Regulation of Phosphoenolpyruvate Carboxykinase and Insulin-like Growth Factor-binding Protein-1 Gene Expression by Insulin

Winged helix/forkhead (Fox) transcription factors have been implicated in the regulation of a number of insulin-responsive genes. The insulin response elements (IREs) of the phosphoenolpyruvate carboxykinase (PEPCK) and insulin-like growth factor-binding protein-1 (IGFBP-1) genes bind members of the FKHR and HNF3 subclasses of Fox proteins. Previous mutational analyses of the PEPCK and IGFBP-1 IREs revealed mutations which do not affect the binding of HNF3 proteins to these elements but do eliminate the ability of the IREs to mediate an insulin response. This dissociation of binding and function provided compelling evidence that HNF3 proteins,per se, are not insulin response proteins. The same approach was used here to determine if FKHRL1, a member of the FKHR subclass of Fox proteins, binds to the PEPCK and IGFBP-1 IREs in a manner that correlates with the ability of these elements to mediate an insulin response. Overexpression of FKHRL1 stimulates transcription from transfected reporter constructs that contain a multimerized PEPCK IRE or an IGFBP-1 IRE and this stimulation is repressed by insulin. There is a direct correlation between the ability of mutant versions of the PEPCK and IGFBP-1 IREs to bind FKHRL1 and their ability to mediate FKHRL1-induced transcription when FKHRL1 is overexpressed. However, under conditions where FKHRL1 is not overexpressed, there is a lack of correlation between FKHRL1 binding to mutant versions of the PEPCK and IGFBP-1 IREs and the ability of these elements to mediate an insulin response. Therefore, the PEPCK and IGFBP-1 IREs mediate FKHRL1-induced transcription and its inhibition by insulin when this protein is overexpressed, but at the normal cellular concentration of FKHRL1 the insulin response mediated by these elements must involve another protein. Winged helix/forkhead (Fox) transcription factors have been implicated in the regulation of a number of insulin-responsive genes. The insulin response elements (IREs) of the phosphoenolpyruvate carboxykinase (PEPCK) and insulin-like growth factor-binding protein-1 (IGFBP-1) genes bind members of the FKHR and HNF3 subclasses of Fox proteins. Previous mutational analyses of the PEPCK and IGFBP-1 IREs revealed mutations which do not affect the binding of HNF3 proteins to these elements but do eliminate the ability of the IREs to mediate an insulin response. This dissociation of binding and function provided compelling evidence that HNF3 proteins,per se, are not insulin response proteins. The same approach was used here to determine if FKHRL1, a member of the FKHR subclass of Fox proteins, binds to the PEPCK and IGFBP-1 IREs in a manner that correlates with the ability of these elements to mediate an insulin response. Overexpression of FKHRL1 stimulates transcription from transfected reporter constructs that contain a multimerized PEPCK IRE or an IGFBP-1 IRE and this stimulation is repressed by insulin. There is a direct correlation between the ability of mutant versions of the PEPCK and IGFBP-1 IREs to bind FKHRL1 and their ability to mediate FKHRL1-induced transcription when FKHRL1 is overexpressed. However, under conditions where FKHRL1 is not overexpressed, there is a lack of correlation between FKHRL1 binding to mutant versions of the PEPCK and IGFBP-1 IREs and the ability of these elements to mediate an insulin response. Therefore, the PEPCK and IGFBP-1 IREs mediate FKHRL1-induced transcription and its inhibition by insulin when this protein is overexpressed, but at the normal cellular concentration of FKHRL1 the insulin response mediated by these elements must involve another protein. Regulation of phosphoenolpyruvate carboxykinase and insulin-like growth factor-binding protein-1 gene expression by insulin. The role of winged helix/forkhead proteins.Journal of Biological ChemistryVol. 276Issue 25PreviewPage 30171, Fig. 2: The top part of the figure should appear as follows. Full-Text PDF Open Access insulin response element phosphoenolpyruvate carboxykinase insulin-like growth factor-binding protein-1 protein kinase B glutathione S-transferase wild type electrophoretic mobility shift assay chloramphenicol acetyltransferase thymidine kinase luciferase phosphatidylinositol gAF2, glucocorticoid accessory factor 2 Insulin affects the expression of over 100 genes (1O'Brien R.M. Granner D.K. Biochem. J. 1991; 278: 609-619Crossref PubMed Scopus (247) Google Scholar, 2O'Brien R.M. Granner D.K. Physiol. Rev. 1996; 76: 1109-1161Crossref PubMed Scopus (439) Google Scholar). Many of these genes are regulated by insulin at the transcriptional level, but the molecular details by which this regulation is achieved are poorly understood. Progress in this area has been hampered by the fact that there is no consensus insulin response element (IRE)1 that can account for the regulation of all insulin-responsive genes. However, an IRE with a T(G/A)TTT(T/G)(G/T) core sequence has been associated with insulin-induced transcriptional repression of a number of metabolic genes, including those that encode phosphoenolpyruvate carboxykinase (PEPCK), insulin-like growth factor-binding protein-1 (IGFBP-1), tyrosine aminotransferase, glucose-6-phosphatase, apolipoprotein C III, and aspartate aminotransferase (3O'Brien R.M. Lucas P.C. Forest C.D. Magnuson M.A. Granner D.K. Science. 1990; 249: 533-537Crossref PubMed Scopus (289) Google Scholar, 4Suwanichkul A. Morris S.L. Powell D.R. J. Biol. Chem. 1993; 268: 17063-17068Abstract Full Text PDF PubMed Google Scholar, 5Ganss R. 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Gustafsson J.A. Granner D.K. Mol. Cell. Biol. 1990; 10: 4712-4719Crossref PubMed Scopus (244) Google Scholar). The gAF2 element is a component of the PEPCK glucocorticoid response unit and is required for a full glucocorticoid response by the PEPCK gene (10Granner D. O'Brien R. Imai E. Forest C. Mitchell J. Lucas P. Rec. Prog. Horm. Res. 1991; 47: 319-346PubMed Google Scholar, 11Sugiyama T. Scott D.K. Wang J.C. Granner D.K. Mol. Endocrinol. 1998; 12: 1487-1498Crossref PubMed Scopus (55) Google Scholar). Winged helix/forkhead (Fox) proteins of the HNF3 subclass are responsible for the accessory activity of gAF2 (12O'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, 13Wang J.C. Stromstedt P.E. O'Brien R.M. Granner D.K. Mol. Endocrinol. 1996; 10: 794-800PubMed Google Scholar). The co-localization of the IRE with gAF2 led to the hypothesis that HNF3 proteins might also be involved in mediating the insulin response (2O'Brien R.M. Granner D.K. Physiol. Rev. 1996; 76: 1109-1161Crossref PubMed Scopus (439) Google Scholar,12O'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). However, point mutation analysis of the gAF2 element showed that there is no correlation between the binding of HNF3 proteins and the ability of the element to confer insulin responsiveness to a heterologous promoter (13Wang J.C. Stromstedt P.E. O'Brien R.M. Granner D.K. Mol. Endocrinol. 1996; 10: 794-800PubMed Google Scholar). The IGFBP-1 IRE, like the PEPCK IRE, is involved in the regulation of IGFBP-1 gene expression by glucocorticoids, and HNF3 proteins mediate this function (12O'Brien R.M. Noisin E.L. Suwanichkul A. Yamasaki T. Lucas P.C. Wang J.C. Powell D.R. Granner D.K. 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Members of the FKHR subclass of the Fox family of transcription factors, namely FKHR, AFX, and FKHRL1, may be involved in the regulation of PEPCK and IGFBP-1 gene expression by insulin (17Tang E.D. Nunez G. Barr F.G. Guan K.L. J. Biol. Chem. 1999; 274: 16741-16746Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar, 18Nakae J. Park B.C. Accili D. J. Biol. Chem. 1999; 274: 15982-15985Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 19Guo S. Rena G. Cichy S. He X. Cohen P. Unterman T. J. Biol. Chem. 1999; 274: 17184-17192Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 20Durham 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, 21Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. 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Toker A. Thomas J.H. Ruvkun G. Genes Dev. 1999; 13: 1438-1452Crossref PubMed Scopus (323) Google Scholar, 29Tissenbaum H.A. Ruvkun G. Genetics. 1998; 148: 703-717PubMed Google Scholar). This pathway consists of DAF-2, AGE-1, PDK-1, and AKT 1, 2 which are the C. eleganshomologs of the insulin receptor, the catalytic subunit of PI 3-kinase, the 3-phosphoinositide-dependent kinase-1, and protein kinase B (PKB), respectively. Genetic complementation experiments showed that activation of this pathway leads to the inactivation of the C. elegans of The of as a Fox family member led to the that FKHR proteins might be of in and might be responsible for mediating the regulation of gene expression by insulin. the FKHR proteins are by in and in and FKHR proteins can bind the PEPCK and IGFBP-1 IREs (17Tang E.D. Nunez G. Barr F.G. Guan K.L. J. Biol. Chem. 1999; 274: 16741-16746Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar, 18Nakae J. Park B.C. Accili D. J. Biol. Chem. 1999; 274: 15982-15985Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 21Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar, 22Biggs III, W.H. Meisenhelder J. Hunter T. Cavenee W.K. Arden K.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7421-7426Crossref PubMed Scopus (946) Google Scholar, S. A. S. A. Unterman T.G. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the of FKHRL1, the protein used in this and in but a to be by a of and in FKHRL1 leads to the of the protein in the from its of in the A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar). have shown that the of FKHR proteins in leads to a stimulation of transfected reporter constructs the IGFBP-1 IRE, and that this is by of the transfected with insulin (17Tang E.D. Nunez G. Barr F.G. Guan K.L. J. Biol. Chem. 1999; 274: 16741-16746Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar, 19Guo S. Rena G. Cichy S. He X. Cohen P. Unterman T. J. Biol. Chem. 1999; 274: 17184-17192Abstract Full Text Full Text PDF PubMed Scopus (469) Google 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). However, expression of FKHR proteins in the of another protein that the insulin response under Therefore, in the and mutant versions of the PEPCK and IGFBP-1 IREs for their ability to confer insulin responsiveness to a heterologous promoter in the or of There is a direct correlation between the binding of FKHRL1 to the PEPCK and IGFBP-1 IREs and the ability of these elements to mediate when FKHRL1 is overexpressed. Insulin the transcriptional activation by However, the insulin response mediated by the PEPCK and IGFBP-1 IREs in the of FKHRL1 not with FKHRL1 that another factor must be required for the PEPCK and IGFBP-1 insulin response when is at in the in with the as described 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 that the to to the The transfected with of reporter where with of FKHRL1 expression or The transfected in and in the or of insulin. to the of S.K. III, 6: PubMed Scopus Google with Scott D.K. Noisin E.L. Lucas P.C. Granner D.K. Mol. Cell. Biol. 12: PubMed Scopus Google Scholar). the luciferase assay The the thymidine kinase promoter of the gene J.L. Cell. Full Text PDF PubMed Scopus Google Scholar). the PEPCK IRE sequence or a mutant of the the of to the of (3O'Brien R.M. Lucas P.C. Forest C.D. Magnuson M.A. Granner D.K. Science. 1990; 249: 533-537Crossref PubMed Scopus (289) Google Scholar). from or was to the of which contain a luciferase reporter The of the of with the of This was from by which a at of the promoter in the of in the was by a and a was of the PEPCK IRE in The was with to the IRE and this was to a or mutant of the IRE the of to the The and by a of the PEPCK IRE by The and for and have been described A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google and provided by and to a at for The protein was to a for at the was with the for at and at was for and a assay was the and from FKHRL1 was a from and was from a for to those used for 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). of was in a 100 2 2 of and of the of protein. The was at for was at a in The of FKHRL1 in was by analysis an FKHRL1 FKHRL1 as a protein an and and contain a protein that with FKHRL1 The in the of the is not to a in the of the a insulin was The are to an insulin-induced in the and of FKHRL1 are of A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar). The of with insulin in a of as by 1, B and This is PI as the of in the of the PI the insulin-induced that contain FKHRL1 and that this protein is in a manner with that in of the PEPCK IRE that affect binding of FKHRL1 should also affect the ability of FKHRL1 to and mediate an insulin response this mutations the that the PEPCK IRE to this The and mutant versions of the PEPCK IRE of the in the reporter to the family of R.M. Lucas P.C. Forest C.D. Magnuson M.A. Granner D.K. Science. 1990; 249: 533-537Crossref PubMed Scopus (289) Google and transfected with this of and the ability of insulin to transcription from the promoter was by the of activity of the PEPCK IRE insulin responsiveness to the promoter that insulin of a of activity as with mutation of of the IRE has no the ability of the IRE to mediate an insulin response, mutations at or of the core at a the ability of the IRE to mediate an insulin response in the of and mutation of or insulin responsiveness The binding of was by that the PEPCK IRE was used as a and the the IRE an IRE at or an IRE at The and with the for binding of the poorly when the binding of HNF3 proteins to the PEPCK IRE was (12O'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 J.C. Stromstedt P.E. O'Brien R.M. Granner D.K. Mol. Endocrinol. 1996; 10: 794-800PubMed Google which that members of the Fox family of transcription factors may this sequence in a The ability of to bind to the as as to the a dissociation of binding and function the mutation the ability of the IRE to mediate an insulin response Fig. The dissociation of binding from function 2 that FKHRL1 is not involved in the regulation of the PEPCK gene that FKHR proteins, when overexpressed, are activity is repressed by insulin. Therefore, the ability of FKHRL1 to and mediate an insulin response PEPCK IRE constructs was of a FKHRL1 expression with has no luciferase gene expression from Insulin the activity of FKHRL1 is or the and constructs are to insulin in C and Therefore, of FKHRL1 cannot insulin responsiveness to or FKHRL1 binds to the as as binds to the Fig. mutant of FKHRL1, which has an in the of the at and was also for an the reporter This protein has no the activity of and in the or of insulin is that the PEPCK IRE in the luciferase gene expression is not to mediate FKHRL1 activity in this Therefore, another of luciferase reporter constructs and all contain of the or mutant versions of the PEPCK IRE of the Insulin reporter gene expression by the Overexpression of FKHRL1 a stimulation of activity and this stimulation is by of the with insulin. The of also stimulates but this stimulation is not by the of the The in the is to mediate an insulin response and not mediate FKHRL1-induced This is with the of and to to insulin or be by FKHRL1, and with the that FKHRL1 binds poorly to the Fig. The the ability to mediate and FKHRL1 binds the as as the the of of and However, the fact that insulin in the of FKHRL1 a dissociation between the binding of FKHRL1 and the ability of insulin to gene transcription the PEPCK The same approach was used to determine if the IGFBP-1 IRE is of mediating an insulin response in a a that the IGFBP-1 IRE sequence as a or mutant versions of the IGFBP-1 IRE used as The of the mutations the IGFBP-1 IRE to the PEPCK IRE are shown in Fig. at and of the and B of the IGFBP-1 can bind a of FKHRL1 of binding to the IGFBP-1 was in the of IGFBP-1 IRE or PEPCK IRE and the was by was with IGFBP-1 IRE in the of of or mutant IGFBP-1 IRE The by and as described in Fig. The the of The a between the of the IGFBP-1 as and that the IGFBP-1 IRE as the mutation as as an the IGFBP-1 IRE for binding of to the The not for the the but not as as the or of reporter in which the IGFBP-1 IRE the same of the promoter used for the PEPCK IRE as described in experiments The IGFBP-1 IRE insulin responsiveness the of this with in a of reporter gene and this was by insulin. The fact that of FKHRL1 stimulates reporter gene expression from constructs the IGFBP-1 gene IRE, but not from constructs a of the PEPCK gene IRE, the of the IGFBP-1 The of repression by insulin was when was the ability of insulin to FKHRL1 stimulation is at the in of the at of the and B in the IGFBP-1 IRE in a of responsiveness to insulin and FKHRL1 Thus, these are for insulin and FKHRL1 The reporter a of insulin responsiveness in the of However, this is when is Therefore, the like the mutation in the PEPCK IRE, a dissociation between binding of FKHRL1 and the ability of the IGFBP-1 IRE to mediate an insulin response. The dissociation of FKHRL1 binding and insulin repression is by this FKHRL1 binds the IGFBP-1 IRE at as as to the IGFBP-1 there is no repression by insulin of in the of Therefore, the and mutant versions of the IGFBP-1 IRE FKHRL1 binding and FKHRL1-induced when is overexpressed, but have the ability to mediate an insulin response in the of that insulin can gene transcription the PEPCK and IGFBP-1 IREs in an and FKHRL1, when overexpressed, binds to the PEPCK and IGFBP-1 IREs and This is by but the insulin response mediated by the PEPCK and IGFBP-1 to be of of This is the binding and which that mutations in the PEPCK and IGFBP-1 IREs that do not affect FKHRL1 binding but the ability of the IREs to mediate an insulin response. Therefore, is that FKHRL1 is the insulin response protein for the PEPCK and IGFBP-1 genes. The same mutation in HNF3 from as an insulin response protein to be in the analysis of FKHRL1 the PEPCK IRE 13Wang J.C. Stromstedt P.E. O'Brien R.M. Granner D.K. Mol. Endocrinol. 1996; 10: 794-800PubMed Google Scholar). The and reporter constructs do not to insulin in transfected However, binding that FKHRL1 binds the of the PEPCK IRE as as binds the The ability of this mutation to the insulin response the PEPCK IRE is not to a promoter also the insulin response of The of these with constructs that contain the PEPCK IRE, in with their normal of FKHRL1, a dissociation of FKHRL1 binding from insulin-induced transcriptional when FKHRL1 is in transfected this and reporter gene expression is by FKHRL1 and insulin this This is not as FKHRL1 binds the PEPCK as as binds the PEPCK Thus, a correlation exists between the ability of FKHRL1 to bind to and mutant versions of the PEPCK IRE and insulin-induced transcriptional repression when the protein is in of the The IGFBP-1 IRE by FKHR, AFX, and in the IGFBP-1 IRE to and the with the PEPCK The mutations at and in of the IGFBP-1 IRE to the and mutations in the PEPCK The mutation at was is for IGFBP-1 IRE function but not PEPCK IRE function (13Wang J.C. Stromstedt P.E. O'Brien R.M. Granner D.K. Mol. Endocrinol. 1996; 10: 794-800PubMed Google Scholar, 19Guo S. Rena G. Cichy S. He X. Cohen P. Unterman T. J. Biol. Chem. 1999; 274: 17184-17192Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar). There is a correlation between the of luciferase gene expression from the and constructs and the binding of FKHRL1 to the and IREs when this transcription factor is overexpressed. with the PEPCK IRE, insulin this However, FKHRL1 binds to the and mutant reporter genes these elements no response to insulin when transfected normal of the reporter gene was repressed by insulin in the of Therefore, mutant versions of the IGFBP-1 IRE that bind FKHRL1 cannot mediate an insulin response in the of This dissociation of FKHRL1 binding from insulin the IGFBP-1 IRE evidence that FKHRL1, and FKHR proteins, are not the of insulin-induced transcriptional activation by or or the expression of of mediated by FKHR proteins (17Tang E.D. Nunez G. 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Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, M. A. J. Accili D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Barr Accili D. J. PubMed Scopus Google Scholar). The of in the regulation of FKHRL1 activity by insulin is by that a mutant of FKHRL1 in which the consensus and are not to insulin. of these must be for insulin to FKHRL1 A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar). constructs of the PEPCK IRE or a of the IGFBP-1 IRE, binding and are not by the The of this and that FKHRL1 binds the PEPCK and IGFBP-1 transcription these and that this activation is by insulin. However, these appear to FKHRL1 in of the normal cellular FKHRL1 is at insulin repression of transcription in an and for for in the and of this and for and