Das Falguni, Bera Amit, Ghosh-Choudhury Nandini, Abboud Hanna E, Kasinath Balakuntalam S, Choudhury Goutam Ghosh
Departments of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.
Department of Pathology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America; VA Research, South Texas Veterans Health Care System, San Antonio, Texas, United States of America.
PLoS One. 2014 Oct 15;9(10):e109608. doi: 10.1371/journal.pone.0109608. eCollection 2014.
Enhanced TGFβ activity contributes to the accumulation of matrix proteins including collagen I (α2) by proximal tubular epithelial cells in progressive kidney disease. Although TGFβ rapidly activates its canonical Smad signaling pathway, it also recruits noncanonical pathway involving mTOR kinase to regulate renal matrix expansion. The mechanism by which chronic TGFβ treatment maintains increased mTOR activity to induce the matrix protein collagen I (α2) expression is not known. Deptor is an mTOR interacting protein that suppresses mTOR activity in both mTORC1 and mTORC2. In proximal tubular epithelial cells, TGFβ reduced deptor levels in a time-dependent manner with concomitant increase in both mTORC1 and mTORC2 activities. Expression of deptor abrogated activity of mTORC1 and mTORC2, resulting in inhibition of collagen I (α2) mRNA and protein expression via transcriptional mechanism. In contrast, neutralization of endogenous deptor by shRNAs increased activity of both mTOR complexes and expression of collagen I (α2) similar to TGFβ treatment. Importantly, downregulation of deptor by TGFβ increased the expression of Hif1α by increasing translation of its mRNA. TGFβ-induced deptor downregulation promotes Hif1α binding to its cognate hypoxia responsive element in the collagen I (α2) gene to control its protein expression via direct transcriptional mechanism. Interestingly, knockdown of raptor to specifically block mTORC1 activity significantly inhibited expression of collagen I (α2) and Hif1α while inhibition of rictor to prevent selectively mTORC2 activation did not have any effect. Critically, our data provide evidence for the requirement of TGFβ-activated mTORC1 only by deptor downregulation, which dominates upon the bystander mTORC2 activity for enhanced expression of collagen I (α2). Our results also suggest the presence of a safeguard mechanism involving deptor-mediated suppression of mTORC1 activity against developing TGFβ-induced renal fibrosis.
在进行性肾病中，增强的转化生长因子β（TGFβ）活性促使近端肾小管上皮细胞积累包括胶原蛋白I（α2）在内的基质蛋白。尽管TGFβ能迅速激活其经典的Smad信号通路，但它也会募集涉及mTOR激酶的非经典通路来调节肾脏基质扩张。慢性TGFβ处理维持mTOR活性增加以诱导基质蛋白胶原蛋白I（α2）表达的机制尚不清楚。Deptor是一种与mTOR相互作用的蛋白，可抑制mTORC1和mTORC2中的mTOR活性。在近端肾小管上皮细胞中，TGFβ以时间依赖性方式降低Deptor水平，同时mTORC1和mTORC2活性均增加。Deptor的表达消除了mTORC1和mTORC2的活性，通过转录机制导致胶原蛋白I（α2）的mRNA和蛋白表达受到抑制。相反，用短发夹RNA（shRNAs）中和内源性Deptor会增加两种mTOR复合物的活性以及胶原蛋白I（α2）的表达，类似于TGFβ处理。重要的是，TGFβ导致的Deptor下调通过增加其mRNA的翻译来增加缺氧诱导因子1α（Hif1α）的表达。TGFβ诱导的Deptor下调促进Hif1α与其在胶原蛋白I（α2）基因中的同源缺氧反应元件结合，通过直接转录机制控制其蛋白表达。有趣的是，敲低猛禽（raptor）以特异性阻断mTORC1活性可显著抑制胶原蛋白I（α2）和Hif1α的表达，而抑制rictor以选择性防止mTORC2激活则没有任何效果。至关重要的是，我们的数据证明仅通过Deptor下调才需要TGFβ激活的mTORC1，这在旁观者mTORC2活性之上主导了胶原蛋白I（α2）的增强表达。我们的结果还表明存在一种涉及Deptor介导的对mTORC1活性抑制的保护机制，以对抗TGFβ诱导的肾纤维化发展。
