Howard K. Surks

ROCK Isoform Regulation of Myosin Phosphatase and Contractility in Vascular Smooth Muscle Cells

Abnormal vascular smooth muscle cell (VSMC) contraction plays an important role in vascular diseases. The RhoA/ROCK signaling pathway is now well recognized to mediate vascular smooth muscle contraction in response to vasoconstrictors by inhibiting myosin phosphatase (MLCP) activity and increasing myosin light chain phosphorylation. Two ROCK isoforms, ROCK1 and ROCK2, are expressed in many tissues, yet the isoform-specific roles of ROCK1 and ROCK2 in vascular smooth muscle and the mechanism of ROCK-mediated regulation of MLCP are not well understood. In this study, ROCK2, but not ROCK1, bound directly to the myosin binding subunit of MLCP, yet both ROCK isoforms regulated MLCP and myosin light chain phosphorylation. Despite that both ROCK1 and ROCK2 regulated MLCP, the ROCK isoforms had distinct and opposing effects on VSMC morphology and ROCK2, but not ROCK1, had a predominant role in VSMC contractility. These data support that although the ROCK isoforms both regulate MLCP and myosin light chain phosphorylation through different mechanisms, they have distinct roles in VSMC function.

High blood pressure arising from a defect in vascular function

Hypertension, a major cardiovascular risk factor and cause of mortality worldwide, is thought to arise from primary renal abnormalities. However, the etiology of most cases of hypertension remains unexplained. Vascular tone, an important determinant of blood pressure, is regulated by nitric oxide, which causes vascular relaxation by increasing intracellular cGMP and activating cGMP-dependent protein kinase I (PKGI). Here we show that mice with a selective mutation in the N-terminal protein interaction domain of PKGIα display inherited vascular smooth muscle cell abnormalities of contraction, abnormal relaxation of large and resistance blood vessels, and increased systemic blood pressure. Renal function studies and responses to changes in dietary sodium in the PKGIα mutant mice are normal. These data reveal that PKGIα is required for normal VSMC physiology and support the idea that high blood pressure can arise from a primary abnormality of vascular smooth muscle cell contractile regulation, suggesting a new approach to the diagnosis and therapy of hypertension and cardiovascular diseases.

Myosin Phosphatase-Rho Interacting Protein A NEW MEMBER OF THE MYOSIN PHOSPHATASE COMPLEX THAT DIRECTLY BINDS RhoA

Regulation of vascular smooth muscle cell contractile state is critical for the maintenance of blood vessel tone. Abnormal vascular smooth muscle cell contractility plays an important role in the pathogenesis of hypertension, blood vessel spasm, and atherosclerosis. Myosin phosphatase, the key enzyme controlling myosin light chain dephosphorylation, regulates smooth muscle cell contraction. Vasoconstrictor and vasodilator pathways inhibit and activate myosin phosphatase, respectively. G-protein-coupled receptor agonists can inhibit myosin phosphatase and cause smooth muscle cell contraction by activating RhoA/Rho kinase, whereas NO/cGMP can activate myosin phosphatase and cause smooth muscle cell relaxation by activation of cGMP-dependent protein kinase. We have used yeast two-hybrid screening to identify a 116-kDa human protein that interacts with both myosin phosphatase and RhoA. This myosin phosphatase-RhoA interacting protein, or M-RIP, is highly homologous to murine p116RIP3, is expressed in vascular smooth muscle, and is localized to actin myofilaments. M-RIP binds directly to the myosin binding subunit of myosin phosphatase in vivo in vascular smooth muscle cells by an interaction between coiled-coil and leucine zipper domains in the two proteins. An adjacent domain of M-RIP directly binds RhoA in a nucleotide-independent manner. M-RIP copurifies with RhoA and Rho kinase, colocalizes on actin stress fibers with RhoA and MBS, and is associated with Rho kinase activity in vascular smooth muscle cells. M-RIP can assemble a complex containing both RhoA and MBS, suggesting that M-RIP may play a role in myosin phosphatase regulation by RhoA.

cGMP-Dependent Protein Kinase I and Smooth Muscle Relaxation A Tale of Two Isoforms

See related article, pages 1096–1103 The maintenance of vascular tone is central to the regulation of blood pressure and tissue perfusion and plays a role in the pathogenesis of hypertension and atherosclerosis. Vascular tone is determined by the balance of vasodilator and vasoconstrictor stimuli. After several decades of research, the NO/cGMP/cGMP-dependent protein kinase (cGK) pathway is now recognized as an important mediator of vasodilation. However, the mechanisms by which cGK causes smooth muscle relaxation continue to be an important question. Smooth muscle contraction and relaxation are tightly coupled to the phosphorylation and dephosphorylation, respectively, of the regulatory myosin light chain.1 Myosin light chain phosphorylation state is determined by the relative activities of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). MLCK phosphorylates MLC leading to contraction,2 and MLCP dephosphorylates MLC, leading to relaxation3 (Figure). Both MLCK and MLCP activities are highly regulated. MLCK activity is activated by the binding of calcium/calmodulin and thus is the primary mechanism linking intracellular calcium concentration to smooth muscle contractility.4 MLCP activity is regulated by both vasodilator and vasoconstrictor stimuli, and is therefore responsible for much of the calcium-independent regulation of contractility (reviewed in5). Figure. MLC phosphorylation determines smooth muscle contractility. Contractile agonists lead to inositol 1,4,5 triphosphate (IP3) production or activation of RhoA (RhoA-GTP). IP3 binding to its receptor in the sarcoplasmic reticulum leads to release of Ca2+. Ca2+/calmodulin binds to and activates MLCK, which in turn phosphorylates MLC (calcium-dependent contraction). Activated RhoA binds to and activates ROCK, leading to phosphorylation and inhibition of MLCP, inhibiting MLC dephosphorylation (calcium-independent contraction). cGKI mediates relaxation by inhibiting both calcium-dependent and -independent contraction. cGKIα activates MLCP by a direct interaction and by inhibition of RhoA, and activates RGS2 to inhibit Gαq …

Effects of PCSK9 Inhibition With Alirocumab on Lipoprotein Metabolism in Healthy Humans

Background: Alirocumab, a monoclonal antibody to proprotein convertase subtilisin/kexin type 9 (PCSK9), lowers plasma low-density lipoprotein (LDL) cholesterol and apolipoprotein B100 (apoB). Although studies in mice and cells have identified increased hepatic LDL receptors as the basis for LDL lowering by PCSK9 inhibitors, there have been no human studies characterizing the effects of PCSK9 inhibitors on lipoprotein metabolism. In particular, it is not known whether inhibition of PCSK9 has any effects on very low-density lipoprotein or intermediate-density lipoprotein (IDL) metabolism. Inhibition of PCSK9 also results in reductions of plasma lipoprotein (a) levels. The regulation of plasma Lp(a) levels, including the role of LDL receptors in the clearance of Lp(a), is poorly defined, and no mechanistic studies of the Lp(a) lowering by alirocumab in humans have been published to date. Methods: Eighteen (10 F, 8 mol/L) participants completed a placebo-controlled, 2-period study. They received 2 doses of placebo, 2 weeks apart, followed by 5 doses of 150 mg of alirocumab, 2 weeks apart. At the end of each period, fractional clearance rates (FCRs) and production rates (PRs) of apoB and apo(a) were determined. In 10 participants, postprandial triglycerides and apoB48 levels were measured. Results: Alirocumab reduced ultracentrifugally isolated LDL-C by 55.1%, LDL-apoB by 56.3%, and plasma Lp(a) by 18.7%. The fall in LDL-apoB was caused by an 80.4% increase in LDL-apoB FCR and a 23.9% reduction in LDL-apoB PR. The latter was due to a 46.1% increase in IDL-apoB FCR coupled with a 27.2% decrease in conversion of IDL to LDL. The FCR of apo(a) tended to increase (24.6%) without any change in apo(a) PR. Alirocumab had no effects on FCRs or PRs of very low-density lipoproteins-apoB and very low-density lipoproteins triglycerides or on postprandial plasma triglycerides or apoB48 concentrations. Conclusions: Alirocumab decreased LDL-C and LDL-apoB by increasing IDL- and LDL-apoB FCRs and decreasing LDL-apoB PR. These results are consistent with increases in LDL receptors available to clear IDL and LDL from blood during PCSK9 inhibition. The increase in apo(a) FCR during alirocumab treatment suggests that increased LDL receptors may also play a role in the reduction of plasma Lp(a). Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01959971.

Anacetrapib, a Novel CETP Inhibitor: Pursuing a New Approach to Cardiovascular Risk Reduction

Cholesteryl ester transfer protein (CETP) inhibition is a promising experimental strategy to raise high‐density lipoprotein cholesterol (HDL‐C) and reduce cardiovascular risk. This review focuses on the highly selective and potent CE TP inhibitor anacetrapib and discusses the available preclinical and clinical information pertaining to it. We also describe strategies to target HDL‐C, discuss the mechanism underlying CETP inhibition and its effects on lipid biology, and give an overview of other CETP inhibitors that are currently in development.

Dimerization of cGMP-dependent protein kinase 1α and the myosin-binding subunit of myosin phosphatase: role of leucine zipper domains

Nitric oxide (NO) and nitrovasodilators induce vascular smooth muscle cell relaxation in part by cGMP-dependent protein kinase (cGK)-mediated activation of myosin phosphatase, which dephosphorylates myosin light chains. We recently found that cGMP-dependent protein kinase 1alpha binds directly to the myosin-binding subunit (MBS) of myosin phosphatase via the leucine/isoleucine zipper of cGK. We have now studied the role of the leucine zipper domain of MBS in dimerization with cGK and the leucine/isoleucine zipper and leucine zipper domains of both proteins in homodimerization. Mutagenesis of the MBS leucine zipper domain disrupts cGKIalpha-MBS dimerization. Mutagenesis of the MBS leucine zipper eliminates MBS homodimerization, while similar disruption of the cGKIalpha leucine/isoleucine zipper does not prevent formation of cGK dimers. The MBS leucine zipper domain is phosphorylated by cGK, but this does not have any apparent effect on heterodimer formation between the two proteins. MBS LZ mutants that are unable to bind cGK were poor substrates for cGK. These data support the theory that the MBS leucine zipper domain is necessary and sufficient to mediate both MBS homodimerization and binding of the protein to cGK. In contrast, the leucine/isoleucine zipper of cGK is required for binding to MBS, but not for cGK homodimerization. These data support that the MBS and cGK leucine zipper domains mediate the interaction between these two proteins. The contribution of these domains to both homodimerization and their specific interaction with each other suggest that additional regulatory mechanisms involving these domains may exist.

Targeting by myosin phosphatase‐RhoA interacting protein mediates RhoA/ROCK regulation of myosin phosphatase

Vascular smooth muscle cell contractile state is the primary determinant of blood vessel tone. Vascular smooth muscle cell contractility is directly related to the phosphorylation of myosin light chains (MLCs), which in turn is tightly regulated by the opposing activities of myosin light chain kinase (MLCK) and myosin phosphatase. Myosin phosphatase is the principal enzyme that dephosphorylates MLCs leading to relaxation. Myosin phosphatase is regulated by both vasoconstrictors that inhibit its activity to cause MLC phosphorylation and contraction, and vasodilators that activate its activity to cause MLC dephosphorylation and relaxation. The RhoA/ROCK pathway is activated by vasoconstrictors to inhibit myosin phosphatase activity. The mechanism by which RhoA and ROCK are localized to and interact with myosin light chain phosphatase (MLCP) is not well understood. We recently found a new member of the myosin phosphatase complex, myosin phosphatase‐rho interacting protein, that directly binds to both RhoA and the myosin‐binding subunit of myosin phosphatase in vitro, and targets myosin phosphatase to the actinomyosin contractile filament in smooth muscle cells. Because myosin phosphatase‐rho interacting protein binds both RhoA and MLCP, we investigated whether myosin phosphatase‐rho interacting protein was required for RhoA/ROCK‐mediated myosin phosphatase regulation. Myosin phosphatase‐rho interacting protein silencing prevented LPA‐mediated myosin‐binding subunit phosphorylation, and inhibition of myosin phosphatase activity. Myosin phosphatase‐rho interacting protein did not regulate the activation of RhoA or ROCK in vascular smooth muscle cells. Silencing of M‐RIP lead to loss of stress fiber‐associated RhoA, suggesting that myosin phosphatase‐rho interacting protein is a scaffold linking RhoA to regulate myosin phosphatase at the stress fiber. J. Cell. Biochem. 103: 1158–1170, 2008.

M-RIP Targets Myosin Phosphatase to Stress Fibers to Regulate Myosin Light Chain Phosphorylation in Vascular Smooth Muscle Cells

Vascular smooth muscle cell contraction and relaxation are directly related to the phosphorylation state of the regulatory myosin light chain. Myosin light chains are dephosphorylated by myosin phosphatase, leading to vascular smooth muscle relaxation. Myosin phosphatase is localized not only at actin-myosin stress fibers where it dephosphorylates myosin light chains, but also in the cytoplasm and at the cell membrane. The mechanisms by which myosin phosphatase is targeted to these loci are incompletely understood. We recently identified myosin phosphatase-Rho interacting protein as a member of the myosin phosphatase complex that directly binds both the myosin binding subunit of myosin phosphatase and RhoA and is localized to actin-myosin stress fibers. We hypothesized that myosin phosphatase-Rho interacting protein targets myosin phosphatase to the contractile apparatus to dephosphorylate myosin light chains. We used RNA interference to silence the expression of myosin phosphatase-Rho interacting protein in human vascular smooth muscle cells. Myosin phosphatase-Rho interacting protein silencing reduced the localization of the myosin binding subunit to stress fibers. This reduction in stress fiber myosin phosphatase-Rho interacting protein and myosin binding subunit increased basal and lysophosphatidic acid-stimulated myosin light chain phosphorylation. Neither cellular myosin phosphatase, myosin light chain kinase, nor RhoA activities were changed by myosin phosphatase-Rho interacting protein silencing. Furthermore, myosin phosphatase-Rho interacting protein silencing resulted in marked phenotypic changes in vascular smooth muscle cells, including increased numbers of stress fibers, increased cell area, and reduced stress fiber inhibition in response to a Rho-kinase inhibitor. These data support the importance of myosin phosphatase-Rho interacting protein-dependent targeting of myosin phosphatase to stress fibers for regulating myosin light chain phosphorylation state and morphology in human vascular smooth muscle cells.

Probing the Interaction between the Coiled Coil Leucine Zipper of cGMP-dependent Protein Kinase Iα and the C Terminus of the Myosin Binding Subunit of the Myosin Light Chain Phosphatase

Nitric oxide and nitrovasodilators induce vascular smooth muscle cell relaxation in part by cGMP-dependent protein kinase I (PKG-Iα)-mediated activation of myosin phosphatase (MLCP). Mechanistically it has been proposed that protein-protein interactions between the N-terminal leucine zipper (LZ) domain of PKG-Iα ((PKG-Iα1-59) and the LZ and/or coiled coil (CC) domain of the myosin binding subunit (MBS) of MLCP are localized in the C terminus of MBS. Although recent studies have supported these interactions, the critical amino acids responsible for these interactions have not been identified. Here we present structural and biophysical data identifying that the LZ domain of PKG-Iα1-59 interacts with a well defined 42-residue CC motif (MBSct42) within the C terminus of MBS. Using glutathione S-transferase pulldown experiments, chemical cross-linking, size exclusion chromatography, circular dichroism, and isothermal titration calorimetry we identified a weak dimer-dimer interaction between PKG-Iα1-59 and this C-terminal CC domain of MBS. The Kd of this non-covalent complex is 178.0 ± 1.5 μm. Furthermore our 1H-15N heteronuclear single quantum correlation NMR data illustrate that this interaction is mediated by several PKG-Iα residues that are on the a, d, e, and g hydrophobic and electrostatic interface of the C-terminal heptad layers 2, 4, and 5 of PKG-Iα. Taken together these data support a role for the LZ domain of PKG-Iα and the CC domain of MBS in this requisite contractile complex.