Human Urotensin II–Induced Contraction and Arterial Smooth Muscle Cell Proliferation Are Mediated by RhoA and Rho-Kinase
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.
The 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 ≈6- 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 Ca2+ 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
Wistar 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 Distribution
Endothelium-denuded aortic rings were stimulated with 0.1 μmol/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 Purification
cDNA 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 Staining
Rat 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 μg/mL) and Texas Red–labeled DNase I (10 μg/mL) was used to label monomeric G-actin. The ratio of fluorescence (F- to G-actin ratio) was used to quantify actin cytoskeleton organization as previously described.8
Proliferation was assessed by counting and 5-bromo-2′-deoxyuridine (BrdU) labeling (Roche Diagnostics) of SMCs incubated with or without hU-II, in the presence or absence of RhoA and Rho-kinase inhibitors for 48 hours or 72 hours.
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
hU-II–Induced Contraction Involves RhoA and Rho-Kinase Activation
The recombinant TAT-C3 protein was used to analyze the involvement of Rho GTPase and the effect of hU-II in arterial tissues. TAT-C3 (50 μg/mL) induced a time-dependent disassembly of actin stress fibers in aortic SMCs (Figure 1A⇓) and a time-dependent inhibition of the noradrenaline (NA)-induced contraction of aortic rings, known to involve the RhoA/Rho-kinase pathway10 11 (Figure 1B⇓). C3 exoenzyme had no effect on NA-induced contraction of aortic rings. In addition, TAT-C3 did not modify the KCl-induced contraction, indicating that its inhibitory effect did not result from a nonspecific inhibition of contractile process. TAT-C3 inhibited actin organization with a concentration yielding a half-maximal effect (IC50) of 2.3 μg/mL (Figure 1C⇓). The inhibition of NA-induced contraction displayed a similar concentration dependency (not shown). The decrease in the electrophoretic mobility of RhoA detected by Western blot analysis12 indicated that TAT-C3 had efficiently ADP-ribosylated RhoA in arterial rings (Figure 1C⇓). Therefore, the involvement of RhoA and the effect of hU-II have been assessed in arterial rings treated for 5 hours with TAT-C3 (50 μg/mL) before hU-II stimulation.
Stimulation of endothelium-denuded arterial rings with hU-II induced a dose-dependent rise in tension (Figure 2A⇓). The maximal hU-II–induced tension corresponded to 91.2±3.9% (n=11) and 58.7±5.5% (n=5) of the phenylephrine (1 μmol/L)–induced contraction recorded in thoracic aorta and pulmonary artery, respectively. The half-maximal effect of hU-II was obtained at 4.7 nmol/L and 9.3 nmol/L in pulmonary artery (Figure 2B⇓) and in aorta (not shown), respectively. TAT-C3 (50 μg/mL, 5 hours) inhibited the hU-II–induced rise in tension (Figures 2A⇓ and 2B⇓), suggesting that hU-II induced activation of RhoA. The stimulation with 0.1 μmol/L hU-II increased the amount of RhoA in the pellet fraction, attesting its activation (Figure 2C⇓). The downstream RhoA effector Rho-kinase inhibitor Y-27632 dose-dependently inhibited the hU-II (0.1 μmol/L)–induced contraction with IC50 of 1.9 μmol/L (Figure 2D⇓). To further analyze the Rho-kinase–sensitive component, hU-II–induced contraction was measured in the presence of the voltage-gated Ca2+ channel inhibitor methoxyverapamil (D600) and the Ca2+ store–depleting agent thapsigargin (TSG) to suppress the agonist-induced rise in [Ca2+]i.8 The TSG/D600-resistant component of hU-II–induced contraction was dose-dependently inhibited by Y-27632 with IC50 value of 0.92 μmol/L. These results suggest that hU-II–induced contraction involved both a rise in [Ca2+]i and Ca2+ sensitization of the contractile apparatus through the activation of RhoA and Rho-kinase. However, the complete inhibition of the contraction by Y-27632 suggests that when Ca2+ sensitization was prevented, the U-II–induced [Ca2+]i rise was not sufficient to produce increase in tension.
U-II Induces Actin Cytoskeleton Organization in Aortic SMCs
hU-II induced stimulation of actin stress fiber formation in aortic SMCs (Figure 3A⇓) that corresponded to a 2.5±0.1-fold increase (n=4) in the F- to G-actin ratio (Figure 3B⇓), attributable to an increase in F-actin and a concomitant decrease in G-actin (online Figure 1⇑; see data supplement available at http://www.circresaha.org). This effect was inhibited by treatment with TAT-C3 or Y-27632 (Figures 3A⇓ and 3B⇓) indicating that hU-II controls actin cytoskeleton organization via the RhoA/Rho-kinase–dependent pathway.
U-II Stimulates Aortic SMC Proliferation
Figure 3C⇑ shows that hU-II stimulated aortic SMC proliferation determined by cell counting and DNA synthesis assessed by BrdU assay. The hU-II–induced SMC proliferation was inhibited by TAT-C3 or Y-27632 indicating that RhoA and Rho-kinase mediate the stimulation of vascular SMC growth.
The present study demonstrates that hU-II activates the small GTPase RhoA and its target Rho-kinase in arterial SM. Activation of the RhoA/Rho-kinase–dependent signaling pathway is involved in the hU-II–induced contraction, actin cytoskeleton organization, and proliferation of arterial SMCs.
The involvement of RhoA in the contracting effect of hU-II in intact arterial ring has been demonstrated by the use of the fusion protein TAT-C3. The Clostridium botulinum exoenzyme C3 that specifically ADP-ribosylates and inactivates Rho proteins does not easily enter cells and therefore could not be used in intact tissues. In the present report, we show that the transactivation domain of the HIV TAT protein fused to C3 rapidly carries the enzyme into the cells in multicellular preparations. TAT-C3 fusion protein is therefore a useful tool to analyze Rho-dependent signaling in intact tissues. The inhibitory action of Y-27632 indicates that Rho-kinase is the downstream RhoA effector involved in the hU-II–induced contraction. The RhoA/Rho-kinase–dependent contracting effect of hU-II is ascribed to the phosphorylation and the consequent inhibition of the myosin light chain phosphatase, leading to an increased myosin light chain phosphorylation and tension at constant Ca2+ concentration (Ca2+ sensitization).7 This signaling pathway is also likely to be responsible for hU-II–induced actin stress fiber formation.
In addition to its vasoconstrictor effect, we show that hU-II induced arterial SMC proliferation. Mitogenic activity, involving several intracellular mechanisms including extracellular signal–regulated kinase, c-Jun N-terminal kinase, or phosphatidylinositol 3-kinase–dependent signaling pathways, has been demonstrated for other vasoactive peptides such as angiotensin II and endothelin-1.13 14 15 Our results show that (1) hU-II induced vascular SMC proliferation and (2) this effect is mediated by the RhoA/Rho-kinase pathway. RhoA/Rho-kinase signaling has been shown to promote SMC proliferation during neointimal formation through the downregulation of the cyclin-dependent kinase inhibitor p27kip1.16 17 Preliminary results indicate that hU-II induced Y-27632–sensitive downregulation of p27kip1 that is dependent on actin cytoskeleton organization.
The significance of hU-II in cardiovascular regulation has not yet been elucidated. However, diffuse hU-II immunostaining has been observed in coronary atherosclerotic plaque.3 Our results showing that hU-II induced RhoA-dependent vascular SMC proliferation, a phenomenon associated with development of atherosclerosis, provide new clues to the understanding of the functions of hU-II and lead to the hypothesis that it could play a role in the formation of atherosclerotic plaques.
This work is supported by grants from Institut National de la Santé et de la Recherche Médicale (INSERM), Région Pays de Loire, Institut de Recherches Internationales Servier (IRIS), and Action Cibles Thérapeutiques et Médicaments (INSERM-CNRS).
Original received January 19, 2001; resubmission received March 15, 2001; revised resubmission received April 24, 2001; accepted April 24, 2001.
Presented in part at the 73rd Scientific Sessions of the American Heart Association, New Orleans, La, November 12–15, 2000, and published in abstract form (Circulation. 2000;102[suppl II]:II-315).
- © 2001 American Heart Association, Inc.
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