Growth hormone- and prolactin-induced proliferation of insulinoma cells, INS-1, depends on activation of STAT5 (signal transducer and activator of transcription 5).
GH and PRL stimulate proliferation and insulin production of pancreatic beta-cells. Whereas GH- and PRL-regulated transcription of the insulin gene in insulinoma cells has been shown to depend on STAT5 (signal transducer and activator of transcription 5), the signaling pathways involved in GH/PRL-induced beta-cell replication are unknown. The roles of various signaling pathways in human GH (hGH)-induced DNA synthesis were studied by analysis of the effect of specific inhibitors in both the insulin-producing cell line, INS-1, and in primary beta-cells. The mitogen-activated protein kinase kinase (MEK)-inhibitor, PD98059, as well as the mitogen-activated protein kinase p38 (MAPKp38) inhibitor, SB203580, partially inhibited hGH- induced proliferation in INS-1 cells but had no significant effect in primary beta-cells. Staurosporine, a protein kinase C (PKC) and protein kinase A (PKA) inhibitor, blocked both basal and hGH-induced proliferation in INS-1 cells, but had no inhibitory effect in primary beta-cells. Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, inhibited hGH-induced proliferation neither in INS-1 cells nor in primary beta-cells, whereas the tyrosine kinase inhibitor, genistein, completely inhibited hGH- induced proliferation in both primary beta-cells and INS-1 cells. To analyze the possible role of STAT5 in hGH-induced proliferation, a dominant negative STAT5 mutant, STAT5Delta749, was expressed in INS-1 cells under the control of a doxycycline- inducible promoter by stable transfection. Two clones were found to exhibit dose-dependent, doxycycline-inducible expression of STAT5Delta749 and suppression of hGH-stimulated transcriptional activation of a STAT5-regulated PRL receptor (PRLR) promoter-reporter construct. Furthermore, induction of STAT5Delta749 expression completely inhibited hGH-induced DNA synthesis. Analysis of endogenous gene expression revealed a doxycycline-dependent inhibition of hGH-stimulated PRLR and cyclin D2 mRNA levels. Our results suggest that GH/PRL-induced beta-cell proliferation is dependent on the Janus Kinase2 (JAK2)/STAT5 signaling pathway but not the MAPK, PI3K, and PKC signaling pathways. Furthermore, the cell cycle regulator cyclin D2 may be a crucial target gene for STAT5 in this process.
Friedrichsen
B N
BN
The Hagedorn Research Institute, Department of Cell Biology 2820 Gentofte, Denmark.
Galsgaard
E D
ED
Nielsen
J H
JH
Møldrup
A
A
D000818:Animals; D000831:Animals, Newborn; D002455:Cell Division / Q000187:drug effects*; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D056742:Cyclin D2; D016213:Cyclins / Q000032:analysis / Q000235:genetics; D004247:DNA / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D019382:Human Growth Hormone / Q000494:pharmacology*; D006801:Humans; D007328:Insulin / Q000096:biosynthesis; D007340:Insulinoma / Q000473:pathology*; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects; D048728:MAP Kinase Kinase Kinase 1; D008894:Milk Proteins; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D009154:Mutation; D010190:Pancreatic Neoplasms / Q000473:pathology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011388:Prolactin / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011981:Receptors, Prolactin / Q000235:genetics; D050799:STAT5 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured; D048051:p38 Mitogen-Activated Protein Kinases
C080336:CCND2 protein, human; C486197:Ccnd2 protein, rat; D056742:Cyclin D2; D016213:Cyclins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D007328:Insulin; D008894:Milk Proteins; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012333:RNA, Messenger; D011981:Receptors, Prolactin; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D019382:Human Growth Hormone; D011388:Prolactin; D004247:DNA; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human