Exendin-4 as a stimulator of rat insulin I gene promoter activity via bZIP/CRE interactions sensitive to serine/threonine protein kinase inhibitor Ro 31-8220.
Signal transduction properties of exendin-4 (Ex-4) underlying its ability to stimulate rat insulin I gene promoter (RIP1) activity were assessed in the pancreatic beta-cell line INS-1. Ex-4 acted via glucagon-like peptide-1 receptors to stimulate RIP1 in a glucose-dependent manner, as measured in cells transfected with a -410-bp RIP1-luciferase construct (RIP1-Luc). The action of Ex-4 was independent of cAMP and PKA because it was not blocked by cotransfection with dominant-negative G alpha(s), was unaffected by pretreatment with the membrane-permeant cAMP antagonist 8-Br-Rp-cAMPS, and remained apparent after treatment with PKA inhibitors H-89 or KT 5720. Similarly, cotransfection with a dominant-negative isoform of the type-2 cAMP-regulated guanine nucleotide exchange factor (Epac2) failed to alter the response to Ex-4. Ro 31-8220, a serine/threonine protein kinase inhibitor that targets PKC as as well as the 90-kDa ribosomal S6 kinase (RSK) and mitogen- and stress-activated protein kinase (MSK) family of cAMP response element-binding protein (CREB) kinases, blocked the stimulatory action of Ex-4 at RIP1-Luc. However, selective inhibition of PKC using K-252c, prolonged exposure to phorbol 1,2-myristate-13-acetate, or cotransfection with dominant-negative atypical PKC-zeta, was without effect. A-CREB, a dominant-negative inhibitor of basic region-leucine zipper transcription factors (bZIPs) related in structure to CREB, inhibited the action of Ex-4 at RIP1-Luc, whereas A-ATF-2 was ineffective. Similarly, introduction of deletions at the RIP1 cAMP response element (CRE), or truncation of RIP1 to remove the CRE, nearly abolished the action of Ex-4. Inactivating mutations introduced at the A4/A3 elements, binding sites for the glucose-regulated homeodomain transcription factor PDX-1, did not diminish the response to Ex-4, although a marked reduction of basal promoter activity was observed. The glucose-dependent stimulation of RIP1-Luc by Ex-4 was reproduced using a synthetic reporter (RIP1-CRE-Luc) incorporating multimerized CREs of the RIP1 nonpalindromic sequence 5'-TGACGTCC-3'. It is concluded that the bZIP and CRE-mediated stimulation of RIP1 by Ex-4 explains, at least in part, how this insulinotropic hormone facilitates transcriptional activity of the rat insulin I gene.
Chepurny
Oleg G
OG
Department of Physiology, New York University School of Medicine, New York, New York 10016, USA.
Hussain
Mehboob A
MA
Holz
George G
GG
D000818:Animals; D050976:Basic-Leucine Zipper Transcription Factors; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004268:DNA-Binding Proteins / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D000077270:Exenatide; D051790:G-Box Binding Factors; D005947:Glucose / Q000502:physiology; D006801:Humans; D007202:Indicators and Reagents; D007211:Indoles / Q000494:pharmacology*; D007328:Insulin / Q000235:genetics; D007334:Insulin-Like Growth Factor I / Q000235:genetics*; D008156:Luciferases; D010455:Peptides / Q000494:pharmacology*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D013268:Stimulation, Chemical; D014157:Transcription Factors / Q000235:genetics*; D014162:Transfection; D014688:Venoms
D050976:Basic-Leucine Zipper Transcription Factors; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D051790:G-Box Binding Factors; D007202:Indicators and Reagents; D007211:Indoles; D007328:Insulin; D010455:Peptides; D014157:Transcription Factors; D014688:Venoms; D007334:Insulin-Like Growth Factor I; D000077270:Exenatide; D000242:Cyclic AMP; D008156:Luciferases; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D005947:Glucose; C064758:Ro 31-8220