MKL1 mediates TGF-β1-induced α-smooth muscle actin expression in human renal epithelial cells
MKL1 mediates TGF-β1-induced α-smooth muscle actin expression in human renal epithelial cells
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DOI：
10.1152/ajprenal.00142.2007
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发表时间：
2008-05-01
影响因子：
4.2
通讯作者：
Turman, Martin A.
中科院分区：
文献类型：
--
作者：
Elberg, Gerard;Chen, Lijuan;Turman, Martin A.
已知转化生长因子-β1(TGF-β1)可诱导肾脏上皮-间充质转化，这一过程与肾小管间质纤维化有关。我们假设血清反应因子(SRF)的共激活物--巨核细胞白血病因子-1(MKL1)能刺激原代培养的肾小管上皮细胞(RTC)转录α-平滑肌肌动蛋白(α-SMA)，在转化生长因子-β1作用下，RTC转化为肌成纤维细胞。在此，我们研究MKL1表达对这些细胞α-SMA的影响。我们证明了转化生长因子-β1对α-SMA转录的刺激是通过已知与SRF结合的CC(A/T)(6)富含GG元件介导的。这些元件还介导MKL1效应，该效应在无血清介质中显著激活α-SMA转录。与绿色荧光蛋白融合的MKL1定位于细胞核，并诱导α-SMA的表达，而不受转化生长因子-β1的影响。使用蛋白酶体抑制剂，我们还证明了蛋白水解性泛素途径调节MKL1的表达。这些数据表明，MKL1的过表达足以诱导α-SMA的表达。小干扰RNA抑制MKL1的内源性表达可取消转化生长因子-β1对α-SMA表达的刺激。因此，MKL1也是转化生长因子-β1刺激α-SMA表达所必需的。Western印迹和免疫荧光分析表明，未刺激的RTC中高表达和内源性的MKL1定位于胞核。染色质免疫沉淀分析表明，转化生长因子-β1诱导内源性SRF和MKL1与染色质中的α-SMA启动子结合。由于MKL1是一个强大的调节α-SMA表达的因子，内源性MKL1的表达或活性的调节可能对肾脏肌成纤维细胞的形成和功能产生深远的影响。
Transforming growth factor-beta 1 (TGF-beta 1) is known to induce epithelial-mesenchymal transition in the kidney, a process involved in tubulointerstitial fibrosis. We hypothesized that a coactivator of the serum response factor (SRF), megakaryoblastic leukemia factor-1 (MKL1), stimulates alpha-smooth muscle actin (alpha-SMA) transcription in primary cultures of renal tubular epithelial cells (RTC), which convert into myofibroblasts on treatment with TGF-beta 1. Herein, we study the effect of MKL1 expression on alpha-SMA in these cells. We demonstrate that TGF-beta 1 stimulation of alpha-SMA transcription is mediated through CC(A/T)(6)-rich GG elements known to bind to SRF. These elements also mediate the MKL1 effect that dramatically activates alpha-SMA transcription in serum-free media. MKL1 fused to green fluorescent protein localizes to the nucleus and induces alpha-SMA expression regardless of treatment with TGF-beta 1. Using proteasome inhibitors, we also demonstrate that the proteolytic ubiquitin pathway regulates MKL1 expression. These data indicate that MKL1 overexpression is sufficient to induce alpha-SMA expression. Inhibition of endogenous expression of MKL1 by small interfering RNA abolishes TGF-beta 1 stimulation of alpha-SMA expression. Therefore, MKL1 is also absolutely required for TGF-beta 1 stimulation of alpha-SMA expression. Western blot and immunofluorescence analysis show that overexpressed and endogenous MKL1 are located in the nucleus in non-stimulated RTC. Chromatin immunoprecipitation assay demonstrates that TGF-beta 1 induces binding of endogenous SRF and MKL1 to the alpha-SMA promoter in chromatin. Since MKL1 constitutes a potent factor regulating alpha-SMA expression, modulation of endogenous MKL1 expression or activity may have a profound effect on myofibroblast formation and function in the kidney.