Exendin-4-mediated cell proliferation in beta-cells: focusing on the transcription factors

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1 Exendin-4-mediated cell proliferation in beta-cells: focusing on the transcription factors Seo-Yoon Chang, Jae Min Cho, Yang-Hyeok Jo, Myung-Jun Kim Departments of Physiology, College of Medicine, The Catholic University of Korea

2 INS-1 cell - rat insulinoma cell line - glucose-responsive insulin secretion cf) RINm5F cell: glucose non-responsive due to the lack of GLUT2 Glucagon-like peptide-1 (GLP-1) - from enteroendocrine L-cell of ileum and colon in response to nutrient intake - in addition to glucose-dependent insulinotrophic action, inhibit glucagon secretion, gastric emptying, food intake - insulin biosysnthesis, insulin gene expression - proliferation, survival and growth of beta-cell

3 Proglucagon processing DNA mrna Protein Posttranslational processing cf) GLP-2: growth factor for gut Adapted from Gastroenterology 2007;132:

4 GLP-1 s action in peripheral target tissues Adapted from Cell Metab 2006;3:

5 GLP-1 receptor - coupled to G-protein - widely expressed in pancreatic islets, brain, heart, kidney, and the GI tract - only a single receptor isolated to date Signaling mechanism of GLP-1 receptor activation in beta-cells Adapted from Current Opin Pharmacol 2006; 6:

6 Signaling mechanism of GLP-1 receptor activation in beta-cells Adapted from Pharmacol Ther 2007;113:

7 Short half-life of GLP-1 (several minutes) Adapted from Gastroenterology 2007;132:

8 Exendins - from the venom of lizard Heloderma species; exendin-3 and exendin-4 - these peptides were named exendin since they were isolated from exocrine gland and were shown to have endocrine actions Exendin-4 - a potent agonist of glucagon-like peptide-1 (GLP-1) receptor - similar properties to native GLP-1 - in experimental diabetes model, improve glucose level, insulin secretion, weight loss, and food intake - stimulates islet mass increase and beta-cell proliferation - BYETTA (Amylin and Lilly)

9 Effects of glucagon-like peptide-1 on INS-1 cell proliferation. Adapted from Diabetologia 2004;47: Adapted from Diabetes 2003;52:

10 Despite many reports about the GLP-1 s role in beta-cell proliferation, there was no report about the effect of GLP-1 on cyclin proteins. Cyclin D1 - a major regulator of G1 to S phases progression

11 EX induces cyclin D1 expression in INS-1 cells. Protein RNA Published J Endocrinol 2006;188:

12 GLP-1 receptor expression (A) and specificity (B). GLP1R antagonist: exendin-(9-39) amide GIPR antagonist: GIP-(6-30) from Dr. C M Isale (Georgia University, USA)

13 Effect of ERK1/2 on cyclin D1 expression. Though exendin-4 activates Raf-ERK pathway, there was no direct evidence in the involvement of ERK in exendin-4-induced cyclin D1 induction.

14 Mechanism of hormone action via camp as second messenger

15 Effects of forskolin and H-89 on cyclin D1 protein expression. FK: forskolin, camp increasing agent H89: a PKA antagonist Exendin-4-induced cyclin D1 expression seems to be dependent on intracellular camp concentration.

16 Involvement of CRE site in EX-induced cyclin D1 transcription. pcd1-96mt: mutated CRE site

17 CREB interacts with the putative CRE site of cyclin D1 promoter. mcre: mutated type wcre: wild type pcd1-96/vector: cotransfection of pcd1 and empty vector pcd1-96/creb: cotransfection of pcd1 and CREB expression vector pcd1-96/kcreb: cotransfection of pcd1 and dominant negative vector

18 Enhanced binding activity of phospho-creb to the putative CRE site of the rat cyclin D1 promoter in vivo.

19 Exendin-4 induces cyclin D1 expression via camp-pka signaling-mediated activation of CRE site within cyclin D1 promoter, and the interaction of phospho-creb with CRE site is implicated in both basal and exendin-4-induced cyclin D1 transcription.

20 Scheme of rat cyclin D1 promoter deletion constructs (A) and promoter analysis of cyclin D1 deletion construct (B). Published Diabetologia 2006;49:

21 Characterization of other transcription factors in rat cyclin D1 expression. - Candidate: EGR1 E2F Sp1 Oct1 EGR/Sp1 CRE Luciferase gene EGR1(5 ) EGR1(3 ) EGR1 (early growth response-1) - one of immediate early gene, zinc-finger transcription factor - a upstream regulator of growth, proliferation and differentiation - involved in glucose-induced beta-cell growth

22 The relevance of EGR1 in beta-cell proliferation Differential gene expression between Zucker Fatty rats and Zucker Diabetic Fatty rats: a potential role for the immediate-early gene Egr-1 in regulation of beta cell proliferation. J Mol Endocrinol 2005;35: Activation of serum response factor in the depolarization induction of Egr-1 transcription in pancreatic Islet beta-cells. J Biol Chem 2000;275: Glucose induces early growth response gene (EGR1) expression in pancreatic beta cells. Diabetologia 1999;42: Egr-1 gene transcription and translation is rapidly induced by glucose treatment. Adapted from J Mol Endocrinol 2005;35:13-25

23 Proposed model for the regulation of PDX-1 expression by Egr-1 Adapted from J Biol Chem 2007;282:

24 Interaction of nuclear proteins with putative EGR1 binding sites. Exendin-4 Exendin-4

25 Characterization of the binding activity of 5 putative EGR1 site: Supershift assay (A) and Competition assay (B). wt: wild type EGR1 mut: mutated EGR1

26 Effect of mutation of the 5 putative EGR1 site on the cyclin D1 induction *, P < 0.05 vs. empty vector (pgl3 basic vector) #, P < 0.05 vs. pcd1-827

27 Recruitment of EGR1 to cyclind1 promoter in vivo.

28 Effect of EGR1 on cyclind1 promoter activity. Relative lu uciferase activity pcr3.1 PCR3.1-EGR1 * 0 pcr3.1 pcd1-827 pcr3.1-egr-1

29 Exendin-4 rapidly induces transcription factor EGR-1 and this was bound the 5 EGR-1 binding site of rat cyclin D1 promoter. Through the EGR-1-mediated induction of cyclin D1 transcription, exendin-4 may contribute to beta-cell proliferation.

30 EX-induced EGR1 expression. A EX (min) B EGR1 EGR1 (721 bp) GAPDH (452 bp) 82 kda EX (min) Protein mrna C Fold Induction of EGR protein mrna 0.5 EX (min) Published AJP-Endo & Metab 2007;292:E215-E222

31 Deletion analysis of rat EGR1 promoter. Fold induction p p-324 SRE ETS CRE p-243 p-159 p-124 p-73 p relative luciferase activity (fold induction)

32 Functional significance of proximal and distal CRE in EX-induced EGR1 expression. wild-462 distal CRE proximal CRE Fold induction 2.1 mpcre-462 mdcre wild mpcre-73 # relative luciferase activity (fold induction)

33 Activation of proximal CRE site through binding of phospho-creb on Ser 133 by EX. consensus SP1 A B no competitor consensus CRE wild CRE mutant CRE consensus AP1 EX (min)

34 Activation of proximal CRE site through binding of phospho-creb on Ser 133 by EX. no antibody anti-creb anti-pcreb anti-ap1 anti-sp1 no antibody anti-creb anti-pcreb anti-ap1 anti-sp1 C untreated exendin-4

35 Requirement of CREB phosphorylation on Ser 133 for EX induction of EGR-1 promoter. Vector: cotransfection of EGR-1 promoter and empty vector CREB: cotransfection of EGR-1 promoter and CREB expression vector Ser 133 Ala: cotransfection of EGR-1 promoter and mutant vector KCREB: cotransfection of EGR-1 promoter and dominant negative vector *, P < 0.05 vs. Vector, P < 0.05 vs. CREB

36 For the induction of EGR-1 transcription by exendin-4, the proximal CRE site of EGR-1 promoter is critical and the binding with CREB phosphorylated on Ser 133 is necessary.

37 EX-induced EGR expression via GLP-1 receptor. Fold Induction Fold Induction Egr-1 β-tubulin EX-4 (nm) 82 kda 0.5 Egr-1 β-tubulin CON EX-4 EX-4+ EX-4+ GLPA GIPA 82 kda, P<0.05 vs. untreated control value, P<0.05 vs. 1 nm, P<0.05 vs. untreated control value Published J Cell Biochem 2008;104:

38 H-89 and PD inhibit EX-induced Egr-1 protein expression. A Fold Induction CON EX-4 FK PD H89 H89+ H89+ PD+ EX-4 FK EX-4 B Egr-1 28S 18S H89+ H89+ PD+ CON EX-4 FK PD H89 EX-4 FK EX-4 Egr-1 β-tubulin 82 kda, P<0.05 vs. CON, P<0.05 vs. EX-4

39 The involvement of camp and ERK in EX-induced Egr-1 expression. C1 PKA (nm) C2 ERK1 (nm) Scr Scr PKA Erk1 44 kda 42 kda EX-4 Scr PKA EX-4 Scr ERK Egr-1 82 kda Egr-1 82 kda β-tubulin β-tubulin Fold Induction Fold induction *, P < 0.05 vs. untreated;, P < 0.05 vs. EX-4 in Scr. 0.5 *, P < 0.05 vs. untreated;, P < 0.05 vs. EX-4 in Scr.

40 ccgcggag cctcagctct acgcgcctgg cgccctccct acgcgggcgt -463 ccccgactcc cgcgcgcgtt caggctccgg gttgggaacc aaggaggggg SRE1 agggtgggtg cgccgacccg gaaacaccat ataaggagca ggaaggatcc SRE2 SRE3 cccgccggaa cagaccttat ttgggcagcg ccttatatgg agtggcccaa tatggccctg ccgcttccgg ctctgggagg aggggcgaac gggggttggg gcgggggcaa gctgggaact ccaggagcct agcccgggag gccactgccg ctgttccaat actaggcttt ccaggagcct gagcgctcag ggtgccggag CRE ccggtcgcag ggtggaagcg cccaccgctc ttggatggga ggtcttcacg SRE4 YY-1 tcactccggg tcctcccggt cggtccttcc atattagggc ttcctgcttc SRE5 CRE ccatatatgg ccatgtacgt cacggcggag gcgggcccgt gctgtttcag +1 acccttgaaa tagaggccga ttcggggagt cgcgagagat cccagcgcgc +26 agaacttgg

41 Transcription factor: regulate transcription (activator or repressor) Zinc finger protein Adapted from Nucleic Acids Res 1994;22:

42 Identification of SRE clusters implicated in transcriptional regulation of rat Egr-1 gene by EX. A pegr-462 dsres CRE psres CRE +1 Fold induction 2.09 pegr-324 * 1.76 pegr-462 p * 1.72, P<0.05 vs. control of pegr-462, P<0.05 vs. control of pegr-324 and pegr-p462 p pegr-324 p * CON EX , P<0.05 vs. EX-4-treated pegr-462, P<0.05 vs. EX-4-treated pegr-324 p and pegr-462 p B relative luciferase activity relative luciferase activity (fold induction) psg5 psg5-yy1 0.0 pegr-124 pegr-46

43 Gel mobility shift assay of SRE site on rat Egr-1 promoter. From -113, 5 -cggtccttccatattagggcttcctgc-3 A1 EX-4 (min) A2 EX-4 (min) A3 Com Com SRF SP1 Rela ative density (S SRF/SP1) EX-4 (min)

44 Gel mobility shift assay of SRE site on rat Egr-1 promoter. B C Competitor WT MT CONS YY1 Ab SRF p-srf Elk YY1 Sp1

45 EX-4-induced Egr-1 expression appears to be largely dependent on camp/pka signaling pathway in β-cells. The interaction of both SRF and phospho-srf with SRE site of the Egr-1 promoter may be implicated in both basal and EX-4-induced Egr-1 transcription.

46 Exendin-4 increased cyclin D1 expression via activation of CRE site and EGR1 binding site within cyclin D1 promoter in INS-1 beta-cells. Exendin-4 increased EGR1 expression via interaction of 1) CREB to proximal CRE site and 2) SRF to SRE site of EGR1 promoter. Exendin-4 serves as a growth factor regulating beta-cell proliferation.

47 Lab Member Prof. Yang-Hyeok Jo Postdoc. Jung-Hoon Kang (NIH) Assistant Seo-Yoon Chang Student Jae Min Cho In collaboration with Assist. Prof. In-Kyung Jeong (Dept. of Internal Medicine, Kyung Hee Univ.) Assist. Prof. Seung Hyun Ko (Dept. of Internal Medicine, Catholic Univ.) Gyeong Ryul Ryu, Ph. D. (Dept. of Internal Medicine, Catholic Univ.) Human cyclin D1 pomoter: Dr. O. Tetsu and Dr. F. McCormick (Univ. of Calif.-San Francisco) pcmv-erk1: Dr. P. E. Shaw (Univ. of Nottingham, UK) Mutant CREB(Ser 133 Ala) and KCREB: Dr. R.H.Goodman (Oregon Health and Science Univ.) Financial support Korea Science and Engineering Foundation (R ) Korean Diabetes Association (BAYER, 2005)

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