We have previously shown that overexpression of SKI, an endogenous TGF-β₁ 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 SKI，一种内源性 TGF- _β1阻遏物，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 通过激活 Hippo 肿瘤抑制途径和抑制具有 PDZ 结合基序（TAZ 或 WWTR1）的转录共激活因子也独立于 SMAD/TGF-β 信号传导发挥作用。SKI 靶向 TAZ 抑制心脏成纤维细胞活化和纤维形成的机制仍未确定。使用心肌梗死后大鼠模型检测急性纤维化和慢性心力衰竭期间 TAZ 的表达。然后，在塑料和惰性弹性基质上进行的原代大鼠心脏成纤维细胞培养，以及使用 siRNA 和腺病毒表达载体用于 SKI、YAP 和 TAZ 的活性形式，证实了结果。通过 qPCR 和荧光素酶测定检查基因表达，而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 和质谱法在人类心脏成纤维细胞中捕获了 SKI 和 TAZ 相互作用组。在体外研究了潜在的相互作用物，以揭示 SKI 的新作用机制。对弹性底物的体外试验揭示了 TAZ 能够克服环境刺激并诱导超合成心脏肌成纤维细胞活化的能力。进一步的基于细胞的测定表明，SKI 导致 TAZ 的特异性蛋白酶体降解，但不是 YAP，并改变肌动蛋白细胞骨架动力学以抑制肌成纤维细胞活化。这些发现得到了在 MI 后重塑和纤维化过程中鉴定 TAZ 在体内的双相表达的支持。人类心脏成纤维细胞中基于 BioID2 的相互作用组学表明，SKI 与肌动蛋白修饰蛋白和含 LIM 结构域的蛋白 1 (LIMD1) 相互作用，LIMD1 是 Hippo 信号传导的负调节因子。此外，我们发现 LATS2 与 TAZ 相互作用，而 LATS1 没有，并且 LATS2 敲低阻止了 SKI 过表达导致 TAZ 下调。我们的研究结果表明，SKI 调节心脏成纤维细胞活化的能力部分是由 Hippo 信号传导介导的。我们假设心脏成纤维细胞中 SKI 和 TAZ 之间的相互作用由 LIMD1 仲裁，LIMD1 是粘着斑相关信号通路中的重要中介。