Department of Internal Medicine, Division of Cardiovascular Health and Disease, University of Cincinnati College of Medicine, Cincinnati, OH, United States of America; Department of Pharmacology & Systems Physiology, University of Cincinnati College of Medicine, Cincinnati, OH, United States of America.
Department of Internal Medicine, Division of Cardiovascular Health and Disease, University of Cincinnati College of Medicine, Cincinnati, OH, United States of America.
J Mol Cell Cardiol. 2023 Jan;174:38-46. doi: 10.1016/j.yjmcc.2022.10.007. Epub 2022 Nov 11.
Cardiac fibrosis is regulated by the activation and phenotypic switching of quiescent cardiac fibroblasts to active myofibroblasts, which have extracellular matrix (ECM) remodeling and contractile functions which play a central role in cardiac remodeling in response to injury. Here, we show that expression and activity of the RNA binding protein HuR is increased in cardiac fibroblasts upon transformation to an active myofibroblast. Pharmacological inhibition of HuR significantly blunts the TGFβ-dependent increase in ECM remodeling genes, total collagen secretion, in vitro scratch closure, and collagen gel contraction in isolated primary cardiac fibroblasts, suggesting a suppression of TGFβ-induced myofibroblast activation upon HuR inhibition. We identified twenty-four mRNA transcripts that were enriched for HuR binding following TGFβ treatment via photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP). Eleven of these HuR-bound mRNAs also showed significant co-expression correlation with HuR, αSMA, and periostin in primary fibroblasts isolated from the ischemic-zone of infarcted mouse hearts. Of these, WNT1-inducible signaling pathway protein-1 (Wisp1; Ccn4), was the most significantly associated with HuR expression in fibroblasts. Accordingly, we found Wisp1 expression to be increased in cardiac fibroblasts isolated from the ischemic-zone of mouse hearts following ischemia/reperfusion, and confirmed Wisp1 expression to be HuR-dependent in isolated fibroblasts. Finally, addition of exogenous recombinant Wisp1 partially rescued myofibroblast-induced collagen gel contraction following HuR inhibition, demonstrating that HuR-dependent Wisp1 expression plays a functional role in HuR-dependent MF activity downstream of TGFβ. In conclusion, HuR activity is necessary for the functional activation of primary cardiac fibroblasts in response to TGFβ, in part through post-transcriptional regulation of Wisp1.
心肌纤维化是由静止的心肌成纤维细胞向活跃的肌成纤维细胞的激活和表型转换所调节的，肌成纤维细胞具有细胞外基质（ECM）重塑和收缩功能，在损伤后心脏重构中起着核心作用。在这里，我们发现 RNA 结合蛋白 HuR 的表达和活性在心肌成纤维细胞向活跃的肌成纤维细胞转化时增加。HuR 的药理学抑制显著减弱 TGFβ 依赖性 ECM 重塑基因、总胶原分泌、体外划痕闭合和分离的原代心肌成纤维细胞中胶原凝胶收缩的增加，表明 HuR 抑制抑制 TGFβ 诱导的肌成纤维细胞激活。我们通过光活化核碱基增强交联和免疫沉淀（PAR-CLIP）鉴定了在 TGFβ 处理后与 HuR 结合的二十四个 mRNA 转录本。这 11 个 HuR 结合的 mRNA 也与原代纤维母细胞中 TGFβ 诱导的肌成纤维细胞激活的αSMA 和periostin 有显著的共表达相关性。在从缺血性梗死小鼠心脏缺血区分离的原代成纤维细胞中，其中 WNT1 诱导信号通路蛋白-1（Wisp1; Ccn4）与 HuR 的相关性最显著。因此，我们发现缺血/再灌注后从缺血性梗死小鼠心脏缺血区分离的心肌成纤维细胞中 Wisp1 的表达增加，并证实了在分离的成纤维细胞中，Wisp1 的表达依赖于 HuR。最后，外源性重组 Wisp1 的添加部分挽救了 HuR 抑制后肌成纤维细胞诱导的胶原凝胶收缩，表明 HuR 依赖性 Wisp1 表达在 TGFβ 下游的 HuR 依赖性 MF 活性中发挥功能作用。总之，HuR 活性对于 TGFβ 刺激下原代心肌成纤维细胞的功能激活是必需的，部分原因是通过 Wisp1 的转录后调节。
