Exendin-4 induction of cyclin D1 expression in INS-1 beta-cells: involvement of cAMP-responsive element.
Glucagon-like peptide-1 (GLP-1) and its analog exendin-4 (EX) have been considered as a growth factor implicated in pancreatic islet mass increase and beta-cell proliferation. This study aimed to investigate the effect of EX on cyclin D1 expression, a key regulator of the cell cycle, in the pancreatic beta-cell line INS-1. We demonstrated that EX significantly increased cyclin D1 mRNA and subsequently its protein levels. Although EX induced phosphorylation of Raf-1 and extracellular-signal-regulated kinase (ERK), both PD98059 and exogenous ERK1 had no effect on the cyclin D1 induction by EX. Instead, the cAMP-elevating agent forskolin induced cyclin D1 expression remarkably and this response was inhibited by pretreatment with H-89, a protein kinase A (PKA) inhibitor. Promoter analyses revealed that the cAMP-responsive element (CRE) site (at position -48; 5'-TAACGTCA-3') of cyclin D1 gene was required for both basal and EX-induced activation of the cyclin D1 promoter, which was confirmed by site-directed mutagenesis study. For EX to activate the cyclin D1 promoter effectively, CRE-binding protein (CREB) should be phosphorylated and bound to the putative CRE site, according to the results of electrophoretic mobility shift and chromatin immunoprecipitation assays. Lastly, a transfection assay employing constitutively active or dominant-negative CREB expression plasmids clearly demonstrated that CREB was largely involved in both basal and EX-induced cyclin D1 promoter activities. Taken together, EX-induced cyclin D1 expression is largely dependent on the cAMP/PKA signaling pathway, and EX increases the level of phosphorylated CREB and more potently trans-activates cyclin D1 gene through binding of the CREB to the putative CRE site, implicating a potential mechanism underlying beta-cell proliferation by EX.
Kim
M-J
MJ
Departments of Physiology, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea.
Kang
J-H
JH
Park
Y G
YG
Ryu
G R
GR
Ko
S H
SH
Jeong
I-K
IK
Koh
K-H
KH
Rhie
D-J
DJ
Yoon
S H
SH
Hahn
S J
SJ
Kim
M-S
MS
Jo
Y-H
YH
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000235:genetics* / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019938:Cyclin D1 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D000077270:Exenatide; D005419:Flavonoids / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D000067757:Glucagon-Like Peptide-1 Receptor; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000378:metabolism*; D007546:Isoquinolines / Q000494:pharmacology; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology*; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D018027:Receptors, Glucagon / Q000378:metabolism; D020218:Response Elements; D013449:Sulfonamides / Q000494:pharmacology; D014688:Venoms / Q000378:metabolism / Q000494:pharmacology*
D017362:Cyclic AMP Response Element-Binding Protein; D005419:Flavonoids; C000593835:Glp1r protein, rat; D000067757:Glucagon-Like Peptide-1 Receptor; D007546:Isoquinolines; D010455:Peptides; D012333:RNA, Messenger; D018027:Receptors, Glucagon; D013449:Sulfonamides; D014688:Venoms; D019938:Cyclin D1; D005576:Colforsin; D000077270:Exenatide; D000242:Cyclic AMP; D019908:Proto-Oncogene Proteins c-raf; D017868:Cyclic AMP-Dependent Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one