36-003 Sigma-AldrichAnti-phospho-MYPT1 (Thr850) Antibody

Chronic pulmonary hypertension in infancy and childhood is characterized by a fixed and progressive increase in pulmonary arterial pressure and resistance, pulmonary arterial remodeling, and right ventricular hypertrophy and systolic dysfunction. These abnormalities are replicated in neonatal rats chronically exposed to hypoxia from birth in which increased activity of Rho-kinase (ROCK) is critical to injury, as evidenced by preventive effects of ROCK inhibitors. Our objective in the present study was to examine the reversing effects of a late or rescue approach to treatment with a ROCK inhibitor on the pulmonary and cardiac manifestations of established chronic hypoxic pulmonary hypertension. Rat pups were exposed to air or hypoxia (13% O(2)) from postnatal day 1 and were treated with Y-27632 (15 mg/kg) or saline vehicle by twice daily subcutaneous injection commencing on day 14, for up to 7 days. Treatment with Y-27632 significantly attenuated right ventricular hypertrophy, reversed arterial wall remodeling, and completely normalized right ventricular systolic function in hypoxia-exposed animals. Reversal of arterial wall remodeling was accompanied by increased apoptosis and attenuated content of endothelin (ET)-1 and ET(A) receptors. Treatment of primary cultured juvenile rat pulmonary artery smooth muscle cells with Y-27632 attenuated serum-stimulated ROCK activity and proliferation and increased apoptosis. Smooth muscle apoptosis was also induced by short interfering RNA-mediated knockdown of ROCK-II, but not of ROCK-I. We conclude that sustained rescue treatment with a ROCK inhibitor reversed both the hemodynamic and structural abnormalities of chronic hypoxic pulmonary hypertension in juvenile rats and normalized right ventricular systolic function. Attenuated expression and activity of ET-1 and its A-type receptor on pulmonary arterial smooth muscle was a likely contributor to the stimulatory effects of ROCK inhibition on apoptosis. In addition, our data suggest that ROCK-II may be dominant in enhancing survival of pulmonary arterial smooth muscle.

Rho-mediated inhibition of myosin light chain (MLC) phosphatase (MLCP), together with Ca(2+)-dependent MLC kinase activation, constitutes the major signaling mechanisms for vascular smooth muscle contraction. We recently unveiled the involvement of Ca(2+)-induced, phosphoinositide 3-kinase (PI3K) class IIα isoform (PI3K-C2α)-dependent Rho activation and resultant Rho kinase-dependent MLCP suppression in membrane depolarization- and receptor agonist-induced contraction. It is unknown whether Ca(2+)- and PI3K-C2α-dependent regulation of MLCP is altered in vascular smooth muscle of hypertensive animals and is involved in hypertension. Therefore, we studied the role of the Ca(2+)-PI3K-C2α-Rho-MLCP pathway in spontaneously hypertensive rats (SHRs). PI3K-C2α was readily detected in various vascular beds of Wistar-Kyoto rats and activated by high KCl. High KCl also stimulated vascular Rho activity and phosphorylation of the MLCP regulatory subunit MYPT1 at Thr(853) in a PI3K inhibitor wortmannin-sensitive manner. In mesenteric and other vessels of SHRs at the hypertensive but not the prehypertensive stage, the activity of PI3K-C2α but not class I PI3K p110α was elevated with concomitant rises of Rho activity and Thr(853)-phosphorylation of MYPT1, as compared with normotensive controls. Infusion of the Ca(2+) channel antagonist nicardipine reduced blood pressure with suppression of vascular activity of PI3K-C2α-Rho and phosphorylation of MYPT1 in hypertensive SHRs. Infusion of wortmannin lowered blood pressure with inhibition of PI3K-C2α-Rho activities and MYPT1 phosphorylation in hypertensive SHRs. These observations suggest that an increased activity of the Ca(2+)-PI3K-C2α-Rho signaling pathway with resultant augmented MLCP suppression contributes to hypertension in SHRs. The Ca(2+)- and PI3K-C2α-dependent Rho stimulation in vascular smooth muscle may be a novel, promising target for treating hypertension.

Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that has pleiotropic effects in a variety of cell types including ECs, SMCs, and macrophages, all of which are central to the development of atherosclerosis. It may therefore exert stimulatory and inhibitory effects on atherosclerosis. Here, we investigated the role of the S1P receptor S1PR2 in atherosclerosis by analyzing S1pr2-/- mice with an Apoe-/- background. S1PR2 was expressed in macrophages, ECs, and SMCs in atherosclerotic aortas. In S1pr2-/-Apoe-/- mice fed a high-cholesterol diet for 4 months, the area of the atherosclerotic plaque was markedly decreased, with reduced macrophage density, increased SMC density, increased eNOS phosphorylation, and downregulation of proinflammatory cytokines compared with S1pr2+/+Apoe-/- mice. Bone marrow chimera experiments indicated a major role for macrophage S1PR2 in atherogenesis. S1pr2-/-Apoe-/- macrophages showed diminished Rho/Rho kinase/NF-κB (ROCK/NF-κB) activity. Consequently, they also displayed reduced cytokine expression, reduced oxidized LDL uptake, and stimulated cholesterol efflux associated with decreased scavenger receptor expression and increased cholesterol efflux transporter expression. S1pr2-/-Apoe-/- ECs also showed reduced ROCK and NF-κB activities, with decreased MCP-1 expression and elevated eNOS phosphorylation. Pharmacologic S1PR2 blockade in S1pr2+/+Apoe-/- mice diminished the atherosclerotic plaque area in aortas and modified LDL accumulation in macrophages. We conclude therefore that S1PR2 plays a critical role in atherogenesis and may serve as a novel therapeutic target for atherosclerosis.

Many biochemical factors regulating progenitor cell differentiation have been examined in detail; however, the role of the local mechanical environment on stem cell fate has only recently been investigated. In this study, we examined whether oscillatory fluid flow, an exogenous mechanical signal within bone, regulates osteogenic, adipogenic or chondrogenic differentiation of C3H10T1/2 murine mesenchymal stem cells by measuring Runx2, PPARgamma and SOX9 gene expression, respectively. Furthermore, we hypothesized that the small GTPase RhoA and isometric tension within the actin cytoskeleton are essential in flow-induced differentiation. We found that oscillatory fluid flow induces the upregulation of Runx2, Sox9 and PPARgamma, indicating that it has the potential to regulate transcription factors involved in multiple unique lineage pathways. Furthermore, we demonstrate that the small GTPase RhoA and its effector protein ROCKII regulate fluid-flow-induced osteogenic differentiation. Additionally, activated RhoA and fluid flow have an additive effect on Runx2 expression. Finally, we show RhoA activation and actin tension are negative regulators of both adipogenic and chondrogenic differentiation. However, an intact, dynamic actin cytoskeleton under tension is necessary for flow-induced gene expression.

Many agents constrict isolated afferent arterioles only at concentrations higher than their physiological levels. Here we determined if norepinephrine, as released by sympathetic nerve activity, could influence the angiotensin II responsiveness of isolated mouse afferent arterioles. Pretreatment of the arterioles for short periods with norepinephrine significantly increased the ability of 10 picomolar angiotensin II to constrict the vessels, an effect inhibited by the alpha receptor blockers prazosin (alpha-1) or yohimbine (alpha-2). Although the intracellular calcium transients induced by angiotensin were not different, phosphorylation of the 20 kDa myosin light chain was significantly increased in the presence of norepinephrine. Phosphorylation of the p38 mitogen-activated protein kinase was not changed. Phosphorylation of the myosin phosphatase targeting subunit at Thr696, but not at Thr850, was significantly enhanced by, norepinephrine pretreatment, thus increasing the calcium sensitivity of the arteriolar smooth muscle. Our results show that norepinephrine increases afferent arteriolar sensitivity to angiotensin II by means of alpha receptor activation, causing increased calcium sensitivity through phosphorylation of the myosin phosphatase targeting subunit.

The patho-physiological hypothesis of mental retardation caused by the deficiency of the RhoGAP Oligophrenin1 (OPHN1), relies on the well-known functions of Rho GTPases on neuronal morphology, i.e. dendritic spine structure. Here, we describe a new function of this Bin/Amphiphysin/Rvs domain containing protein in the control of clathrin-mediated endocytosis (CME). Through interactions with Src homology 3 domain containing proteins involved in CME, OPHN1 is concentrated to endocytic sites where it down-regulates the RhoA/ROCK signaling pathway and represses the inhibitory function of ROCK on endocytosis. Indeed disruption of Ophn1 in mice reduces the endocytosis of synaptic vesicles and the post-synaptic alpha-amino-3-hydroxy-5-methylisoazol-4-propionate (AMPA) receptor internalization, resulting in almost a complete loss of long-term depression in the hippocampus. Finally, pharmacological inhibition of this pathway by ROCK inhibitors fully rescued not only the CME deficit in OPHN1 null cells but also synaptic plasticity in the hippocampus from Ophn1 null model. Altogether, we uncovered a new patho-physiological mechanism for intellectual disabilities associated to mutations in RhoGTPases linked genes and also opened new directions for therapeutic approaches of congenital mental retardation.

Although stimulation with sphingosylphosphorylcholine (SPC) or sphingosine-1-phosphate (S1P) generally leads to similar vascular responses, the contractile patterns and their underlying signalling mechanisms are often distinct. We investigated the different reliance upon Ca2+-dependent and Ca2+-sensitizing mechanisms of constriction in response to SPC or S1P in coronary arteries.

The LRRK2 (leucine-rich repeat protein kinase-2) is mutated in a significant number of Parkinson's disease patients, but little is known about its regulation and function. A common mutation changing Gly2019 to serine enhances catalytic activity, suggesting that small-molecule inhibitors might have utility in treating Parkinson's disease. We employed various approaches to explore the substrate-specificity requirements of LRRK2 and elaborated a peptide substrate termed Nictide, that had 20-fold lower Km and nearly 2-fold higher Vmax than the widely deployed LRRKtide substrate. We demonstrate that LRRK2 has marked preference for phosphorylating threonine over serine. We also observed that several ROCK (Rho kinase) inhibitors such as Y-27632 and H-1152, suppressed LRRK2 with similar potency to which they inhibited ROCK2. In contrast, GSK429286A, a selective ROCK inhibitor, did not significantly inhibit LRRK2. We also identified a mutant LRRK2[A2016T] that was normally active, but resistant to H-1152 and Y-27632, as well as sunitinib, a structurally unrelated multikinase inhibitor that, in contrast with other compounds, suppresses LRRK2, but not ROCK. We have also developed the first sensitive antibody that enables measurement of endogenous LRRK2 protein levels and kinase activity as well as shRNA (short hairpin RNA) methods to reduce LRRK2 expression. Finally, we describe a pharmacological approach to validate whether substrates are phosphorylated by LRRK2 and use this to provide evidence that LRRK2 may not be rate-limiting for the phosphorylation of the proposed substrate moesin. The findings of the present study will aid with the investigation of LRRK2.