Mobility Shift Assay (EMSA)—EMSA was performed using radiolabeling double-stranded oligonucleotide corresponding to the Sp1 consensus sequence (Promega). The protein-DNA binding reaction was performed at room temperature for 20 min in a volume of 20 μl. The reaction mixture contained 5 mg of nuclear protein extract, 1 mg of poly(dI:dC), 10 mm Hepes (pH 7.9), 60 mm KCl, 1 mm EDTA, 7% glycerol, and 100,000 cpm labeled probe. After the incubation, samples were loaded onto 5% polyacrylamide gels in 0.25 mm Tris borate-EDTA buffer and run at 150 V for 1.5 h. The gels were dried, and the bands were visualized by autoradiography. For competition assay, nonlabeled oligonucleotides were added at a 50-fold molar excess to the reaction mixture before the addition of nuclear extract protein as described (19Sekine O. Nishio Y. Egawa K. Nakamura T. Maegawa H. Kashiwagi A. J. Biol. Chem. 2002; 277: 36631-36639Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Generation of Mutant SREBP-1a and SREBP-1c Promoter—The putative Sp1 binding site in the proximal promoter region of human SREBP-1a gene was mutated using the oligonucleotides 5′-CTCTCTCAAGGTGttGCGGGGCCGTCTATCTG-3′ and 5′-GTCTATCTGCCGGGGCGttGTGGAACTCTCTC-3′ as primers in the in vitro mutagenesis reaction. The putative Sp1 binding site in the proximal promoter region of rat Srebp-1c gene was also mutated using the oligonucleotides 5′-CACCCGAGGGGCGGttCACGGGGGCGCTTC-3′ and 5′-CTTCGCGGGGGCACtt GGCGGGGAGCCCAC-3′ as primers. Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Statistical Analysis—Data are expressed as means ± S.E. of four separate experiments unless stated otherwise. Scheffe's multiple comparison test was used to determine the significance of any differences among more than three groups. p < 0.05 was considered significant. Coexistence of High Expression of PTP1B and SREBP-1 mRNA in the Liver of High Fructose-fed Rats—High fructose feeding for 8 weeks induced postprandial hyperinsulinemia and hypertriglyceridemia in rats as reported previously (6Nagai Y. Nishio Y. Nakamura T. Maegawa H. Kikkawa R. Kashiwagi A. Am. J. Physiol.. 2002; 282: E1180-E1190Crossref PubMed Scopus (174) Google Scholar). These rats also revealed several characteristic features of the insulin resistance/metabolic syndrome. Moreover, we found the coexistence of high expression of PTP1B and SREBP-1 mRNA in the liver (Fig. 1). Overexpression of PTP1B Enhances SREBP-1 mRNA but Impairs Insulin Signaling in Isolated Rat Hepatocytes—We next examined the effect of PTP1B overexpression on both SREBP-1 mRNA expression and insulin signaling in isolated hepatocytes by the adenovirus-mediated gene transfer technique. PTP1B overexpression markedly enhanced SREBP-1 mRNA expression to a level comparable with that seen after insulin stimulation alone. We did not observe any synergic effect of PTP1B on insulin-stimulated SREBP-1 mRNA expression. Furthermore, mRNA expression of fatty-acid synthase, one of the SREBP-1-responsive lipogenic genes, was also increased (Fig. 2a). On the other hand, overexpression of PTP1B led to decreased insulin-induced tyrosine phosphorylation of the insulin receptor and insulin receptor substrates 1 and 2 (IRS-1 and IRS-2), as shown in Fig. 2b. The insulin-stimulated phosphorylation of Akt and Erk was also attenuated in cells overexpressing PTP1B (Fig. 2b). Thus, PTP1B regulates "off"-signaling of insulin action in hepatocytes, as is also reported to occur in Fao hepatoma cells, L6 myocytes, and 3T3-L1 adipocytes (13Egawa K. Maegawa H. Shimizu S. Morino K. Nishio Y. Bryer-Ash M. Cheung A.T. Kolls J.K. Kikkawa R. Kashiwagi A. J. Biol. Chem. 2001; 276: 10207-10211Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 21Shimizu S. Maegawa H. Egawa K. Shi K. Bryer-Ash M. Kashiwagi A. Endocrinology. 2002; 143: 4563-4569Crossref PubMed Scopus (22) Google Scholar). Effect of PTP1B Overexpression on SREBP-1 Gene Expression in Rat Hepatocytes—Although insulin signaling was inhibited by overexpression of PTP1B, its overexpression markedly enhanced SREBP-1 mRNA expression. This PTP1B effect was mediated by tyrosine-phosphatase activity, because a phosphatase-defective PTP1B C/S mutant had no such effect (Fig. 3a). PTP1B overexpression enhanced SREBP-1 mRNA expression in a time- and dose-dependent manner (data not shown). Although insulin selectively increases SREBP-1c mRNA expression (16Shimomura I. Bashmakov Y. Ikemoto S. Horton J.D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13656-13661Crossref PubMed Scopus (628) Google Scholar), RNase protection assay revealed that PTP1B increased the mRNA contents of both SREBP-1a and -1c in rat hepatocytes (Fig. 3b). Consistently, PTP1B overexpression increased transcriptional activities of both SREBP-1a and SREBP-1c promoter-luciferase constructs, but a phosphatase-defective PTP1B C/S mutant was without effect (Fig. 3, c and d). These results indicate that tyrosine phosphatase activity is required for up-regulation of SREBP-1 expression. Identical findings were also observed in Fao hepatoma cells. PTP1B and Activation of SREBP-1 Expression in Rat Hepatocytes—To clarify the molecular mechanism for PTP1B-induced SREBP-1 gene expression, we selected as a candidate molecule, protein phosphatase 2A (PP2A), which is activated by dephosphorylation of the tyrosine residue and might regulate SREBP-1 gene expression, because catalytic A subunit of PP2A is inactivated by in vitro phosphorylation of Tyr307 by receptor tyrosine kinases (22Chen J. Parsons S. Brautigan D.L. J. Biol. Chem. 1994; 269: 7957-7962Abstract Full Text PDF PubMed Google Scholar). Thus, we hypothesized that PP2A would be activated by tyrosine dephosphorylation when PTP1B is overexpressed. In fact, we found that PTP1B overexpression enhanced PP2A activity in a dose-dependent manner, whereas a phosphatase-defective PTP1B C/S mutant had no such effect (Fig. 4a). Furthermore, we confirmed that overexpression of SV40 small t antigen (ST), a selective inhibitor of PP2A (15Sontag E. Fedorov S. Kamibayashi C. Robbins D. Cobb M. Mumby M. Cell. 1993; 75: 887-897Abstract Full Text PDF PubMed Scopus (461) Google Scholar, 20Ugi S. Imamura T. Ricketts W. Olefsky J.M. Mol. Cell. Biol. 2002; 22: 2375-2387Crossref PubMed Scopus (84) Google Scholar), inhibited PP2A activity in hepatocytes (Fig. 4a). Moreover, co-transfection of ST and PTP1B gene blocked the PTP1B-induced increase in SREBP-1 mRNA expression in hepatocytes (Fig. 4b). To further confirm the association of PP2A activity with SREBP-1 expression, we co-transfected ST and PTP1B genes into hepatocytes and found that the increases in PTP1B-induced SREBP-1a and -1c promoter activities were inhibited (Fig. 4, c and d). These results indicate that PTP1B enhances the activity of the SREBP-1 promoter through increased PP2A activity. PTP1B/PP2A Signaling Is Required for Activation of Sp1 Promoter Activities of SREBP-1 Gene—In EMSA, we found that PTP1B overexpression markedly increased nuclear Sp1 binding in both the basal and insulin-stimulated states (Fig. 5a). When the Sp1 binding sites in the SREBP-1a and -1c promoters were mutated from GCGGGG to GCGGTT by in vitro mutagenesis, we were unable to detect any stimulation of SREBP-1a and -1c promoter activities by PTP1B overexpression in hepatocytes (Fig. 5b). It is clear that these sites are important for the PTP1B effect on the regulation of the activities of SREBP-1a and -1c promoters (2Shimano H. Prog. Lipid Res. 2001; 40: 439-452Crossref PubMed Scopus (587) Google Scholar, 17Deng X. Cagen L.M. Wilcox H.G. Park E.A. Raghow R. Elam M.B. Biochem. Biophys. Res. Commun. 2002; 290: 256-262Crossref PubMed Scopus (95) Google Scholar, 18Miserez A.R. Cao G. Probst L.C. Hobbs H.H. Genomics. 1997; 40: 31-40Crossref PubMed Scopus (90) Google Scholar, 23Amemiya-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 (213) Google Scholar). Thus, we conclude that activated PP2A may enhance Sp1 transcriptional activity when PTP1B is overexpressed. These results indicate that PTP1B enhances the activity of the SREBP-1 promoter by modulating Sp1 transcriptional activity through increased PP2A activity. PTP1B is a major negative regulator of insulin signal transduction and is associated with the development of insulin resistance (13Egawa K. Maegawa H. Shimizu S. Morino K. Nishio Y. Bryer-Ash M. Cheung A.T. Kolls J.K. Kikkawa R. Kashiwagi A. J. Biol. Chem. 2001; 276: 10207-10211Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 21Shimizu S. Maegawa H. Egawa K. Shi K. Bryer-Ash M. Kashiwagi A. Endocrinology. 2002; 143: 4563-4569Crossref PubMed Scopus (22) Google Scholar, 24Maegawa H. Ide R. Hasegawa M. Ugi S. Egawa K. Iwanishi M. Kikkawa R. Shigeta Y. Kashiwagi A. J. Biol. Chem. 1995; 270: 7724-7730Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Furthermore, we and others have observed that hyperinsulinemia as well as increased carbohydrate intake stimulate its gene expression (6Nagai Y. Nishio Y. Nakamura T. Maegawa H. Kikkawa R. Kashiwagi A. Am. J. Physiol.. 2002; 282: E1180-E1190Crossref PubMed Scopus (174) Google Scholar, 24Maegawa H. Ide R. Hasegawa M. Ugi S. Egawa K. Iwanishi M. Kikkawa R. Shigeta Y. Kashiwagi A. J. Biol. Chem. 1995; 270: 7724-7730Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 25Kenner K.A. Anyanwu E. Olefsky J.M. Kusari J. J. Biol. Chem. 1996; 271: 19810-19816Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 26Obata T. Maegawa H. Kashiwagi A. Pillay T.S. Kikkawa R. J Biochem. (Tokyo). 1998; 123: 813-820Crossref PubMed Scopus (22) Google Scholar). Moreover, we observed that hepatic PTP1B content was increased by 3-fold in high fructose-fed rats and that exposure of isolated hepatocytes to 25 mm fructose for 8 h led to doubled PTP1B mRNA content (data not shown). Thus, a vicious metabolic cycle may be initiated, in which PTP1B plays a key role. A mouse model lacking PTP1B consistently exhibited augmented insulin sensitivity and was resistant to the development of obesity induced by high fat feeding (10Elchebly M. Payette P. Michaliszyn E. Cromlish W. Collins S. Loy A.L. Normandin D. Cheng A. Himms-Hagen J. Chan C.C. Ramachandran C. Gresser M.J. Tremblay M.L. Kennedy B.P. Science. 1999; 283: 1544-1548Crossref PubMed Scopus (1926) Google Scholar, 11Klaman L.D. Boss O. Peroni O.D. Kim J.K. Martino J.L. Zabolotny J.M. Moghal N. Lubkin M. Kim Y.B. Sharpe A.H. Stricker-Krongrad A. Shulman G.I. Neel B.G. Kahn B.B. Mol. Cell. Biol. 2000; 20: 5479-5489Crossref PubMed Scopus (1130) Google Scholar). Therefore, PTP1B is postulated to be an important therapeutic target to reduce insulin resistance (9Johnson T.O. Ermolieff J. Jirousek M.R. Nat. Rev. Drug. Discov. 2002; 1: 696-709Crossref PubMed Scopus (567) Google Scholar) as well as resistance to diet-induced obesity. Postprandial (diet-induced) hypertriglyceridemia is thought to be a central feature of the insulin resistance/metabolic syndrome, and enhanced expression of SREBP-1c mRNA is responsible in part for these abnormalities (1Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Investig. 2002; 109: 1125-1131Crossref PubMed Scopus (3838) Google Scholar, 2Shimano H. Prog. Lipid Res. 2001; 40: 439-452Crossref PubMed Scopus (587) Google Scholar). In insulinresistant fatless mice, IRS-2 protein has been shown to be diminished, but SREBP-1 expression is continuously activated (4Shimomura I. Matsuda M. Hammer R.E. Bashmakov Y. Brown M.S. Goldstein J.L. Mol. Cell. 2000; 6: 77-86Abstract Full Text Full Text PDF PubMed Scopus (708) Google Scholar). Thus, despite the presence of insulin resistance in both the peripheral tissues and liver in these mice, the compensatory hyperinsulinemia maintains a high level of SREBP-1c gene transcription (3Shimomura I. Bashmakov Y. Horton J.D. J. Biol. Chem. 1999; 274: 30028-30032Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar, 4Shimomura I. Matsuda M. Hammer R.E. Bashmakov Y. Brown M.S. Goldstein J.L. Mol. Cell. 2000; 6: 77-86Abstract Full Text Full Text PDF PubMed Scopus (708) Google Scholar, 5Tobe K. Suzuki R. Aoyama M. Yamauchi T. Kamon J. Kubota N. Terauchi Y. Matsui J. Akanuma Y. Kimura S. Tanaka J. Abe M. Ohsumi J. Nagai R. Kadowaki T. J. Biol. Chem. 2001; 276: 38337-38340Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Therefore, there may be another mechanism for the continued strong expression of hepatic SREBP-1c mRNA in insulin-resistant mice. We found coexistent strong expression of PTP1B and SREBP-1 in rats that were insulin-resistant by a high fructose diet. Overexpression of PTP1B protein has been also observed in insulin-resistant db/db mice, and treatment with antisense PTP1B in db/db mice has been reported to improve hyperglycemia and hyperinsulinemia (27Zinker B.A. Rondinone C.M. Trevillyan J.M. Gum R.J. Clampit J.E. Waring J.F. Xie N. Wilcox D. Jacobson P. Frost L. Kroeger P.E. Reilly R.M. Koterski S. Opgenorth T.J. Ulrich R.G. Crosby S. Butler M. Murray S.F. McKay R.A. Bhanot S. Monia B.P. Jirousek M.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11357-11362Crossref PubMed Scopus (404) Google Scholar). Furthermore, this antisense treatment in ob/ob mice also ameliorates adiposity and attenuates increased lipogenic gene expression in adipose tissue (28Rondinone C.M. Trevillyan J.M. Clampit J. Gum R.J. Berg C. Kroeger P. Frost L. Zinker B.A. Reilly R. Ulrich R. Butler M. Monia B.P. Jirousek M.R. Waring J.F. Diabetes. 2002; 51: 2405-2411Crossref PubMed Scopus (150) Google Scholar). However, in the study, it is unclear whether the effect of PTP1B inhibition on SREBP-1 expression was mediated by a direct interaction or mediated by improvements of hyperinsulinemia and hyperglycemia. We hypothesized that PTP1B might directly regulate SREBP-1 gene expression and modulate hepatic lipogenesis in insulin-resistant states. In the current study, we found that overexpression of PTP1B directly enhanced SREBP-1 gene expression, revealing a new aspect of PTP1B action. Furthermore, we propose a unique new molecular mechanism for the activation of SREBP-1 gene expression through activation of PP2A by PTP1B overexpression. Phosphorylation of the tyrosine residue of PP2A was found to decrease its enzymatic activity in an in vitro experiment (22Chen J. Parsons S. Brautigan D.L. J. Biol. Chem. 1994; 269: 7957-7962Abstract Full Text PDF PubMed Google Scholar), suggesting that dephosphorylation of PP2A may stimulate its phosphatase activity. In the present study, we have demonstrated that PTP1B increases PP2A activity, resulting in increased activity of the SREBP-1 promoter. Furthermore, overexpression of ST, a specific inhibitor of PP2A, blocks the effect of PTP1B on SREBP-1 gene expression by inhibiting PP2A activity. These results indicate that PTP1B activates SREBP-1 gene expression via increased PP2A activity. The Sp1 sites are located at about the –90 bp region of the SREBP-1a and -1c promoters and are thought to be important sites in the regulation of its gene expression (2Shimano H. Prog. Lipid Res. 2001; 40: 439-452Crossref PubMed Scopus (587) Google Scholar, 17Deng X. Cagen L.M. Wilcox H.G. Park E.A. Raghow R. Elam M.B. Biochem. Biophys. Res. Commun. 2002; 290: 256-262Crossref PubMed Scopus (95) Google Scholar, 18Miserez A.R. Cao G. Probst L.C. Hobbs H.H. Genomics. 1997; 40: 31-40Crossref PubMed Scopus (90) Google Scholar, 23Amemiya-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 (213) Google Scholar). In the current study, a defective mutation of the Sp1 binding sites in SREBP-1a and -1c promoters led to the cancellation of PTP1B effect on its promoter activity (Fig. 5b). These results indicate that the Sp1 site is important for the PTP1B effect on SREBP-1 gene expression. With regard to the regulation of Sp1 transcriptional activity (29Black A.R. Black J.D. Azizkhan-Clifford J. J. Cell. Physiol. 2001; 188: 143-160Crossref PubMed Scopus (904) Google Scholar, 30Samson S.L. Wong N.C. J. Mol. Endocrinol. 2002; 29: 265-279Crossref PubMed Scopus (137) Google Scholar), increased Sp1 glycosylation is responsible for increased gene expression in hyperglycemic condition by an enhanced hexosamine pathway (31Du X.L. Edelstein D. Rossetti L. Fantus I.G. Goldberg H. Ziyadeh F. Wu J. Brownlee M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12222-12226Crossref PubMed Scopus (900) Google Scholar). However, we did not observe increased glycosylation of Sp1 protein in PTP1B-overexpressed cells (data not shown). PP2A is known to activate Sp1 transcriptional activity in nonhepatic cultured cells (32Lacroix I. Lipcey C. Imbert J. Kahn-Perles B. J. Biol. Chem. 2002; 277: 9598-9605Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). SREBP-1 promoter activities were well correlated with PP2A activities. Thus, it is possible that PTP1B may activate SREBP-1 gene expression by enhancing Sp1 transcriptional activity via increased PP2A activity. It has been reported that PP2A dephosphorylates Sp1 protein and increases DNA binding. However, the dephosphorylation site(s) of Sp1 by PP2A has not been determined (29Black A.R. Black J.D. Azizkhan-Clifford J. J. Cell. Physiol. 2001; 188: 143-160Crossref PubMed Scopus (904) Google Scholar, 30Samson S.L. Wong N.C. J. Mol. Endocrinol. 2002; 29: 265-279Crossref PubMed Scopus (137) Google Scholar). In the current study, we did not detect any significant changes in phosphorylation levels of Sp1 protein in cells overexpressing PTP1B (data not shown), although subtle change might not have been detectable by the methods used. Thus, the dephosphorylation site(s) by PP2A needs to be determined in further studies. We propose that the molecular mechanism for the activation of SREBP-1 gene expression by activation of Sp1 transcription factor is through activation of PP2A. We demonstrate here a unique serial activation of the PTP1B-PP2A axis as a novel mechanism for the regulation of gene expression in the biosynthesis of triglyceride. Therefore, PTP1B is a crucial molecule in the pathogenesis of postprandial hypertriglyceridemia and the insulin resistance/metabolic syndrome (Fig. 6). Further in vivo study is necessary to confirm the in vitro findings and to clarify the clinical significance of PTP1B roles in insulin-resistant states. In summary, we demonstrate here that another important action of PTP1B stimulates SREBP-1 gene expression, hepatic triglyceride synthesis, and postprandial hypertriglyceridemia, even though PTP1B is postulated to be an important target to reduce resistance to insulin and leptin in obesity and other insulin-resistant states (9Johnson T.O. Ermolieff J. Jirousek M.R. Nat. Rev. Drug. Discov. 2002; 1: 696-709Crossref PubMed Scopus (567) Google Scholar). We therefore propose that PTP1B represents a novel therapeutic target for the amelioration of postprandial (diet-induced) hypertriglyceridemia through modulation of SREBP-1a and -1c expression in the liver by activation of the PTP1B-PP2A axis. We are grateful to Dr. C. R. Kahn (Joslin Diabetes Center) and Dr. M. Mumby (University of Texas Southwestern Medical Center) for providing Fao cells and ST vector. We are grateful to Dr. I. Shimomura (Osaka University) for helpful discussion.

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