Growth Hormone- and Prolactin-Induced Proliferation of Insulinoma Cells, INS-1, Depends on Activation of STAT5 (Signal Transducer and Activator of Transcription 5)

Abstract

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.

S731 in the transactivation domain modulates STAT5b activity

“…Phosphorylation of Y699 results in an upshift of the STAT5b band, as seen in the STAT5b immunoblot (lower panel). Similar band shifts of STAT5b upon phosphorylation have been reported by others [27][28][29]. Both the serine and tyrosine phosphorylation of STAT5b are sustained through 3 hours (180 minutes), at which point both phosphorylations begin to decrease.…”

Beneficial effects of growth hormone-releasing hormone agonists on rat INS-1 cells and on streptozotocin-induced NOD/SCID mice

“…The results suggested that at least three signaling pathways, MAPK/ERK, PI3K/AKT, and cAMP/PKA are involved in the action of GHRH agonists upon their binding to GHRH receptors on INS-1 cells. GH and various growth factors stimulate proliferation and survival of β cells; these well-characterized growth factors include IGF1 and glucagon-like peptide (GLP-1) (8,11,(28)(29)(30). It has been reported that the binding of GH to GH-receptor triggers downstream signal transduction through the JAK2-STAT5a/b pathway, which leads to increase β-cell proliferation and survival (30,31).…”

Intrauterine programming of fetal islet gene expression in rats?effects of maternal protein restriction during gestation revealed by proteome analysis

“…This protein is also up-regulated when embryonic stem cells commit to neural differentiation [98], suggesting that low-protein-diet islet cells could be less differentiated. In addition G1/S-specific cyclin D2 is a target gene for Signal Transducer and Activator of Transcription 5 (STAT5), which activates transcription [99]. FBP 2 is a transcription factor which participates in the regulation of c-myc expression [78] and loss of FBP function arrests cellular proliferation [79].…”