Inhibition Of Myosin Phosphatase Research Articles

Smoothelin-like protein 1 promotes insulin sensitivity and modulates the contractile properties of endometrial epithelial cells with insulin resistance.

The incidence of infertility is significantly higher in women with diseases linked to impaired glucose homeostasis, such as insulin resistance. Defective glucose metabolism interferes with fertilization; however, the molecular mechanism underlying this interference is unclear. Smoothelin-like protein 1 (SMTNL1) was isolated from muscle and steroid hormone-responsive tissues and regulates the contractile functions of various cell types through the inhibition of myosin phosphatase (MP) holoenzyme. In addition, SMTNL-1 after phosphorylation at Ser301 by protein kinase A translocates to the nucleus and functions as a transcriptional co-activator of the progesterone receptor-B. SMTNL1 null mice exhibit reduced reproductive fitness and are more prone to type 2 diabetes mellitus. However, the role of SMTNL1 in endometrial epithelial cells is not known. The effect of SMTNL1 overexpression was investigated in pregnancy and in gestational diabetic endometrial epithelial cell models by immunofluorescent staining, cell migration, and semi quantitative Western blot analysis and glucose uptake assay. We show that SMTNL1 promotes the differentiation of endometrial epithelial cells in a progesterone-dependent manner to attenuate insulin resistance. Furthermore, SMTNL1 hampers the migration capacity of epithelial cells in a gestational diabetes model by inhibiting the expression of MYPT1, the regulatory subunit of MP, and the activity of the holoenzyme, resulting in increased phosphorylation of the 20 kDa regulatory myosin light chain. SMTNL1 also acts as an insulin-sensitizing agent by increasing the gene expression of PP2A and DUPS9 protein phosphatases, resulting in decreased ERK1/2 activity and, hence, decreasing the phosphorylation of IRS-1 at Ser612 under gestational diabetes conditions. SMTNL1 may have therapeutic relevance to the progesterone-dependent inhibition of endometrial epithelial cell migration under hyperglycemic conditions and insulin sensitivity in the endometrium in gestational diabetes or other metabolic disorders.

Itga8-Cre-mediated deletion of YAP and TAZ impairs bladder contractility with minimal inflammation and chondrogenic differentiation.

A role of Yes1-associated transcriptional regulator (YAP) and WW domain-containing transcription regulator 1 (TAZ) in vascular and gastrointestinal contractility due to control of myocardin (Myocd) expression, which in turn activates contractile genes, has been demonstrated. Whether this transcriptional hierarchy applies to the urinary bladder is unclear. We found that YAP/TAZ are expressed in human detrusor myocytes and therefore exploited the Itga8-CreERT2 model for the deletion of YAP/TAZ. Recombination occurred in detrusor, and YAP/TAZ transcripts were reduced by >75%. Bladder weights were increased (by ≈22%), but histology demonstrated minimal changes in the detrusor, while arteries in the mucosa were inflamed. Real-time quantitative reverse transcription PCR (RT-qPCR) using the detrusor demonstrated reductions of Myocd (-79 ± 18%) and serum response factor (Srf) along with contractile genes. In addition, the cholinergic receptor muscarinic 2 (Chrm2) and Chrm3 were suppressed (-80 ± 23% and -80 ± 10%), whereas minute increases of Il1b and Il6 were seen. Unlike YAP/TAZ-deficient arteries, SRY (sex-determining region Y)-box 9 (Sox9) did not increase, and no chondrogenic differentiation was apparent. Reductions of smooth muscle myosin heavy chain 11 (Myh11), myosin light-chain kinase gene (Mylk), and Chrm3 were seen at the protein level. Beyond restraining the smooth muscle cell (SMC) program of gene expression, YAP/TAZ depletion silenced SMC-specific splicing, including exon 2a of Myocd. Reduced contractile differentiation was associated with weaker contraction in response to myosin phosphatase inhibition (-36%) and muscarinic activation (reduced by 53% at 0.3 µM carbachol). Finally, short-term overexpression of constitutively active YAP in human embryonic kidney 293 (HEK293) cells increased myocardin (greater than eightfold) along with archetypal target genes, but contractile genes were unaffected or reduced. YAP and TAZ thus regulate myocardin expression in the detrusor, and this is important for SMC differentiation and splicing as well as for contractility.NEW & NOTEWORTHY This study addresses the hypothesis that YAP and TAZ have an overarching role in the transcriptional hierarchy in the smooth muscle of the urinary bladder by controlling myocardin expression. Using smooth muscle-specific and inducible deletion of YAP and TAZ in adult mice, we find that YAP and TAZ control myocardin expression, contractile differentiation, smooth muscle-specific splicing, and bladder contractility. These effects are largely independent of inflammation and chondrogenic differentiation.

N6-methyladenosine-modified microRNA-675 advances the development of gastrointestinal stromal tumors via inhibiting myosin phosphatase targeting protein 1

RNA N6-methyladenosine (m6A) modifications influence gastrointestinal stromal tumors (GISTs) development, but the detailed molecular mechanisms have not been fully studied. Here, microRNA-675 was found to be aberrantly elevated in cancerous tissues and cells of GISTs, compared to the corresponding normal counterparts, and GISTs patients with high-expressed microRNA-675 have worse outcomes. Additional experiments confirmed that silencing of microRNA-675 hindered cell division, mobility and tumorigenesis in vitro and in vivo, whereas triggered apoptotic cell death in GISTs cells. Furthermore, microRNA-675-ablation increased the expression levels of myosin phosphatase targeting protein 1 (MYPT1) to inactivate the tumor-initiating RhoA/NF2/YAP1 signal pathway, and downregulation of MYPT1 recovered the malignant phenotypes in microRNA-675-silenced GISTs cells. In addition, we evidenced that METTL3-mediated m6A modifications were essential for sustaining the stability of microRNA-675, and silencing of METTL3 restrained tumorigenesis of GISTs cells by regulating the microRNA-675/MYPT1 axis. To summarize, theMETTL3/m6A/microRNA-675/MYPT1 axis could be used as novel biomarkers for the diagnosis and treatment of GISTs.

Phosphodiesterase 4D promotes angiotensin II-induced hypertension in mice via smooth muscle cell contraction

Hypertension is a common chronic disease, which leads to cardio-cerebrovascular diseases, and its prevalence is increasing. The cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway participates in multiple cardiovascular diseases. Phosphodiesterase (PDE) 4 has been shown to regulate PKA activity via cAMP specific hydrolysis. However, whether PDE4-cAMP-PKA pathway influences hypertension remains unknown. Herein, we reveal that PDE4D (one of PDE4 isoforms) expression is upregulated in the aortas of experimental hypertension induced by angiotensin II (Ang II). Furthermore, knockout of Pde4d in mouse smooth muscle cells (SMCs) attenuates Ang II-induced hypertension, arterial wall media thickening, vascular fibrosis and vasocontraction. Additionally, we find that PDE4D deficiency activates PKA-AMP-activated protein kinase (AMPK) signaling pathway to inhibit myosin phosphatase targeting subunit 1 (MYPT1)-myosin light chain (MLC) phosphorylation, relieving Ang II-induced SMC contraction in vitro and in vivo. Our results also indicate that rolipram, a PDE4 inhibitor, may be a potential drug for hypertension therapy.

Possible roles of N- and C-terminal unstructured tails of CPI-17 in regulating Ca2+ sensitization force of smooth muscle.

CPI-17 regulates the myosin phosphatase and mediates the agonist-induced contraction of smooth muscle. PKC and ROCK phosphorylate CPI-17 at Thr38 leading to a conformational change of the central inhibitory domain (PHIN domain). The N- and C-terminal tails of CPI-17 are predicted as unstructured loops and their sequences are conserved among mammals. Here we characterized CPI-17 N- and C-terminal unstructured tails using recombinant proteins that lack the potions. Recombinant CPI-17 proteins at a physiologic level (10 µM) were doped into beta-escin-permeabilized smooth muscle strips for Ca2+ sensitization force measurement. The ectopic full-length CPI-17 augmented the PDBu-induced Ca2+ sensitization force at pCa6.3, indicating myosin phosphatase inhibition. Deletion of N- and C-terminal tails of CPI-17 attenuated the extent of PDBu-induced Ca2+-sensitization force. The N-terminal deletion dampened phosphorylation at Thr38 by protein kinase C (PKC), and the C-terminal truncation lowered the affinity to the myosin phosphatase. Under the physiologic conditions, PKC and myosin phosphatase may recognize CPI-17 N-/C-terminal unstructured tails inducing Ca2+ sensitization force in smooth muscle cells.

CDK14 Promotes Axon Regeneration by Regulating the Noncanonical Wnt Signaling Pathway in a Kinase-Independent Manner.

The postinjury regenerative capacity of neurons is known to be mediated by a complex interaction of intrinsic regenerative pathways and external cues. In Caenorhabditis elegans, the initiation of axon regeneration is regulated by the nonmuscle myosin light chain-4 (MLC-4) phosphorylation signaling pathway. In this study, we have identified svh-16/cdk-14, a mammalian CDK14 homolog, as a positive regulator of axon regeneration in motor neurons. We then isolated the CDK-14-binding protein MIG-5/Disheveled (Dsh) and found that EGL-20/Wnt and the MIG-1/Frizzled receptor (Fz) are required for efficient axon regeneration. Further, we demonstrate that CDK-14 activates EPHX-1, the C. elegans homolog of the mammalian ephexin Rho-type GTPase guanine nucleotide exchange factor (GEF), in a kinase-independent manner. EPHX-1 functions as a GEF for the CDC-42 GTPase, inhibiting myosin phosphatase, which maintains MLC-4 phosphorylation. These results suggest that CDK14 activates the RhoGEF–CDC42–MLC phosphorylation axis in a noncanonical Wnt signaling pathway that promotes axon regeneration.SIGNIFICANCE STATEMENT Noncanonical Wnt signaling is mediated by Frizzled receptor (Fz), Disheveled (Dsh), Rho-type GTPase, and nonmuscle myosin light chain (MLC) phosphorylation. This study identified svh-16/cdk-14, which encodes a mammalian CDK14 homolog, as a regulator of axon regeneration in Caenorhabditis elegans motor neurons. We show that CDK-14 binds to MIG-5/Dsh, and that EGL-20/Wnt, MIG-1/Fz, and EPHX-1/RhoGEF are required for axon regeneration. The phosphorylation-mimetic MLC-4 suppressed axon regeneration defects in mig-1, cdk-14, and ephx-1 mutants. CDK-14 mediates kinase-independent activation of EPHX-1, which functions as a guanine nucleotide exchange factor for CDC-42 GTPase. Activated CDC-42 inactivates myosin phosphatase and thereby maintains MLC phosphorylation. Thus, the noncanonical Wnt signaling pathway controls axon regeneration via the CDK-14–EPHX-1–CDC-42–MLC phosphorylation axis.

Glabridin Relaxes Vascular Smooth Muscles by Activating BKCa Channels and Inhibiting Phosphodiesterase in Human Saphenous Vein.

The aim of the current study was to investigate the pharmacological activity of glabridin on the isolated human saphenous vein (SV) and explore the underlying mechanisms. Samples of patients' SVs were removed during bypass surgery, and 4-mm lengths of the vessels were placed in Krebs solution at +4°C and hung in an isolated organ bath to assess their contraction/relaxation responses. The contraction/relaxation responses were recorded to observe if the cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) pathway mediates the relaxant effect of glabridin after treatment with blockers like ODQ (a guanylate cyclase inhibitor), KT5823 (a PKG inhibitor), isobutylmethylxanthine [IBMX, a phosphodiesterase (PDE) inhibitor], and cantharidin [Cant, a myosin light-chain phosphatase (MLCP) inhibitor]. Moreover, nitric oxide (NO), cGMP, and PKG levels in SV tissues were determined by ELISA after incubation with glabridin, N(ω)-nitro-L-arginine methyl ester (L-Name, a NO synthetase inhibitor), phenylephrine (PE), ODQ, IBMX, and KT5823. The results showed that glabridin relaxed the vascular smooth muscle of human SV pretreated with PE in a dose-dependent manner, which was independent of the endothelium. The vasorelaxant effect of glabridin was only inhibited by iberiotoxin (IbTX), Cant, and KT5823. Glabridin increased cGMP and PKG levels in SV homogenates, whereas it did not alter the NO level. The enhancing effects of cGMP and PKG levels by glabridin were abolished by ODQ and KT5823. In conclusion, glabridin has a vasorelaxant effect, which is associated with the activation of BKCa channels and inhibition of PDE.

Myosin Phosphatase Is Implicated in the Control of THP-1 Monocyte to Macrophage Differentiation.

Monocyte to macrophage differentiation is characterized by the activation of various signal transduction pathways, which may be modulated by protein phosphorylation; however, the impact of protein kinases and phosphatases is not well understood yet. It has been demonstrated that actomyosin rearrangement during macrophage differentiation is dependent on Rho-associated protein kinase (ROCK). Myosin phosphatase (MP) target subunit-1 (MYPT1) is one of the major cellular substrates of ROCK, and MP is often a counter enzyme of ROCK; therefore, MP may also control macrophage differentiation. Changes in MP activity and the effects of MP activation were studied on PMA or l,25(OH)2D3-induced differentiation of monocytic THP-1 cells. During macrophage differentiation, phosphorylation of MYPT1 at Thr696 and Thr853 increased significantly, resulting in inhibition of MP. The ROCK inhibitor H1152 and the MP activator epigallocatechin-3-gallate (EGCG) attenuated MYPT1 phosphorylation and concomitantly decreased the extent of phosphorylation of 20 kDa myosin light chain. H1152 and EGCG pretreatment also suppressed the expression of CD11b and weakened the PMA-induced adherence of the cells. Our results indicate that MP activation/inhibition contributes to the efficacy of monocyte to macrophage differentiation, and this enzyme may be a target for pharmacological interventions in the control of disease states that are affected by excessive macrophage differentiation.

A microtubule-LUZP1 association around tight junction promotes epithelial cell apical constriction.

Apical constriction is critical for epithelial morphogenesis, including neural tube formation. Vertebrate apical constriction is induced by di‐phosphorylated myosin light chain (ppMLC)‐driven contraction of actomyosin‐based circumferential rings (CRs), also known as perijunctional actomyosin rings, around apical junctional complexes (AJCs), mainly consisting of tight junctions (TJs) and adherens junctions (AJs). Here, we revealed a ppMLC‐triggered system at TJ‐associated CRs for vertebrate apical constriction involving microtubules, LUZP1, and myosin phosphatase. We first identified LUZP1 via unbiased screening of microtubule‐associated proteins in the AJC‐enriched fraction. In cultured epithelial cells, LUZP1 was found localized at TJ‐, but not at AJ‐, associated CRs, and LUZP1 knockout resulted in apical constriction defects with a significant reduction in ppMLC levels within CRs. A series of assays revealed that ppMLC promotes the recruitment of LUZP1 to TJ‐associated CRs, where LUZP1 spatiotemporally inhibits myosin phosphatase in a microtubule‐facilitated manner. Our results uncovered a hitherto unknown microtubule‐LUZP1 association at TJ‐associated CRs that inhibits myosin phosphatase, contributing significantly to the understanding of vertebrate apical constriction.

The Evolution of Duplicated Genes of the Cpi-17/Phi-1 (ppp1r14) Family of Protein Phosphatase 1 Inhibitors in Teleosts.

The Cpi-17 (ppp1r14) gene family is an evolutionarily conserved, vertebrate specific group of protein phosphatase 1 (PP1) inhibitors. When phosphorylated, Cpi-17 is a potent inhibitor of myosin phosphatase (MP), a holoenzyme complex of the regulatory subunit Mypt1 and the catalytic subunit PP1. Myosin phosphatase dephosphorylates the regulatory myosin light chain (Mlc2) and promotes actomyosin relaxation, which in turn, regulates numerous cellular processes including smooth muscle contraction, cytokinesis, cell motility, and tumor cell invasion. We analyzed zebrafish homologs of the Cpi-17 family, to better understand the mechanisms of myosin phosphatase regulation. We found single homologs of both Kepi (ppp1r14c) and Gbpi (ppp1r14d) in silico, but we detected no expression of these genes during early embryonic development. Cpi-17 (ppp1r14a) and Phi-1 (ppp1r14b) each had two duplicate paralogs, (ppp1r14aa and ppp1r14ab) and (ppp1r14ba and ppp1r14bb), which were each expressed during early development. The spatial expression pattern of these genes has diverged, with ppp1r14aa and ppp1r14bb expressed primarily in smooth muscle and skeletal muscle, respectively, while ppp1r14ab and ppp1r14ba are primarily expressed in neural tissue. We observed that, in in vitro and heterologous cellular systems, the Cpi-17 paralogs both acted as potent myosin phosphatase inhibitors, and were indistinguishable from one another. In contrast, the two Phi-1 paralogs displayed weak myosin phosphatase inhibitory activity in vitro, and did not alter myosin phosphorylation in cells. Through deletion and chimeric analysis, we identified that the difference in specificity for myosin phosphatase between Cpi-17 and Phi-1 was encoded by the highly conserved PHIN (phosphatase holoenzyme inhibitory) domain, and not the more divergent N- and C- termini. We also showed that either Cpi-17 paralog can rescue the knockdown phenotype, but neither Phi-1 paralog could do so. Thus, we provide new evidence about the biochemical and developmental distinctions of the zebrafish Cpi-17 protein family.

Activation of Myosin Phosphatase by Epigallocatechin-Gallate Sensitizes THP-1 Leukemic Cells to Daunorubicin.

The Myosin Phosphatase (MP) holoenzyme is composed of a Protein Phosphatase type 1 (PP1) catalytic subunit and a regulatory subunit termed Myosin Phosphatase Target subunit 1 (MYPT1). Besides dephosphorylation of myosin, MP has been implicated in the control of cell proliferation via dephosphorylation and activation of the tumor suppressor gene products, retinoblastoma protein (pRb) and merlin. Inhibition of MP was shown to attenuate the drug-induced cell death of leukemic cells by chemotherapeutic agents, while activation of MP might have a sensitizing effect. Recently, Epigallocatechin-Gallate (EGCG), a major component of green tea, was shown to activate MP by inducing the dephosphorylation of MYPT1 at phospho-Thr696 (MYPT1pT696), which might confer enhanced chemosensitivity to cancer cells. THP-1 leukemic cells were treated with EGCG and Daunorubicin (DNR) and cell viability was analyzed. Phosphorylation of tumor suppressor proteins was detected by Western blotting. EGCG or DNR (at sub-lethal doses) alone had moderate effects on cell viability, while the combined treatment caused a significant decrease in the number of viable cells by enhancing apoptosis and decreasing proliferation. EGCG plus DNR decreased the phosphorylation level of MYPT1pT696, which was accompanied by prominent dephosphorylation of pRb. In addition, significant dephosphorylation of merlin was observed when EGCG and DNR were applied together. Our results suggest that EGCG-induced activation of MP might have a regulatory function in mediating the chemosensitivity of leukemic cells via dephosphorylation of tumor suppressor proteins.

Improving Small Intestinal Motility in Experimental Acute Necrotising Pancreatitis by Modulating the CPI-17/MLCP Pathway Using Chaiqin Chengqi Decoction.

Protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), a specific inhibitor of myosin light-chain phosphatase (MLCP) regulated by proinflammatory cytokines, is central for calcium sensitisation. We investigated the effects of chaiqin chengqi decoction (CQCQD) on the CPI-17/MLCP pathway in the small intestinal smooth muscle cells (SMCs) and strips (SMS) in an AP model. Necrotising AP was induced in rats by intraperitoneal injections (IPI) of L-ornithine (3.0 g/kg, pH 7.0; hourly × 2) at 1 hour apart; controls received saline. In treatment groups, carbachol (CCh; 60 μg/kg, IPI) or CQCQD (20 g/kg; 2-hourly × 3, intragastric) was administered. The necrotising AP model was associated with systemic inflammation (serum IL-1β and TNF-α) and worsened jejunum histopathology and motility (serum vasoactive intestinal peptide and intestinal fatty acid-binding protein) as the disease progressed. There was decreased intracellular calcium concentration ([Ca2+]i) SMCs. Contractile function of isolated SMCs was reduced and associated with down-regulated expression of key mRNAs and proteins of the CPI-17/MLCP pathway as well as increased IL-1β and TNF-α. CQCQD and CCh significantly reversed these changes and the disease severity. These data suggest that CQCQD can improve intestinal motility by modulating the CPI-17/MLCP pathway in small intestinal smooth muscle during AP.

TIMAP inhibits endothelial myosin light chain phosphatase by competing with MYPT1 for the catalytic protein phosphatase 1 subunit PP1cβ

Transforming growth factor-β membrane associated protein (TIMAP) is an endothelial cell (EC)–predominant PP1 regulatory subunit and a member of the myosin phosphatase target (MYPT) protein family. The MYPTs preferentially bind the catalytic protein phosphatase 1 subunit PP1cβ, forming myosin phosphatase holoenzymes. We investigated whether TIMAP/PP1cβ could also function as a myosin phosphatase. Endogenous PP1cβ, myosin light chain 2 (MLC2), and myosin IIA heavy chain coimmunoprecipitated from EC lysates with endogenous TIMAP, and endogenous MLC2 colocalized with TIMAP in EC projections. Purified recombinant GST-TIMAP interacted directly with purified recombinant His-MLC2. However, TIMAP overexpression in EC enhanced MLC2 phosphorylation, an effect not observed with a TIMAP mutant that does not bind PP1cβ. Conversely, MLC2 phosphorylation was reduced in lung lysates from TIMAP-deficient mice and upon silencing of endogenous TIMAP expression in ECs. Ectopically expressed TIMAP slowed the rate of MLC2 dephosphorylation, an effect requiring TIMAP–PP1cβ interaction. The association of MYPT1 with PP1cβ was profoundly reduced in the presence of excess TIMAP, leading to proteasomal MYPT1 degradation. In the absence of TIMAP, MYPT1-associated PP1cβ readily bound immobilized microcystin-LR, an active-site inhibitor of PP1c. By contrast, TIMAP-associated PP1cβ did not interact with microcystin-LR, indicating that the active site of PP1cβ is blocked when it is bound to TIMAP. Thus, TIMAP inhibits myosin phosphatase activity in ECs by competing with MYPT1 for PP1cβ and blocking the PP1cβ active site.

Smoothelin-like 1 deletion enhances myogenic reactivity of mesenteric arteries with alterations in PKC and myosin phosphatase signaling

The role of the smoothelin-like 1 (SMTNL1) protein in mediating vascular smooth muscle contractile responses to intraluminal pressure was examined in resistance vessels. Mesenteric arterioles from wild type (WT) and SMTNL1 global knock-out (KO) mice were examined with pressure myography. SMTNL1 deletion was associated with enhanced myogenic tone in vessels isolated from male, but not female, mice. Intraluminal pressures greater than 40 mmHg generated statistically significant differences in myogenic reactivity between WT and KO vessels. No overt morphological differences were recorded for vessels dissected from KO animals, but SMTNL1 deletion was associated with loss of myosin phosphatase-targeting protein MYPT1 and increase in the myosin phosphatase inhibitor protein CPI-17. Additionally, we observed altered contractile responses of isolated arteries from SMTNL1 KO mice to phenylephrine, KCl-dependent membrane depolarization and phorbol 12,13-dibutyrate (PDBu). Using pharmacological approaches, myogenic responses of both WT and KO vessels were equally affected by Rho-associated kinase (ROCK) inhibition; however, augmented protein kinase C (PKC) signaling was found to contribute to the increased myogenic reactivity of SMTNL1 KO vessels across the 60–120 mmHg pressure range. Based on these findings, we conclude that deletion of SMTNL1 contributes to enhancement of pressure-induced contractility of mesenteric resistance vessels by influencing the activity of myosin phosphatase.

Механизмы утеростимулирующего и других эффектов окситоцина

The activation of the G-protein coupled oxytocin receptors by oxytocin, causes various physiological effects. This article is focused on the mechanisms underlying the uterine-contracting effect of the oxytocin. The role of Gq-, Gi- and Gs-proteins associated with the oxytocin receptors, as well as phospholipase C, inositol triphosphate, diacylglycerol, sarcoplasmic reticulum, Са2+-channels of the reticulum and plasma membrane (including the SOC-channels), Са2+-pumps of the reticulum and plasma membrane, as well as potassium channels and Ca-regulated chloride channels of plasma membrane is examined. The ability of oxytocin to cause the Ca2+-sensitization of myosin, i.e. to increase the myosin light chains (MCL) sensitivity to Ca2+ ions, is reported. This phenomenon involves the oxytocin-induced Ca2+-independent phosphorylation of the MCL by noncanonical protein kinases, including Rho-kinase. The ability of the oxytocin to inhibit the myosin phosphatase through the activation of the Rho-kinase and protein kinase C, as well as its contribution to the Са2+-independent myosin phosphorylation and the myosin phosphatase inhibition to the uterine-contracting effect of the oxytocin (including its tonic component) is discussed. The data on the role of the MAPК (mitogen-activated protein kinase) signaling pathway in the oxytocin action leading to the increased synthesis of prostaglandins, MCL, connexin-43 and Rho-kinase, and regulating the cell proliferation and prenatal and postnatal neuronal maturation in the brain, is provided.

Regulation of mitogen-activated protein kinase by protein kinase C and mitogen-activated protein kinase phosphatase-1 in vascular smooth muscle

Vascular smooth muscle contraction is primarily regulated by phosphorylation of myosin light chain. There are also modulatory pathways that control the final level of force development. We tested the hypothesis that protein kinase C (PKC) and mitogen-activated protein (MAP) kinase modulate vascular smooth muscle activity via effects on MAP kinase phosphatase-1 (MKP-1). Swine carotid arteries were mounted for isometric force recording and subjected to histamine stimulation in the presence and absence of inhibitors of PKC [bisindolylmaleimide-1 (Bis)], MAP kinase kinase (MEK) (U0126), and MKP-1 (sanguinarine) and flash frozen for measurement of MAP kinase, PKC-potentiated myosin phosphatase inhibitor 17 (CPI-17), and caldesmon phosphorylation levels. CPI-17 was phosphorylated in response to histamine and was inhibited in the presence of Bis. Caldesmon phosphorylation levels increased in response to histamine stimulation and were decreased in response to MEK inhibition but were not affected by the addition of Bis. Inhibition of PKC significantly increased p42 MAP kinase, but not p44 MAP kinase. Inhibition of MEK with U0126 inhibited both p42 and p44 MAP kinase activity. Inhibition of MKP-1 with sanguinarine blocked the Bis-dependent increase of MAP kinase activity. Sanguinarine alone increased MAP kinase activity due to its effects on MKP-1. Sanguinarine increased MKP-1 phosphorylation, which was inhibited by inhibition of MAP kinase. This suggests that MAP kinase has a negative feedback role in inhibiting MKP-1 activity. Therefore, PKC catalyzes MKP-1 phosphorylation, which is reversed by MAP kinase. Thus the fine tuning of vascular contraction is due to the concerted effort of PKC, MAP kinase, and MKP-1.

OxLDL induce endothelial cell stiffening through Rho/ROCK signaling pathway

Our lab demonstrated that Oxidized LDL (OxLDL) induce endothelial cell stiffening. The signal transduction pathways leading to this increase in stiffness remain poorly understood. We hypothesize that the Rho/ROCK pathway plays a role in oxLDL induced endothelial cell stiffening. To test our hypothesis, human aortic endothelial cells (HAEC) were treated with 50µg/ml oxLDL and Rho/ROCK signaling was probed by western‐blot. First, our results show that RhoA is significantly activated by 50ug/ml oxLDL stimulation. The activated RhoA level increased 1.9 times vs control after 15 minutes incubation with oxLDL, and activated RhoA level return to control level after 60 minutes of incubation with oxLDL. Second, incubation with oxLDL increase phospho‐myosin phosphatase target subunit 1 (p‐MYPT1 (Thr853)) and phosphor‐Myosin ligh chain 2 (p‐MLC2 (Thr18/Ser19) expression level vs control. We further test that if oxLDL could affect myosin phosphorylation. Our data showed p‐MLC2 increased 1.7 times vs control after 15 minutes incubation with oxLDL, and increased 2.6 times vs control after 60 minutes incubation. The level of p‐MYPT1, which indicate the inhibition of myosin phosphatase, increased to 1.7 times vs control after 15 minutes incubation with oxLDL. Third, to further test our hypothesis, we treated HAECs with RhoA inhibitor (C3) or ROCK inhibitor (Y27632) before oxLDL incubation. Our data showed that both C3 and Y27632 attenuate oxLDL induced increase of p‐MLC2 and p‐MYPT1. Lastly, our data showed that increase of cell stiffening is also attenuated by C3 or Y27632 when treated with oxLDL.ConclusionoxLDL induce cell stiffening through increase RhoA activity, active Rho/ROCK pathway, inhibition myosin phosphatase, thereby increasing phosphorylation of myosin and leading to cell stiffening.

A tale of two threonines: myosin phosphatase inhibition and calcium sensitization of smooth muscle.

A tale of two threonines: myosin phosphatase inhibition and calcium sensitization of smooth muscle.

Emodin inhibits tonic tension through suppressing PKCδ-mediated inhibition of myosin phosphatase in rat isolated thoracic aorta.

Dysregulated tonic tension and calcium sensitization in blood vessels has frequently been observed in many cardiovascular diseases. Despite a huge therapeutic potential, little is known about natural products targeting tonic tension and calcium sensitization. We screened natural products for inhibitory effects on vasoconstriction using the rat isolated thoracic aorta and found that an anthraquinone derivative, emodin, attenuated tonic tension. Organ bath system, primary vascular smooth muscle cells, confocal microscopy and Western blot analysis were employed to demonstrate the suppressive effects of emodin on PKCδ-mediated myosin phosphatase inhibition. Emodin, an active ingredient of Polygonum multiflorum extract, inhibited phenylephrine-induced vasoconstriction in rat isolated thoracic aorta, and inhibited vasoconstriction induced by 5-HT and endothelin-1. It also generally suppressed vasoconstrictions mediated by voltage-operated, store-operated calcium channels and intracellular calcium store. However, emodin did not affect agonist-induced calcium increases in primary smooth muscle cells. In contrast, post-treatment with emodin following phenylephrine stimulation potently suppressed tonic tension in rat aortic rings. Western blot analysis revealed that emodin inhibited phenylephrine-induced phospho-myosin light chain (pMLC) and the phosphorylation of myosin-targeting subunit and C-kinase-activated protein phosphatase-1 inhibitor (CPI-17). This was mediated by selective inhibition of PKCδ, whereas PKCα was not involved. Emodin attenuates tonic tension through the blockade of PKCδ and CPI-17-mediated MLC-phosphatase inhibition. This new mode of action for the suppression of tonic tension and structural insights into PKCδ inhibition revealed by emodin may provide new information for the development of modulators of tonic tension and for the treatment of hypertension.

Neural programming of mesenteric and renal arteries.

There is evidence for developmental origins of vascular dysfunction yet little understanding of maturation of vascular smooth muscle (VSM) of regional circulations. We measured maturational changes in expression of myosin phosphatase (MP) and the broader VSM gene program in relation to mesenteric small resistance artery (SRA) function. We then tested the role of the sympathetic nervous system (SNS) in programming of SRAs and used genetically engineered mice to define the role of MP isoforms in the functional maturation of the mesenteric circulation. Maturation of rat mesenteric SRAs as measured by qPCR and immunoblotting begins after the second postnatal week and is not complete until maturity. It is characterized by induction of markers of VSM differentiation (smMHC, γ-, α-actin), CPI-17, an inhibitory subunit of MP and a key target of α-adrenergic vasoconstriction, α1-adrenergic, purinergic X1, and neuropeptide Y1 receptors of sympathetic signaling. Functional correlates include maturational increases in α-adrenergic-mediated force and calcium sensitization of force production (MP inhibition) measured in first-order mesenteric arteries ex vivo. The MP regulatory subunit Mypt1 E24+/LZ- isoform is specifically upregulated in SRAs during maturation. Conditional deletion of mouse Mypt1 E24 demonstrates that splicing of E24 causes the maturational reduction in sensitivity to cGMP-mediated vasorelaxation (MP activation). Neonatal chemical sympathectomy (6-hydroxydopamine) suppresses maturation of SRAs with minimal effect on a conduit artery. Mechanical denervation of the mature rat renal artery causes a reversion to the immature gene program. We conclude that the SNS captures control of the mesenteric circulation by programming maturation of the SRA smooth muscle.