Epidermal growth factor induces fibroblast contractility and motility via a protein kinase C δ-dependent pathway
Epidermal growth factor induces fibroblast contractility and motility via a protein kinase C δ-dependent pathway
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DOI：
10.1074/jbc.m311981200
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发表时间：
2004-04-09
影响因子：
4.8
通讯作者：
Wells, A
中科院分区：
文献类型：
--
作者：
Iwabu, A;Smith, K;Wells, A
基于肌球蛋白的细胞收缩力被认为是细胞运动的关键过程。然而，对于表皮生长因子（EGF）诱导的成纤维细胞迁移，EGF受体（EGFR）激活和力产生之间的分子联系尚未阐明。在此，我们证明，EGF刺激增加肌球蛋白轻链（MLC）磷酸化，收缩力的标志物，伴随着蛋白激酶C（PKC）的活性在小鼠成纤维细胞表达人EGFR的构建。有趣的是，PKC δ是最强烈的磷酸化亚型，并且优先的PKC δ抑制剂rottlerin在很大程度上阻止了EGF诱导的PKC底物和MARCKS的磷酸化。MEK-1抑制剂U 0126和磷脂酰肌醇3-激酶抑制剂LY 294002对PKC δ激活没有影响，而PLC γ信号传导的缺乏导致延迟的PKC δ激活，这一事实表明EGFR激活PKC δ的途径。EGF增强的MLC磷酸化被特定的MLC激酶抑制剂ML-7和PKC抑制剂chelerythrine chloride和rottlerin阻止。进一步表明PKC δ是必需的，显性负性PKC δ构建体或RNAi介导的PKC δ耗竭也阻止了MLC磷酸化。在PLC信号传导的情况下，MLC磷酸化和细胞力产生延迟类似于PKC δ激活。所有阻断PKC δ激活或MLC磷酸化的干预措施都消除了EGF诱导的细胞收缩力产生和运动。我们的研究结果表明，PKC δ激活是负责EGF诱导的成纤维细胞收缩力的产生的主要部分。因此，我们在这里确定了一个新的途径，有助于管理细胞运动，PLC信号在激活PKC δ，促进EGF诱导的MLC激活的急性期发挥作用。
Myosin-based cell contractile force is considered to be a critical process in cell motility. However, for epidermal growth factor (EGF)-induced fibroblast migration, molecular links between EGF receptor ( EGFR) activation and force generation have not been clarified. Herein, we demonstrate that EGF stimulation increases myosin light chain (MLC) phosphorylation, a marker for contractile force, concomitant with protein kinase C (PKC) activity in mouse fibroblasts expressing human EGFR constructs. Interestingly, PKCdelta is the most strongly phosphorylated isoform, and the preferential PKCdelta inhibitor rottlerin largely prevented EGF-induced phosphorylation of PKC substrates and MARCKS. The pathway through which EGFR activates PKCdelta is suggested by the fact that the MEK-1 inhibitor U0126 and the phosphatidylinositol 3-kinase inhibitor LY294002 had no effect on PKCdelta activation, whereas lack of PLCgamma signaling resulted in delayed PKCdelta activation. EGF-enhanced MLC phosphorylation was prevented by a specific MLC kinase inhibitor ML-7 and the PKC inhibitors chelerythrine chloride and rottlerin. Further indicating that PKCdelta is required, a dominant-negative PKCdelta construct or RNAi-mediated PKCdelta depletion also prevented MLC phosphorylation. In the absence of PLC signaling, MLC phosphorylation and cell force generation were delayed similarly to PKCdelta activation. All of the interventions that blocked PKCdelta activation or MLC phosphorylation abrogated EGF-induced cell contractile force generation and motility. Our results suggest that PKCdelta activation is responsible for a major part of EGF-induced fibroblast contractile force generation. Hence, we identify here a new pathway helping to govern cell motility, with PLC signaling playing a role in activation of PKCdelta to promote the acute phase of EGF-induced MLC activation.