Friedrichsen B N, Galsgaard E D, Nielsen J H, Møldrup A
The Hagedorn Research Institute, Department of Cell Biology 2820 Gentofte, Denmark.
Mol Endocrinol. 2001 Jan;15(1):136-48. doi: 10.1210/mend.15.1.0576.
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.
生长激素（GH）和催乳素（PRL）可刺激胰腺β细胞的增殖及胰岛素分泌。虽然已证明胰岛素瘤细胞中生长激素和催乳素调节的胰岛素基因转录依赖于信号转导及转录激活因子5（STAT5），但生长激素/催乳素诱导β细胞复制所涉及的信号通路尚不清楚。通过分析特异性抑制剂对胰岛素分泌细胞系INS-1和原代β细胞的作用，研究了各种信号通路在人生长激素（hGH）诱导的DNA合成中的作用。促分裂原活化蛋白激酶激酶（MEK）抑制剂PD98059以及促分裂原活化蛋白激酶p38（MAPKp38）抑制剂SB203580可部分抑制INS-1细胞中hGH诱导的增殖，但对原代β细胞无显著影响。星形孢菌素是一种蛋白激酶C（PKC）和蛋白激酶A（PKA）抑制剂，可阻断INS-1细胞中的基础增殖及hGH诱导的增殖，但对原代β细胞无抑制作用。渥曼青霉素是一种磷脂酰肌醇3激酶（PI3K）抑制剂，对INS-1细胞和原代β细胞中的hGH诱导增殖均无抑制作用，而酪氨酸激酶抑制剂染料木黄酮可完全抑制原代β细胞和INS-1细胞中hGH诱导的增殖。为了分析STAT5在hGH诱导增殖中的可能作用，通过稳定转染，在强力霉素诱导型启动子的控制下，在INS-1细胞中表达了显性负性STAT5突变体STAT5Delta749。发现两个克隆表现出剂量依赖性、强力霉素诱导型的STAT5Delta749表达，并抑制了hGH刺激的STAT5调节的催乳素受体（PRLR）启动子-报告基因构建体的转录激活。此外，STAT5Delta749表达的诱导完全抑制了hGH诱导的DNA合成。对内源基因表达的分析显示，强力霉素依赖性抑制了hGH刺激的PRLR和细胞周期蛋白D2 mRNA水平。我们的结果表明，生长激素/催乳素诱导的β细胞增殖依赖于Janus激酶2（JAK2）/STAT5信号通路，而不是MAPK、PI3K和PKC信号通路。此外，细胞周期调节因子细胞周期蛋白D2可能是此过程中STAT5的关键靶基因。
