Expression of CPI-17 in smooth muscle during embryonic development and in neointimal lesion formation

Ca²⁺ sensitivity of smooth muscle (SM) contraction is determined by CPI-17, an inhibitor protein for myosin light chain phosphatase (MLCP). CPI-17 is highly expressed in mature SM cells, but the expression level varies under pathological conditions. Here, we determined the expression of CPI-17 in embryonic SM tissues and arterial neointimal lesions using immunohistochemistry. As seen in adult animals, the predominant expression of CPI-17 was detected at SM tissues on mouse embryonic sections, whereas MLCP was ubiquitously expressed. Compared with SM α-actin, CPI-17 expression doubled in arterial SM from embryonic day E10 to E14. Like SM α-actin and other SM marker proteins, CPI-17 was expressed in embryonic heart, and the expression was down-regulated at E17. In adult rat, CPI-17 expression level was reduced to 30% in the neointima of injured rat aorta, compared with the SM layers, whereas the expression of MLCP was unchanged in both regions. Unlike other SM proteins, CPI-17 was detected at non-SM organs in the mouse embryo, such as embryonic neurons and epithelium. Thus, CPI-17 expression is reversibly controlled in response to the phenotype transition of SM cells that restricts the signal to differentiated SM cells and particular cell types. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

PHI-1 induced enhancement of myosin phosphorylation in chicken smooth muscle

Herein, we provide evidence that in chicken smooth muscle, G-protein stimulation by a Rho-kinase pathway leads to an increase in myosin light chain phosphorylation. Additionally, G-protein stimulation did not increase MYPT1 phosphorylation at Thr695 or Thr850, and CPI-17, was not expressed in chicken smooth muscle. However, PHI-1 was present in chicken smooth muscle tissues. Both agonist and GTP(gamma)S stimulation result in an increase in PHI-1 phosphorylation, which is inhibited by inhibitors to both Rho-kinase (Y-27632) and (PKC) GF109203x. These data suggest that PHI-1 may act as a CPI-17 analog in chicken smooth muscle and inhibit myosin phosphatase activity during G-protein stimulation to produce Ca2+ sensitization.     

Decreased phosphatase activity, increased Ca2+ sensitivity, and myosin light chain phosphorylation in urinary bladder smooth muscle of newborn mice

Developmental changes in the regulation of smooth muscle contraction were examined in urinary bladder smooth muscle from mice. Maximal active stress was lower in newborn tissue compared with adult, and it was correlated with a lower content of actin and myosin. Sensitivity to extracellular Ca2+ during high-K+ contraction, was higher in newborn compared with 3-wk-old and adult bladder strips. Concentrations at half maximal tension (EC50) were 0.57 +/- 0.01, 1.14 +/- 0.12, and 1.31 +/- 0.08 mM. Force of the newborn tissue was inhibited by approximately 45% by the nonmuscle myosin inhibitor Blebbistatin, whereas adult tissue was not affected. The calcium sensitivity in newborn tissue was not affected by Blebbistatin, suggesting that nonmuscle myosin is not a primary cause for increased calcium sensitivity. The relation between intracellular [Ca2+] and force was shifted toward lower [Ca2+] in the newborn bladders. This increased Ca2+ sensitivity was also found in permeabilized muscles (EC50: 6.10 +/- 0.07, 5.77 +/- 0.08, and 5.55 +/- 0.02 pCa units, in newborn, 3-wk-old, and adult tissues). It was associated with an increased myosin light chain phosphorylation and a decreased rate of dephosphorylation. No difference was observed in the myosin light chain phosphorylation rate, whereas the rate of myosin light chain phosphatase-induced relaxation was about twofold slower in the newborn tissue. The decreased rate was associated with a lower expression of the phosphatase regulatory subunit MYPT-1 in newborn tissue. The results show that myosin light chain phosphatase activity can be developmentally regulated in mammalian urinary bladders. The resultant alterations in Ca2+ sensitivity may be of importance for the nervous and myogenic control of the newborn bladders.     

A functional interaction between CPI-17 and RACK1 proteins in bronchial smooth muscle cells

CPI-17 is a phosphorylation-dependent inhibitor of smooth muscle myosin light chain. Using yeast two-hybrid system, we have identified the receptor for activated C kinase 1 (RACK1) as a novel interaction partner of CPI-17. The direct interaction and co-localization of CPI-17 with RACK1 were confirmed by immunoprecipitation and confocal microscopy analysis, respectively. An in vitro assay system using recombinant/purified proteins revealed that the PKC-mediated phosphorylation of CPI-17 was augmented in the presence of RACK1. These results suggest that RACK1 may play a role in PKC/CPI-17 signaling pathway.     

Regulation of fibronectin matrix assembly and capillary morphogenesis in endothelial cells by Rho family GTPases

Fibronectin (FN) fibrillogenesis is an essential biological process mediated by α5β1 integrin and cellular contractile forces. Assembly of a FN matrix by activated endothelial cells occurs during angiogenic blood vessel remodeling and signaling components that control this event represent attractive therapeutic targets. Here we examined the role of individual Rho GTPases in FN matrix remodeling by selectively attenuating their expression in cultured endothelial cells. Whereas pharmacological ablation of myosin-regulated contractility abrogated matrix assembly, no significant decrease was detected in the amount of FN deposited by RhoA, RhoB-, RhoC-, Rac1-, or Cdc42-depleted cells. Rather, distinct differences in fiber arrangement were observed. Most strikingly, RhoA silenced cells assembled a fine FN meshwork beneath α5β1 integrin-based fibrillar adhesions, in the absence of classical focal adhesions and actin stress fibers, indicating that α5β1 integrin translocation and FN fibril elongation can occur in low tension states such as those encountered by newly-forming vessels in tissue. In contrast, highly contractile Cdc42-deficient cells deposited FN globules and Rac-deficient cells assembled long arrays, reflecting their increased motility. We propose that regulation of FN scaffolds by Rho GTPase signaling impacts bidirectional communications and mechanical interactions between endothelial cells and their extracellular matrix during vascular morphogenesis.     

高血压大鼠不同血管平滑肌肌球蛋白磷酸化和脱磷酸化酶的变化

本文比较了正常和高血压大鼠不同动脉血管肌球蛋白轻链激酶（ＭＬＣＫ）和依赖Ｃａ＾２＋的钙调素磷酸酶（Ｃａ＾２＋／ＣａＭ－ＰＰ）活性的变化。结果表明：在自发性高血压大鼠（ＳＨＲ）不同血管ＭＬＣＫ的活性不同,依次为主动脉（Ａ）〉尾动脉（ＣＡ）〉肠系膜动脉（ＭＡ）;而在ＷＫＹ大鼠,该酶在不同血管的活性依次为Ａ〈〈ＣＡ〈〈ＡＭ。在ＷＫＹ大鼠,ＭＡ Ｃａ＾２＋／ＣａＭ－ＰＰ活性明显高于ＳＨＲ。在肾性高血压大鼠     

Bi-directional regulation of emodin and quercetin on smooth muscle myosin of gizzard

This study is to reveal the characteristics of bidirectional regulation of emodin (1,3,8-trihydroxy-6-methyl-anthraquinone) and quercetin on gizzard smooth muscle myosin. Our results indicate that: (a) emodin demonstrates stimulatory effects, and quercetin produces inhibitory effects on myosin phosphorylation and Mg(2+)-ATPase activities of Ca(2+)/calmodulin-dependent phosphorylated myosin in a dose-dependent manner; (b) a combination of emodin and quercetin enhances phosphorylation and Mg(2+)-ATPase activities for partially phosphorylated myosin and inhibits those activities for fully phosphorylated myosin; (c) 1-(5-Chloronaphthalene-1-sulfonyl)-1H2-hexahydro-1,4-diazepine inhibits myosin phosphorylation in the presence of emodin and/or quercetin. A combination of emodin and quercetin indicates its potential for modulating gastric-intestinal smooth muscle.     

The B2 alternatively spliced isoform of nonmuscle myosin II-B lacks actin-activated MgATPase activity and in vitro motility

We report the initial biochemical characterization of an alternatively spliced isoform of nonmuscle heavy meromyosin (HMM) II-B2 and compare it with HMM II-B0, the nonspliced isoform. HMM II-B2 is the HMM derivative of an alternatively spliced isoform of endogenous nonmuscle myosin (NM) II-B, which has 21-amino acids inserted into loop 2, near the actin-binding region. NM II-B2 is expressed in the Purkinje cells of the cerebellum as well as in other neuronal cells [X. Ma, S. Kawamoto, J. Uribe, R.S. Adelstein, Function of the neuron-specific alternatively spliced isoforms of nonmuscle myosin II-B during mouse brain development, Mol. Biol. Cell 15 (2006) 2138-2149]. In contrast to any of the previously described isoforms of NM II (II-A, II-B0, II-B1, II-C0 and II-C1) or to smooth muscle myosin, the actin-activated MgATPase activity of HMM II-B2 is not significantly increased from a low, basal level by phosphorylation of the 20 kDa myosin light chain (MLC-20). Moreover, although HMM II-B2 can bind to actin in the absence of ATP and is released in its presence, it cannot propel actin in the sliding actin filament assay following MLC-20 phosphorylation. Unlike HMM II-B2, the actin-activated MgATPase activity of a chimeric HMM with the 21-amino acid II-B2 sequence inserted into the homologous location in the heavy chain of HMM II-C is increased following MLC-20 phosphorylation. This indicates that the effect of the II-B2 insert is myosin heavy chain specific.     

Regulation of phosphorylation at Ser of GluN2B receptor in the postsynaptic density

Neuronal N-methyl-D-aspartate subtype of ionotropic glutamate receptor (NMDAR) that plays essential roles in excitatory synaptic transmission is regulated by phosphorylation. However, the kinases and phosphatases involved in this regulation are not completely known. We show that the GluN2B subunit of NMDAR is phosphorylated at Ser¹³⁰³ by protein kinase C (PKC) and is dephosphorylated by protein phosphatase 1 (PP1), but not protein phosphatase 2A (PP2A) in isolated postsynaptic density (PSD). Although PSD is known to harbor PKC, PP1 and PP2A, their ability to regulate phosphorylation of GluN2B-Ser¹³⁰³ would depend on the accessibility of GluN2B-Ser¹³⁰³ to these proteins. Since PSD preparation is likely to maintain the organization of its component proteins as inside neurons, accessibility of kinases and phosphatases to GluN2B-Ser¹³⁰³in vivo would be addressed by experiments using this system. Using an antibody specific for the phosphorylated state of GluN2B-Ser¹³⁰³ we demonstrate that PP1 is the major phosphatase in rat brain PSD that can dephosphorylate the GluN2B-Ser¹³⁰³ endogenous to PSD. We also show that PKC present in PSD can phosphorylate GluN2B-Ser¹³⁰³. The events reported here might be important in regulating GluN2B-Ser¹³⁰³ phosphorylation in vivo.     

Ca2+-induced Ca2+ desensitization of myosin light chain phosphorylation and contraction in phasic smooth muscle

The temporal relationship between Ca2+-induced contraction and phosphorylation of 20 kDa myosin light chain (MLC) during a step increase in Ca2+ was investigated using permeabilized phasic smooth muscle from rabbit portal vein and guinea-pig ileum at 25°C. We describe here a Ca2+-induced Ca2+ desensitization phenomenon in which a transient rise in MLC phosphorylation is followed by a transient rise in contractile force. During and after the peak contraction, the force to phosphorylation ratio remained constant. Further treatment with cytochalasin D, an actin fragmenting agent, did not affect the transient increase in phosphorylation, but blocked force development. Together, these results indicate that the transient phosphorylation causes the transient contraction and that neither inhomogeneous contractility nor reduced thin filament integrity effects the transient phosphorylation. Lastly, we show that known inhibitors to MLC kinase kinases and to a Ca2+-dependent protein phosphatase did not eliminate the desensitized contractile force. This study suggests that the Ca2+-induced Ca2+ desensitization phenomenon in phasic smooth muscle does not result from any of the known intrinsic mechanisms involved with other aspects of smooth muscle contractility.     

Cytokinesis and cancer:Polo loves ROCK'n' Rho(A)

Cytokinesis is the last step of the M (mitosis) phase,yet it is crucial for the faithful division of one cell into two.Cytokinesis failure is often associated with cancer.Cytokinesis can be morphologically divided into four steps:cleavage furrow initiation,cleavage furrow ingression,midbody formation and abscission.Molecular studies have revealed that RhoA as well as its regulators and effectors are important players to ensure a successful cytokinesis.At the same time,Polo-like kinase 1 (Plk1) is an important kinase that can target many substrates and carry out different functions during mitosis,including cytokinesis.Recent studies are beginning to unveil a closer tie between Plk1 and RhoA networks.More specifically,Plk1 phosphorylates the centralspindlin complex Cyk4 and MKLP1/CHO1,thus recruiting RhoA guanine nucleotide-exchange factor (GEF) Ect2 through its phosphopeptide-binding BRCT domains.Ect2 itself can be phosphorylated by Plk1 in vitro.Plk1 can also phosphorylate another GEF MyoGEF to regulate RhoA activity.Once activated,RhoA-GTP will activate downstream effectors,including ROCK1 and ROCK2.ROCK2 is among the proteins that associate with Plk1 Polo-binding domain (PBD) in a large proteomic screen,and Plk1 can phosphorylate ROCK2 in vitro.We review current understandings of the interplay between Plk1,RhoA proteins and other proteins (e.g.,NudC,MKLP2,PRC1,CEP55) involved in cytokinesis,with partitular emphasis of its clinical implications in cancer.     

Involvement of Fyn tyrosine kinase in actin stress fiber formation in fibroblasts

Lysophosphatidic acid (LPA) and sphingosylphosphorylcholine (SPC) activated Fyn tyrosine kinase and induced stress fiber formation, which was blocked by pharmacological inhibition of Fyn, gene silencing of Fyn, or dominant negative Fyn. Overexpressed constitutively active Fyn localized at both ends of F-actin bundles and triggered stress fiber formation, only the latter of which was abolished by Rho-kinase (ROCK) inhibition. SPC, but not LPA, induced filopodia-like protrusion formation, which was not mediated by Fyn and ROCK. Thus, Fyn appears to act downstream of LPA and SPC to specifically stimulate stress fiber formation mediated by ROCK in fibroblasts.     

Myosin light chains are not a physiological substrate of AMPK in the control of cell structure changes

The kinetics of myosin regulatory light chain (MLC) phosphorylation by recombinant AMP-activated protein kinase (AMPK) were compared with commercial AMPK from rat liver and smooth muscle myosin light chain kinase (smMLCK). With identical amounts of activity units, initial rates of phosphorylation of MLC were at least 100-fold less with recombinant AMPK compared to smMLCK, whereas with rat liver AMPK significant phosphorylation was seen. In Madin-Darby Canine Kidney cells, AMPK activation led to an increase in MLC phosphorylation, which was decreased by a Rho kinase inhibitor without affecting AMPK activation. Therefore, MLC phosphorylation during energy deprivation does not result from direct phosphorylation by AMPK.

Structured summary

MINT-6800264: smMLCK (uniprotkb:P11799) phosphorylates (MI:0217) MLC (uniprotkb:P08590) by protein kinase assay (MI:0424)

MINT-6800252: AMPK (uniprotkb:Q13131) phosphorylates (MI:0217) ACC2 (uniprotkb:000763) by protein kinase assay (MI:0424)

     

Regulation of myofibroblast activities: Calcium pulls some strings behind the scene

Myofibroblast-induced remodeling of collagenous extracellular matrix is a key component of our body's strategy to rapidly and efficiently repair damaged tissues; thus myofibroblast activity is considered crucial in assuring the mechanical integrity of vital organs and tissues after injury. Typical examples of beneficial myofibroblast activities are scarring after myocardial infarct and repair of damaged connective tissues including dermis, tendon, bone, and cartilage. However, deregulation of myofibroblast contraction causes the tissue deformities that characterize hypertrophic scars as well as organ fibrosis that ultimately leads to heart, lung, liver and kidney failure. The phenotypic features of the myofibroblast, within a spectrum going from the fibroblast to the smooth muscle cell, raise the question as to whether it regulates contraction in a fibroblast- or muscle-like fashion. In this review, we attempt to elucidate this point with a particular focus on the role of calcium signaling. We suggest that calcium plays a central role in myofibroblast biological activity not only in regulating contraction but also in mediating intracellular and extracellular mechanical signals, structurally organizing the contractile actin-myosin cytoskeleton, and establishing lines of intercellular communication.     

A molecular model of phosphorylation-based activation and potentiation of tarantula muscle thick filaments

Myosin filaments from many muscles are activated by phosphorylation of their regulatory light chains (RLCs). To elucidate the structural mechanism of activation, we have studied RLC phosphorylation in tarantula thick filaments, whose high-resolution structure is known. In the relaxed state, tarantula RLCs are ∼ 50% non-phosphorylated and 50% mono-phosphorylated, while on activation, mono-phosphorylation increases, and some RLCs become bi-phosphorylated. Mass spectrometry shows that relaxed-state mono-phosphorylation occurs on Ser35, while Ca²⁺-activated phosphorylation is on Ser45, both located near the RLC N-terminus. The sequences around these serines suggest that they are the targets for protein kinase C and myosin light chain kinase (MLCK), respectively. The atomic model of the tarantula filament shows that the two myosin heads (“free” and “blocked”) are in different environments, with only the free head serines readily accessible to kinases. Thus, protein kinase C Ser35 mono-phosphorylation in relaxed filaments would occur only on the free heads. Structural considerations suggest that these heads are less strongly bound to the filament backbone and may oscillate occasionally between attached and detached states (“swaying” heads). These heads would be available for immediate actin interaction upon Ca²⁺ activation of the thin filaments. Once MLCK becomes activated, it phosphorylates free heads on Ser45. These heads become fully mobile, exposing blocked head Ser45 to MLCK. This would release the blocked heads, allowing their interaction with actin. On this model, twitch force would be produced by rapid interaction of swaying free heads with activated thin filaments, while prolonged exposure to Ca²⁺ on tetanus would recruit new MLCK-activated heads, resulting in force potentiation.     

MRS2578 通过P2Y6-ROCK-MILC信号通路抑制海马CA3区域小胶质细胞吞噬作用并加重小鼠癫痫发作

许多研究表明,癫痫过程中P2Y6受体表达增加。MRS2578作为P2Y6受体的不可逆抑制剂,其在癫痫中作用尚不明确。本研究旨在阐明MRS2578在癫痫中的作用及其潜在的分子机制。研究发现,小鼠脑室注射海人酸诱导癫痫模型,同时注射MRS2578能加重癫痫发作,此作用呈现浓度依赖效应;MRS2578增加海马CA3区域神经元丢失（FJB+神经元由71.67±8.88增加至98.45±9.97个/视野）;同时抑制小胶质细胞的激活以及转变为阿米巴样细胞（小胶质细胞密度由66.18±5.86下降至55.22±10.85个/视野）,小胶质细胞CD68表达也相应降低;共聚焦显微镜检测发现,MRS2578处理后,CA3区域小胶质细胞吞噬细胞所形成的“吞噬杯”结构减少;Western 印迹检测证实,MRS2578抑制小胶质细胞P2Y6、Rho相关激酶（ROCK）、p-MLC-Ser19的分子表达。上述结果提示,MRS2578加重海人酸诱导的癫痫发作,伴随小胶质细胞激活的抑制和细胞吞噬功能下调,此过程依赖P2Y6-ROCK-MLC信号途径。     

PHI-1 interacts with the catalytic subunit of myosin light chain phosphatase to produce a Ca(2+) independent increase in MLC(20) phosphorylation and force in avian smooth muscle

In avian smooth muscles, GTPgammaS produces a Rho kinase mediated increase in PHI-1 phosphorylation and force, but whether this correlation is causal is unknown. We examined the effect of phosphorylated PHI-1 (P-PHI-1) on force and myosin light chain (MLC(20)) phosphorylation at a constant [Ca(2+)]. P-PHI-1, but not PHI-1, increased MLC(20) phosphorylation and force, and phosphorylation of PHI-1 increased the interaction of PHI-1 with PP1c. Microcystin induced a dose-dependent reduction in the binding of PHI-1 to PP1c. These results suggest PHI-1 inhibits myosin light chain phosphatase by interacting with the active site of PP1c to produce a Ca(2+) independent increase in MLC(20) phosphorylation and force.