Essential Role for Class II Phosphoinositide 3-kinase -Isoform in Ca -Induced, Rho- and Rho Kinase-Dependent Regulation of Myosin Phosphatase and Contraction in Isolated Vascular Smooth Muscle Cells

The laser confocal fluorescent microscope-based observation of contractile responses in green fluorescent protein-expressing differentiated vascular smooth muscle cells, combined with the RNA interference-mediated gene-silencing technique, allowed us to determine the role of phosphoinositide 3-kinase (PI3K) class II -isoform (PI3K-C2 ) as a novel, Ca -dependent regulator of myosin light-chain phosphatase (MLCP) and contraction. The Ca -ionophore ionomycin induced a robust contractile response with an increase in the intracellular free Ca concentration ([Ca ]i). The PI3K-C2 -specific short interfering RNA (siRNA) induced a selective and marked reduction in PI3K-C2 protein expression. The siRNA-mediated knockdown of PI3K-C2 , but not class I PI3K p110 , suppressed ionomycin-induced contraction without altering Ca mobilization. PI3K-C2 is uniquely less sensitive to the PI3K inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) than the other PI3K members, including p110 . Ionomycin-induced contraction was inhibited only by a relatively high concentration of LY294002. Consistent with our previous observations showing that ionomycin and membrane depolarization induced Rho activation in vascular smooth muscle tissues in a Ca -dependent manner, ionomycin-induced contraction was dependent on Rho and Rho-kinase. Ionomycin induced phosphorylation of the MLCP-regulatory subunit myosin targeting protein 1(MYPT1) at Thr and the 20-kDa myosin light chain (MLC) in a Rho kinase-dependent manner. Knockdown of PI3K-C2 suppressed phosphorylation of both MYPT1 and MLC. The receptor agonist noradrenaline, which induced a rapid increase in the [Ca ]i and Ca 2 -dependent contraction, stimulated phosphorylation of MYPT1 and MLC, which was also dependent on Ca , PI3K-C2 , and Rho-kinase. These observations indicate that PI3K-C2 is necessary for Ca -induced Rhoand Rho kinase-dependent negative regulation of MLCP and consequently MLC phosphorylation and contraction. Membrane depolarization and excitatory receptor agonists, including noradrenaline, induce an increase in the cytoplasmic free Ca concentration ([Ca ]i) in vascular smooth muscle, resulting in the activation of calmodulin-dependent myosin light-chain kinase (MLCK) and phosphorylation of the 20-kDa myosin light chain (MLC) (Morgan and Suematsu, 1990; Somlyo and Somlyo, 1994; Kamm and Stull, 2001). Excitatory receptor agonists also exert inhibitory regulation on the MLC dephosphorylating enzyme, myosin lightchain phosphatase (MLCP), which acts to potentiate Ca induced MLC phosphorylation and contraction (Pfitzer, 2001; Somlyo and Somlyo, 2003; Sward et al., 2003; Takuwa et al., 2005). Accumulating evidence implicates the small GTPase Rho and the Rho effector Rho-kinase in the negative regulation of MLCP by excitatory receptor agonists; excitatory receptor agonists trigger Rho activation (Sakurada et al., 2001), leading to MLCP inhibition through mechanisms involving Rho kinase-dependent phosphorylation of the 110This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan, and Novartis Pharma. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.032599. □S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: MLCK, myosin light-chain kinase; MLCP, myosin light-chain phosphatase; MLC, 20-kDa myosin light chain; PI3K-C2 , phosphoinositide 3-kinase class II isoform; MYPT1, myosin targeting protein 1; CPI-17, 17-kDa protein kinase C-potentiated inhibitory protein of PP1; GFP, enhanced green fluorescent protein; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N,N -tetraacetic acid tetra (acetoxymethyl) ester; VSMC, vascular smooth muscle; PI3K, phosphoinositide 3-kinase; siRNA, short interfering RNA; C2 -siRNA, phosphoinositide 3-kinase-C2 -specific short interfering RNA; EGFP, enhanced green fluorescent protein; sc-siRNA, scrambled short interfering RNA; Y27632, N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide dihydrochloride. 0026-895X/07/7103-912–920$20.00 MOLECULAR PHARMACOLOGY Vol. 71, No. 3 Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics 32599/3184071 Mol Pharmacol 71:912–920, 2007 Printed in U.S.A. 912 http://molpharm.aspetjournals.org/content/suppl/2006/12/20/mol.106.032599.DC1 Supplemental material to this article can be found at: at A PE T Jornals on Jne 2, 2017 m oharm .aspeurnals.org D ow nladed from kDa myosin targeting subunit MYPT1/MBS of MLCP at Thr and/or Thr (numbering of chicken M133 isoform) (Noda et al., 1995; Kimura et al., 1996; Hartshorne et al., 2004) and of the smooth muscle-specific MLCP inhibitor protein CPI-17 (Kitazawa et al., 2000; Niiro et al., 2003). CPI-17 may also be phosphorylated by a protein kinase C-dependent mechanism (Eto et al., 2001). Thus, Rho acts as a switch molecule to negatively regulate MLCP in smooth muscle. We and others have demonstrated that membrane depolarization and ionomycin induce Rho activation and MLCP inhibition in a Ca -dependent manner in vascular smooth muscle (Mita et al., 2002; Sakamoto et al., 2003; Sakurada et al., 2003; Wang et al., 2006). Thus, it seems that an increase in the [Ca ]i not only activates MLCK but also inhibits MLCP in membrane depolarizationand ionomycin-stimulated muscle, like the case of excitatory receptor agonist stimulation. We also have shown that excitatory receptor agonist-induced Rho activation is Ca -dependent (Wang et al., 2006), suggesting that the Ca -dependent Rho activation mechanism, together with the receptor-coupled G12/13dependent mechanism (Somlyo and Somlyo, 2003), seems to operate in receptor agonist-stimulated smooth muscle. We demonstrated recently in vascular smooth muscle that phosphoinositide 3-kinase (PI3K) inhibitors suppress membrane depolarizationand receptor agonist noradrenaline-induced Rho activation and MYPT1 phosphorylation and MLC phosphorylation and contraction (Wang et al., 2006), suggesting that a PI3K plays a critical role in the activation of the Rho signaling pathway. We showed evidence that class II PI3K enzyme PI3K-C2 , which characteristically exhibits relatively lower sensitivities to PI3K inhibitors compared with other isoforms (Domin et al., 1997; Stein and Waterfield, 2000), is a PI3K isoform that is responsible for the receptor agonist noradrenaline-induced, PI3K inhibitor-sensitive Rho activation. However, it is not yet established whether PI3KC2 is involved in Ca -induced Rho activation and MLCP inhibition in vascular smooth muscle, although high concentrations of PI3K inhibitors inhibit membrane depolarizationinduced Rho activation. In the present study, we addressed this question by taking advantage of RNA interference-mediated gene-silencing technique (Sharp, 2001) and differentiated vascular smooth muscle cells (VSMCs), which maintain contractile responses to various vasoactive substances (see the videos showing contractile responses in Supplementary Videos S1–S8). Materials and Methods Materials. LY294002, ionomycin, and BAPTA-AM were purchased from Merck-Calbiochem Biosciences (Darmstadt, Germany). Noradrenaline was bought from Sigma (St. Louis, MO). Insulin-like growth factor-I was bought from PeproTech (Rocky Hill, NJ). Laminin was bought from Asahi Techno Glass (Funabashi, Japan). Y27632 was donated by WelFide Corporation (Osaka, Japan). Fluo-4 acetoxymethyl ester was bought from Molecular Probes (Eugene, OR). Monoclonal antibody to PI3K-C2 (611046) was bought from BD Biosciences (San Jose, CA). Monoclonal antibodies to MLC (MY21), smooth muscle -actin (1A4), and MLCK (K36) were from Sigma. Rabbit polyclonal antibodies to phospho-MYPT1 (Thr) (36–003) and MYPT1 (PRB-457C) were bought from Upstate (Charlottesville, VA) and Covance Research Products (Berkeley, CA), re-