Institute of Cardiovascular Sciences, St. Boniface Hospital Albrechtsen Research Centre, 351 Taché Avenue, Winnipeg, MB, Canada.
Department of Physiology and Pathophysiology, University of Manitoba, Winnipeg, Canada.
Basic Res Cardiol. 2021 Apr 13;116(1):25. doi: 10.1007/s00395-021-00865-9.
We have previously shown that overexpression of SKI, an endogenous TGF-β repressor, deactivates the pro-fibrotic myofibroblast phenotype in the heart. We now show that SKI also functions independently of SMAD/TGF-β signaling, by activating the Hippo tumor-suppressor pathway and inhibiting the Transcriptional co-Activator with PDZ-binding motif (TAZ or WWTR1). The mechanism(s) by which SKI targets TAZ to inhibit cardiac fibroblast activation and fibrogenesis remain undefined. A rat model of post-myocardial infarction was used to examine the expression of TAZ during acute fibrogenesis and chronic heart failure. Results were then corroborated with primary rat cardiac fibroblast cell culture performed both on plastic and on inert elastic substrates, along with the use of siRNA and adenoviral expression vectors for active forms of SKI, YAP, and TAZ. Gene expression was examined by qPCR and luciferase assays, while protein expression was examined by immunoblotting and fluorescence microscopy. Cell phenotype was further assessed by functional assays. Finally, to elucidate SKI's effects on Hippo signaling, the SKI and TAZ interactomes were captured in human cardiac fibroblasts using BioID2 and mass spectrometry. Potential interactors were investigated in vitro to reveal novel mechanisms of action for SKI. In vitro assays on elastic substrates revealed the ability of TAZ to overcome environmental stimuli and induce the activation of hypersynthetic cardiac myofibroblasts. Further cell-based assays demonstrated that SKI causes specific proteasomal degradation of TAZ, but not YAP, and shifts actin cytoskeleton dynamics to inhibit myofibroblast activation. These findings were supported by identifying the bi-phasic expression of TAZ in vivo during post-MI remodeling and fibrosis. BioID2-based interactomics in human cardiac fibroblasts suggest that SKI interacts with actin-modifying proteins and with LIM Domain-containing protein 1 (LIMD1), a negative regulator of Hippo signaling. Furthermore, we found that LATS2 interacts with TAZ, whereas LATS1 does not, and that LATS2 knockdown prevented TAZ downregulation with SKI overexpression. Our findings indicate that SKI's capacity to regulate cardiac fibroblast activation is mediated, in part, by Hippo signaling. We postulate that the interaction between SKI and TAZ in cardiac fibroblasts is arbitrated by LIMD1, an important intermediary in focal adhesion-associated signaling pathways. This study contributes to the understanding of the unique physiology of cardiac fibroblasts, and of the relationship between SKI expression and cell phenotype.
我们之前已经证明，过表达内源性 TGF-β 抑制剂 SKI 可使心脏中的促纤维化肌成纤维细胞表型失活。我们现在表明，SKI 还可以独立于 SMAD/TGF-β 信号通路发挥作用，通过激活 Hippo 肿瘤抑制途径并抑制带有 PDZ 结合基序的转录共激活因子（TAZ 或 WWTR1）。SKI 靶向 TAZ 以抑制心肌成纤维细胞激活和纤维化的机制尚不清楚。使用心肌梗死后大鼠模型来检查 TAZ 在急性纤维化和慢性心力衰竭期间的表达。然后，使用塑料和惰性弹性底物上的原代大鼠心肌成纤维细胞培养物，以及使用 SKI、YAP 和 TAZ 的活性形式的 siRNA 和腺病毒表达载体，对结果进行了验证。通过 qPCR 和荧光素酶测定法检查基因表达，通过免疫印迹和荧光显微镜检查蛋白质表达。通过功能测定进一步评估细胞表型。最后，为了阐明 SKI 对 Hippo 信号传导的影响，使用 BioID2 和质谱法在人心脏成纤维细胞中捕获 SKI 和 TAZ 的相互作用组。在体外研究潜在的相互作用体以揭示 SKI 的新作用机制。在弹性底物上的体外测定表明，TAZ 能够克服环境刺激并诱导超合成心肌成纤维细胞的激活。进一步的细胞测定表明，SKI 导致 TAZ 的特异性蛋白酶体降解，但不导致 YAP，并且改变肌动蛋白细胞骨架动力学以抑制肌成纤维细胞激活。这些发现通过在心肌梗死后重塑和纤维化过程中体内 TAZ 的双相表达得到支持。基于 BioID2 的人心脏成纤维细胞相互作用组学表明，SKI 与肌动蛋白修饰蛋白和 LIM 结构域蛋白 1（LIMD1）相互作用，LIMD1 是 Hippo 信号通路的负调节剂。此外，我们发现 LATS2 与 TAZ 相互作用，而 LATS1 不与 TAZ 相互作用，并且 LATS2 敲低可防止 SKI 过表达时 TAZ 的下调。我们的研究结果表明，SKI 调节心肌成纤维细胞激活的能力部分由 Hippo 信号传导介导。我们假设 SKI 和 TAZ 在心脏成纤维细胞中的相互作用由 LIMD1 介导，LIMD1 是粘着斑相关信号通路中的重要中介物。这项研究有助于理解心肌成纤维细胞的独特生理学，以及 SKI 表达与细胞表型之间的关系。
