TRPV1 potentiates TGFβ-induction of corneal myofibroblast development through an oxidative stress-mediated p38-SMAD2 signaling loop.
TRPV1 potentiates TGFβ-induction of corneal myofibroblast development through an oxidative stress-mediated p38-SMAD2 signaling loop.
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DOI：
10.1371/journal.pone.0077300
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Reinach PS
中科院分区：
文献类型：
--
作者：
Yang Y;Wang Z;Yang H;Wang L;Gillespie SR;Wolosin JM;Bernstein AM;Reinach PS
碱损伤小鼠角膜引起肌成纤维细胞表达，导致组织混浊。然而，在瞬时受体电位香草样蛋白1通道（TRPV1-/-）敲除小鼠中，愈合导致透明度恢复。由于TGFβ是肌成纤维细胞表型的主要诱导剂，我们研究了TRPV1影响TGFβ诱导的肌成纤维细胞发育的机制。实验分别在猪角膜和人角膜成纤维细胞（HCFs）中进行。采用α-平滑肌肌动蛋白（α-SMA）应激纤维免疫组化染色观察成纤维细胞。Western blotting检测蛋白和磷蛋白。siRNA转染使TRPV1基因表达沉默。用活性氧（ROS）报告染料流式细胞术分析细胞内ROS。通过在HCF中加载fura2来测量[Ca2+]I。在器官培养的角膜中，TRPV1拮抗剂辣椒平可显著减少75%的伤口诱导的肌成纤维细胞发育。在HCF细胞培养中，TGF-β1诱导Ca2+内流快速增加，SMAD2和MAPKs （ERK1/2、JNK1/2和p38）磷酸化，ROS生成，并在72小时后肌成纤维细胞发育。SMAD2和p38的激活持续超过16小时，而p-ERK1/2和p-JNK1/2在90分钟内减弱。长寿命的SMAD2激活依赖于激活的p38，反之亦然，这是产生α-SMA蛋白> 13倍增加和完全发育的肌成纤维细胞表型所必需的。这些后期的变化通过抑制TRPV1或降低ROS生成速率而显著减少。综上所述，我们的研究结果表明，在角膜源性成纤维细胞中，TGFβ-诱导的肌成纤维细胞发育高度依赖于一个正反馈回路，其中p-SMAD2诱导的ROS激活TRPV1， TRPV1引起p38的激活，后者反过来进一步增强SMAD2的激活，从而建立一个循环循环，大大延长了驱动肌成纤维细胞发育的SMAD2激活状态的驻留时间。
Injuring mouse corneas with alkali causes myofibroblast expression leading to tissue opacification. However, in transient receptor potential vanilloid 1 channel (TRPV1-/-) knockout mice healing results in transparency restoration. Since TGFβ is the primary inducer of the myofibroblast phenotype, we examined the mechanism by which TRPV1 affects TGFβ-induced myofibroblast development. Experiments were performed in pig corneas and human corneal fibroblasts (HCFs). Immunohistochemical staining of α-smooth muscle actin (α-SMA) stress fibers was used to visualize myofibroblasts. Protein and phosphoprotein were determined by Western blotting. siRNA transfection silenced TRPV1 gene expression. Flow cytometry with a reactive oxygen species (ROS) reporting dye analyzed intracellular ROS. [Ca2+]I was measured by loading HCF with fura2. In organ cultured corneas, the TRPV1 antagonist capsazepine drastically reduced by 75% wound-induced myofibroblast development. In HCF cell culture, TGF-β1 elicited rapid increases in Ca2+ influx, phosphorylation of SMAD2 and MAPKs (ERK1/2, JNK1/2 and p38), ROS generation and, after 72 hrs myofibroblast development. SMAD2 and p38 activation continued for more than 16 h, whereas p-ERK1/2 and p-JNK1/2 waned within 90 min. The long-lived SMAD2 activation was dependent on activated p38 and vice versa, and it was essential to generate a > 13-fold increase in α-SMA protein and a fully developed myofibroblast phenotype. These later changes were markedly reduced by inhibition of TRPV1 or reduction of the ROS generation rate. Taken together our results indicate that in corneal derived fibroblasts, TGFβ- induced myofibroblast development is highly dependent on a positive feedback loop where p-SMAD2-induced ROS activates TRPV1, TRPV1 causes activation of p38, the latter in turn further enhances the activation of SMAD2 to establish a recurrent loop that greatly extends the residency of the activated state of SMAD2 that drives myofibroblast development.
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DOI：
10.1073/pnas.0902675106
发表时间：
2009-11-24
影响因子：
11.1
作者：
Chuang, Huai-hu;Lin, Stephanie
通讯作者：
Lin, Stephanie
影响因子：
4.8
作者：
Hao, N;Yildirim, N;Dohlman, HG
通讯作者：
Dohlman, HG
影响因子：
4.8
作者：
Jain, Manu;Rivera, Stephanie;Chandel, Navdeep S.
通讯作者：
Chandel, Navdeep S.
DOI：
10.1073/pnas.93.9.4219
发表时间：
1996-04-30
影响因子：
11.1
作者：
Masur, SK;Dewal, HS;Petridou, S
通讯作者：
Petridou, S
影响因子：
3.4
作者：
Jester, JV;Huang, JY;Cavanagh, HD
通讯作者：
Cavanagh, HD