Regulation of Insulin Gene Transcription by ERK1 and ERK2 in Pancreatic β Cells

Abstract

We show that the mitogen-activated protein kinases ERK1/2 are components of the mechanism by which glucose stimulates insulin gene expression. ERK1/2 activity is required for glucose-dependent transcription from both the full-length rat insulin I promoter and the glucose-sensitive isolated E2A3/4 promoter element in intact islets and β cell lines. Dominant negative ERK2 and MEK inhibitors suppress glucose stimulation of the rat insulin I promoter and the E2A3/4 element. Overexpression of ERK2 is sufficient to stimulate transcription from the E2A3/4 element. The glucose-induced response is dependent upon ERK1/2 phosphorylation of a subset of transcription factors that include Beta2 (also known as NeuroD1) and PDX-1. Phosphorylation increases their functional activity and results in a cumulative transactivation of the promoter. Thus, ERK1/2 act at multiple points to transduce a glucose signal to insulin gene transcription. We show that the mitogen-activated protein kinases ERK1/2 are components of the mechanism by which glucose stimulates insulin gene expression. ERK1/2 activity is required for glucose-dependent transcription from both the full-length rat insulin I promoter and the glucose-sensitive isolated E2A3/4 promoter element in intact islets and β cell lines. Dominant negative ERK2 and MEK inhibitors suppress glucose stimulation of the rat insulin I promoter and the E2A3/4 element. Overexpression of ERK2 is sufficient to stimulate transcription from the E2A3/4 element. The glucose-induced response is dependent upon ERK1/2 phosphorylation of a subset of transcription factors that include Beta2 (also known as NeuroD1) and PDX-1. Phosphorylation increases their functional activity and results in a cumulative transactivation of the promoter. Thus, ERK1/2 act at multiple points to transduce a glucose signal to insulin gene transcription. Circulating insulin is produced by the β cells in the pancreatic islets of Langerhans in adult mammals. Insulin regulates glucose metabolism and in turn glucose regulates synthesis and secretion of insulin by the β cells. Insulin synthesis is stimulated by glucose at several steps, including transcription of the insulin gene (1Brunstedt J. Chan S.J. Biochem. Biophys. Res. Commun. 1982; 106: 1383-1389Crossref PubMed Scopus (67) Google Scholar, 2Giddings S.J. Chirgwin J. Permutt M.A. Diabetes. 1982; 31: 624-629Crossref PubMed Google Scholar, 3Nielsen D.A. Welsh M. Casadaban M.J. Steiner D.F. J. Biol. Chem. 1985; 260: 13585-13589Abstract Full Text PDF PubMed Google Scholar, 4Redmon J.B. Towle H.C. Robertson R.P. Diabetes. 1994; 43: 546-551Crossref PubMed Scopus (32) Google Scholar). Insulin mRNA is extremely stable with a half-life of more than 24 h (5Welsh M. Nielsen D.A. MacKrell A.J. Steiner D.F. J. Biol. Chem. 1985; 260: 13590-13594Abstract Full Text PDF PubMed Google Scholar). Thus, many studies have focused not on regulation of insulin gene transcription but on understanding the translation of its mRNA and its processing to insulin (6Welsh M. Scherberg N. Gilmore R. Steiner D.F. Biochem. J. 1986; 235: 459-467Crossref PubMed Scopus (145) Google Scholar, 7Alarcon C. Wicksteed B. Prentki M. Corkey B.E. Rhodes C.J. Diabetes. 2002; 51: 2496-2504Crossref PubMed Scopus (75) Google Scholar). Nevertheless, increased initiation of insulin gene transcription occurs within 10 min of an elevation in blood glucose concentration (8Leibiger B. Moede T. Schwarz T. Brown G.R. Kohler M. Leibiger I.B. Berggren P.O. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9307-9312Crossref PubMed Scopus (94) Google Scholar). Thus, insulin gene transcription, which is obviously essential for homeostasis of mRNA stores, is also sensitive to acute stimuli. The organization of the insulin promoter is complex, allowing both tissue-restricted transcription of the insulin gene and levels of control for contextual regulation by nutrients, hormones, neurotransmitters, and other agents (9German M. Ashcroft S. Docherty K. Edlund H. Edlund T. Goodison S. Imura H. Kennedy G. Madsen O. Melloul D. Moss L. Olson K. Permutt M.A. Philippe J. Robertson B.P. Rutter W.J. Serup P. Stein R. Steiner D. Tsai M.J. Walker M.D. Diabetes. 1995; 44: 1002-1004Crossref PubMed Scopus (148) Google Scholar, 10Dumonteil E. Philippe J. Diabetes Metab. 1996; 22: 164-173PubMed Google Scholar, 11Ohneda K. Ee H. German M. Semin. Cell Dev. Biol. 2000; 11: 227-233Crossref PubMed Scopus (106) Google Scholar). The proximal promoter, the sequences within a few hundred base pairs of the transcription start site, has been studied in detail for the rat and human insulin genes (for a review see Ref. 10Dumonteil E. Philippe J. Diabetes Metab. 1996; 22: 164-173PubMed Google Scholar). The proximal promoter contains multiple sequence elements capable of responding to glucose (12German M.S. Wang J. Mol. Cell Biol. 1994; 14: 4067-4075Crossref PubMed Scopus (150) Google Scholar, 13German M.S. Moss L.G. Rutter W.J. J. Biol. Chem. 1990; 265: 22063-22066Abstract Full Text PDF PubMed Google Scholar, 14Melloul D. Ben-Neriah Y. Cerasi E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3865-3869Crossref PubMed Scopus (160) Google Scholar, 15Odagiri H. Wang J. German M.S. J. Biol. Chem. 1996; 271: 1909-1915Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 16Sander M. Griffen S.C. Huang J. German M.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11572-11577Crossref PubMed Scopus (46) Google Scholar, 17Sharma A. Stein R. Mol. Cell Biol. 1994; 14: 871-879Crossref PubMed Google Scholar), the best studied being the juxtaposed E and A elements, which together can function as a glucose-responsive minienhancer (13German M.S. Moss L.G. Rutter W.J. J. Biol. 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E elements are binding sites for heterodimeric complexes formed by the neuroendocrine basic helix-loop-helix (bHLH) 1The abbreviations used are: bHLH, basic helix-loop-helix; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MEK, MAP kinase/ERK kinase; CAT, chloramphenicol acetyltransferase; SAPK, stress-activated protein kinase; CMV, cytomegalovirus; GST, glutathione S-transferase; AD2, second activation domain. protein Beta2 (18Lee J.E. Hollenberg S.M. Snider L. Turner D.L. Lipnick N. Weintraub H. Science. 1995; 268: 836-844Crossref PubMed Scopus (939) Google Scholar, 19Naya F.J. Stellrecht C.M. Tsai M.J. Genes Dev. 1995; 9: 1009-1019Crossref PubMed Scopus (525) Google Scholar) and a ubiquitous bHLH protein such as E47 (20Aronheim A. Ohlsson H. Park C.W. Edlund T. Walker M.D. Nucleic Acids Res. 1991; 19: 3893-3899Crossref PubMed Scopus (66) Google Scholar, 21Cordle S.R. Henderson E. Masuoka H. Weil P.A. Stein R. Mol. 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A elements bind any of several homeodomain transcription factors found in β cells, the most abundant being PDX-1 (25Leonard J. Peers B. Johnson T. Ferreri K. Lee S. Montiminy M.R. Mol. Endocrinol. 1993; 7: 1275-1283Crossref PubMed Google Scholar, 26Macfarlane W.M. Read M.L. Gilligan M. Bujalska I. Docherty K. Biochem. J. 1994; 303: 625-631Crossref PubMed Scopus (124) Google Scholar, 27Miller C.P. McGehee Jr., R.E. Habener J.F. EMBO J. 1994; 13: 1145-1156Crossref PubMed Scopus (378) Google Scholar, 28Ohlsson H. Karlsson K. Edlund T. EMBO J. 1993; PubMed Scopus Google Scholar). to the glucose-induced increase in bHLH heterodimer binding to the E PDX-1 binding increases in response to a in glucose concentration W.M. Read M.L. Gilligan M. Bujalska I. Docherty K. Biochem. J. 1994; 303: 625-631Crossref PubMed Scopus (124) Google Scholar, S. H. Cerasi E. Melloul D. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). The increase in binding by PDX-1 is to phosphorylation that is dependent on and the MAP W.M. Read M.L. Gilligan M. Bujalska I. Docherty K. Biochem. J. 1994; 303: 625-631Crossref PubMed Scopus (124) Google Scholar, W.M. P. Docherty K. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), found to PDX-1. also PDX-1 to the W.M. C.M. H. Docherty K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, I. Kennedy Rutter J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) and increases its activation M. Philippe J. Stein R. Madsen Serup P. 1998; PubMed Scopus Google Scholar). diabetes of the has been to PDX-1 D.A. J. Habener J.F. 1997; PubMed Scopus Google Scholar). PDX-1 in a a in the activation domain. for this are to diabetes of the insulin promoter and control of insulin gene transcription by factors that bind to promoter elements, the glucose and insulin gene transcription has We and have that glucose the MAP kinases ERK1/2 in cells M. N. E. C. Prentki M. E. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, C. H. C. J. R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, D. B. S. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (110) Google Scholar). MAP also known as are components of for extracellular to MAP kinases have been in many from and to cell in Res. 1998; PubMed Google and L. G. B. A. C. Chem. PubMed Scopus Google Scholar). ERK1/2 regulate the but the most function is the control of gene transcription. ERK1/2 are stimulated by glucose in and pancreatic β cell M. N. E. C. Prentki M. E. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, C. H. C. J. R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, D. B. S. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (110) Google Scholar). ERK1/2 are the glucose from to as that insulin of insulin secretion ERK1/2 ERK1/2 are not required for insulin glucose increased the and activity of ERK1/2 in the of β cells, that ERK1/2 regulate gene transcription in these cells S. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). this show that negative ERK2 and inhibitors of the ERK1/2 the suppress transcription of the rat insulin I promoter in cells, cells, and ERK2 stimulates transcription from in the of glucose the glucose-responsive element. is at in ERK2 bHLH transcription and and the transcription PDX-1. Phosphorylation of Beta2 and PDX-1 regulates their transactivation phosphorylation of regulates its with Beta2 and that ERK1/2 are components of the mechanism of glucose-responsive insulin gene transcription. of from adult isolated as C. Rhodes C.J. J. 1995; 95: PubMed Scopus Google Scholar), in for and as for min at The islets in S. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). cells as in Ref. S. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google with or negative ERK2 at a of of for and ERK2 as in Ref. T. Cell Biol. 1994; 43: PubMed Scopus Google The cells glucose for 24 h and with glucose for 24 isolated of on a of at for and at with at The in in and in for min at The a in rat insulin I promoter a The minienhancer of from the rat insulin I promoter to a rat insulin I promoter of a chloramphenicol or cells to a of and 24 h of with 10 of and ERK2 or the cells in or in with for 24 h or glucose for The for and 10 of or with 10 of ERK2 or or or as as Mol. 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D.A. 1995; Full Text PDF PubMed Scopus Google The and the cell the in and through of of and of used to cells. of for to cells for 3–5 and with the the protein with as and by of A by The promoter by ERK1/2 in of that glucose ERK1/2 in cells S. Proc. Natl. Acad. Sci. U. S. 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ERK1/2 from the Insulin I in β of insulin mRNA levels by ERK2 through an on the of insulin mRNA or on the initiation of insulin gene transcription. effects on transcription, glucose-dependent in the of a gene to the rat insulin I promoter with and of ERK1/2 ERK2 transcription from the promoter the the promoter Y. H. M.A. Diabetes. 1994; 43: PubMed Scopus Google Scholar) by ERK2 not and inhibitors of glucose activation of ERK1/2 in cells and rat insulin I promoter activity S. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google and and are consistent with the results with that ERK1/2 regulate rat insulin I activity in cells. a of ERK1/2 in insulin gene transcription in the of ERK2 to transcription from a rat insulin I promoter in isolated adult rat these the The stimulation of transcription from the rat insulin I promoter by glucose by ERK2 and transcription from the promoter in rat islets also by and results in rat cells that ERK1/2 are of insulin gene transcription in pancreatic β cells. ERK1/2 Insulin a the of the rat insulin promoter that is by the effects of ERK1/2 on a glucose-responsive element E2A3/4 ERK2 and the MEK the glucose-dependent activity of E2A3/4 in cells that regulation of insulin promoter activity by ERK1/2 occurs at in through this glucose-responsive of the promoter. results also found in cells We also ERK1/2 regulate insulin gene transcription the glucose-sensitive element in ERK2 on the glucose-sensitive element at the of glucose to stimulate transcription by E2A3/4 at glucose results are consistent with in β cell that ERK1/2 regulate insulin gene transcription at in through ERK1/2 are sufficient for transcription from the insulin gene promoter of the glucose of and of ERK1/2 stimulated of the gene by E2A3/4 of ERK2 of its used of ERK2 to the of its or to with a and ERK2 activity M.J. E. M.A. Biol. 1998; Full Text Full Text PDF PubMed Google Scholar). the of glucose transcription from this of ERK2 stimulated transcription from this by the glucose control Thus, ERK2 is sufficient to gene transcription a glucose-responsive element as as or than glucose results the that are within the glucose-responsive element. ERK2 and PDX-1 in to the mechanism of of the glucose-responsive the of ERK2 and other MAP to several factors known to bind to the A and E E elements bind to factors to the bHLH bHLH transcription factors bind to as or as formed the A of the bHLH such as M.S. Blanar M.A. Nelson C. Moss J.B. Rutter W. Mol. Endocrinol. 1991; 5: 292-299Crossref PubMed Scopus (110) Google Scholar), and the tissue-restricted bHLH The known bHLH protein in β cells is Beta2 (18Lee J.E. Hollenberg S.M. Snider L. Turner D.L. Lipnick N. Weintraub H. Science. 1995; 268: 836-844Crossref PubMed Scopus (939) Google Scholar, 19Naya F.J. Stellrecht C.M. Tsai M.J. Genes Dev. 1995; 9: 1009-1019Crossref PubMed Scopus (525) Google Scholar). of the A in the rat insulin promoter are sites for homeodomain transcription factors that bind these A from β cells include PDX-1 and H. Karlsson K. Edlund T. EMBO J. 1993; PubMed Scopus Google Scholar, A. H. Rutter W. German M.S. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar, B. J. S. G. M.R. Mol. Endocrinol. 1994; Google Scholar). and used as in for MAP kinases A. S. J. P. E. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). ERK2 and PDX-1 but not in and not E47 and PDX-1 also by and the other Beta2 and not by of these of Beta2 that ERK2 The sequence of Beta2 and that a ERK2 phosphorylation and that to of which in the most ERK2 phosphorylation the phosphorylation of Beta2 to than of the that is the for of the other sites Beta2 phosphorylation by or that is a ERK2 phosphorylation of phosphorylation to than of the that are or more E47 on and by ERK2 phosphorylation and in human are in its second activation these to in phosphorylation by ERK2 with E47 Phosphorylation of in the phosphorylation Thus, E47 is by ERK2 on these and that not that PDX-1 on by ERK2 The phosphorylation and to PDX-1 also by and in with of kinases for not PDX-1 with this of both phosphorylation by and for in Phosphorylation of in Phosphorylation of Beta2 by ERK2 in a in its as of on the Beta2 in Beta2 consistent with the that phosphorylation of the Beta2 is in cells, of the in of Beta2 as a of its phosphorylation of from cells Beta2 of the in cells Beta2 the The of Beta2 are most of the consistent with the idea that Beta2 is on multiple sites including in cells. Phosphorylation of phosphorylation sites in both E47 and Beta2 are in their activation Thus, phosphorylation of the ERK2 sites is required for the activity of the transcription factors of of and the Beta2 activation of has been to functional in β cells (20Aronheim A. Ohlsson H. Park C.W. Edlund T. Walker M.D. Nucleic Acids Res. 1991; 19: 3893-3899Crossref PubMed Scopus (66) Google Scholar). the activity of the phosphorylation with in cells or cells not transactivation by Beta2 in the of glucose A and Beta2 a activity of of the other ERK2 sites transactivation to of of the transactivation activity of Beta2 to of that the other sites than results in cells Beta2 transactivation is dependent on activation of ERK1/2 in β cells, ERK1/2 activation suppress the activity of Beta2 in the of the of the MEK the activity of Beta2 to than of that in its The of the transactivation of the and to than of the control that has the on but that the sites also to the activity of Phosphorylation of PDX-1 of the in the activation of PDX-1 its activity in β cells of or to the transactivation activity of the to and of the in the of of both than of that phosphorylation of is more and required for the glucose-induced transactivation of PDX-1. the of transactivation is by consistent with the that ERK2 PDX-1 in response to glucose to increase its activity PDX-1 is also by effects of the on its The a on that glucose not regulate the activity of PDX-1 through in cells. and Binding of E47 and by ERK2 also the effects on E47 and binding by E47 to the from the E Binding by phosphorylation of E47 in by ERK2 Beta2 by or by not bind not of E47 and to an E47 and an these the The of the complexes by with and with in the E sequence not by the that the with the are not The of the increased E47 or Beta2 by both E47 and Beta2 by ERK2 in Phosphorylation also the of the E47 ERK1/2 an essential in the through which glucose regulates insulin gene transcription. The that ERK1/2 in this by that both kinases are by glucose in cell M. N. E. C. Prentki M. E. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), that have to The that ERK1/2 activation with inhibitors or ERK1/2 activity with a negative glucose stimulation of the insulin promoter a for ERK1/2 in the as has been C. J. R. Biochem. J. PubMed Scopus Google Scholar). ERK2 is sufficient to transcription by E2A3/4 in the of The of the glucose-responsive E2A3/4 minienhancer transcription factors that act through E and A elements. of these and are for ERK1/2 and are upon phosphorylation by ERK2 Beta2 at multiple sites within its activation that its glucose-sensitive Phosphorylation of Beta2 by ERK2 also its to with and bind to The that ERK1/2 activity inhibitors or also transactivation by Beta2 the that ERK1/2 regulate insulin transcription through effects on Stein R. Serup P. 2002; PubMed Scopus Google Scholar) have that the of of also ERK1/2 in this phosphorylation of the sites on has on its ERK2 phosphorylation of increases its to and to bind to Phosphorylation of E47 also to the of E47 such complexes in insulin transcription in β cells. ERK1/2 phosphorylation also regulates of E47 in other cell in which ERK2 also in its activation domain. Phosphorylation of PDX-1 increases its binding and transactivation M. Philippe J. Stein R. Madsen Serup P. 1998; PubMed Scopus Google Scholar). of ERK1/2 phosphorylation sites on PDX-1 its transactivation and of ERK1/2 activity reduces transactivation to that in the of that phosphorylation by ERK1/2 its glucose-dependent Thus, ERK1/2 act at multiple within the that insulin gene transcription to transduce by Phosphorylation stimulate the Beta2 and PDX-1 activation in any of several The for such as H. F.J. Tsai M.J. Leiter A.B. Genes Dev. 1998; PubMed Scopus Google Scholar, Y. A. Stein R. Mol. Cell Biol. 1998; PubMed Scopus Google Scholar) and with Phosphorylation also such as the PDX-1 and the bHLH heterodimer B. J. S. G. M.R. Mol. Endocrinol. 1994; Google Scholar). Phosphorylation of PDX-1 is by a in a W.M. P. Docherty K. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The regulate the or binding of PDX-1 but has on glucose-dependent transactivation on PDX-1 is by and that PDX-1 signals from multiple MAP the sites as that increase PDX-1 PDX-1 activity is not by the is by ERK1/2 is by glucose S. Proc. Natl. Acad. Sci. U. S. 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R. 1997; PubMed Scopus Google Scholar, D. M. A. H. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, H. J. 1996; PubMed Google Scholar). ERK1/2 have been to both through the of by and by from the of an lacking the in of C. H.P. E. R. 1997; PubMed Scopus Google Scholar, D. M. A. H. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The ERK1/2 activation and glucose concentration that these kinases glucose to that insulin both and control to a for We show that ERK1/2 to a function in β cells to that in cells by to and in the to insulin We at and the of the and for the and the and for