Elmo1介导TGF-β1活化Rac1的机制及其在肾间质纤维化中的作用研究

Tubulointerstitial fibrosis is the common final outcome of almost all progressive chronic kidney diseases. And excessive deposition of extracellularmatrix is the most striking and name-lending feature of tubulointerstitial fibrosis. In the human renal tubular epithelial cell line HKC, Rac1 activity is required for TGF-β1-induced ECM production. But how TGF-β1activates Rac1 is still unclear. Our preliminary study suggests that in NRK52E cells TGF-β1 not only induces higher Rac1 activity but also stimulates the expression of Elmo1. Overexpression of wild-type Elmo1 promotes TGF-β1-stimulated ECM synthesis and Rac1 GTP-loading. These observations suggest that TGF-β1-Elmo1-Rac1 pathway was involved in the regulation of ECM deposition. In order to prove this hypothesis, we unmask the manner on which TGF-β1 regulates the expression of Elmo1. Elmo1 depletion by RNA interference impairs TGF-β1 stimulation of Rac1 activation and ECM deposition. At basal levels, Elmo1 is autoinhibited due to intramolecular EID/EAD interactions. Phosphorylation of Elmo1 could disrupt the autoinhibitory interaction. Tyr18 and Tyr 216, which locate in the EID domain, have been identified as the phosphorylation sites. Mutant forms of Elmo1 lacking these sites were defective in their ability to promote Rac1 activity. Overexpression of these mutants would give a lowered activation of Rac1. Moreover, in vivo, analyses reveals a crucial role for Elmo1 in the progress of tubulointerstitial fibrosis through compared with Elmo1-/- mice. Collectively, these studies will uncover a selective requirement for Elmo1 in TGF-β1-induced Rac1 activity and ECM deposition.

肾间质纤维化（TIF）是各种慢性肾脏病发展至终末期肾病的共同途径，细胞外基质（ECM）沉积是其重要特征。TGF-β1活化Rac1可促进小管上皮细胞合成ECM，但TGF-β1活化Rac1的机制尚不明。申请者前期工作发现TGF-β1以时间依赖模式上调具活化Rac1功能的Elmo1的表达，过表达Elmo1增强TGF-β1对Rac1的活化及ECM的合成，提示TGF-β1-Elmo1-Rac1途径参与调控ECM沉积。为证实上述假说，本项目拟：1）探讨TGF-β1调控Elmo1表达的机制；2）利用Elmo1或Rac1 siRNA，证实Elmo1-Rac1途径参与TGF-β1诱导的ECM的合成；3）构建Elmo1磷酸化位点的突变体，阐明TGF-β1通过促进Elmo1的磷酸化解除其自身抑制，从而活化Rac1。4）利用Elmo1敲基因鼠，进一步证实Elmo1在TIF中的作用，从而为防治慢性肾脏病提供新靶标。

肾间质纤维化（TIF）是各种慢性肾脏病发展至终末期肾病的共同途径，细胞外基质（ECM）沉积是其重要特征。TGF-β1活化Rac1可促进小管上皮细胞合成ECM，但TGF-β1活化Rac1的机制尚不明。申请者前期工作发现TGF-β1以时间依赖模式上调具活化Rac1功能的Elmo1的表达，过表达Elmo1增强TGF-β1对Rac1的活化及ECM的合成，提示TGF-β1-Elmo1-Rac1途径参与调控ECM沉积。进一步研究发现，TGF-β1上调Elmo1表达的同时，亦增强其活性，从而活化Rac1，诱导细胞表达过多的Collagen Ⅰ和fibronectin；Elmo1的EID结构域中18、216、395和511四个酪氨酸磷酸化位点联合突变后， TGF-β1诱导的Rac1的活化显著降低。过表达Elmo1除增加ECM的表达外，还促进细胞迁移。我们前期工作发现Nck-1可以与Elmo1结合，且依赖于Nck-1的SH2结构域。进一步研究发现，Elmo1 N端的495个氨基酸介导了Elmo1与Nck-SH2结构域的相互作用；Elmo1和Nck-SH2的相互作用依赖Elmo1的酪氨酸磷酸化，Y18、Y216、Y395和Y511四个酪氨酸磷酸化位点对于两者的结合至关重要；Nck-SH2与Elmo1结合后增强Elmo1与活化的RhoG之间的结合，提高Elmo1/DOCK180复合物的GEF活性，进一步活化Rac1，促进Elmo1/DOCK180复合物介导的细胞迁移。Numb是一种高度进化保守的蛋白，广泛存在于哺乳动物中，Numb具有调节Rac1活性的功能。我们发现，UUO模型中，随着造模时间的延长，Numb在近端肾小管上皮细胞中的表达逐渐升高；特异性敲低近端肾小管上皮细胞中Numb的表达，可以显著改善UUO模型中肾间质纤维化的情况。体外实验进一步证实，TGF-β1可以诱导Numb的表达，过表达Numb可以使肾小管上皮细胞阻滞于G2/M期，诱导细胞分泌促纤维化因子，使细胞合成分泌大量的细胞外基质，从而促进肾间质纤维化。本项目从上述几个方面探讨几种可能参与肾脏纤维化的分子机制，为有效的防治慢性肾脏病提供新的干预策略。