Mechanical strain on osteoblasts activates autophosphorylation of focal adhesion kinase and proline-rich tyrosine kinase 2 tyrosine sites involved in ERK activation
Mechanical strain on osteoblasts activates autophosphorylation of focal adhesion kinase and proline-rich tyrosine kinase 2 tyrosine sites involved in ERK activation
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DOI：
10.1074/jbc.m313244200
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发表时间：
2004-07-16
影响因子：
4.8
通讯作者：
Lafage-Proust, MH
中科院分区：
文献类型：
--
作者：
Boutahar, N;Guignandon, A;Lafage-Proust, MH
机械负荷诱导骨形成增加的机制尚不清楚。在这项研究中，我们发现循环应变（CS）（10分钟，1%拉伸，0.25 Hz）刺激了隔夜血清饥饿的ROS 17/2.8成骨样细胞在I型胶原涂层硅胶膜上的增殖。这种增加被MEK抑制剂PD-98059阻断。然后通过Western blotting和共免疫沉淀在一个CS期后的0分钟、30分钟和4小时评估信号事件。CS快速且时间依赖性地促进了tyr1 -187位点ERK2和tyr1 -397和tyr1 -925位点focal adhesion kinase （FAK）的磷酸化，从而激活Ras/Raf/MEK通路。细胞转染tyr3 - 97突变的FAK完全阻断ERK2 tyr1 -187磷酸化。磷酸化酪氨酸残基的定量免疫荧光分析显示，在紧张的细胞中，局灶黏附斑块的数量和大小增加。CS还诱导Src- tir -418磷酸化和Src与FAK的关联。使用选择性Src家族激酶抑制剂pyrazolopy嘧啶2治疗并不能阻止cs诱导的FAK- tyr -397磷酸化，这表明FAK的活化与Src无关。CS还激活了富含脯氨酸的酪氨酸激酶2 (PYK2)，这是一种与FAK高度同源的酪氨酸激酶，位于402磷酸化位点，并以时间依赖性的方式促进其与FAK的关联。Tyr-402位点PYK2突变仅在4小时内阻止ERK2磷酸化。细胞内和细胞外钙螯合剂仅在4小时内阻止PYK2激活。总之，我们的数据表明成骨细胞对有丝分裂CS的反应是通过MEK途径激活介导的。后者是在FAK和PYK2磷酸化的控制下，以一种时间依赖性的方式介导的ERK2磷酸化诱导的。
The mechanisms involved in the mechanical loading-induced increase in bone formation remain unclear. In this study, we showed that cyclic strain (CS) (10 min, 1% stretch at 0.25 Hz) stimulated the proliferation of overnight serum-starved ROS 17/2.8 osteoblast-like cells plated on type I collagen-coated silicone membranes. This increase was blocked by MEK inhibitor PD-98059. Signaling events were then assessed 0 min, 30 min, and 4 h after one CS period with Western blotting and co-immunoprecipitation. CS rapidly and time-dependently promoted phosphorylation of both ERK2 at Tyr-187 and focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, leading to the activation of the Ras/Raf/MEK pathway. Cell transfection with FAK mutated at Tyr-397 completely blocked ERK2 Tyr-187 phosphorylation. Quantitative immunofluorescence analysis of phosphotyrosine residues showed an increase in focal adhesion plaque number and size in strained cells. CS also induced both Src-Tyr-418 phosphorylation and Src to FAK association. Treatment with the selective Src family kinase inhibitor pyrazolopyrimidine 2 did not prevent CS-induced FAK-Tyr-397 phosphorylation suggesting a Src-independent activation of FAK. CS also activated proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase highly homologous to FAK, at the 402 phosphorylation site and promoted its association to FAK in a time-dependent manner. Mutation of PYK2 at the Tyr-402 site prevented the ERK2 phosphorylation only at 4 h. Intra and extracellular calcium chelators prevented PYK2 activation only at 4 h. In summary, our data showed that osteoblast response to mitogenic CS was mediated by MEK pathway activation. The latter was induced by ERK2 phosphorylation under the control of FAK and PYK2 phosphorylation orchestrated in a time-dependent manner.