An mRNA Splice Variant of the AFX Gene with Altered Transcriptional Activity
2002; Elsevier BV; Volume: 277; Issue: 10 Linguagem: Inglês
10.1074/jbc.m106091200
ISSN1083-351X
AutoresZhenyu Yang, James Whelan, Robert Babb, Benjamin R. Bowen,
Tópico(s)Pancreatic function and diabetes
ResumoSeveral studies indicate that FKHR and AFX, mammalian homologues of the Caenorhabditis elegans forkhead transcription factor DAF-16, function in the insulin signaling pathway. Here we describe the discovery of a novel AFX isoform, which we designated AFXζ, in which the first 16 amino acids of the forkhead domain are not present. PCR analysis showed that this isoform is most abundant in the liver, kidney, and pancreas. In HepG2 cells, overexpressed AFXζ induced reporter gene activity through the insulin-responsive sequences of the phosphoenolpyruvate carboxykinase (PEPCK), IGFBP-1, andG6Pase promoters. AFXζ−mediated stimulation was repressed by insulin treatment, by bisperoxovanadate treatment, and by overexpression of constitutively active protein kinase B (PKB). Insulin treatment and PKB overexpression resulted in phosphorylation of AFXζ. Furthermore, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), an AMP-activated protein kinase activator, repressed AFXζ-dependent reporter activation. Taken together, these findings suggest that AFXζ is a downstream target of both the phosphatidylinositol 3-kinase/PKB insulin signaling pathway and an AMP-activated protein kinase-dependent pathway. Several studies indicate that FKHR and AFX, mammalian homologues of the Caenorhabditis elegans forkhead transcription factor DAF-16, function in the insulin signaling pathway. Here we describe the discovery of a novel AFX isoform, which we designated AFXζ, in which the first 16 amino acids of the forkhead domain are not present. PCR analysis showed that this isoform is most abundant in the liver, kidney, and pancreas. In HepG2 cells, overexpressed AFXζ induced reporter gene activity through the insulin-responsive sequences of the phosphoenolpyruvate carboxykinase (PEPCK), IGFBP-1, andG6Pase promoters. AFXζ−mediated stimulation was repressed by insulin treatment, by bisperoxovanadate treatment, and by overexpression of constitutively active protein kinase B (PKB). Insulin treatment and PKB overexpression resulted in phosphorylation of AFXζ. Furthermore, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), an AMP-activated protein kinase activator, repressed AFXζ-dependent reporter activation. Taken together, these findings suggest that AFXζ is a downstream target of both the phosphatidylinositol 3-kinase/PKB insulin signaling pathway and an AMP-activated protein kinase-dependent pathway. phosphoenolpyruvate carboxykinase insulin-like growth factor-binding protein insulin-responsive sequence(s) phosphatidylinositol 3-kinase protein kinase B glucose-6-phosphatase AMP-activated protein kinase potassium bisperoxo(1,10-phenanthroline)oxovanadate 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside secreted alkaline phosphatase PEPCK promoter myristate An important role of insulin in glucose homeostasis is regulating the transcription of genes critical in glucose metabolism (1O'Brien R.M. Granner D.K. Physiol. Rev. 1996; 76: 1109-1161Crossref PubMed Scopus (439) Google Scholar). For example, insulin inhibits the expression of genes such asphosphoenolpyruvatecarboxykinase (PEPCK)1 (2Sasaki K. Cripe T.P. Koch S.R. Andreone T.L. Petersen D.D. Beale E.G. Granner D.K. J. Biol. 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Diabetes. 1999; 48: 1885-1889Crossref PubMed Scopus (100) Google Scholar). Proteins involved in the insulin signaling pathway include the insulin receptor, phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB), and downstream transcription factors. Genetic studies suggest that an insulin-like signaling pathway exists in Caenorhabditis elegans. In the nematode, the pathway is composed of DAF-2, AGE-1, and AKT1/AKT2, which are considered the orthologs of mammalian insulin receptor (12Kimura K.D. Tissenbaum H.A. Liu Y. Ruvkun G. Science. 1997; 277: 942-946Crossref PubMed Scopus (1742) Google Scholar), PI3K (13Morris J.Z. Tissenbaum H.A. Ruvkun G. Nature. 1996; 382: 536-539Crossref PubMed Scopus (706) Google Scholar), and PKB (14Paradis S. Ruvkun G. Genes Dev. 1998; 12: 2488-2498Crossref PubMed Scopus (558) Google Scholar), respectively. Together, the products of these genes negatively regulate the activity of DAF-16 (15Ogg S. Paradis S. Gottlieb S. Patterson G.I. Lee L. Tissenbaum H.A. Ruvkun G. Nature. 1997; 389: 994-999Crossref PubMed Scopus (1551) Google Scholar), a forkhead transcription factor that binds IRSs (16Lin K. Dorman J.B. Rodan A. Kenyon C. Science. 1997; 278: 1319-1322Crossref PubMed Scopus (1214) Google Scholar). Because the major target of DAF-2/AGE-1 signaling in C. elegans is DAF-16 (14Paradis S. Ruvkun G. Genes Dev. 1998; 12: 2488-2498Crossref PubMed Scopus (558) Google Scholar, 17Gottlieb S. Ruvkun G. Genetics. 1994; 137: 107-120Crossref PubMed Google Scholar), the orthologs of DAF-16 may represent distal effectors of insulin signaling in mammalian cells (12Kimura K.D. Tissenbaum H.A. Liu Y. Ruvkun G. Science. 1997; 277: 942-946Crossref PubMed Scopus (1742) Google Scholar, 15Ogg S. Paradis S. Gottlieb S. Patterson G.I. Lee L. Tissenbaum H.A. Ruvkun G. Nature. 1997; 389: 994-999Crossref PubMed Scopus (1551) Google Scholar, 18Durham S.K. Suwanichkul A. Scheimann A.O. Yee D. Jackson J.G. Barr F.G. Powell D.R. Endocrinology. 1999; 140: 3140-3146Crossref PubMed Scopus (133) Google Scholar). The DAF-16 forkhead domain is most similar to those of human FKHR (67% identities) and AFX proteins (64% identities) (15Ogg S. Paradis S. Gottlieb S. Patterson G.I. Lee L. Tissenbaum H.A. Ruvkun G. Nature. 1997; 389: 994-999Crossref PubMed Scopus (1551) Google Scholar, 16Lin K. Dorman J.B. Rodan A. Kenyon C. Science. 1997; 278: 1319-1322Crossref PubMed Scopus (1214) Google Scholar). Therefore, human forkhead factors FKHR and/or AFX may be downstream targets of the insulin-activated PI3K-PKB signaling pathway and also may be responsible for mediating insulin regulation of gene expression (12Kimura K.D. Tissenbaum H.A. Liu Y. Ruvkun G. Science. 1997; 277: 942-946Crossref PubMed Scopus (1742) Google Scholar,14Paradis S. Ruvkun G. Genes Dev. 1998; 12: 2488-2498Crossref PubMed Scopus (558) Google Scholar, 15Ogg S. Paradis S. Gottlieb S. Patterson G.I. Lee L. Tissenbaum H.A. Ruvkun G. Nature. 1997; 389: 994-999Crossref PubMed Scopus (1551) Google Scholar, 16Lin K. Dorman J.B. Rodan A. Kenyon C. Science. 1997; 278: 1319-1322Crossref PubMed Scopus (1214) Google Scholar). AFX was originally identified on chromosome X as an oncogenic fusion protein in acute lymphoblastic leukemia (19Parry P. Wei Y. Evans G. Genes Chromosomes Cancer. 1994; 11: 79-84Crossref PubMed Scopus (139) Google Scholar, 20Corral J. Forster A. Thompson S. Lampert F. Kaneko Y. Slater R. Kroes W.G. van der Schoot C.E. Ludwig W.D. Karpas A. et al.Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8538-8542Crossref PubMed Scopus (156) Google Scholar) and is involved in the regulation of the cell-cycle (21Medema R.H. Kops G.J. Bos J.L. Burgering B.M. Nature. 2000; 404: 782-787Crossref PubMed Scopus (1231) Google Scholar, 22Collado M. Medema R.H. Garcia-Cao I. Dubuisson M.L. Barradas M. Glassford J. Rivas C. Burgering B.M. Serrano M. Lam E.W. J. Biol. Chem. 2000; 275: 21960-21968Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar), apoptosis (23Takaishi H. Konishi H. Matsuzaki H. Ono Y. Shirai Y. Saito N. Kitamura T. Ogawa W. Kasuga M. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11836-11841Crossref PubMed Scopus (217) Google Scholar), and tumorigenesis (24Graff J.R. Konicek B.W. McNulty A.M. Wang Z. Houck K. Allen S. Paul J.D. Hbaiu A. Goode R.G. Sandusky G.E. Vessella R.L. Neubauer B.L. J. Biol. Chem. 2000; 275: 24500-24505Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). The gene for AFX consists of three exons and is reported to encode a protein of 501 amino acids (25Borkhardt A. Repp R. Haas O.A. Leis T. Harbott J. Kreuder J. Hammermann J. Henn T. Lampert F. Oncogene. 1997; 14: 195-202Crossref PubMed Scopus (213) Google Scholar). It is expressed ubiquitously with high levels in placenta and skeletal muscle (19Parry P. Wei Y. Evans G. Genes Chromosomes Cancer. 1994; 11: 79-84Crossref PubMed Scopus (139) Google Scholar). AFX binds the IRS element from IGFBP-1 and induces a pronounced increase in the activity of a reporter gene under the control of theIGFBP-1 promoter. This transcriptional activation requires an intact IRS, and insulin treatment suppresses the activation through the PI3K-PKB signaling pathway (26Kops G.J. De de Ruiter N.D. Vries-Smits A.M. Powell D.R. Bos J.L. Burgering B.M. Nature. 1999; 398: 630-634Crossref PubMed Scopus (953) Google Scholar). Indeed, AFX contains three putative PKB phosphorylation sites and can be phosphorylated by PKB both in vitro and in vivo (23Takaishi H. Konishi H. Matsuzaki H. Ono Y. Shirai Y. Saito N. Kitamura T. Ogawa W. Kasuga M. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11836-11841Crossref PubMed Scopus (217) Google Scholar, 26Kops G.J. De de Ruiter N.D. Vries-Smits A.M. Powell D.R. Bos J.L. Burgering B.M. Nature. 1999; 398: 630-634Crossref PubMed Scopus (953) Google Scholar). Phosphorylation by PKB alters the nuclear import of AFX, shifting its localization from the nucleus to the cytoplasm and thereby inhibiting AFX transcriptional activity (27Brownawell A.M. Kops G.J. Macara I.G. Burgering B.M. Mol. Cell. Biol. 2001; 21: 3534-3546Crossref PubMed Scopus (269) Google Scholar). The PI3K-PKB pathway is an important but not unique route by which the activity of forkhead transcription factors are regulated (28Kops G.J. Burgering B.M. J. Mol. Med. 1999; 77: 656-665Crossref PubMed Scopus (253) Google Scholar, 29Tomizawa M. Kumar A. Perrot V. Nakae J. Accili D. Rechler M.M. Kumaro A. J. Biol. Chem. 2000; 275: 7289-7295Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). A PI3K inhibitor or a dominant-negative PKB mutant decreases but does not abolish insulin-induced phosphorylation of AFX (26Kops G.J. De de Ruiter N.D. Vries-Smits A.M. Powell D.R. Bos J.L. Burgering B.M. Nature. 1999; 398: 630-634Crossref PubMed Scopus (953) Google Scholar), suggesting the existence of an alternate signaling pathway. A candidate for this alternative pathway is the mammalian AMP-activated protein kinase (AMPK) cascade. AMPK influences many metabolic processes that become dysregulated in the diabetic state (30Winder W.W. Hardie D.G. Am. J. Physiol. 1999; 277: E1-E10PubMed Google Scholar). However, the mechanism by which AMPK modulates transcriptional activity is unknown due to the fact that the transcription factor(s) involved remains unidentified. In this study, we investigated the involvement of AFX in the mammalian insulin signaling pathway. We discovered a novel AFX isoform, AFXζ, characterized its tissue distribution, DNA binding ability, and transcriptional activity. Reporter gene assays demonstrated that AFXζ is a potent transcription activator with properties distinct from those of the previously described isoform, AFXα, and that AFXζ is regulated by the insulin signaling pathway and by an agent known to affect AMPK activity. AFXζ may represent an integration point of the insulin signaling and AMPK pathways, allowing these pathways to regulate downstream gene expression cooperatively. Oligonucleotides for AFX amplification were based on the sequence in GenBankTM accession number X93996. All primers were synthesized by Life Technologies, Inc. The sequences added to facilitate cloning are represented in lowercase: 5–1: 5′-GAAGACTGGCAGGAATGTGCCTCCTGG-3′; 3–1: 5′-CGCCTGGCTCCACATCTGAAGCAGG-3′; 5–2: 5′-gacgtcgacctATGGATCCGGGGAATGAG-3′; 3–2: 5′-gacgtcgacTCAGGGATCTGGCTCAAAG-3′; 5–3: 5′-CTGTGGCAGGCTTCACTGAAC-3′; and 3–3: 5′-GCAAGTGTCAGTCGCTTCTC-3′. AFX cDNA was amplified using primers 5–1 and 3–1 from Marathon-Ready human liver and heart cDNA libraries (CLONTECH). Nested primers 5–2 and 3–2 were used to perform a second round PCR. Conditions for the PCR reactions were: 94 °C for 1 min; 94 °C for 15 s, 68 °C for 2 min, repeat for 30 cycles; and 68 °C for 3 min and dwell at 15οC. The PCR products were digested withSalI and cloned into the XhoI site of pcDNA6/His A (Invitrogen, Carlsbad, CA) and separately into theSalI site of pET-30a (Novagen, Madison, WI). The sequences of the constructs were confirmed using dRhodamine Terminator Cycle Sequencing Kits on an ABI 377 machine (Applied Biosystems). Human genomic DNA from four individuals were kindly provided by Dr. Chack Yung Yu (Columbus, Ohio). Human MTC cDNA panels I and II were purchased from CLONTECH. The Expand High Fidelity PCR System (Roche Molecular Biochemicals) was used to perform PCR amplifications with primers 5–3 and 3–3. PCR products were separated on 2% agarose gels. DNA was stained with Vistra Green and was visualized on the Storm FluorImager system (Molecular Dynamics, Sunnyvale, CA). The sequences of genomic PCR products were analyzed using programs in the GCG package. His-tagged AFXζ was expressed in an Escherichia coli BL21 strain after induction by 0.4 mmisopropyl-1-thio-β-d-galactopyranoside at 30 °C for 4 h. The bacterial fusion protein was purified using TALON metal affinity columns (CLONTECH) according to the manufacturer's instructions. AFXα and AFXζ proteins were translated in vitro from pcDNA6/His-AFXα and pcDNA6/His-AFXζ templates, respectively. The TNT T7 quick coupled transcription/translation system (Promega, Madison, WI) was utilized according to the manufacturer's protocol. One microgram of the expression construct was used in each reaction. The DNA binding abilities of in vitro translated AFXα, AFXζ and purified bacterial fusion protein His-tagged AFXζ were tested in a gel shift assay system (Promega). IRS elements from PEPCK,IGFBP1, and G6Pase promoters were used as probes. Briefly, oligonucleotides for the sense and antisense strand of each IRS were annealed, and double-stranded fragments were end-labeled using T4 polynucleotide kinase. The manufacturer's protocol was followed except that [γ-33P]ATP (NEN Life Science Products, Boston, MA) was used in substitution of the suggested [γ-32P]ATP to label the DNA fragment. About 2 × 104-cpm probe was used in each binding reaction. For competition experiments, a 50 molar excess of each corresponding unlabeled oligonucleotide was added to the reaction. One hundred nanograms of in vitro translated AFX protein or 200 ng of purified His-tagged AFXζ was used in each binding reaction. The DNA-protein complexes were separated on 6% DNA retardation gel (Invitrogen). Gels were dried prior to autoradiography. Double-stranded DNA fragments containing each of the IRS elements from the promoters ofPEPCK, IGFBP-1, and G6Pase were cloned into the NheI/BglII site of the pSEAP2-promoter vector (CLONTECH). Dissected fragments of thePEPCK and IGFBP-1 IRS elements, referred to asPEPCK-a and PEPCK-b, and IGFBP-1a andIGFBP-1b, respectively, were cloned into the same site of the pSEAP2-promoter. In addition, a 501-bp fragment corresponding to the human PEPCK 5′ regulatory sequence (−458–+43 relative to the transcription start site, abbreviated as PEPCK p) was cloned into the BglII/EcoRI site of the same vector. The human hepatoma cell line HepG2 was cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (Life Technologies). Transient transfection experiments were performed in Dulbecco's modified Eagle's medium supplemented with 10% dialyzed fetal bovine serum (HyClone Laboratories) in 24-well plates. HepG2 cells were transfected using LipofectAMINE 2000 (Life Technologies) with 0.2 μg (unless otherwise specified) of an appropriate SEAP reporter construct, CMV-β, and either pcDNA6/His-AFXα or -AFXζ expression plasmid. Each set of reporter assays includes a transfection in which no AFX expression construct was added. In experiments where PKB effects were tested, 0.2 μg of myr-Akt1 in pUSEamp(+) (Upstate Biotechnology, Lake Placid, NY), which expresses constitutively activated PKB, was included. For experiments in which the cells were treated with soluble agents, the medium was changed 24 h after the transfection and was replaced with medium containing appropriate concentrations of bpV (potassium bisperoxo(1,10-phenanthroline) oxovanadate, Alexis Biochemicals, San Diego, CA), porcine insulin, or AICAR (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside, Sigma) for another 15–18 h. Secreted alkaline phosphatase (SEAP) and β-galactosidase activities were measured using Phospha-Light and Galacto-Star systems (Tropix Inc., Bedford, MA), respectively. HepG2 cells were grown in 100-mm dishes and were transfected with 4 μg of pCDNA6/His-AFXζ and/or myr-Akt1 or were treated for 12 h with insulin (100 nm). Cells were washed with phosphate-buffered saline, were removed gently from the plate by scraping, and were pelleted by centrifugation. Cells were lysed for 1 min on ice in 0.2 ml of mild extraction buffer (10 mmHEPES, pH 8, 1.5 mm MgCl2, 10 mmKCl, 5 mm dithiothreitol, 0.5% Nonidet P-40, complete protease inhibitor mixture (Boehringer), and 1 mm sodium orthovanadate.) The lysate was transferred to a microcentrifuge tube and was spun for 3 min on setting 3 in a Eppendorf 5415C Microfuge. The resulting supernatant was collected and labeled the cytosolic fraction, and the residual nuclear pellet was resuspended in 0.1 ml of nuclear extraction buffer (10 mm HEPES, pH 8, 25% glycerol, 420 mm NaCl, 0.2 mm EDTA, complete protease inhibitor mixture, and 1 mm sodium orthovanadate) and placed on ice for 15 min. The tubes subsequently were spun for 10 min in a microcentrifuge at maximum speed, and the resulting nuclear extract was transferred to a new tube for storage. Protein concentrations were determined using the DC Protein Assay (Bio-Rad, Hercules, CA). For Western blotting, 20 μg of cellular extract was subjected to SDS-PAGE with subsequent blotting to nitrocellulose. AFXζ and phospho-AFXζ proteins were detected using an AFX antibody (Santa Cruz Biotechnology sc5224) or a phospho-AFX (Ser-193) antibody (NEB 9471) according to the manufacturers' recommendations. We amplified AFX cDNAs by PCR from a human liver cDNA library using a primer set based on GenBankTM sequence X93996 (25Borkhardt A. Repp R. Haas O.A. Leis T. Harbott J. Kreuder J. Hammermann J. Henn T. Lampert F. Oncogene. 1997; 14: 195-202Crossref PubMed Scopus (213) Google Scholar). In all cloned amplicons, a single A nucleotide was found to be absent 20 nucleotides downstream of the previously designated initiation codon (Fig.1 B). This same “deletion” was also identified in PCR products using human heart cDNA library as DNA template. Using primers 5–3 and 3–3, we amplified the identical sequence from four independent human genomic DNA samples. Further confirmation for the absence of this A nucleotide comes from GenBankTM sequence Y11284, the gene sequence of human AFX exon 1. Correction of the sequence leads to slight changes of the predicted amino acid sequence of the protein N terminus as the initiation codon is shifted upstream (Fig. 1 B, sequence 2) to the most likely initiation codon as predicted by the ATGpr program (31Salamov A.A. Nishikawa T. Swindells M.B. Bioinformatics. 1998; 14: 384-390Crossref PubMed Scopus (141) Google Scholar). The coding region of human AFX thus encodes 505 amino acids (Fig. 1 C). The predicted forkhead domain is located at amino acids 97–184 of the revised sequence. Further analyses of the cloned AFX cDNAs revealed the presence of a shorter splice variant that encodes a shorter protein lacking amino acids 58–112 (Fig.1 C); we designate this alternate form as AFXζ and will refer to the longer form as AFXα. As shown in Fig. 1 A, the termini of the 165-bp cDNA region that is not present in AFXζ conform to the consensus sequences of splice junctions, suggesting that this deletion represents a cryptic intron. The amino acids previously identified as PKB phosphorylation sites in AFXα are retained in AFXζ as Thr-32, Ser-142, and Ser-207, but the Myb DNA binding homology domain of AFXα is absent in AFXζ. To investigate the tissue distribution of the AFXζ alternate splice form, PCR reactions were performed on first-strand cDNAs from different tissues using primer set 5–3 and 3–3 (Fig. 1 D) that generates a 424-bp product for AFXα and a 259-bp product for AFXζ. Among PCR amplifications from 16 different tissues, the 424-bp fragment representing AFXα was ubiquitous, while the 259-bp fragment from AFXζ was more tissue-specific. The greatest expression of AFXζ was observed in liver, kidney, and pancreas RNA (lanes 6,8 and 9). It was also readily detectable in heart and placenta (lanes 2 and 4). Lung, skeletal muscle, spleen, thymus, and small intestine expressed AFXζ at lower levels (lanes 5, 7, 10, 11, and 15). AFXζ transcripts were not detected in RNA samples from brain, prostate, testis, ovary, colon, and leukocyte (lanes 3, 12, 13, 14, 16, and17). Because part of the forkhead DNA binding domain (amino acids 97–184 in AFXα, Fig. 1 C) is absent in AFXζ, we examined whether AFXζ protein can bind DNA fragments that harbor IRSs. In electrophoretic mobility shift assays, a His-tagged AFXζ bacterial fusion protein bound the IRS elements from the PEPCK (Fig. 2 B,lane 2), IGFBP1 (lane 5) andG6Pase (lane 8) promoters. A single DNA-protein complex was detected in each experiment. Corresponding unlabeled DNA fragments competed for binding of the probes (lanes 3,6, and 9). In vitro translated proteins for AFXζ (Fig. 2 C) and AFXα (Fig.2 D) were also used as the protein sources for gel shift assays, and a variety of shifted bands were observed. Multiple DNA-protein complexes have been shown previously in the binding of GST-AFXα and Trx-AFXα to an IRS within the IGFBP-1promoter (26Kops G.J. De de Ruiter N.D. Vries-Smits A.M. Powell D.R. Bos J.L. Burgering B.M. Nature. 1999; 398: 630-634Crossref PubMed Scopus (953) Google Scholar, 32Furuyama T. Nakazawa T. Nakano I. Mori N. Biochem. J. 2000; 349: 629-634Crossref PubMed Scopus (558) Google Scholar). Therefore, AFXζ protein binds IRS fragments even though it lacks the first 16 amino acids of the forkhead domain as identified in AFXα. To test the transcriptional activity of AFXα and AFXζ, cellular assay systems were developed. Reporter constructs were generated by inserting the fragments spanning the IRS elements of the PEPCK,IGFBP-1, and G6Pase promoters (Fig.2 A) or an expanded DNA fragment of the PEPCK p (−458–+43) regulatory sequence into the pSEAP2-promoter vector. First, we determined the transcriptional role of AFXζ on the expression of a reporter driven by the PEPCK IRS (−425–−399) and the full-length promoter (−458–+43) ofPEPCK (PEPCK p). Fig.3 A shows that in HepG2 cells, AFXζ activated both PEPCK IRS and PEPCKp-enhanced reporter transcription in a dose-dependent manner and to a similar extent, suggesting that transactivation at this single IRS accounts for the entire stimulation. We next compared the ability of AFXζ and AFXα to activate reporters containing previously characterized IRSs in HepG2 cells. As shown in Fig. 3 B, AFXα expression activated the IGFBP-1reporter ∼4-fold. This result is consistent with previous observations that AFXα induces a 6-fold increase of reporter chloramphenicol acetyltransferase activity when under the control of the IGFBP-1 promoter (26Kops G.J. De de Ruiter N.D. Vries-Smits A.M. Powell D.R. Bos J.L. Burgering B.M. Nature. 1999; 398: 630-634Crossref PubMed Scopus (953) Google Scholar). AFXα failed to activate the reporters containing the IRS elements fromPEPCK and G6Pase promoters or the reporter containing the extended PEPCK promoter region (−458–+43). On the other hand, AFXζ not only stimulated IGFBP-1reporter transcription to a high level (∼7-fold, Fig. 3 B), AFXζ also increased transcription of the IRS containingPEPCK and G6Pase and extended thePEPCK promoter constructs to 7–9-fold. Therefore, AFXζ appeared to have a broader and more potent ability to activate transcription when compared with AFXα. The PEPCK IRS overlaps with the consensus binding sequences for HNF3 and C/EBP; the IGFBP-1 IRS also overlaps with an HNF3 site (33Imai E. Stromstedt P.E. Quinn P.G. Carlstedt-Duke J. Gustafsson J.A. Granner D.K. Mol. Cell. Biol. 1990; 10: 4712-4719Crossref PubMed Scopus (244) Google Scholar, 34Mitchell J. Noisin E. Hall R. O'Brien R. Imai E. Granner D. Mol. Endocrinol. 1994; 8: 585-594PubMed Google Scholar, 35O'Brien R.M. Noisin E.L. Suwanichkul A. Yamasaki T. Lucas P.C. Wang J.C. Powell D.R. Granner D.K. Mol. Cell. Biol. 1995; 15: 1747-1758Crossref PubMed Google Scholar, 36Unterman T.G. Fareeduddin A. Harris M.A. Goswami R.G. Porcella A. Costa R.H. Lacson R.G. Biochem. Biophys. Res. Commun. 1994; 203: 1835-1841Crossref PubMed Scopus (82) Google Scholar). To investigate in more detail the sequence elements mediating transcriptional activation by both AFXα and AFXζ, a number of reporter constructs were generated by inserting dissectedPEPCK and IGFBP-1 IRS fragments (Fig.2 A) into the pSEAP2-promoter vector. These reporter constructs were cotransfected into HepG2 cells with either the AFXα or the AFXζ expression plasmids. Again, AFXζ activated all reporter constructs tested to a degree greater than that achieved with AFXα (Fig. 3 B). It is noteworthy that AFXζ activated bothPEPCK-a (IRS and HNF-3 sites) and PEPCK-b (IRS and C/EBP sites) ∼4-fold, while the effect on PEPCK (all three sites) appeared to be higher (∼8-fold). This suggested that AFXζ, unlike AFXα, can act cooperatively with factors binding at both the HNF3 and C/EBP sites. In the case of the IGFBP-1promoter, AFXα appeared to require both IRS sites to mediate transcriptional activation. These observations suggested that AFXζ may be less stringent or have altered DNA binding affinity compared with AFXα. We tested whether the transactivational properties of AFXζ respond to insulin treatment. In HepG2 cells, AFXζ activated the reporter transcription under the control of PEPCKpromoter (Fig. 3). When the cells were treated with increasing concentrations of insulin, the induced PEPCKp-SEAP activity decreased to 50% compared with the untreated cells (Fig. 4 A). Insulin also inhibited the AFXζ-induced PEPCK IRS-SEAP activity in a dose-dependent manner (Fig. 4 B), decreasing reporter activity to the basal level at 200 nminsulin. Additionally, we observed that insulin repressedPEPCK IRS-SEAP in cells in which exogenous AFXζ had not been introduced (data not shown). Because reverse transcription-PCR analysis showed that the AFXζ mRNA splice variant is expressed in HepG2 cells (data not shown), this insulin-dependent inhibition may be exerted through endogenous AFXζ. Our data suggest that insulin suppressesPEPCK expression and that AFXζ can mediate this effect through IRS. We also investigated the effect of bisperoxovanadate (bpV), an inhibitor of phosphatases (37Gordon J.A. Methods Enzymol. 1991; 201: 477-482Crossref PubMed Scopus (528) Google Scholar) that can deactivate components of the insulin signaling pathway by dephosphorylation (38Wijkander J. Holst L.S. Rahn T. Resjo S. Castan I. Manganiello V. Belfrage P. Degerman E. J. Biol. Chem. 1997; 272: 21520-21526Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Similar to the effect seen with insulin, bpV reduced the AFXζ-induced PEPCK p-SEAP activity in a dose-dependent manner(Fig.5 A). The addition of bpV repressed reporter gene activity at concentrations as low as 0.5 μm. More significantly, the reporter activity was diminished by 5 μm bpV to below the basal level. We observed toxicity of bpV in HepG2 cells above 5 μm as evidenced by the reduction of the control β-galactosidase activity. The data suggest that the suppressive effect of insulin on the transcriptional activity of AFXζ may be mediated through a phosphatase-sensitive pathway. To assess the role of the PI3K/PKB insulin signaling pathway in the suppression of AFXζ transactivation, a plasmid encoding constitutively active PKB was cotransfected with an AFXζ expression construct and the PEPCK p-SEAP reporter construct. Expression of
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