Introduction

The implantation of biomaterials into soft tissue leads to the development of foreign body response, a non-specific inflammatory condition that is characterized by the presence of fibrotic tissue. Epithelial–mesenchymal transition (EMT) is a key event in development, fibrosis, and oncogenesis. Emerging data support a role for both a mechanical signal and a biochemical signal in EMT. We hypothesized that transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive channel, is a mediator of EMT.

Methods

Normal human primary epidermal keratinocytes (NHEKs) were seeded on collagen-coated plastic plates or varied stiffness polyacrylamide gels in the presence or absence of TGFβ1. Immunofluorescence, immunoblot, and polymerase chain reaction analysis were performed to determine expression level of EMT markers and signaling proteins. Knock-down of TRPV4 function was achieved by siRNA transfection or by GSK2193874 treatment.

Results

We found that knock-down of TRPV4 blocked both matrix stiffness- and TGFβ1-induced EMT in NHEKs. In a murine skin fibrosis model, TRPV4 deletion resulted in decreased expression of the mesenchymal marker, α-SMA, and increased expression of epithelial marker, E-cadherin. Mechanistically, our data showed that: (i) TRPV4 was essential for the nuclear translocation of TAZ in response to matrix stiffness and TGFβ1; (ii) Antagonism of TRPV4 inhibited both matrix stiffness-induced and TGFβ1-induced expression of TAZ proteins; and (iii) TRPV4 antagonism suppressed both matrix stiffness-induced and TGFβ1-induced activation of Smad2/3, but not of AKT.

Conclusions

These data identify a novel role for TRPV4-TAZ mechanotransduction signaling axis in regulating EMT in NHEKs in response to both matrix stiffness and TGFβ1.

中文翻译：

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基质刚度和 TGFβ1 诱导的上皮-间质转化中的 TRPV4-TAZ 机械转导信号轴。

介绍

将生物材料植入软组织会导致异物反应的发展，这是一种以存在纤维化组织为特征的非特异性炎症状况。上皮-间质转化（EMT）是发育、纤维化和肿瘤发生的关键事件。新兴数据支持机械信号和生化信号在 EMT 中的作用。我们假设瞬时受体电位香草素 4 (TRPV4) 是一种机械敏感通道，是 EMT 的介质。

方法

在存在或不存在 TGFβ1 的情况下，将正常人原代表皮角质形成细胞 (NHEK) 接种在胶原涂层塑料板上或不同硬度的聚丙烯酰胺凝胶上。进行免疫荧光、免疫印迹和聚合酶链反应分析以确定 EMT 标记和信号蛋白的表达水平。TRPV4 功能的敲低是通过 siRNA 转染或 GSK2193874 处理实现的。

结果

我们发现 TRPV4 的敲除阻断了 NHEK 中基质刚度和 TGFβ1 诱导的 EMT。在小鼠皮肤纤维化模型中，TRPV4 缺失导致间充质标志物 α-SMA 的表达降低，而上皮标志物 E-钙粘蛋白的表达增加。从机制上讲，我们的数据表明：（i）TRPV4 对 TAZ 的核转位是必不可少的，以响应基质刚度和 TGFβ1；(ii) TRPV4 的拮抗作用抑制基质刚度诱导和 TGFβ1 诱导的 TAZ 蛋白表达；(iii) TRPV4 拮抗抑制基质刚度诱导和 TGFβ1 诱导的 Smad2/3 激活，但不抑制 AKT。

结论

这些数据确定了 TRPV4-TAZ 机械转导信号轴在调节 NHEK 中 EMT 以响应基质刚度和 TGFβ1 的新作用。