title: Signal transducer and activator of transcription 5 activation is sufficient to drive transcriptional induction of cyclin D2 gene and proliferation of rat pancreatic beta-cells.
abstract: Signal transducer and activator of transcription 5 (STAT5) activation plays a central role in GH- and prolactin-mediated signal transduction in the pancreatic beta-cells. In previous experiments we demonstrated that STAT5 activation is necessary for human (h)GH-stimulated proliferation of INS-1 cells and hGH-induced increase of mRNA-levels of the cell cycle regulator cyclin D2. In this study we have further characterized the role of STAT5 in the regulation of cyclin D expression and beta-cell proliferation by hGH. Cyclin D2 mRNA and protein levels (but not cyclin D1 and D3) were induced in a time-dependent manner by hGH in INS-1 cells. Inhibition of protein synthesis by coincubation with cycloheximide did not affect the hGH-induced increase of cyclin D2 mRNA levels at 4 h. Expression of a dominant negative STAT5 mutant, STAT5aDelta749, partially inhibited cyclin D2 protein levels. INS-1 cells transiently transfected with a cyclin D2 promoter-reporter construct revealed a 3- to 5-fold increase of transcriptional activity in response to hGH stimulation. Furthermore, coexpression of a constitutive active STAT5 mutant (either CA-STAT5a or CA-STAT5b) was sufficient to drive transactivation of the promoter. CA-STAT5b was stably expressed in INS-1 cells under the control of a doxycycline-inducible promoter. Gel retardation experiments using a probe representing a putative STAT5 binding site in the cyclin D2 promoter revealed binding of the doxycycline-induced CA-STAT5b. Furthermore, induction of CA-STAT5b stimulated transcriptional activation of the cyclin D2 promoter and induced hGH-independent proliferation in these cells. In primary beta-cells, adenovirus-mediated expression of CA-STAT5b profoundly stimulated DNA-synthesis (5.3-fold over control) in the absence of hGH. Our studies indicate that STAT5 activation is sufficient to drive proliferation of the beta-cells and that cyclin D2 may be a critical target gene for STAT5 in this process.
authors.affiliation: Department of Islet Discovery Research, Novo Nordisk A/S, Bagsvaerd, Denmark. bttm@novonordisk.com
authors.forename: Birgitte N
authors.initials: BN
authors.lastname: Friedrichsen
authors.forename: Henrijette E
authors.initials: HE
authors.lastname: Richter
authors.forename: Johnny A
authors.initials: JA
authors.lastname: Hansen
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Rhodes
authors.forename: Jens H
authors.initials: JH
authors.lastname: Nielsen
authors.forename: Nils
authors.initials: N
authors.lastname: Billestrup
authors.forename: Annette
authors.initials: A
authors.lastname: Møldrup
mesh_terms: D000818:Animals; D001665:Binding Sites; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D056742:Cyclin D2; D016213:Cyclins / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004318:Doxycycline / Q000494:pharmacology; D005786:Gene Expression Regulation; D019382:Human Growth Hormone / Q000494:pharmacology; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D008894:Milk Proteins; D009154:Mutation; D011401:Promoter Regions, Genetic; D051381:Rats; D050799:STAT5 Transcription Factor; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
chemical_list: C486196:Ccnd2 protein, mouse; C486197:Ccnd2 protein, rat; D056742:Cyclin D2; D016213:Cyclins; D004268:DNA-Binding Proteins; D008894:Milk Proteins; D050799:STAT5 Transcription Factor; C494093:Stat5a protein, mouse; C494094:Stat5a protein, rat; C494096:Stat5b protein, mouse; C494097:Stat5b protein, rat; D015534:Trans-Activators; D019382:Human Growth Hormone; D004318:Doxycycline