Silva C M, Lu H, Day R N
Department of Medicine, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Mol Endocrinol. 1996 May;10(5):508-18. doi: 10.1210/mend.10.5.8732682.
The interaction of GH with its receptor has been shown to lead to the phosphorylation of the signal transducer and activator of transcription (STAT) family of transcription factors. We demonstrate here that GH activates the tyrosine phosphorylation of STAT5 in the human IM-9 lymphocyte cell line. Western blotting indicates that GH also activates STAT5 in human embryonic kidney cells (293), which stably express the rabbit GH receptor. Although it has been shown previously that GH activates both STATs 1 and 3 in the 3T3-F442A mouse preadipocyte cell line, we demonstrate that GH also activates STAT5 in these cells. Using electrophoretic mobility shift assay, we examined the interaction of proteins with DNA elements containing consensus STAT-binding sequences. Proteins prepared from GH-treated 3T3-F442A cells bound to the c-sis inducible element of the human c-fos gene (m67 SIE), whereas proteins from GH-treated IM-9 cells did not. However, proteins from GH-treated IM-9 cells did interact with oligonucleotides containing either an interferon response element or the lactogenic hormone-responsive region. Treatment of IM-9 cells with interferon-gamma also induced protein interactions with these elements although the complexes were distinctly different than those seen with GH treatment. Using STAT-specific antibodies, we demonstrate that the GH-induced DNA-protein complex formed with the lactogenic hormone-responsive region contained STAT5, while the interferon-gamma-induced complex contained STAT1. These results implicate STAT5 as a downstream mediator of GH action in IM-9 cells. We report here the cloning of two forms of STAT5, STAT5A and STAT5B, from an IM-9 cDNA library. Northern blot analysis demonstrated multiple-forms of STAT5 mRNA in IM-9 cells.
生长激素（GH）与其受体的相互作用已被证明可导致转录因子信号转导子和转录激活子（STAT）家族的磷酸化。我们在此证明，GH可激活人IM-9淋巴细胞系中STAT5的酪氨酸磷酸化。蛋白质印迹法表明，GH还可激活稳定表达兔GH受体的人胚肾细胞（293）中的STAT5。尽管先前已表明GH可激活3T3-F442A小鼠前脂肪细胞系中的STAT1和STAT3，但我们证明GH也可激活这些细胞中的STAT5。使用电泳迁移率变动分析，我们检测了蛋白质与含有共有STAT结合序列的DNA元件的相互作用。从经GH处理的3T3-F442A细胞制备的蛋白质与人类c-fos基因的c-sis诱导元件（m67 SIE）结合，而经GH处理的IM-9细胞的蛋白质则不结合。然而，经GH处理的IM-9细胞的蛋白质确实与含有干扰素反应元件或催乳激素反应区域的寡核苷酸相互作用。用γ干扰素处理IM-9细胞也诱导了蛋白质与这些元件的相互作用，尽管复合物与GH处理所见的复合物明显不同。使用STAT特异性抗体，我们证明与催乳激素反应区域形成的GH诱导的DNA-蛋白质复合物含有STAT5，而γ干扰素诱导的复合物含有STAT1。这些结果表明STAT5是IM-9细胞中GH作用的下游介质。我们在此报告从IM-9 cDNA文库中克隆出两种形式的STAT5，即STAT5A和STAT5B。Northern印迹分析表明IM-9细胞中存在多种形式的STAT5 mRNA。
