Ebong Samuel, Chepelinsky Ana B, Robinson Michael L, Zhao Haotian, Yu Cheng-Rong, Egwuagu Charles E
Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892-1857, USA.
Mol Vis. 2004 Feb 19;10:122-31.
IGF-1 and PDGF are implicated in regulating lens proliferation and/or providing spatial cues that restrict lens proliferation to germinative and transition zones of the lens. However, very little is known about how IGF-1- or PDGF-induced signals are transduced and coupled to gene transcription in lens cells. Therefore, we examined whether these growth factors mediate their effects in the lens through the evolutionarily conserved JAK/STAT signal transduction pathway and if STAT signaling is essential for mammalian lens development.
Expression of STAT1 and STAT3 was analyzed in mouse lens and lens epithelial cells by RT-PCR and western blot analysis. Activation of the STAT signaling pathway was examined by a combination of gel-shift, super-shift, and western blotting assays. Regulation of lens proliferation and gene transcription by STAT pathways was assessed by 3H-Thymidine incorporation or RT-PCR assays with lens explants treated or untreated with Genistein or the JAK2 and STAT3 inhibitor, AG-490. Mice with targeted deletion of STAT3 in the lens were generated by Cre/lox recombination and STAT1-/-, STAT3-/- deficient as well as normal lenses were characterized by histology.
We show that PDGF and IGF1 induce proliferation in 1AMLE6 lens cells and couple their extracellular signals to gene transcription, in part through activation of STAT3 and to a lesser extent STAT1 signal transduction pathways. We further show that targeted deletion of STAT3 in E10.5 lens does not produce overt developmental lens defects. STAT1 knockout mice also exhibit a normal lens phenotype.
Our results showing that deletion of either STAT1 or STAT3 does not affect the normal development of the lens is surprising in view of the fact that STAT pathways are activated in developing chick or mouse lens and inappropriate activation of STAT1 pathway in the lens by ectopic lens expression of IFN? inhibits lens differentiation and induces cataract in transgenic mice. Our data thus suggest that although STAT-signaling pathways may contribute to activation of gene transcription in the lens, it may not be essential for normal lens development or STAT proteins may be functionally redundant during lens development. However, because several growth factors and cytokines present in the lens activate STATs in mouse lens explants and 1AMLE6 lens epithelial cells, it may well be that this evolutionarily conserved signaling pathway is under stringent control in the mammalian lens. Whereas deficiency in any particular STAT pathway can be compensated for by any of the functionally redundant STAT proteins induced by a wide array of growth factors in the lens, chronic or prolonged activation of a particular STAT protein may perturb homeostatic balance in STAT-dependent growth factor signaling, culminating in pathologic lens changes.
胰岛素样生长因子-1（IGF-1）和血小板衍生生长因子（PDGF）参与调节晶状体增殖和/或提供空间线索，将晶状体增殖限制在晶状体的生发区和过渡区。然而，关于IGF-1或PDGF诱导的信号如何在晶状体细胞中进行转导并与基因转录偶联，我们了解得还很少。因此，我们研究了这些生长因子是否通过进化上保守的JAK/STAT信号转导途径在晶状体中介导其作用，以及STAT信号传导对于哺乳动物晶状体发育是否必不可少。
通过逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹分析，检测小鼠晶状体和晶状体上皮细胞中STAT1和STAT3的表达。通过凝胶迁移、超迁移和蛋白质免疫印迹分析相结合的方法，检测STAT信号通路的激活情况。采用3H-胸腺嘧啶核苷掺入法或RT-PCR法，评估用染料木黄酮或JAK2和STAT3抑制剂AG-490处理或未处理的晶状体外植体中，STAT通路对晶状体增殖和基因转录的调节作用。通过Cre/lox重组技术构建晶状体中STAT3靶向缺失的小鼠，通过组织学方法对STAT1-/-、STAT3-/-缺陷型以及正常晶状体进行特征分析。
我们发现，PDGF和IGF1可诱导1AMLE6晶状体细胞增殖，并将其细胞外信号与基因转录偶联，部分是通过激活STAT3，在较小程度上是通过激活STAT1信号转导途径。我们进一步发现，在胚胎第10.5天的晶状体中靶向缺失STAT3，并不会导致明显的晶状体发育缺陷。STAT1基因敲除小鼠也表现出正常的晶状体表型。
我们的结果表明，缺失STAT1或STAT3均不影响晶状体的正常发育，鉴于在发育中的鸡或小鼠晶状体中STAT通路被激活，以及在转基因小鼠中通过异位表达干扰素在晶状体中不适当激活STAT1通路会抑制晶状体分化并诱发白内障，这一结果令人惊讶。因此，我们的数据表明，尽管STAT信号通路可能有助于晶状体中基因转录的激活，但它对于正常晶状体发育可能并非必不可少，或者STAT蛋白在晶状体发育过程中可能具有功能冗余性。然而，由于晶状体中存在的几种生长因子和细胞因子可在小鼠晶状体外植体和1AMLE6晶状体上皮细胞中激活STAT，很可能这条进化上保守的信号通路在哺乳动物晶状体中受到严格调控。虽然任何特定STAT通路的缺陷都可以由晶状体中多种生长因子诱导的任何功能冗余的STAT蛋白来补偿，但特定STAT蛋白的慢性或长期激活可能会扰乱STAT依赖性生长因子信号传导中的稳态平衡，最终导致晶状体发生病理性变化。
