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

Tension Measurements

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

Cell Proliferation

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 pathway^{10 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.

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Figure 1.

Effects of TAT-C3. Time course of the inhibitory action of TAT-C3 (50 μg/mL) on actin cytoskeleton organization of aortic SMCs (A) and NA-induced contraction (B) recorded in thoracic aorta (▴) and pulmonary artery (▪). Contraction induced by 60 mmol/L KCl (○) was not inhibited. C3 (50 μg/mL) did not inhibit NA-induced contraction (•). C, Concentration response for the inhibitory action of TAT-C3 on the aortic SMC actin cytoskeleton organization. D, Western blotting for RhoA in aortic SM treated (+) or untreated (−) with TAT-C3 (50 μg/mL for 5 hours).

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.

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Figure 2.

hU-II induced contraction and RhoA activation. A, Traces showing the contracting effect of hU-II and the inhibition by TAT-C3 in pulmonary artery. B, Concentration-response curves for effect of hU-II in the absence (▪) and presence of 50 μg/mL TAT-C3 (•). C, Representative blot showing the RhoA increase in the pellet fraction of hU-II–stimulated aortic SM. D, Concentration-response curves showing inhibitory effect of Y-27632 on hU-II (0.1 μmol/L)–induced tension under control condition (•) or after treatment with 2 μmol/L thapsigargin and 20 μmol/L D600 (▾) in thoracicaorta.

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.

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Figure 3.

hU-II (0.1 μmol/L) induced actin cytoskeleton organization and proliferation in aortic SMCs. A, F-actin staining in aortic SMCs under control conditions or stimulated with hU-II alone and in the presence of Y-27632 or TAT-C3. B, hU-II– induced increase in F- to G-actin ratio is inhibited by 10 μmol/L Y-27632 or 50 μg/mL TAT-C3. C, hU-II stimulated cell proliferation (top) and DNA synthesis (bottom). Cells were cultured in the absence (control) or presence of hU-II, with or without Y-27632 (10 μmol/L) or TAT-C3 (10 μg/mL) for 48 hours (white column) or 72 hours (black column). Cell number and BrdU-positive cells were expressed as percentage of control. Cells cultured in 10% FCS were used as controls.

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.