VA Research, South Texas Veterans Health Care System, San Antonio, Texas, USA; Department of Medicine, UT Health, San Antonio, Texas, USA.
Department of Pathology, UT Health, San Antonio, Texas, USA.
J Biol Chem. 2022 Sep;298(9):102246. doi: 10.1016/j.jbc.2022.102246. Epub 2022 Jul 11.
Proximal tubular epithelial cells respond to transforming growth factor β (TGFβ) to synthesize collagen I (α2) during renal fibrosis. The oncoprotein DJ-1 has previously been shown to promote tumorigenesis and prevent apoptosis of dopaminergic neurons; however, its role in fibrosis signaling is unclear. Here, we show TGFβ-stimulation increased expression of DJ-1, which promoted noncanonical mTORC1 and mTORC2 activities. We show DJ-1 augmented the phosphorylation/activation of PKCβII, a direct substrate of mTORC2. In addition, coimmunoprecipitation experiments revealed association of DJ-1 with Raptor and Rictor, exclusive subunits of mTORC1 and mTORC2, respectively, as well as with mTOR kinase. Interestingly, siRNAs against DJ-1 blocked TGFβ-stimulated expression of collagen I (α2), while expression of DJ-1 increased expression of this protein. In addition, expression of dominant negative PKCβII and siRNAs against PKCβII significantly inhibited TGFβ-induced collagen I (α2) expression. In fact, constitutively active PKCβII abrogated the effect of siRNAs against DJ-1, suggesting a role of PKCβII downstream of this oncoprotein. Moreover, we demonstrate expression of collagen I (α2) stimulated by DJ-1 and its target PKCβII is dependent on the transcription factor hypoxia-inducible factor 1α (Hif1α). Finally, we show in the renal cortex of diabetic rats that increased TGFβ was associated with enhanced expression of DJ-1 and activation of mTOR and PKCβII, concomitant with increased Hif1α and collagen I (α2). Overall, we identified that DJ-1 affects TGFβ-induced expression of collagen I (α2) via an mTOR-, PKCβII-, and Hif1α-dependent mechanism to regulate renal fibrosis.
近端肾小管上皮细胞在肾纤维化过程中对转化生长因子β（TGFβ）作出反应，合成胶原 I（α2）。先前已经表明癌蛋白 DJ-1 促进肿瘤发生并防止多巴胺能神经元凋亡；然而，其在纤维化信号中的作用尚不清楚。在这里，我们显示 TGFβ 刺激增加了 DJ-1 的表达，这促进了非典型 mTORC1 和 mTORC2 活性。我们显示 DJ-1 增强了 PKCβII 的磷酸化/激活，PKCβII 是 mTORC2 的直接底物。此外，共免疫沉淀实验表明 DJ-1 与 Raptor 和 Rictor 相关，Raptor 和 Rictor 分别是 mTORC1 和 mTORC2 的独特亚基，以及与 mTOR 激酶相关。有趣的是，针对 DJ-1 的 siRNA 阻断了 TGFβ 刺激的胶原 I（α2）的表达，而 DJ-1 的表达增加了该蛋白的表达。此外，表达显性负 PKCβII 和针对 PKCβII 的 siRNA 显著抑制了 TGFβ 诱导的胶原 I（α2）的表达。事实上，组成型激活的 PKCβII 消除了针对 DJ-1 的 siRNA 的作用，表明 PKCβII 是该癌蛋白下游的一个作用因子。此外，我们证明了 DJ-1 及其靶标 PKCβII 刺激的胶原 I（α2）的表达依赖于转录因子缺氧诱导因子 1α（Hif1α）。最后，我们在糖尿病大鼠的肾皮质中显示，增加的 TGFβ 与 DJ-1 和 mTOR 和 PKCβII 的激活相关，伴随着 Hif1α 和胶原 I（α2）的增加。总体而言，我们确定 DJ-1 通过 mTOR、PKCβII 和 Hif1α 依赖性机制影响 TGFβ 诱导的胶原 I（α2）的表达，从而调节肾纤维化。
