title: [Serum response factor participates in RhoA-induced endothelial cell F-actin rearrangements].
abstract: 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 regulates 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.
authors.affiliation: Department of Cardiology, General Hospital of Shenyang, The Institute of Cardiovascular Research, PLA, Shenyang 110016, China. hanyal@mail.sy.ln.cn
authors.forename: Ya-Ling
authors.initials: YL
authors.lastname: Han
authors.forename: Hai-Bo
authors.initials: HB
authors.lastname: Yu
authors.forename: Cheng-Hui
authors.initials: CH
authors.lastname: Yan
authors.forename: Zi-Min
authors.initials: ZM
authors.lastname: Meng
authors.forename: Xiao-Lin
authors.initials: XL
authors.lastname: Zhang
authors.forename: Jian
authors.initials: J
authors.lastname: Kang
authors.forename: Shao-Hua
authors.initials: SH
authors.lastname: Li
authors.forename: Shi-Wen
authors.initials: SW
authors.lastname: Wang
mesh_terms: D000199:Actins / Q000096:biosynthesis* / Q000235:genetics; D003599:Cytoskeleton / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D026362:Serum Response Factor / Q000096:biosynthesis* / Q000235:genetics; D014471:Umbilical Veins / Q000166:cytology; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000502:physiology*
chemical_list: D000199:Actins; D047908:Intracellular Signaling Peptides and Proteins; D026362:Serum Response Factor; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein