Elberg Gerard, Chen Lijuan, Elberg Dorit, Chan Michael D, Logan Charlotte J, Turman Martin A
Section of Nephrology, Department of Pediatrics, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA .
Am J Physiol Renal Physiol. 2008 May;294(5):F1116-28. doi: 10.1152/ajprenal.00142.2007. Epub 2008 Mar 12.
Transforming growth factor-beta1 (TGF-beta1) 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-beta1. Herein, we study the effect of MKL1 expression on alpha-SMA in these cells. We demonstrate that TGF-beta1 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-beta1. 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-beta1 stimulation of alpha-SMA expression. Therefore, MKL1 is also absolutely required for TGF-beta1 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-beta1 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.
已知转化生长因子-β1（TGF-β1）可诱导肾脏中的上皮-间质转化，这一过程参与肾小管间质纤维化。我们推测，血清反应因子（SRF）的共激活因子巨核母细胞白血病因子-1（MKL1）可刺激肾小管上皮细胞（RTC）原代培养物中α-平滑肌肌动蛋白（α-SMA）的转录，而RTC在用TGF-β1处理后会转化为肌成纤维细胞。在此，我们研究了MKL1表达对这些细胞中α-SMA的影响。我们证明，TGF-β1对α-SMA转录的刺激是通过已知与SRF结合的富含CC(A/T)(6)的GG元件介导的。这些元件也介导了MKL1的作用，MKL1可在无血清培养基中显著激活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表达，无论是否用TGF-β1处理。使用蛋白酶体抑制剂，我们还证明了蛋白水解泛素途径调节MKL1的表达。这些数据表明，MKL1的过表达足以诱导α-SMA的表达。用小干扰RNA抑制MKL1的内源性表达可消除TGF-β1对α-SMA表达的刺激。因此，MKL1也是TGF-β1刺激α-SMA表达所绝对必需的。蛋白质印迹和免疫荧光分析表明，过表达的和内源性的MKL1在未受刺激的RTC中位于细胞核中。染色质免疫沉淀试验表明，TGF-β1可诱导内源性SRF和MKL1与染色质中的α-SMA启动子结合。由于MKL1是调节α-SMA表达的一个有效因子，调节内源性MKL1的表达或活性可能对肾脏中肌成纤维细胞的形成和功能产生深远影响。
