Signal transducer and activator of transcription 5 activation is sufficient to drive transcriptional induction of cyclin D2 gene and proliferation of rat pancreatic β-cells
Signal transducer and activator of transcription 5 activation is sufficient to drive transcriptional induction of cyclin D2 gene and proliferation of rat pancreatic β-cells
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DOI：
10.1210/me.2002-0356
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发表时间：
2003-05-01
影响因子：
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通讯作者：
Moldrup, A
中科院分区：
文献类型：
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作者：
Friedrichsen, BN;Richter, HE;Moldrup, A
信号转导子和转录激活子5（STAT 5）激活在胰腺β细胞中GH和催乳素介导的信号转导中起核心作用。在先前的实验中，我们证明了STAT 5激活对于人（h）GH刺激的INS-1细胞增殖和hGH诱导的细胞周期调节剂细胞周期蛋白D2的mRNA水平增加是必需的。在这项研究中，我们进一步表征了STAT 5在hGH调节细胞周期蛋白D表达和β细胞增殖中的作用。在INS-1细胞中，hGH以时间依赖性方式诱导细胞周期蛋白D2 mRNA和蛋白水平（但不诱导细胞周期蛋白D1和D3）。抑制蛋白质。与放线菌酮共孵育的合成在4 h时不影响hGH诱导的细胞周期蛋白D2 mRNA水平的增加。显性负性STAT 5突变体STAT 5aDelta 749的表达部分抑制细胞周期蛋白D2蛋白水平。INS-1细胞瞬时转染细胞周期蛋白D2启动子-报告构建体揭示了3- 5倍的转录活性增加响应hGH刺激。此外，组成型活性STAT 5突变体（CA-STAT 5a或CA-STAT 5 b）的共表达足以驱动启动子的反式激活。CA-STAT 5 b在强力霉素诱导型启动子的控制下在INS-1细胞中稳定表达。凝胶阻滞实验中使用的探针代表一个假定的细胞周期蛋白D2启动子中的STAT 5结合位点，揭示了多西环素诱导的CA-STAT 5 b的结合。此外，CA-STAT 5 b的诱导刺激细胞周期蛋白D2启动子的转录激活，并诱导这些细胞中的hGH非依赖性增殖。在原代β-细胞中，在不存在hGH的情况下，腺病毒介导的CA-STAT 5 b表达显著刺激DNA合成（超过对照的5.3倍）。我们的研究表明，STAT 5的激活足以驱动β细胞的增殖，并且细胞周期蛋白D2可能是STAT 5在此过程中的关键靶基因。
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.