Human Urotensin II–Induced Contraction and Arterial Smooth Muscle Cell Proliferation Are Mediated by RhoA and Rho-Kinase

Abstract

The aim of this work was to investigate the coupling of human urotensin II (hU-II) to RhoA activation and regulation of RhoA-dependent functions. The use of the Rho-kinase inhibitor Y-27632 and the development of a membrane-permeant RhoA inhibitor (TAT-C3) allowed us to demonstrate that hU-II induced arterial smooth muscle contraction, actin stress fiber formation, and proliferation through the activation of the small GTPase RhoA and its downstream effector Rho-kinase.T he human homologue of the fish dodecapeptide urotensin II (hU-II) has been recently cloned. 1 Prepro-U-II mRNA was highly expressed in spinal cord but also found in the adrenal glands, kidney, and spleen. 1,2 hU-II-like immunoreactivity was detected in the vasculature and a diffuse staining was observed in the heart. 3 hU-II induced vasoconstriction of arteries from both rat and human. [3][4][5] With a potency Ϸ6-to 28-fold greater than endothelin-1 in nonhuman primate arteries, hU-II is the most potent mammalian vasoconstrictor identified so far. 3 hU-II has been defined as the ligand for the orphan receptor GPR14, 2,3 predominantly expressed in cardiovascular tissues. 3 Recombinant GPR14 coupled to Ca 2ϩ mobilization, and hU-II has been reported to produce a phospholipase C-dependent increase in inositol phosphates. 6 However, the intracellular signaling pathways of hU-II are not fully established.The small GTPase RhoA is now recognized as a major regulator of smooth muscle (SM) contraction involved in the control of arterial tone. 7 Thus, we postulate that hU-II should activate RhoA and regulate RhoA-dependent functions in vascular smooth muscle cells (SMCs). Materials and Methods Tension MeasurementsWistar rats (Janvier, France) were stunned and then killed by cervical dislocation. Isometric tension of endothelium-denuded arterial rings of thoracic aorta from the 2-cm portion proximal to the carotid bifurcation and pulmonary artery was measured as previously described. 8 Measurement of RhoA DistributionEndothelium-denuded aortic rings were stimulated with 0.1 mol/L hU-II. When maximal tension was raised, rings were rapidly frozen in liquid nitrogen then homogenized in lysis buffer. Membrane and cytosolic fractions were prepared and analyzed by Western blot using a mouse monoclonal anti-RhoA antibody (Santa Cruz Biotechnology) as previously described. 8 All experiments were approved by the local ethics committee. Plasmid Constructions and TAT-C3 Protein PurificationcDNA encoding for Clostridium botulinum C3 exoenzyme was cloned in frame, in the C-terminal of the HIV TAT protein transduction domain (AA 47-57) in vector pTAT-HA (kindly provided by S. Dowdy, Washington University, St. Louis, Mo). 9 Recombinant TAT-C3 protein was produced in Escherichia coli and purified as previously described. 9 SMC Culture and Actin StainingRat SMCs from the proximal segment of thoracic aorta were isolated by enzymatic dissociation and cultured as previously described. 8 Polymerized (F) actin was stained with FITC-conjugated phalloidin (5 g/mL) and Texas Red-la...

Urotensin‐II regulates intracellular calcium in dissociated rat spinal cord neurons

“…The peptide induced an increase of [Ca 2+ ] i in 68% of the cells investigated, suggesting the existence of different neuronal subpopulations with respect to U-II sensitivity. The estimated EC 50 of 6.2 · 10 )9 M is close to that reported for activation of contraction and Rho-kinase in arterial smooth muscle (Sauzeau et al 2001). Modulation of [Ca 2+ ] i by U-II in the same neurons where it is produced suggests the possibility that the peptide might contribute to an autocrine controlling system of motoneuron functions, as has been described for calcitonin gene-related peptide on motoneurons (Skofitsch and Jacobowitz 1985).…”

“…The finding that cAMP is the major second messenger system activated by U-II in neurons, as in the case of plasma membrane Ca 2+ fluxes, suggests that U-II activates a variety of signal transduction mechanisms that are target cell/tissue specific. For example, U-II activates protein kinase C in vascular smooth muscle (Sauzeau et al 2001), and protein kinase A cascade in spinal neurons, as shown here.…”

“…From the experiments using specific inhibitors, we concluded that increases in [Ca 2+ ] i induced by U-II are mainly mediated by Ca 2+ influx through N-type Ca 2+ channels. In the arterial smooth muscle, 60% of the contractile effect is associated with Ca 2+ release from intracellular stores and Ca 2+ influx through L-type Ca 2+ channels (Sauzeau et al 2001). These differences suggest that U-II receptors may couple to different Ca 2+ channels in vascular smooth muscle and motoneurons.…”

“…The cyclic peptide has been shown to be a potent vascular constricting and proliferative agent Sauzeau et al 2001), but it also has relaxing properties in resistance arteries (Bottrill et al 2000). In addition, U-II is involved in cardiac remodeling (Zhang et al 2002), acting synergistically with oxidized low-density lipoprotein (LDL; Watanabe et al 2001).…”

C3 exoenzymes, novel insights into structure and action of Rho-ADP-ribosylating toxins

“…Several studies have shown that the Cterminal fusion of short transport peptide sequences enhances the cellular uptake of C3 (Sauzeau et al 2001;Park et al 2003;Winton et al 2002;Sahai and Olson 2006). In many studies, the C3 gene was introduced into eukaryotic target cells by transient and stable transfection using plasmids or by viral infection (Fujisawa et al 1998;Hill et al 1995;Caron and Hall 1998;Henning et al 1997;Meacci et al 1999;Genot et al 1996).…”