512: Involvement of Rho-Kinase in the Contractile Mechanism of Human Ureteral Smooth Muscle

It is clear that RhoA activates the DRF (diaphanous-related formin) mDia2 by disrupting the molecular interaction between the DAD (diaphanous autoregulatory domain) and the DID (diaphanous inhibitory domain). Previous studies indicate that a basic motif within the DAD contributes to mDia2 auto-inhibition, and results shown in the present study suggest these residues bind a conserved acidic region within the DID. Furthermore, we demonstrate that mDia2 is phosphorylated by ROCK (Rho-kinase) at two conserved residues (Thr1061 and Ser1070) just C-terminal to the DAD basic region. Phosphomimetic mutations to these residues in the context of the full-length molecule enhanced mDia2 activity as measured by increased actin polymerization, SRF (serum response factor)-dependent smooth muscle-specific gene transcription, and nuclear localization of myocardin-related transcription factor B. Biochemical and functional data indicate that the T1061E/S1070E mutation significantly inhibited the ability of DAD to interact with DID and enhanced mDia2 activation by RhoA. Taken together, the results of the present study indicate that ROCK-dependent phosphorylation of the mDia2 DAD is an important determinant of mDia2 activity and that this signalling mechanism affects actin polymerization and smooth muscle cell-specific gene expression.

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EFFECTS OF RHO-KINASE INHIBITION ON ADRENERGIC AND CALCIUM SENSITIVITY IN VASCULAR SMOOTH MUSCLE ASSOCIATED WITH DIABETES MELLITUS: PP.17.135

Journal of Hypertension ◽

10.1097/01.hjh.0000379061.77394.3a ◽

2010 ◽

Vol 28 ◽

pp. e290-e291

Author(s):

I Kizub ◽

C Jonson ◽

O Pavlova ◽

A Soloviev ◽

A Zholos

Keyword(s):

Diabetes Mellitus ◽

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Rho Kinase ◽

Calcium Sensitivity ◽

Kinase Inhibition

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Abstract 641: Expression Of GLUT4 In Vascular Smooth Muscle Affects Endothelial Function In Hypertension.

Hypertension ◽

10.1161/hyp.62.suppl_1.a641 ◽

2013 ◽

Vol 62 (suppl_1) ◽

Author(s):

Kevin B Atkins ◽

Jharna Saha ◽

Frank C Brosius

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Kinase Inhibitor ◽

Rho Kinase ◽

Interesting Phenomenon ◽

Glut4 Expression ◽

Endothelial Denudation ◽

Rho Kinase Inhibitor ◽

Total Body ◽

Rats And Mice

Expression of GLUT4 is decreased in arterial smooth muscle of hypertensive rats and mice, and total body overexpression of GLUT4 in mice prevents enhanced arterial reactivity. To demonstrate that the effect on vascular response to GLUT4 overexpression is vascular rather than systemic in origin we utilized smooth muscle-specific GLUT4 transgenic mice (SMG4). GLUT4 expression in aortae of SMG4 compared to WT mice was increased 2-3 fold. Adult wild-type (WT) and SMG4 mice were made hypertensive or not through implantation of angiotensin II (AngII; 1.4mg/kg/d for 2 wks) or vehicle containing osmotic mini-pumps. Both WT and SMG4 mice AngII-treated mice exhibited significantly increased systolic blood pressure. In AngII-treated WT mice (WT-AngII) aortic GLUT4 expression was significantly decreased, whereas GLUT4 expression in aortae of AngII-treated SMG4 mice (SMG4-AngII) was maintained. The phosphorylation of ERM and MYPT1(Thr850) were significantly increased in aortae of WT-AngII compared to WT-Sham and SMG4-AngII mice. Responsiveness to the contractile agonists, phenylephrine, 5-HT, and PGF 2 was significantly increased in endothelium-intact aortic rings from WT-AngII mice, but remained normal in aortae of SMG4-AngII mice. Following pretreatment with Rho-kinase inhibitor Y-27632, relative inhibition of contractility to 5-HT was equal in aortae from WT-AngII and SMG4-AngII-treated mice. With endothelial denudation, contractility to 5-HT was equally enhanced in aortae of WT-AngII and SMG4-AngII-treated mice. Interestingly, whereas acetylcholine stimulated relaxation was significantly decreased in aortic rings of WT-AngII mice, relaxation in rings from SMG4-AngII mice was not significantly different from WT or SMG4. These results demonstrate an interesting phenomenon whereby decreased expression of GLUT4 in vascular smooth muscle leads to an endothelial dysfunction that not only impairs relaxation, but also enhances contractility.

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Urokinase-induced migration of human vascular smooth muscle cells requires coupling of the small GTPases RhoA and Rac1 to the Tyk2/PI3-K signalling pathway

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613478 ◽

2003 ◽

Vol 89 (05) ◽

pp. 904-914 ◽

Cited By ~ 35

Author(s):

Natalia Tkachuk ◽

Hermann Haller ◽

Inna Dumler ◽

Ioulia Kiian

Keyword(s):

Smooth Muscle ◽

Cell Migration ◽

Vascular Smooth Muscle ◽

Kinase Inhibitor ◽

Rho Kinase ◽

Small Gtpases ◽

Janus Kinase ◽

Immunocytochemical Staining ◽

Boyden Chamber ◽

Dependent Manner

SummaryUrokinase-type plasminogen activator (uPA) facilitates cell migration by localizing proteolisys on the cell surface and by inducing intracellular signalling pathways. In human vascular smooth muscle cell (VSMC) uPA stimulates migration via the uPA receptor (uPAR) signalling complex containing the Janus kinase Tyk2 and phosphatidylinositol 3-kinase (PI3-K). We report that active GTP-bound forms of small GTPases RhoA and Rac1, but not Cdc42, are directly associated with Tyk2 and PI3-K in an uPA/uPAR-dependent fashion. Endogenous RhoA, but not Rac1 or Cdc42, was significantly activated in response to uPA. RhoA activation was abolished by cell treatment with two unrelated, structurally distinct, specific inhibitors of PI3-K, wortmannin, and LY294002. Downstream of RhoA, phosphorylation of myosin light chain (MLC) was dramatically upregulated by uPA in a Rho kinase- and PI3-K-dependent manner. Thus, selective Rho kinase inhibitor Y27632 and PI3-K inhibitors wortmannin and LY294002 prevented the uPA-induced stimulation of MLC phosphorylation. Rho kinase inhibition also decreased uPA-stimulated VSMC migration as observed in a Boyden chamber. VSMC immunocytochemical staining demonstrated redistribution of RhoA and Rac1 active forms to the newly formed leading edge of migrating cell. VSMC microinjection with antibodies to either Rho or Rac1 decreased uPA-stimulated cell migration, indicating the involvement of both GTPases in the migration process. Our results provide evidence that the small GTPases RhoA and Rac1, together with Rho kinase, are necessary to mediate the uPA/uPAR-directed migration via the Tyk2/PI3-K signalling complex in human VSMC.

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