韩雅玲, 于海波, 闫承慧, 孟子敏, 张效林, 康建, 李少华, 王士雯
沈阳军区总医院全军心血管病研究所心内科. 辽宁, 沈阳 110016；Robert Wood Johnson医学院病理实验科. 新泽西州 08854, 美国；解放军总医院老年心血管病研究所. 北京 100853
为进一步阐明RhoA调控人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)肌动蛋白骨架重构的分子机制,用逆转录病毒感染并筛选出稳定表达持续活化型RhoA(Q63LRhoA)和主导抑制型RhoA(T19NRhoA)的HUVECs.应用免疫组化和Western blot方法分析去血清前后HUVECs血清反应因子(serum response factor,SRF)的表达及定位，Rhodamine-Phalloidine染色观察F-actin动态变化。结果显示,Q63LRhoA组细胞核中SRF表达增加,F-actin重排形成大量应力纤维;T19NRhoA组中SRF表达较弱,F-actin无明显改变,无应力纤维形成.去血清后,正常HUVECs(对照组)和感染细胞中SRF的表达均显著增加,但其亚细胞定位明显不同.对照组去血清培养3 d,SRF主要定位在细胞核,去血清培养5 d,SRF出核转位入细胞浆.Q63LRhoA组SRF发生核滞留,不随去血清培养时间延长发生出核转位现象.T19NRhoA组SRF的表达主要定位于细胞核周.对照组去血清培养3 d,F-actin表达增加,同时形成大量应力纤维,去血清培养5 d,细胞F-actin表达下调,应力纤维解聚.Q63LRhoA组F-actin重构持续发生并形成大量应力纤维,但不随去血清培养时间延长发生明显解聚.而T19NRhoA组F-actin表达不随去血清时间延长而增加.上述结果提示,RhoA介导HUVECs F-actin的重构与SRF的核转位现象密切相关。
Serum response factor participates in RhoA-induced endothelial cell F-actin rearrangements
Han Yaling, Yu Haibo, Yan Chenghui, Meng Zimin, Zhang Xiaolin, KANG Jian, Li Shaohua, Wang Shiwen
Department of Cardiology, General Hospital of Shenyang, The Institute of Cardiovascular Research, PLA. Shenyang 110016, Liaoning, China；Department of Pathology, Robert Wood Johnson Medical School. New Jersey 08854, USA；Institute of Geriatric Cardiology, General Hospital of PLA. Beijing 100853, China
RhoA is one of the main members of RhoGTPase family involved in cell morphology, smooth muscle contraction, cytoskeletal microfilaments and stress fiber formation. It has been demonstrated that RhoA modulates endothelial cell permeability by its effect on F-actin rearrangement, but the molecular mechanism of rearrangement of actin cytoskeleton remains unclear. Recent studies prove that RhoA/Rho kinase regulats smooth muscle specific actin dynamics by activating serum response factor (SRF)-dependent transcription. To further investigate the molecular mechanism of the rearrangement of vascular endothelial cell actin cytoskeleton, we explored the relationship between the activation of SRF and F-actin rearrangement induced by RhoA in human umbilical vein endothelial cells (HUVECs). HUVECs were infected with the constitutively active forms of RhoA (Q63LRhoA) or the dominant negative forms of RhoA (T19NRhoA) using retrovirus vector pLNCX-Q63LRhoA or pLNCX-T19NRhoA, the positive clone was obtained by G418 selection. The expression and distribution of SRF in normal and infected cells were evaluated by immunohistochemistry and Western blot in complete medium and in serum-free medium. The effect of F-actin polymerization was detected by Rhodamine-Phalloidine staining. Infection of PLNCX-Q63LRhoA induced F-actin rearrangement and stress fiber formation in HUVECs, as well as enhanced the expression of SRF in the nuclei. In contrast, the cells infected with T19NRhoA showed no distinct changes. With serum deprivation, the expression of SRF increased obviously in both normal and infected HUVECs, but the subcellular localization of SRF was evidently different. In HUVECs, the localization of SRF was in the nuclei after 3 d with serum deprivation, but it was redistributed outside the nuclei after 5 d with serum deprivation. In cells infected with Q63LRhoA, the immunolocalization of SRF was always in the nuclei compared with HUVECs infected with T19NRhoA, which was almost always localized in the cytoplasm. In HUVECs, the rearrangement of F-actin and formation of stress fiber increased after 3 d with serum deprivation, but appeared decreased and unpolymerized after 5 d with serum deprivation. The polymerization of F-actin and the formation of stress fiber in HUVECs infected with Q63LRhoA kept during the period of serum-free culture, whereas the rearrangement of F-actin in cells infected with T19NRhoA was not found. These results suggest that RhoA influences endothelial F-actin rearrangement in part by regulating the expression and subcellular localization of SRF.
韩雅玲, 于海波, 闫承慧, 孟子敏, 张效林, 康建, 李少华, 王士雯. 血清反应因子参与RhoA诱导的人血管内皮细胞肌动蛋白骨架重构[J]. 生理学报 2005; 57 (3): .
Han Yaling, Yu Haibo, Yan Chenghui, Meng Zimin, Zhang Xiaolin, KANG Jian, Li Shaohua, Wang Shiwen. Serum response factor participates in RhoA-induced endothelial cell F-actin rearrangements. Acta Physiol Sin 2005; 57 (3): (in Chinese with English abstract).