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Abstract：
During the development of pleural fibrosis, pleural mesothelial cells (PMCs) undergo phenotypic switching from differentiated mesothelial cells to mesenchymal cells (MesoMT). Here, we investigated how external stimuli such as TGF-β induce HPMC-derived myofibroblast differentiation to facilitate the development of pleural fibrosis. TGF-β significantly increased di-phosphorylation but not mono-phosphorylation of myosin II regulatory light chain (RLC) in HPMCs. An increase in RLC di-phosphorylation was also found at the pleural layer of our carbon black bleomycin (CBB) pleural fibrosis mouse model, where it showed filamentous localization that coincided with alpha smooth muscle actin (αSMA) in the cells in the pleura. Among the protein kinases that can phosphorylate myosin II RLC, ZIPK (zipper-interacting kinase) protein expression was significantly augmented after TGF-β stimulation. Furthermore, ZIPK gene silencing attenuated RLC di-phosphorylation, suggesting that ZIPK is responsible for di-phosphorylation of myosin II in HPMCs. Although TGF-β significantly increased the expression of ZIP kinase protein, the change in ZIP kinase mRNA was marginal, suggesting a posttranscriptional mechanism for the regulation of ZIP kinase expression by TGF-β. ZIPK gene knockdown (KD) also significantly reduced TGF-β-induced upregulation of αSMA expression. This finding suggests that siZIPK attenuates myofibroblast differentiation of HPMCs. siZIPK diminished TGF-β-induced contractility of HPMCs consistent with siZIPK-induced decrease in the di-phosphorylation of myosin II RLC. The present results implicate ZIPK in the regulation of the contractility of HPMC-derived myofibroblasts, phenotype switching, and myofibroblast differentiation of HPMCs.NEW & NOTEWORTHY Here, we highlight that ZIP kinase is responsible for di-phosphorylation of myosin light chain, which facilitates stress fiber formation and actomyosin-based cell contraction during mesothelial to mesenchymal transition in human pleural mesothelial cells. This transition has a significant impact on tissue remodeling and subsequent stiffness of the pleura. This study provides insight into a new therapeutic strategy for the treatment of pleural fibrosis.
摘要：
在胸膜纤维化的发展过程中，胸膜间皮细胞(PMCs)经历从分化的间皮细胞到间充质细胞(MesoMT)的表型转换。这里,我们研究了TGF-β等外界刺激如何诱导HPMC衍生的肌成纤维细胞分化以促进胸膜纤维化的发展。TGF-β显着增加HPMC中肌球蛋白II调节轻链（RLC）的双磷酸化而不是单磷酸化。在我们的炭黑博来霉素（CBB）胸膜纤维化小鼠模型的胸膜层也发现了RLC双磷酸化的增加，其中显示丝状定位，与胸膜细胞中的αSMA重合。在可以磷酸化肌球蛋白IIRLC的蛋白激酶中，TGF-β刺激后，ZIPK（拉链相互作用激酶）蛋白表达显着增强。Further,ZIPK基因沉默减弱RLC双磷酸化，表明ZIPK负责HPMC中肌球蛋白II的双磷酸化。而TGF-β显著增加ZIP激酶蛋白的表达,ZIP激酶mRNA的变化是边缘的，提示TGF-β调节ZIP激酶表达的转录后调节机制。ZIPK基因敲低(KD)也显著降低TGF-β诱导的αSMA表达上调。这一发现表明ZIPKKD减弱MesoMT。ZIPKKD降低TGF-β诱导的HPMC收缩性，与ZIPKKD诱导的肌球蛋白IIRLC双磷酸化降低一致。本结果提示ZIPK参与HPMC衍生的肌成纤维细胞的收缩性调节，经由MesoMT的HPMC的表型转换和肌成纤维细胞分化。
