Gan Yujun, Zhang Yue, Buckels Ashiya, Paterson Andrew J, Jiang Jing, Clemens Thomas L, Zhang Zhong-Yin, Du Keyong, Chang Yingzi, Frank Stuart J
MD, University of Alabama at Birmingham, 1530 Third Avenue South, BDB 720, Birmingham, Alabama 35294-0012.
Mol Endocrinol. 2013 Nov;27(11):1969-79. doi: 10.1210/me.2013-1178. Epub 2013 Sep 12.
GH is a potent anabolic and metabolic factor that binds its cell surface receptor (GHR), activating the GHR-associated tyrosine kinase, Janus kinase 2, which phosphorylates and activates the latent transcription factor, signal transducer and activator of transcription 5 (STAT5). Some GH actions are mediated by the elaboration of IGF-1, which exerts effects by binding and activating the heterotetrameric tyrosine kinase growth factor receptor, IGF-1R. In addition to this GH-GHR-IGF-1-IGF-1R scheme, we have demonstrated in primary osteoblasts and in islet β-cells that then deletion or silencing of IGF-1R results in diminished GH-induced STAT5 phosphorylation, suggesting that the presence of IGF-1R may facilitate GH signaling. In this study, we explore potential roles for protein tyrosine phosphatase activity in modulating GH-induced signaling, comparing conditions in which IGF-1R is present or diminished. We confirm that in mouse primary osteoblasts harboring loxP sites flanking the IGF-1R gene, infection with an adenovirus that expresses the Cre recombinase results in IGF-1R deletion and diminished acute GH-induced STAT5 phosphorylation. Furthermore, we present a new model of IGF-1R silencing, in which expression of short hairpin RNA directed at IGF-1R greatly reduces IGF-1R abundance in LNCaP human prostate cancer cells. In both models, treatment with a chemical inhibitor of protein tyrosine phosphatase-1B (PTP-1B), but not one of src homology region 2 domain-containing phosphotase-1 (SHP-1) and SHP-2, reverses the loss of GH-induced STAT5 phosphorylation in cells lacking IGF-1R but has no effect in cells with intact IGF-1R. Furthermore, expression of either a dominant-negative PTP-1B or the PTP-1B-interacting inhibitory protein, constitutive photomorphogenesis 1, also rescues acute GH-induced STAT5 signaling in IGF-1R-deficient cells but has no effect in IGF-1R replete cells. By expressing a substrate-trapping mutant PTP-1B, we demonstrate that tyrosine phosphorylated Janus kinase-2 is a PTP-1B substrate only in cells lacking IGF-1R. Collectively, our data suggest that IGF-1R positively regulates acute GH signaling by preventing access of PTP-1B activity to Janus kinase 2 and thereby preventing PTP-1B-mediated suppression of GH-induced STAT5 activation.
生长激素（GH）是一种强大的合成代谢和代谢因子，它与细胞表面受体（GHR）结合，激活与GHR相关的酪氨酸激酶——Janus激酶2，后者使潜在的转录因子——信号转导子和转录激活子5（STAT5）磷酸化并激活。GH的一些作用是通过胰岛素样生长因子-1（IGF-1）的分泌介导的，IGF-1通过结合并激活异源四聚体酪氨酸激酶生长因子受体——IGF-1R发挥作用。除了这种GH-GHR-IGF-1-IGF-1R模式外，我们在原代成骨细胞和胰岛β细胞中已经证明，IGF-1R的缺失或沉默会导致GH诱导的STAT5磷酸化减少，这表明IGF-1R的存在可能促进GH信号传导。在本研究中，我们探讨蛋白酪氨酸磷酸酶活性在调节GH诱导的信号传导中的潜在作用，比较IGF-1R存在或减少的情况。我们证实，在IGF-1R基因两侧带有loxP位点的小鼠原代成骨细胞中，用表达Cre重组酶的腺病毒感染会导致IGF-1R缺失，并减少急性GH诱导的STAT5磷酸化。此外，我们提出了一种新的IGF-1R沉默模型，其中针对IGF-1R的短发夹RNA的表达大大降低了LNCaP人前列腺癌细胞中IGF-1R的丰度。在这两种模型中，用蛋白酪氨酸磷酸酶-1B（PTP-1B）的化学抑制剂处理，但不是含src同源区2结构域的磷酸酶-1（SHP-1）和SHP-2中的一种，可逆转缺乏IGF-1R的细胞中GH诱导的STAT5磷酸化的丧失，但对IGF-1R完整的细胞没有影响。此外，显性负性PTP-1B或与PTP-1B相互作用的抑制蛋白——组成型光形态建成1的表达，也能挽救IGF-1R缺陷细胞中急性GH诱导的STAT5信号传导，但对IGF-1R充足的细胞没有影响。通过表达一种底物捕获突变体PTP-1B，我们证明酪氨酸磷酸化的Janus激酶-2只是缺乏IGF-1R的细胞中的PTP-1B底物。总的来说，我们的数据表明，IGF-1R通过阻止PTP-1B活性接近Janus激酶2，从而防止PTP-1B介导的对GH诱导的STAT5激活的抑制，来正向调节急性GH信号传导。
