Possible role of Efnb1 protein, a ligand of Eph receptor tyrosine kinases, in modulating blood pressure

Eph kinases constitute the largest receptor tyrosine kinase family, and their ligands, ephrins (Efns), are also cell surface molecules. Although they are ligands, Efns can transduce signals reversely into cells. We have no prior knowledge of the role played by any members of this family of kinases or their ligands in blood pressure (BP) regulation. In the present studies, we investigated the role of Efnb1 in vascular smooth muscle cell (VSMC) contractility and BP regulation. We revealed that reverse signaling through Efnb1 led to a reduction of RhoA activation and VSMC contractility in vitro. Consistent with this finding, ex vivo, there was an increase of RhoA activity accompanied by augmented myosin light chain phosphorylation in mesenteric arteries from mice with smooth muscle-specific conditional Efnb1 gene knock-out (KO). Small interfering RNA knockdown of Grip1, a molecule associated with the Efnb1 intracellular tail, partially eliminated the effect of Efnb1 on VSMC contractility and myosin light chain phosphorylation. In support of these in vitro and ex vivo results, Efnb1 KO mice on a high salt diet showed a statistically significant heightened increment of BP at multiple time points during stress compared with wild type littermates. Our results demonstrate that Efnb1 is a previously unknown negative regulator of VSMC contractility and BP and that it exerts such effects via reverse signaling through Grip1.     

Receptor tyrosine kinase Ephb6 regulates vascular smooth muscle contractility and modulates blood pressure in concert with sex hormones

Eph kinases constitute the largest receptor tyrosine kinase family, and their ligands, ephrins (Efns), are also cell surface molecules. Our study is the first to assess the role of Ephb6 in blood pressure (BP) regulation. We observed that EphB6 and all three of its Efnb ligands were expressed on vascular smooth muscle cells (VSMC) in mice. We discovered that small arteries from castrated Ephb6 gene KO males showed increased contractility, RhoA activation, and constitutive myosin light chain phosphorylation ex vivo compared with their WT counterparts. Consistent with this finding, castrated Ephb6 KO mice presented heightened BP compared with castrated WT controls. In vitro experiments in VSMC revealed that cross-linking Efnbs but not Ephb6 resulted in reduced VSMC contractions, suggesting that reverse signaling through Efnbs was responsible for the observed BP phenotype. The reverse signaling was mediated by an adaptor protein Grip1. Additional experiments demonstrated decreased 24-h urine catecholamines in male Ephb6 KO mice, probably as a compensatory feedback mechanism to keep their BP in the normal range. After castration, however, such compensation was abolished in Ephb6 KO mice and was likely the reason why BP increased overtly in these animals. It suggests that Ephb6 has a target in the nervous/endocrine system in addition to VSMC, regulating a testosterone-dependent catecholamine compensatory mechanism. Our study discloses that Ephs and Efns, in concert with testosterone, play a critical role in regulating small artery contractility and BP.     

Dynamic changes occur in patterns of endometrial EFNB2/EPHB4 expression during the period of spiral arterial modification in mice

Transient, human and murine decidua-associated, Natural Killer lymphocytes (uNK cells) have special, localized roles in early gestational endometrial remodeling and angiogenesis. To determine if uNK cells promote a specific vessel subtype, a histological time-course study of implantation site endothelia was undertaken using normal C57BL/6J (B6) and uNK-deficient B6.129-Rag2 tm1Fwa Il2rg tm2Cgn (alymphoid) mice, a strain lacking pregnancy-induced structural modifications of spiral arteries. Antibodies to EFNB2, EPHB4, and LYVE1, respectively, identified arterial, venous, and lymphatic endothelia. Unexpectedly, many uNK cells in B6 endometrium showed strong EFNB2 expression early in gestation, then became EPHB4+. This molecular transition coincided with structural modifications of spiral arteries that shifted from EFNB2+/EPHB4(-) to EFNB2+/EPHB4+. NK cells from B6 spleen and liver did not express EFNB2. LYVE1 expression was similar in endometrium from B6 and alymphoid mice, but EFNB2 and EPHB4 expression in alymphoid mice was dramatically different. Strong stromal expression of both molecules developed mesometrially, and this was reduced by B6 lymphocyte transfer. Trophoblasts reacted with each marker in both strains. Expression of EFNB2 by uNK cells and trophoblasts may be the key regulatory mechanism that drives their positional association with EFNB2+ arteries and prevents association of both cell types with EPHB4+ veins. Gain of EPHB4 by midgestation spiral arteries may signal completion of pregnancy-induced arterial modification and provide a repulsion mechanism that limits subsequent interactions of the modified vessel with uNK cells and trophoblasts.     

Myosin light chain kinase phosphorylation in tracheal smooth muscle

Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, and the multifunctional calmodulin-dependent protein kinase II. Because phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in 32P-labeled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A in contracting tracheal smooth muscle may play a role in the reported desensitization of contractile elements to activation by Ca2+.     

Myosin Phosphatase Target Subunit 1 (MYPT1) Regulates the Contraction and Relaxation of Vascular Smooth Muscle and Maintains Blood Pressure

Myosin light chain phosphatase with its regulatory subunit, myosin phosphatase target subunit 1 (MYPT1) modulates Ca²⁺-dependent phosphorylation of myosin light chain by myosin light chain kinase, which is essential for smooth muscle contraction. The role of MYPT1 in vascular smooth muscle was investigated in adult MYPT1 smooth muscle specific knock-out mice. MYPT1 deletion enhanced phosphorylation of myosin regulatory light chain and contractile force in isolated mesenteric arteries treated with KCl and various vascular agonists. The contractile responses of arteries from knock-out mice to norepinephrine were inhibited by Rho-associated kinase (ROCK) and protein kinase C inhibitors and were associated with inhibition of phosphorylation of the myosin light chain phosphatase inhibitor CPI-17. Additionally, stimulation of the NO/cGMP/protein kinase G (PKG) signaling pathway still resulted in relaxation of MYPT1-deficient mesenteric arteries, indicating phosphorylation of MYPT1 by PKG is not a major contributor to the relaxation response. Thus, MYPT1 enhances myosin light chain phosphatase activity sufficient for blood pressure maintenance. Rho-associated kinase phosphorylation of CPI-17 plays a significant role in enhancing vascular contractile responses, whereas phosphorylation of MYPT1 in the NO/cGMP/PKG signaling module is not necessary for relaxation.     

Characterization of chicken skeletal muscle myosin light chain kinase. Evidence for muscle-specific isozymes

Myosin light chain kinase purified from chicken white skeletal muscle (Mr = 150,000) was significantly larger than both rabbit skeletal (Mr = 87,000) and chicken gizzard smooth (Mr = 130,000) muscle myosin light chain kinases, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Km and Vmax values with rabbit or chicken skeletal, bovine cardiac, and chicken gizzard smooth muscle myosin P-light chains were very similar for the chicken and rabbit skeletal muscle myosin light chain kinases. In contrast, comparable Km and Vmax data for the chicken gizzard smooth muscle myosin light chain kinase showed that this enzyme was catalytically very different from the two skeletal muscle kinases. Affinity-purified antibodies to rabbit skeletal muscle myosin light chain kinase cross-reacted with chicken skeletal muscle myosin light chain kinase, but the titer of cross-reacting antibodies was approximately 20-fold less than the anti-rabbit skeletal muscle myosin light chain kinase titer. There was no detectable antibody cross-reactivity against chicken gizzard myosin light chain kinase. Proteolytic digestion followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or high performance liquid chromatography showed that these enzymes are structurally very different with few, if any, overlapping peptides. These data suggest that, although chicken skeletal muscle myosin light chain kinase is catalytically very similar to rabbit skeletal muscle myosin light chain kinase, the two enzymes have different primary sequences. The two skeletal muscle myosin light chain kinases appear to be more similar to each other than either is to chicken gizzard smooth muscle myosin light chain kinase.     

Metformin-induced AMP-activated protein kinase activation regulates phenylephrine-mediated contraction of rat aorta

The aim of the present study is to determine the effects and molecular mechanisms by which activation of LKB1-AMP-activated protein kinase (AMPK) by metformin regulates vascular smooth muscle contraction. The essential ability of vascular smooth muscle cells (VSMCs) to contract and relax in response to an elevation and reduction in intravascular pressure is necessary for appropriate blood flow regulation. Thus, vessel contraction is a critical mechanism for systemic blood flow regulation. In cultured rat VSMCs, AMPK activation through LKB1 by metformin-inhibited phenylephrine-mediated myosin light chain kinase (MLCK) and myosin light chain phosphorylation (p-MLC). Conversely, inhibition of AMPK and LKB1 reversed phenylephrine-induced MLCK and p-MLC phosphorylation. Measurement of the tension trace in rat aortic rings also showed that the effect of AMPK activation by metformin decreased phenylephrine-induced contraction. Metformin inhibited PE-induced p-MLC and α-smooth muscle actin co-localization. Our results suggest that activation of AMPK by LKB1 decreases VSMC contraction by inhibiting MLCK and p-MLC, indicating that induction by the AMPK-LKB1 pathway may be a new therapeutic target to lower high blood pressure.     

Substrate specificity of myosin light chain kinases.

Skeletal muscle myosin light chain kinase can phosphorylate myosin light chains isolated from skeletal or smooth muscle. In contrast, smooth muscle myosin light chain kinase specifically phosphorylates light chains isolated from smooth muscle. In this study, we have identified residues within the rabbit smooth and skeletal muscle myosin light chain kinases which may interact with the basic residues that are important substrate determinants in the light chains. Mutation of aspartic acid 270 amino-terminal of the catalytic core of the skeletal muscle myosin light chain kinase increased the Km value for both smooth and skeletal muscle light chains. Although deletions of the analogous region of the smooth muscle myosin light chain kinase (residues 663-678) markedly increased the Km value for light chain, mutation of any single acidic residue within this region did not have a similar effect. Mutation of single residues within the catalytic core of the skeletal muscle (E377 and E421) and smooth muscle (E777 and E821) myosin light chain kinases increased Km values for the smooth muscle light chain at least 35- and 100-fold, respectively. It is proposed that these residues may form ionic interactions with the arginine that is 3 residues amino-terminal of the phosphorylatable serine in the smooth muscle light chain.     

Biochemical properties of chimeric skeletal and smooth muscle myosin light chain kinases.

The molecular and biochemical properties of myosin light chain kinases from chicken skeletal and smooth muscle were investigated by recombinant DNA techniques. Deletion of the amino-terminal region of either the smooth or skeletal muscle myosin light chain kinase resulted in a decrease in Vmax with no significant change in Km values for light chain substrates. Skeletal/smooth muscle chimeric kinases were inactive when a 65-residue region amino-terminal of the catalytic core was exchanged between the two forms. Changing alanine 494 to glutamic acid within this region in the chicken skeletal muscle myosin light chain kinase increased the Km values for light chains 10-fold. These results are consistent with the hypothesis that the region amino-terminal of the catalytic core in myosin light chain kinases is involved in light chain recognition. A skeletal muscle kinase which contained the smooth muscle calmodulin binding domain remained regulated by Ca2+/calmodulin. Thus, the calmodulin binding domains of smooth and skeletal muscle myosin light chain kinases share structural elements necessary for regulation.     

Regulation of myosin-bound protein phosphatase by insulin in vascular smooth muscle cells: evaluation of the role of Rho kinase and phosphatidylinositol-3-kinase-dependent signaling pathways

In this study, we examined the molecular mechanism of myosin-bound protein phosphatase (MBP) regulation by insulin and evaluated the role of MBP in insulin-mediated vasorelaxation. Insulin rapidly stimulated MBP in confluent primary vascular smooth muscle cell (VSMC) cultures. In contrast, VSMCs isolated from diabetic and hypertensive rats exhibited impaired MBP activation by insulin. Insulin-mediated MBP activation was accompanied by a rapid time-dependent reduction in the phosphorylation state of the myosin-bound regulatory subunit (MBS) of MBP. The decrease observed in MBS phosphorylation was due to insulin-induced inhibition of Rho kinase activity. Insulin also prevented a thrombin-mediated increase in Rho kinase activation and abolished the thrombin-induced increase in MBS phosphorylation and MBP inactivation. These data are consistent with the notion that insulin inactivates Rho kinase and decreases MBS phosphorylation to activate MBP in VSMCs. Furthermore, treatment with synthetic inhibitors of phosphatidylinositol-3 kinase (PI3-kinase), nitric oxide synthase (NOS), and cyclic guanosine monophosphate (cGMP) all blocked insulin's effect on MBP activation. We conclude that insulin stimulates MBP via its regulatory subunit, MBS partly by inactivating Rho kinase and stimulating NO/cGMP signaling via PI3-kinase as part of a complex signaling network that controls 20-kDa myosin light chain (MLC20) phosphorylation and VSMC contraction.     

Mammalian skeletal muscle myosin light chain kinases. A comparison by antiserum cross-reactivity

Purified myosin light chain kinases from skeletal muscle are reported to be significantly smaller (Mr = 75,000-90,000) than the kinases purified from smooth muscle (Mr = 130,000-155,000). It has been suggested that the smaller kinases from striated muscle are proteolytic fragments of a larger enzyme which is homologous, if not identical, to myosin light chain kinase from smooth muscle. Therefore, we have used an antiserum to rabbit skeletal muscle myosin light chain kinase and Western blot analysis to compare the subunit molecular weight of the kinase in skeletal muscle extracts of several mammalian species. In rabbit skeletal muscle, the antiserum only recognized a polypeptide of Mr = 87,000, with no indication that this polypeptide was a proteolyzed fragment of a larger protein. The apparent molecular weights observed in different animal species were 75,000 (mouse), 83,000 (guinea pig), 82,000 (rat), 87,000 (rabbit), 100,000 (dog), and 108,000 (steer). The molecular weight of myosin light chain kinase was constant within an animal species, regardless of skeletal muscle fiber type. The antiserum inhibited the catalytic activity of skeletal muscle myosin light chain kinase. Similar antibody dilution curves for inhibition of myosin light chain kinase activity in extracts were observed for all animal species (rabbit, rat, mouse, guinea pig, dog, cat, steer, and chicken) and different fibers (slow twitch oxidative, fast twitch oxidative glycolytic, and fast twitch glycolytic) tested. The antiserum did not inhibit the activity of rabbit smooth muscle myosin light chain kinase. These results suggest that there may be at least two classes of muscle myosin light chain kinase represented in skeletal and smooth muscles, respectively.     

Resident phenotypically modulated vascular smooth muscle cells in healthy human arteries

Vascular interstitial cells (VICs) are non‐contractile cells with filopodia previously described in healthy blood vessels of rodents and their function remains unknown. The objective of this study was to identify VICs in human arteries and to ascertain their role. VICs were identified in the wall of human gastro‐omental arteries using transmission electron microscopy. Isolated VICs showed ability to form new and elongate existing filopodia and actively change body shape. Most importantly sprouting VICs were also observed in cell dispersal. RT‐PCR performed on separately collected contractile vascular smooth muscle cells (VSMCs) and VICs showed that both cell types expressed the gene for smooth muscle myosin heavy chain (SM‐MHC). Immunofluorescent labelling showed that both VSMCs and VICs had similar fluorescence for SM‐MHC and αSM‐actin, VICs, however, had significantly lower fluorescence for smoothelin, myosin light chain kinase, h‐calponin and SM22α. It was also found that VICs do not have cytoskeleton as rigid as in contractile VSMCs. VICs express number of VSMC‐specific proteins and display features of phenotypically modulated VSMCs with increased migratory abilities. VICs, therefore represent resident phenotypically modulated VSMCs that are present in human arteries under normal physiological conditions.     

Myosin Light Chain Kinase Is Necessary for Tonic Airway Smooth Muscle Contraction

Different interacting signaling modules involving Ca²⁺/calmodulin-dependent myosin light chain kinase, Ca²⁺-independent regulatory light chain phosphorylation, myosin phosphatase inhibition, and actin filament-based proteins are proposed as specific cellular mechanisms involved in the regulation of smooth muscle contraction. However, the relative importance of specific modules is not well defined. By using tamoxifen-activated and smooth muscle-specific knock-out of myosin light chain kinase in mice, we analyzed its role in tonic airway smooth muscle contraction. Knock-out of the kinase in both tracheal and bronchial smooth muscle significantly reduced contraction and myosin phosphorylation responses to K⁺-depolarization and acetylcholine. Kinase-deficient mice lacked bronchial constrictions in normal and asthmatic airways, whereas the asthmatic inflammation response was not affected. These results indicate that myosin light chain kinase acts as a central participant in the contractile signaling module of tonic smooth muscle. Importantly, contractile airway smooth muscles are necessary for physiological and asthmatic airway resistance.     

Molecular characterization of a mammalian smooth muscle myosin light chain kinase.

A 5.6-kilobase cDNA clone has been isolated which includes the entire coding region for the myosin light chain kinase from rabbit uterine tissue. This cDNA, expressed in COS cells, encodes a Ca2+/calmodulin-dependent protein kinase with catalytic properties similar to other purified smooth muscle myosin light chain kinases. A module (TLKPVGNIKPAE), repeated sequentially 15 times, has been identified near the N terminus of this smooth muscle kinase. It is not present in chicken gizzard or rabbit skeletal muscle myosin light chain kinases. This repeat module and a subrepeat (K P A/V) are similar in amino acid content to repeated motifs present in other proteins, some of which have been shown to associate with chromatin structures. Immunoblot analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, used to compare myosin light chain kinase present in rabbit, bovine, and chicken smooth and nonmuscle tissues, showed that within each species both tissue types have myosin light chain kinases with indistinguishable molecular masses. These data suggest that myosin light chain kinases present in smooth and nonmuscle tissues are the same protein.     

Role of myosin light chain kinase in regulation of basal blood pressure and maintenance of salt-induced hypertension

Vascular tone, an important determinant of systemic vascular resistance and thus blood pressure, is affected by vascular smooth muscle (VSM) contraction. Key signaling pathways for VSM contraction converge on phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin. This phosphorylation is mediated by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) but Ca(2+)-independent kinases may also contribute, particularly in sustained contractions. Signaling through MLCK has been indirectly implicated in maintenance of basal blood pressure, whereas signaling through RhoA has been implicated in salt-induced hypertension. In this report, we analyzed mice with smooth muscle-specific knockout of MLCK. Mesenteric artery segments isolated from smooth muscle-specific MLCK knockout mice (MLCK(SMKO)) had a significantly reduced contractile response to KCl and vasoconstrictors. The kinase knockout also markedly reduced RLC phosphorylation and developed force. We suggest that MLCK and its phosphorylation of RLC are required for tonic VSM contraction. MLCK(SMKO) mice exhibit significantly lower basal blood pressure and weaker responses to vasopressors. The elevated blood pressure in salt-induced hypertension is reduced below normotensive levels after MLCK attenuation. These results suggest that MLCK is necessary for both physiological and pathological blood pressure. MLCK(SMKO) mice may be a useful model of vascular failure and hypotension.     

PDGF-dependent regulation of regulator of G protein signaling-5 expression and vascular smooth muscle cell functionality

Regulator of G protein signaling (RGS) proteins, and notably members of the RGS-R4 subfamily, control vasocontractility by accelerating the inactivation of Gα-dependent signaling. RGS5 is the most highly and differently expressed RGS-R4 subfamily member in arterial smooth muscle. Expression of RGS5 first appears in pericytes during development of the afferent vascular tree, suggesting that RGS5 is a good candidate for a regulator of arterial contractility and, perhaps, for determining the mass of the smooth muscle coats required to regulate blood flow in the branches of the arterial tree. Consistent with this hypothesis, using cultured vascular smooth muscle cells (VSMCs), we demonstrate RGS5 overexpression inhibits G protein-coupled receptor (GPCR)-mediated hypertrophic responses. The next objective was to determine which physiological agonists directly control RGS5 expression in VSMCs. GPCR agonists failed to directly regulate RGS5 mRNA expression; however, platelet-derived growth factor (PDGF) acutely represses expression. Downregulation of RGS5 results in the induction of migration and the activation of the GPCR-mediated signaling pathways. This stimulation leads to the activation of mitogen-activated protein kinases directly downstream of receptor stimulation, and ultimately VSMC hypertrophy. These results demonstrate that RGS5 expression is a critical mediator of both VSMC contraction and potentially, arterial remodeling.     

Role of myosin light chain kinase and myosin light chain phosphatase in the resistance arterial myogenic response to intravascular pressure

The intrinsic ability of vascular smooth muscle cells (VSMCs) within arterial resistance vessels to respectively contract and relax in response to elevation and reduction of intravascular pressure is essential for appropriate blood flow autoregulation. This fundamental mechanism, referred to as the myogenic response, is dependent on apposite control of myosin regulatory light chain (LC₂₀) phosphorylation, a prerequisite for force generation, through the coordinated activity of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Here, we highlight the molecular basis of the smooth muscle contractile mechanism and review the regulatory pathways demonstrated to participate in the control of LC₂₀ phosphorylation in the myogenic response, with a focus on the Ca²⁺-dependent and Rho-associated kinase (ROK)-mediated regulation of MLCK and MLCP, respectively.     

Phosphorylation of synthetic peptides by skeletal muscle myosin light chain kinases

Substrate determinants for rabbit and chicken skeletal muscle myosin light chain kinases were examined with synthetic peptides. Both skeletal muscle myosin light chain kinases had similar phosphorylation kinetics with synthetic peptide substrates. Average kinetic constants for skeletal muscle myosin light chain heptadecapeptide, (formula; see text) where S(P) is phosphoserine, were Km, 2.3 microM and Vmax, 0.9 mumol/min/mg of enzyme. Km values were 122 and 162 microM for skeletal muscle peptides containing A-A for basic residues at positions 2-3 and 6-7, respectively. Average kinetic constants for smooth muscle myosin light chain peptide, (formula; see text), were Km, 1.4 microM and Vmax 27 mumol/min/mg of enzyme. Average Km values for the smooth muscle peptide, residues 11-23, were 10 microM which increased 6- and 11-fold with substitutions of alanine at residues 12 and 13, respectively. Vmax values decreased and Km values increased markedly by substitution of residue 16 with glutamate in the 11-23 smooth muscle tridecapeptide. Basic residues located 3 and 6-7 residues toward the NH2 terminus from phosphoserine in smooth muscle myosin light chain and 6-8 and 10-11 residues toward the NH2 terminus from phosphoserine in skeletal muscle myosin light chain appear to be important substrate determinants for skeletal muscle myosin light chain kinases. These properties are different from myosin light chain kinase from smooth muscle.     

A novel RhoA/ROCK-CPI-17-MEF2C signaling pathway regulates vascular smooth muscle cell gene expression

Differentiation of vascular smooth muscle cells (VSMC) is a fundamental aspect of normal development and vascular disease. During contraction, VSMCs modulate calcium sensitivity through RhoA/ROCK-mediated inhibition of the myosin light chain phosphatase complex (MLCP). Previous studies have demonstrated that this signaling pathway functions in parallel to increase the expression of smooth muscle genes through the myocardin-family of co-activators. MEF2C fulfills a critical role in VSMC differentiation and regulates myocardin expression, leading us to investigate whether the RhoA/ROCK signaling cascade might regulate MEF2 activity. Depolarization-induced calcium signaling increased the expression of myocardin, which was sensitive to ROCK and p38 MAPK inhibition. We previously identified protein phosphatase 1α (PP1α), a known catalytic subunit of the MLCP in VSMCs, as a potent repressor of MEF2 activity. PP1α inhibition resulted in increased expression of myocardin, while ectopic expression of PP1α inhibited the induction of myocardin by MEF2C. Consistent with these data, shRNA-mediated suppression of a PP1α inhibitor, CPI-17, reduced myocardin expression and inhibited VSMC differentiation, suggesting a pivotal role for CPI-17 in regulating MEF2 activity. These data constitute evidence of a novel signaling cascade that links RhoA-mediated calcium sensitivity to MEF2-dependent myocardin expression in VSMCs through a mechanism involving p38 MAPK, PP1α, and CPI-17.     

Invited review: cGMP-dependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression.

cGMP is a second messenger that produces its effects by interacting with intracellular receptor proteins. In smooth muscle cells, one of the major receptors for cGMP is the serine/threonine protein kinase, cGMP-dependent protein kinase (PKG). PKG has been shown to catalyze the phosphorylation of a number of physiologically relevant proteins whose function it is to regulate the contractile activity of the smooth muscle cell. These include proteins that regulate free intracellular calcium levels, the cytoskeleton, and the phosphorylation state of the regulatory light chain of smooth muscle myosin. Other studies have shown that vascular smooth muscle cells (VSMCs) that are cultured in vitro may cease to express PKG and will, coincidentally, acquire a noncontractile, synthetic phenotype. The restoration of PKG expression to the synthetic phenotype VSMC results in the cells acquiring a more contractile phenotype. These more recent studies suggest that PKG controls VSMC gene expression that, in turn, regulates phenotypic modulation of the cells. Therefore, the regulation of PKG gene expression appears to be linked to phenotypic modulation of VSMC. Because several vascular disorders are related to the accumulation of synthetic, fibroproliferative VSMC in the vessel wall, it is likely that changes in the activity of the nitric oxide/cGMP/PKG pathway is involved the development of these diseases.