Myosin Light Chain Phosphatase Activity Research Articles

FOXO1 promotes endothelial cell elongation and angiogenesis by up-regulating the phosphorylation of myosin light chain 2.

The forkhead box O1 (FOXO1) is an important transcription factor related to proliferation, metabolism, and homeostasis, while the major phenotype of FOXO1-null mice is abnormal vascular morphology, such as vessel enlargement and dilation. In in vitro mouse embryonic stem cell (ESC)-differentiation system, Foxo1-/- vascular endothelial cells (ECs) fail to elongate, and mimic the abnormalities of FOXO1-deficiency in vivo. Here, we identified the PPP1R14C gene as the FOXO1 target genes responsible for elongating using transcriptome analyses in ESC-derived ECs (ESC-ECs), and found that the FOXO1-PPP1R14C-myosin light chain 2 (MLC2) axis is required for EC elongation during angiogenesis. MLC2 is phosphorylated by MLC kinase (MLCK) and dephosphorylated by MLC phosphatase (MLCP). PPP1R14C is an inhibitor of PP1, the catalytic subunit of MLCP. The abnormal morphology of Foxo1-/- ESC-ECs was associated with low level of PPP1R14C and loss of MLC2 phosphorylation, which were reversed by PPP1R14C-introduction. Knockdown of either FOXO1 or PPP1R14C suppressed vascular cord formation and reduced MLC2 phosphorylation in human ECs (HUVECs). The mouse and human PPP1R14C locus possesses an enhancer element containing conserved FOXO1-binding motifs. In vivo chemical inhibition of MLC2 phosphorylation caused dilated vascular structures in mouse embryos. Furthermore, foxo1 or ppp1r14c-knockdown zebrafish exhibited vascular malformations, which were also restored by PPP1R14C-introduction. Mechanistically, FOXO1 suppressed MLCP activity by up-regulating PPP1R14C expression, thereby promoting MLC2 phosphorylation and EC elongation, which are necessary for vascular development. Given the importance of MLC2 phosphorylation in cell morphogenesis, this study may provide novel insights into the role of FOXO1 in control of angiogenesis.

T18/S19 diphosphorylation of myosin regulatory light chain impairs pulmonary artery relaxation in monocrotaline-induced pulmonary hypertensive rats.

Phosphorylation of Ser19 (S19-p) on the myosin regulatory light chain (MLC2) is critical for arterial contraction. It has been shown that elevated RhoA-dependent kinase (ROCK) activity or decreased MLC phosphatase (MLCP) activity leads to further phosphorylation of Thr18 (T18/S19-pp), which has been linked to vasospastic diseases. However, this phenomenon has not yet been studied in the context of pulmonary arterial hypertension (PAH). In the monocrotaline-induced PAH (PAH-MCT) rat model, we observed a significant delay in pulmonary artery (PA) relaxation following high potassium-induced contraction, which persisted even with the use of an L-type calcium channel blocker or in a calcium-free solution. Immunoblot analysis showed increased levels of both S19-p and T18/S19-pp in unstimulated PAs from PAH-MCT rats. Proteomics analysis revealed a reduction in soluble guanylate cyclase (sGC) and protein kinase G (PKG) levels, and immunoblotting confirmed decreased levels of MYPT1 (a component of MLCP) and increased ROCK in PAH-MCT. In the control PAs, the pharmacological inhibition of sGC with ODQ resulted in a prominent delay of relaxation and increased T18/S19-pp as in PAH-MCT. The delayed relaxation and the T18/S19-pp in PAH-MCT were reversed by ROCK inhibitor, Y27632, while not by membrane permeable 8-Br-cGMP. The delayed relaxation and T18/S19-diP in the ODQ-treated control PA were also reversed by Y27632. Taken together, the decreased sGC and MLCP, and increased ROCK increased T18/S19-pp, which leads to the decreased ability of PA to relax in PAH-MCT rats. PA specific inhibition of ROCK or activation of MLCP are expected to serve as potential drugs in the treatment of PAH.

Striated muscle MLCP is regulated by MYPT2 to limit baseline cardiac myosin phosphorylation.

Cardiac myosin is a hexameric protein comprised of two protamers that each have a heavy chain, a regulatory light chain (RLC), and an essential light chain. Constitutive phosphorylation of cardiac myosin in beating hearts at 0.4 mol phosphate/mol RLC is necessary for normal function. This level is maintained by balanced activities of the cardiac myosin light chain kinase (cMLCK) and myosin light chain phosphatase (MLCP). The canonical MLCP is well-studied in smooth muscles, and is defined as a trimeric holoenzyme comprised of a myosin target regulatory subunit MYPT1, a type-1 catalytic subunit PP1cβ, and an accessory protein M21. In striated muscles, the dominantly expressed regulatory subunit is MYPT2. There is limited information on the localization and regulatory contributions of MYPT2. Studies of regulation of cardiac myosin dephosphorylation is hindered by the fact that myosin dephosphorylation occurs rapidly in isolated cardiac myocytes, and cultured “cardiac” cell lines do not express the regulatory proteins at levels comparable to those of freshly isolated cardiac myocytes. We generated a floxed MYPT2 mouse line and conditionally knocked out nearly all detectable MYPT2 protein in cardiac myocytes, and used this model to determine that: 1) MYPT2 localizes to myosin in vivo, 2) MYPT1 expression does not compensate for the reduction in MYPT2 in the cardiac muscle, 3) the cardiac MLCP holoenzyme expression level is partly regulated by stabilization of the catalytic subunit by MYPT2, 4) without MYPT2-regulated MLCP activity, baseline cardiac RLC phosphorylation is increased to 0.6 mol phosphate/mol RLC, and 5) MYPT2 knockout animals have reduced responses to pressure-overload induced cardiac hypertrophy. These findings help to fill knowledge-gaps about the fundamental properties of the cardiac muscle MLCP.

Raptor knockdown concurrently increases the electrical resistance and paracellular permeability of Caco-2 cell monolayers

AimsAs a critical regulatory point of nutrient sensing, growth and metabolism, the mechanistic target of rapamycin complex 1 (mTORC1) is poised to influence intestinal homeostasis under basal conditions and in disease state. Intestinal barrier integrity ensures tissue homeostasis by closely regulating the permeability of the epithelium to lumenal contents. The role of mTORC1 in the regulation of intestinal barrier function and permeability remains to be fully elucidated. Materials and methodsIn this study, we employed lentivirus-mediated knockdown of mTORC1 signaling-associated proteins Raptor (regulatory-associated protein of mTOR) and TSC2 (tuberin) to ascertain the effects of constitutive activation or repression of mTORC1 activity on barrier function in Caco-2 cell monolayers. Key findingsResults showed that the loss of Raptor concomitantly raised the transepithelial electrical resistance (TEER) and para/transcellular permeability leading to a cell monolayer that is leaky for dextran yet electrically resistant to the movement of ions. Paracellular permeability was linked to the downregulation of tight junction protein expression and enhanced autophagy. Raptor-depleted cells had the highest abundance of myosin binding subunit MYPT1 concomitantly with the lowest abundance of p-MYPT1 (Thr696) and phosphorylated myosin light chain (p-MLC, Ser19) implying that MLC phosphatase activity was increased resulting in MLC relaxation. Although rapamycin suppressed mTORC1 activity and decreased the abundance of tight junction proteins in control cells, rapamycin caused a modest increase of TEER compared to Raptor knockdown. SignificanceThe study showed that epithelium paracellular permeability of small molecular weight dextran is dissociated from TEER.

MTOR Inhibition Promotes Pneumonitis through Inducing Endothelial Contraction and Hyperpermeability.

Compromised endothelial-cell (EC) barrier function is a hallmark of inflammatory diseases. mTOR inhibitors, widely applied as clinical therapies, cause pneumonitis through mechanisms that are not yet fully understood. This study aimed to elucidate the EC mechanisms underlying the pathogenesis of pneumonitis caused by mTOR inhibition (mTORi). Mice with EC-specific deletion of mTOR complex components (Mtor, Rptor or Rictor) were administered LPS to induce pulmonary injury. Cultured ECs were treated with pharmacologic inhibitors, siRNA, or overexpression plasmids. EC barrier function was evaluated invivo with Evans blue assay and invitro by measurement of transendothelial electrical resistance and albumin flux. mTORi increased basal and TNFα-induced EC permeability, which was caused by myosin light chain (MLC) phosphorylation-dependent cell contraction. Inactivation of mTOR kinase activity by mTORi triggered PKCδ/p38/NF-κB signaling that significantly upregulated TNFα-induced MLCK (MLC kinase) expression, whereas Raptor promoted the phosphorylation of PKCα/MYPT1 independently of its interaction with mTOR, leading to suppression of MLCP (MLC phosphatase) activity. EC-specific deficiency in mTOR, Raptor or Rictor aggravated lung inflammation in LPS-treated mice. These findings reveal that mTORi induces PKC-dependent endothelial MLC phosphorylation, contraction, and hyperpermeability that promote pneumonitis.

Mechanism of α1-Adrenergic Receptor-Induced Increased Contraction of Rat Mesenteric Artery in Aging Hypertension Rats

Introduction: Vasoconstriction is triggered by an increase in intracellular-free calcium concentration. Growing evidence indicates that contraction is also regulated by calcium-independent mechanisms involving RhoA-Rho kinase (ROCK), protein kinase C (PKC), and so on. In this study, we studied the changes of vascular reactivity as well as the underlying signaling pathways in aging spontaneously hypertensive rats (SHRs). Methods: The artery tension induced by α1-adrenergic receptor activator (α1-AR) phenylephrine (PE) was measured in the absence or presence of myosin light chain kinase (MLCK), PKC, and ROCK inhibitors. The α1-AR, PKC, ROCK, phosphorylation of myosin light chain (MLC), and PKC-potentiated phosphatase inhibitors of 17 kDa (CPI-17) of rat mesenteric arteries were analyzed at the mRNA level or protein level. Results: The vascular tension measurements showed that there was a significant increase in the mesenteric artery contraction induced by PE in old SHR. MLCK inhibitor ML-7 can similarly inhibit PE-induced vasoconstriction. PKC inhibitor GF109203X has the weakest inhibitory effect on PE-induced contraction in old SHR. At the presence of ROCK inhibitor H1152, PE-induced contraction was significantly reduced in young Wistar-Kyoto (WKY) rats, but this phenomenon disappeared in other rats. Furthermore, in old SHR the protein expression of α1-AR decreased and phosphorylation of MLC and CPI-17 were upregulated and MLC phosphatase (MLCP) activity was significantly lower. The expressions of PKC were upregulated in SHR and old rats. In addition, the expression of ROCK-1 was decreased and ROCK-2 was significantly upregulated with age in SHR. Conclusion: In aging hypertension, the expression/activity of PKC or ROCK-2/CPI-17 excessively increased, MLCP activity decreased and MLC phosphorylation enhanced, leading to increased α1-AR-induced vasoconstriction.

Acute glucose influx-induced mitochondrial hyperpolarization inactivates myosin phosphatase as a novel mechanism of vascular smooth muscle contraction

It is well-established that long-term exposure of the vasculature to metabolic disturbances leads to abnormal vascular tone, while the physiological regulation of vascular tone upon acute metabolic challenge remains unknown. Here, we found that acute glucose challenge induced transient increases in blood pressure and vascular constriction in humans and mice. Ex vivo study in isolated thoracic aortas from mice showed that glucose-induced vascular constriction is dependent on glucose oxidation in vascular smooth muscle cells. Specifically, mitochondrial membrane potential (ΔΨm), an essential component in glucose oxidation, was increased along with glucose influx and positively regulated vascular smooth muscle tone. Mechanistically, mitochondrial hyperpolarization inhibited the activity of myosin light chain phosphatase (MLCP) in a Ca2+-independent manner through activation of Rho-associated kinase, leading to cell contraction. However, ΔΨm regulated smooth muscle tone independently of the small G protein RhoA, a major regulator of Rho-associated kinase signaling. Furthermore, myosin phosphatase target subunit 1 (MYPT1) was found to be a key molecule in mediating MLCP activity regulated by ΔΨm. ΔΨm positively phosphorylated MYPT1, and either knockdown or knockout of MYPT1 abolished the effects of glucose in stimulating smooth muscle contraction. In addition, smooth muscle-specific Mypt1 knockout mice displayed blunted response to glucose challenge in blood pressure and vascular constriction and impaired clearance rate of circulating metabolites. These results suggested that glucose influx stimulates vascular smooth muscle contraction via mitochondrial hyperpolarization-inactivated myosin phosphatase, which represents a novel mechanism underlying vascular constriction and circulating metabolite clearance.

Regulation of Myosin Light-Chain Phosphatase Activity to Generate Airway Smooth Muscle Hypercontractility.

Smooth muscle is a central structure involved in the regulation of airway tone. In addition, it plays an important role in the development of some pathologies generated by alterations in contraction, such as hypercontractility and the airway hyperresponsiveness observed in asthma. The molecular processes associated with smooth muscle contraction are centered around myosin light chain (MLC) phosphorylation, which is controlled by a balance in the activity of myosin light-chain kinase (MLCK) and myosin light-chain phosphatase (MLCP). MLCK activation depends on increasing concentrations of intracellular Ca2+, while MLCP activation is independent of Ca2+. MLCP contains a phosphatase subunit (PP1c) that is regulated through myosin phosphatase target subunit 1 (MYPT1) and other subunits, such as glycogen-associated regulatory subunit and myosin-binding subunit 85 kDa. Interestingly, MLCP inhibition may contribute to exacerbation of smooth muscle contraction by increasing MLC phosphorylation to induce hypercontractility. Many pathways inhibiting MLCP activity in airway smooth muscle have been proposed and are focused on inhibition of PP1c, inhibitory phosphorylation of MYPT1 and dissociation of the PP1c-MYPT1 complex.

MLCK and ROCK mutualism in endothelial barrier dysfunction

Myosin activation contributes to the contractile forces that induce disturbances in the vascular endothelial integrity and promote protein-rich edema of the underlying tissues. Myosin light chain kinase (MLCK) and Rho-associated protein kinase (ROCK) have been reported to phosphorylate myosin regulatory light chains (RLC) for myosin activation. However, the relative contribution and roles of these kinases are debatable and not understood in very detail. In this study, using a combinational inhibitory analysis of MLCK, ROCK, and their antagonist, myosin light chain phosphatase (MLCP), we show that the MLCK-dependent RLC mono-(Ser19)phosphorylation (P-RLC) is sufficient to induce the FITC-dextran hyperpermeability in EA.hy926 endothelial cells (EC) in response to thrombin. However, MLCK relies on the ROCK assistance that attenuates MLCP activity. On the other hand, MLCK supplies P-RLC myosin as an intermediate substrate to ROCK thus adding to a faster accumulation of di-(Thr18/Ser19)phosphorylated RLC (PP-RLC) by the latter kinase. ROCK also produces P-RLC but is solely responsible for the thrombin-induced PP-RLC generation in EA.hy926 EC and other cell types. Still, as a direct myosin activator, ROCK contributes less to endothelial hyperpermeability than MLCK. Our findings are consistent with a concerted complementary mutual interplay between ROCK and MLCK to activate endothelial myosin and elicit the thrombin-mediated EC barrier dysfunction.

P116Rip promotes myosin phosphatase activity in airway smooth muscle cells.

Myosin phosphatase-Rho interacting protein (p116Rip ) was originally found as a RhoA-binding protein. Subsequent studies by us and others revealed that p116Rip facilitates myosin light chain phosphatase (MLCP) activity through direct and indirect manners. However, it is unclear how p116Rip regulates myosin phosphatase activity in cells. To elucidate the role of p116Rip in cellular contractile processes, we suppressed the expression of p116Rip by RNA interference in human airway smooth muscle cells (HASMCs). We found that knockdown of p116Rip in HASMCs led to increased di-phosphorylated MLC (pMLC), that is phosphorylation at both Ser19 and Thr18. This was because of a change in the interaction between MLCP and myosin, but not an alteration of RhoA/ROCK signaling. Attenuation of Zipper-interacting protein kinase (ZIPK) abolished the increase in di-pMLC, suggesting that ZIPK is involved in this process. Moreover, suppression of p116Rip expression in HASMCs substantially increased the histamine-induced collagen gel contraction. We also found that expression of the p116Rip was decreased in the airway smooth muscle tissue from asthmatic patients compared with that from non-asthmatic patients, suggesting a potential role of p116Rip expression in asthma pathogenesis.

AdipoRon, adiponectin receptor agonist, improves vascular function in the mesenteric arteries of type 2 diabetic mice

BackgroundAdiponectin is one of the most abundant adipokines secreted from adipose tissue. An orally active synthetic adiponectin receptor agonist, adipoRon has been suggested to ameliorate insulin resistance, myocardial apoptosis, and pancreatic tumor. It has been reported that adiponectin directly induces vascular relaxation however, the chronic effect of adipoRon in the vascular dysfunction in type 2 diabetes has not been studied yet. Thus, in this study, we examined whether adipoRon improves vascular function in type 2 diabetes and what mechanism is involved.MethodsTen to 12‐week old male type 2 diabetic (db−/db−) mice were treated with adiponectin receptor agonist (adipoRon, 10 mg/kg/everyday, by oral gavage) for 2 weeks. Isolated mesenteric arteries were mounted in the arteriography and arterial diameter was measured. And western blot analysis was assessed.ResultsPressure‐induced myogenic response was significantly increased, whereas endothelium‐dependent relaxation was significantly reduced in the mesenteric arteries from type 2 diabetic mice. Interestingly, treatment of adipoRon normalized potentiated myogenic response. However, endothelium‐dependent relaxation was not affected by treatment of adipoRon. The expression levels of adiponectin receptor 1, 2 and adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 1, 2 (APPL 1, 2) were increased in the mesenteric arteries from Type 2 diabetic mice and treatment of adipoRon did not affect them. Interestingly, adipoRon treatment increased the phosphorylation level of AMP‐activated protein kinase (AMPK) and decreased phosphorylation of myosin phosphatase target subunit 1 (MYPT1) in the type 2 diabetic mice while there was no change in the level of eNOS phosphorylation.ConclusionThe treatment of adipoRon improves vascular function in the mesenteric arteries from type 2 diabetic mice through endothelium‐independent mechanism. We suggest that myosin light chain phosphatase (MLCP) activation through reduced phosphorylation of MYPT1 might be the dominant mechanism in the adipoRon‐induced vascular effect.Support or Funding InformationThis work was supported by the Basic Science Research Program of the National Research Foundation (NRF) of Korea funded by the Ministry of Education, Science and Technology (NRF‐2018R1D1A1B07041820).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of epoxyeicosatrienoic acid in splanchnic vasodilation in portal hypertension

Objective To evaluate the role of epoxyeicosatrienoic acid (EETs) in the haemodynamic alterations of experimental cirrhosis, and its relationship with vascular contractile reactivity and calcium sensitivity of mesenteric arteries. Methods Twenty control rats and 40 rats with carbon tetrachloride (CCl4)-induced cirrhosis were studied. The plasma EETs in the mesenteric arteries was evaluated by enzyme linked immunosorbent assay (ELISA). Concentration-response curves to Norepinephrine (NE) and calcium (Ca2+ ) were evaluated in mesenteric arteries pre-incubated with indomethacin (indo), N (G)-nitro-L-arginine-methyl-ester (L-NAME), 1H-[1, 2, 4]oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ), ibetiotoxin (IbTx) and Glibenclamide before and after the epoxygenase inhibitor miconazole. pD2 was calculated. the activity of myosin light chain phosphatase (MLCP) and myosin light chain (MLC20) phosphorylation were observed. Results The plasma EETs in mesenteric arteries were increased more than that in control rats (P<0.01), and the activity of MLCP was increased, the MLC20 phosphorylation decreased; The NE and Ca2+ response were decreased in small mesenteric arteries. Indo, L-NAME, ODQ, IbTx and Glibenclamide blunted the response to NE and Ca2+ more in cirrhotic than that in control rats (P<0.05). Concentration-response curves to miconazole showed an increased NE and Ca2+ response in cirrhotic rats (P<0.05); the activity of MLCP was decreased, the MLC20 phosphorylation was increased by using miconazole. An increased plasma EETs were significantly correlated with NE-pD2, Ca2+ -pD2, the activity of MLCP and the MLC20 phosphorylation in mesenteric arteries. Conclusion The increased plasma EETs play a role in splanchnic vasodilation of PHT through regulating the response to NE and Ca2+ in mesenteric arteries. Key words: Hypertension portal; Splanchnic vasodilation; Epoxyeicosatrienoic acid; Calcium sensitivity; Vascular reactivity

Paying the Toll for Inflammation

Paying the Toll for Inflammation

Selenium Deficiency Affects Uterine Smooth Muscle Contraction Through Regulation of the RhoA/ROCK Signalling Pathway in Mice.

Selenium (Se) is considered one of the essential micronutrients for humans and animals, and its effects on physiological functions are multifaceted. In the present study, we investigated the effects of Se deficiency on uterine smooth muscle contraction in mice by studying G protein Rho (RhoA)/Rho kinase (ROCK) signalling pathway-related molecules. The α-sma in smooth muscle tissue of mice was determined. The extracorporeal contraction curve for uterine smooth muscle in mice was determined. Both of these results indicate that Se deficiency impairs the contractile ability of uterine smooth muscle in mice. The expression of mRNA was measured by real-time quantitative PCR. The results showed that there was no significant change in mRNA expression of RhoA, ROCK, myosin light chain phosphatase (MLCP), or myosin light chain (MLC) in tissues. The protein levels were detected by Western blot. The results showed that there were no significant differences in RhoA, ROCK, MLCP, or MLC expression. However, compared with the CG, the concentration of phosphorylated MLC (P-MLC) increased in the SG and the concentration of P-MLC decreased in the DG. The activity of ROCK and MLCP was tested by liquid scintillation. The results suggest that the lack of Se may affect the regulation of MLCP by ROCK. Cellular experiments were performed to compare with results from tissues. There was no significant difference between the two models. The results indicated that Se deficiency affects uterine smooth muscle contraction by regulating the RhoA/ROCK signalling pathway. As the concentration of Se decreases, the activity of MLCP increases, which promotes the dephosphorylation of P-MLC, causing a decrease in contraction.

Endothelial cell rearrangements during vascular patterning require PI3-kinase-mediated inhibition of actomyosin contractility

Angiogenesis is a dynamic process relying on endothelial cell rearrangements within vascular tubes, yet the underlying mechanisms and functional relevance are poorly understood. Here we show that PI3Kα regulates endothelial cell rearrangements using a combination of a PI3Kα-selective inhibitor and endothelial-specific genetic deletion to abrogate PI3Kα activity during vessel development. Quantitative phosphoproteomics together with detailed cell biology analyses in vivo and in vitro reveal that PI3K signalling prevents NUAK1-dependent phosphorylation of the myosin phosphatase targeting-1 (MYPT1) protein, thereby allowing myosin light chain phosphatase (MLCP) activity and ultimately downregulating actomyosin contractility. Decreased PI3K activity enhances actomyosin contractility and impairs junctional remodelling and stabilization. This leads to overstretched endothelial cells that fail to anastomose properly and form aberrant superimposed layers within the vasculature. Our findings define the PI3K/NUAK1/MYPT1/MLCP axis as a critical pathway to regulate actomyosin contractility in endothelial cells, supporting vascular patterning and expansion through the control of cell rearrangement.

Involvement of inhibitor kappa B kinase 2 (IKK2) in the regulation of vascular tone

Inhibitor kappa B kinase 2 (IKK2) plays an essential role in the activation of nuclear factor kappa B (NF-kB). Recently, it has been suggested that IKK2 acts as a myosin light chain kinase (MLCK) and contributes to vasoconstriction in mouse aorta. However, the underlying mechanisms are still unknown. Therefore, we investigated whether IKK2 acts as a MLCK or regulates the activity of myosin light chain phosphatase (MLCP). Pressure myograph was used to measure vascular tone in rat mesenteric arteries. Immunofluorescence staining was performed to identify phosphorylation levels of MLC (ser19), MYPT1 (thr853 and thr696) and CPI-17 (thr38). SC-514 (IKK2 inhibitor, 50 μM) induced relaxation in the mesenteric arteries pre-contracted with 70 mM high K+ solution or U-46619 (thromboxane analog, 5 μM). The relaxation induced by SC-514 was increased in the arteries pre-contracted with U-46619 compared to arteries pre-contracted with 70 mM high K+ solution. U-46619-induced contraction was decreased by treatment of SC-514 in the presence of MLCK inhibitor, ML-7 (10 μM). In the absence of intracellular Ca2+, U-46619 still induced contraction, which was decreased by treatment of SC-514. Furthermore, phosphorylation levels of MLC (ser19) and MYPT1 (thr853) were decreased by treatment of SC-514. IKK2 is involved in the vascular contraction through regulation of MLCP activity by phosphorylating MYPT1 at thr853 in rat mesenteric arteries. These findings suggest IKK2 could be a new pharmacological target for specific therapies of various vascular diseases.This paper examines whether inhibitor kappa B kinase 2 (IKK2) acts as a myosin light chain kinase (MLCK) or regulates the activity of myosin light chain phosphatase (MLCP), and therefore affects vasoconstriction. The authors found that IKK2 regulates MLCP activity by phosphorylating the MYPT1 subunit. Furthermore, SC-514, an IKK2 inhibitor, induces relaxation of mesenteric arteries. Therefore, IKK2 may be a new pharmacological target for treatment of various vascular diseases.

The dominant protein phosphatase PP1c isoform in smooth muscle cells, PP1cβ, is essential for smooth muscle contraction

Contractile force development of smooth muscle is controlled by balanced kinase and phosphatase activities toward the myosin regulatory light chain (RLC). Numerous biochemical and pharmacological studies have investigated the specificity and regulatory activity of smooth muscle myosin light-chain phosphatase (MLCP) bound to myosin filaments and comprised of the regulatory myosin phosphatase target subunit 1 (MYPT1) and catalytic protein phosphatase 1cβ (PP1cβ) subunits. Recent physiological and biochemical evidence obtained with smooth muscle tissues from a conditional MYPT1 knockout suggests that a soluble, MYPT1-unbound form of PP1cβ may additionally contribute to myosin RLC dephosphorylation and relaxation of smooth muscle. Using a combination of isoelectric focusing and isoform-specific immunoblotting, we found here that more than 90% of the total PP1c in mouse smooth muscles is the β isoform. Moreover, conditional knockout of PP1cα or PP1cγ in adult smooth muscles did not result in an apparent phenotype in mice up to 6 months of age and did not affect smooth muscle contractions ex vivo In contrast, smooth muscle-specific conditional PP1cβ knockout decreased contractile force development in bladder, ileal, and aortic tissues and reduced mouse survival. Bladder smooth muscle tissue from WT mice was selectively permeabilized to remove soluble PP1cβ to measure contributions of total (α-toxin treatment) and myosin-bound (Triton X-100 treatment) phosphatase activities toward phosphorylated RLC in myofilaments. Triton X-100 reduced PP1cβ content by 60% and the rate of RLC dephosphorylation by 2-fold. These results are consistent with the selective dephosphorylation of RLC by both MYPT1-bound and -unbound PP1cβ forms in smooth muscle.

HDAC7 in MLCP‐mediated endothelial barrier preservation

Acute lung injury (ALI) is an acute systemic inflammatory process characterized by pulmonary infiltrates hypoxemia and edema. ALI arises from a wide range of lung injuries such as toxins or inflammatory mediators. Despite the high level of mortality, to date no pharmacological therapies have been proven to improve ALI except from ventilation strategies. A deeper understanding of the pathogenic mechanisms involved remains a barrier to new and effective therapies that will reduce the vascular leak associated with ALI.A major cause of ALI is dysfunction of the pulmonary vascular endothelial barrier. Endothelial cells (EC) are a specialized cell type that line the lumen of the blood vessels serving every major organ system and provide a semi‐selective barrier between the blood and the interstitial space. Epigenetic mechanisms have emerged as a major regulator of enduring changes in cell behavior and the therapeutic potential of inhibiting histone deacetylases (HDACs) for the treatment of cardiovascular and inflammatory diseases is gaining increasing attention. In addition to histones, it has been shown that HDACs can deacetylate many non‐histone proteins to regulate cellular functions such as cytoskeletal acetylation and polymerization, and signal transduction. Further, it has been shown that HDAC inhibition is directly involved in EC barrier preservation, however, the molecular mechanisms of HDAC‐mediated EC barrier regulation remain largely unexplored.Our data indicate that the class IIa histone deacetylases comprising of HDACs 4, 5, 7, 9 are involved in the regulation of pulmonary vascular barrier in vitro and in lipopolysaccharide (LPS)‐induced murine model of ALI. These HDACs are known to function as transcriptional co‐repressors, however, they also are able to shuttle from nucleus to the cytoplasm thus becoming accessible for extra nuclear targets. We demonstrate that HDAC 7 is directly involved in LPS‐induced human lung microvascular EC (HLMVEC) barrier compromise. Further, barrier‐disruptive effects of LPS are accompanied by HDAC7 phosphorylation and its nuclear export. Conversely, agents that protect the EC barrier such as adenosine, decrease its phosphorylation. These data suggest the involvement of phosphatase activity in HDAC7‐mediated EC barrier regulation. Our results show that HDAC7 is a substrate for myosin light chain (MLC) phosphatase (MLCP) in HLMVEC. Conversely, down‐regulation of HDAC7 may affect MLCP activity. However, the physiological role of HDAC7/MLCP crosstalk in EC barrier regulation is currently unexplored. It is known that activation of MLCP leads to dephosphorylation of MLC thus opposing actomyosin contraction. Our newly published results suggest that MLCP activation may be involved in EC barrier preservation via the dephosphorylation of unconventional MLCP cytoskeletal targets such as moesin. Our results demonstrate a direct association between HDAC7 and moesin, suggesting a novel role of a HDAC7/MLCP/moesin signaling complex in the regulation of EC barrier. Collectively, these data demonstrate the involvement of HDAC7 activity in MLCP‐mediated EC barrier preservation and suggest, for the first time, the importance of HDAC7 nuclear export in pulmonary vascular injury induced by edemagenic agents such as LPS.Support or Funding InformationNIH; Contract grant number: HL101902This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Physiological vs. pharmacological signalling to myosin phosphorylation in airway smooth muscle.

Smooth muscle myosin regulatory light chain (RLC) is phosphorylated by Ca2+ /calmodulin-dependent myosin light chain kinase and dephosphorylated by myosin light chain phosphatase (MLCP). Tracheal smooth muscle contains significant amounts of myosin binding subunit 85 (MBS85), another myosin phosphatase targeting subunit (MYPT) family member, in addition to MLCP regulatory subunit MYPT1. Concentration/temporal responses to carbachol demonstrated similar sensitivities for bovine tracheal force development and phosphorylation of RLC, MYPT1, MBS85 and paxillin. Electrical field stimulation releases ACh from nerves to increase RLC phosphorylation but not MYPT1 or MBS85 phosphorylation. Thus, nerve-mediated muscarinic responses in signalling modules acting on RLC phosphorylation are different from pharmacological responses with bath added agonist. The conditional knockout of MYPT1 or the knock-in mutation T853A in mice had no effect on muscarinic force responses in isolated tracheal tissues. MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction. Ca2+ /calmodulin activation of myosin light chain kinase (MLCK) initiates myosin regulatory light chain (RLC) phosphorylation for smooth muscle contraction with subsequent dephosphorylation for relaxation by myosin light chain phosphatase (MLCP) containing regulatory (MYPT1) and catalytic (PP1cδ) subunits. RLC phosphorylation-dependent force development is regulated by distinct signalling modules involving protein phosphorylations. We investigated responses to cholinergic agonist treatment vs. neurostimulation by electric field stimulation (EFS) in bovine tracheal smooth muscle. Concentration/temporal responses to carbachol demonstrated tight coupling between force development and RLC phosphorylation but sensitivity differences in MLCK, MYPT1 T853, MYPT1 T696, myosin binding subunit 85 (MBS85), paxillin and CPI-17 (PKC-potentiated protein phosphatase 1 inhibitor protein of 17 kDa) phosphorylations. EFS increased force and phosphorylation of RLC, CPI-17 and MLCK. In the presence of the cholinesterase inhibitor neostigmine, EFS led to an additional increase in phosphorylation of MYPT1 T853, MYPT1 T696, MBS85 and paxillin. Thus, there were distinct pharmacological vs. physiological responses in signalling modules acting on RLC phosphorylation and force responses, probably related to degenerate G protein signalling networks. Studies with genetically modified mice were performed. Expression of another MYPT1 family member, MBS85, was enriched in mouse, as well as bovine tracheal smooth muscle. Carbachol concentration/temporal-force responses were similar in trachea from MYPT1SM+/+ , MYPT1SM-/- and the knock-in mutant mice containing nonphosphorylatable MYPT1 T853A with no differences in RLC phosphorylation. Thus, MYPT1 T853 phosphorylation was not necessary for regulation of RLC phosphorylation in tonic airway smooth muscle. Furthermore, MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction.

CGMP signaling inhibits platelet shape change through regulation of the RhoA‐Rho Kinase‐MLC phosphatase signaling pathway

Background Platelet shape change, spreading and thrombus stability require activation of the actin cytoskeleton contractile machinery. The mechanisms controlling actin assembly to prevent unwanted platelet activation are unclear. Objectives We examined the effects of nitric oxide on the signaling pathways regulating platelet actin-myosin activation. Results S-nitrosoglutathione (GSNO) inhibited thrombin-induced platelet shape change and myosin phosphorylation of the myosin light chain (MLC). Because thrombin stimulates phospho-MLC through the RhoA/ ROCK dependent inhibition of MLC phosphatase (MLCP) we examined the effects of NO on this pathway. Thrombin caused the GTP loading and activation of RhoA, leading to the ROCK-mediated phosphorylation of MLCP on threonine 853 (thr853 ), which is known to inhibit phosphatase activity. Treatment of platelets with GSNO blocked ROCK-mediated increases in phosphoMLCP-thr853 induced by thrombin. This effect was mimicked by the direct activator of protein kinase G, 8-pCPT-PET-cGMP, and blocked by the inhibition of guanylyl cyclase, but not inhibitors of protein kinase A. Further exploration of the mechanism demonstrated that GSNO stimulated the association of RhoA with protein kinase G (PKG) and the inhibitory phosphorylation (serine188) of RhoA in a cGMP-dependent manner. Consistent with these observations, in vitro experiments revealed that recombinant PKG caused direct phosphorylation of RhoA. The inhibition of RhoA by GSNO prevented ROCK-mediated phosphorylation and inhibition of MLCP activity. Conclusions These data suggest novel crosstalk between the NO-cGMP-PKG and RhoA/ROCK signaling pathways to control platelet actin remodeling.