Zhang Baifang, Peng Fangfang, Wu Dongcheng, Ingram Alistair J, Gao Bo, Krepinsky Joan C
Division of Nephrology, McMaster University, and St. Joseph's Hospital, Hamilton, Ontario, Canada.
Cell Signal. 2007 Aug;19(8):1690-700. doi: 10.1016/j.cellsig.2007.03.005. Epub 2007 Mar 19.
Increased glomerular hydrostatic pressure is an important determinant of glomerulosclerosis and can be modeled in vitro by exposure of mesangial cells (MC) to cyclic mechanical strain. We have recently shown that Akt mediates the stretch-induced production of type I collagen, an important contributor to sclerosis, in MC. Here we studied the upstream mediators of Akt activation. Primary rat MC were exposed to 1 Hz cyclic strain for 10 min, previously shown to induce maximal Akt activation. Neither the integrin inhibitor GRDGSP nor cytoskeletal disruptors had any effect on stretch-induced Akt activation. Akt activation was, however, mediated by transactivation of the epidermal growth factor receptor (EGFR), and this required receptor kinase activity since Akt activation did not occur in cells expressing kinase-dead EGFR (K721A). Src was further shown to be upstream of the EGFR, with its inhibitor SU6656 preventing both EGFR and Akt activation. The membrane microdomains caveolae were found to be required for this signaling to occur. Chemical disruption of caveolae with cyclodextrin or filipin prevented Akt activation, and both EGFR and Akt activation were lost in caveolin-1 (cav-1) knockout MC. The latter was rescued with reexpression of cav-1. Further, Src-mediated phosphorylation of cav-1 on Y14 was required for stretch-induced EGFR and Akt activation, since these were abrogated in MC expressing the nonphosphorylatable cav-1 Y14A mutant. Thus, mechanical strain-induced activation of Akt in MC is independent of integrin activation and the actin cytoskeleton, but depends upon EGFR transactivation. EGFR transactivation requires intact caveolae and the Src-mediated phosphorylation of cav-1 on Y14. These studies define a novel function for cav-1 and caveolae in EGFR transactivation leading to Akt activation by mechanical stress.
肾小球静水压力升高是肾小球硬化的一个重要决定因素，并且可以通过体外将系膜细胞（MC）暴露于周期性机械应变来模拟。我们最近发现，Akt介导了MC中I型胶原蛋白的拉伸诱导产生，而I型胶原蛋白是导致硬化的一个重要因素。在此，我们研究了Akt激活的上游介质。将原代大鼠MC暴露于1Hz的周期性应变10分钟，先前已证明这会诱导最大程度的Akt激活。整合素抑制剂GRDGSP和细胞骨架破坏剂均对拉伸诱导的Akt激活没有任何影响。然而，Akt激活是由表皮生长因子受体（EGFR）的反式激活介导的，并且这需要受体激酶活性，因为在表达激酶失活的EGFR（K721A）的细胞中不会发生Akt激活。进一步表明，Src在EGFR的上游，其抑制剂SU6656可阻止EGFR和Akt激活。发现膜微区小窝是该信号传导发生所必需的。用环糊精或菲律宾菌素对小窝进行化学破坏可阻止Akt激活，并且在小窝蛋白-1（cav-1）基因敲除的MC中EGFR和Akt激活均丧失。通过重新表达cav-1可挽救后者。此外，拉伸诱导的EGFR和Akt激活需要Src介导的cav-1在Y14位点的磷酸化，因为在表达不可磷酸化的cav-1 Y14A突变体的MC中这些激活被消除。因此，机械应变诱导的MC中Akt激活独立于整合素激活和肌动蛋白细胞骨架，但依赖于EGFR反式激活。EGFR反式激活需要完整的小窝以及Src介导的cav-1在Y14位点的磷酸化。这些研究定义了cav-1和小窝在EGFR反式激活中导致机械应激激活Akt的新功能。
