Regulation of LPA-promoted myofibroblast contraction: role of Rho, myosin light chain kinase, and myosin light chain phosphatase

Myofibroblasts generate the contractile force responsible for wound healing and pathological tissue contracture. In this paper the stress-relaxed collagen lattice model was used to study lysophosphatidic acid (LPA)-promoted myofibroblast contraction and the role of the small GTPase Rho and its downstream targets Rho kinase and myosin light chain phosphatase (MLCPPase) in regulating myofibroblast contraction. In addition, the regulation of myofibroblast contraction was compared with that of smooth muscle cells. LPA-promoted myofibroblast contraction was inhibited by the myosin light chain kinase (MLCK) inhibitors KT5926 and ML-7; however, in contrast to that observed in smooth muscle cells, elevation of intracellular calcium alone was not sufficient to promote myofibroblast contraction. These results suggest that Ca(2+)-mediated activation of MLCK, while necessary, is not sufficient to promote myofibroblast contraction. The specific Rho inactivator C3-transferase and the Rho kinase inhibitor Y-27632 inhibited LPA-promoted myofibroblast contraction, suggesting that contraction depends on activation of the Rho/Rho kinase pathway. Calyculin, a type 1 phosphatase inhibitor known to inhibit MLCPPase, could promote myofibroblast contraction in the absence of LPA, as well as restore contraction in the presence of C3-transferase or Y-27632. Together these results support a model whereby Rho/Rho kinase-mediated inhibition of MLCPPase is necessary for LPA-promoted myofibroblast contraction, in contrast to smooth muscle cells in which Ca(2+) activation of MLCK alone is sufficient to promote contraction.     

Microsphere embolism-induced endothelial nitric oxide synthase expression mediates disruption of the blood-brain barrier in rat brain

Microsphere embolism (ME)-induced up-regulation of endothelial nitric oxide synthase (eNOS) in endothelial cells of brain microvessels was observed 2-48 h after ischemia. eNOS induction preceded disruption of the blood-brain barrier (BBB) observed 6-72 h after ischemia. In vascular endothelial cells, ME-induced eNOS expression was closely associated with protein tyrosine nitration, which is a marker of generation of peroxynitrite. Leakage of rabbit IgG from microvessels was also evident around protein tyrosine nitration-immunoreactive microvessels. To determine whether eNOS expression and protein tyrosine nitration in vascular endothelial cells mediates BBB disruption in the ME brain, we tested the effect of a novel calmodulin-dependent NOS inhibitor, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e), which inhibits eNOS activity and, in turn, protein tyrosine nitration. Concomitant with inhibition of protein tyrosine nitration in vascular endothelial cells, DY-9760e significantly inhibited BBB disruption as assessed by Evans blue (EB) excretion. DY-9760e also inhibited cleavage of poly (ADP-ribose) polymerase as a marker of the apoptotic pathway in vascular endothelial cells. Taken together with previous evidence in which DY-9760e inhibited brain edema, ME-induced eNOS expression in vascular endothelial cells likely mediates BBB disruption and, in turn, brain edema.     

Localization and activity of myosin light chain kinase isoforms during the cell cycle

Phosphorylation on Ser 19 of the myosin II regulatory light chain by myosin light chain kinase (MLCK) regulates actomyosin contractility in smooth muscle and vertebrate nonmuscle cells. The smooth/nonmuscle MLCK gene locus produces two kinases, a high molecular weight isoform (long MLCK) and a low molecular weight isoform (short MLCK), that are differentially expressed in smooth and nonmuscle tissues. To study the relative localization of the MLCK isoforms in cultured nonmuscle cells and to determine the spatial and temporal dynamics of MLCK localization during mitosis, we constructed green fluorescent protein fusions of the long and short MLCKs. In interphase cells, localization of the long MLCK to stress fibers is mediated by five DXRXXL motifs, which span the junction of the NH(2)-terminal extension and the short MLCK. In contrast, localization of the long MLCK to the cleavage furrow in dividing cells requires the five DXRXXL motifs as well as additional amino acid sequences present in the NH(2)-terminal extension. Thus, it appears that nonmuscle cells utilize different mechanisms for targeting the long MLCK to actomyosin structures during interphase and mitosis. Further studies have shown that the long MLCK has twofold lower kinase activity in early mitosis than in interphase or in the early stages of postmitotic spreading. These findings suggest a model in which MLCK and the myosin II phosphatase (Totsukawa, G., Y. Yamakita, S. Yamashiro, H. Hosoya, D.J. Hartshorne, and F. Matsumura. 1999. J. Cell Biol. 144:735-744) act cooperatively to regulate the level of Ser 19-phosphorylated myosin II during mitosis and initiate cytokinesis through the activation of myosin II motor activity.     

Normobaric hyperoxia attenuates early blood–brain barrier disruption by inhibiting MMP-9-mediated occludin degradation in focal cerebral ischemia

Early blood–brain barrier (BBB) disruption resulting from excessive neurovascular proteolysis by matrix metalloproteinases (MMPs) is closely associated with hemorrhagic transformation events in ischemic stroke. We have shown that normobaric hyperoxia (NBO) treatment reduces MMP-9 increase in the ischemic brain. The aim of this study was to determine whether NBO could attenuate MMP-9-mediated early BBB disruption following ischemic stroke. Rats were exposed to NBO (95% O₂) or normoxia (30% O₂) during 90-min middle cerebral artery occlusion, followed by 3-hour reperfusion. NBO-treated rats showed a significant reduction in Evan's blue extravasation in the ischemic hemisphere compared with normoxic rats. Topographically, Evan's blue leakage was mainly seen in the subcortical regions including the striatum, which was accompanied by increased gelatinolytic activity and reduced immunostaining for tight-junction protein, occludin. Increased gelatinolytic activities and occludin protein loss were also observed in isolated ischemic microvessels. Gel gelatin zymography identified that MMP-9 was the main enzymatic source in the cerebral microvessels. Incubation of brain slices or isolated microvessels with purified MMP-9 revealed specific degradation of occludin. Inhibition of MMP-9 by NBO or MMP-inhibitor, BB1101, significantly reduced occludin protein loss in ischemic microvessels. These results suggest that NBO attenuates early BBB disruption, and inhibition of MMP-9-mediated occludin degradation is an important mechanism for this protection.     

Alcohol-induced blood-brain barrier dysfunction is mediated via inositol 1,4,5-triphosphate receptor (IP3R)-gated intracellular calcium release

The blood-brain barrier (BBB) formed by brain microvascular endothelial cells (BMVEC), pericytes and astrocytes controls the transport of ions, peptides and leukocytes in and out of the brain. Tight junctions (TJ) composed of TJ proteins (occludin, claudins and zonula occludens) ensure the structural integrity of the BMVEC monolayer. Neuropathologic studies indicated that the BBB was impaired in alcohol abusers; however, the underlying mechanism of BBB dysfunction remains elusive. Using primary human BMVEC, we previously demonstrated that oxidative stress induced by ethanol (EtOH) metabolism in BMVEC activated myosin light chain kinase (MLCK), resulting in the enhanced phosphorylation of either cytoskeletal or TJ proteins, and in BBB impairment. We proposed that EtOH metabolites stimulated inositol 1,4,5-triphosphate receptor (IP(3)R)-operated intracellular calcium (Ca(2+)) release, thereby causing the activation of MLCK in BMVEC. Indeed, treatment of primary human BMVEC with EtOH or its metabolites resulted in the increased expression of IP(3)R protein and IP(3)R-gated intracellular Ca(2+) release. These functional changes paralleled MLCK activation, phosphorylation of cytoskeletal/TJ proteins, loss of BBB integrity, and enhanced leukocyte migration across BMVEC monolayers. Inhibition of either EtOH metabolism or IP(3)R activation prevented BBB impairment. These findings suggest that EtOH metabolites act as signaling molecules for the activation of MLCK via the stimulation of IP(3)R-gated intracellular Ca(2+) release in BMVEC. These putative events can lead to BBB dysfunction in the setting of alcoholism, and to neuro-inflammatory disorders promoting leukocyte migration across the BBB.     

Design of peptidase‐resistant peptide inhibitors of myosin light chain kinase

Myosin light chain kinase (MLCK) is a key regulator of various forms of cell motility including smooth muscle contraction, cell migration, cytokinesis, receptor capping, secretion, etc. Inhibition of MLCK activity in endothelial and epithelial monolayers using cell‐permeant peptide Arg‐Lys‐Lys‐Tyr‐Lys‐Tyr‐Arg‐Arg‐Lys (PIK, P eptide I nhibitor of K inase) allows protecting the barrier capacity, suggesting a potential medical use of PIK. However, low stability of L ‐PIK in a biological milieu prompts for development of more stable L ‐PIK analogues for use as experimental tools in basic and drug‐oriented biomedical research. Previously, we designed PIK1, H‐(N^αMe)Arg‐Lys‐Lys‐Tyr‐Lys‐Tyr‐Arg‐Arg‐Lys‐NH₂, that was 2.5‐fold more resistant to peptidases in human plasma in vitro than L ‐PIK and equal to it as MLCK inhibitor. In order to further enhance proteolytic stability of PIK inhibitor, we designed the set of six site‐protected peptides based on L ‐PIK and PIK1 degradation patterns in human plasma as revealed by ¹H‐NMR analysis. Implemented modifications increased half‐live of the PIK‐related peptides in plasma about 10‐fold, and these compounds retained 25–100% of L ‐PIK inhibitory activity toward MLCK in vitro. Based on stability and functional activity ranking, PIK2, H‐(N^αMe)Arg‐Lys‐Lys‐Tyr‐Lys‐Tyr‐Arg‐D ‐Arg‐Lys‐NH₂, was identified as the most stable and effective L ‐PIK analogue. PIK2 was able to decrease myosin light chain phosphorylation in endothelial cells stimulated with thrombin, and this effect correlated with the inhibition by PIK2 of thrombin‐induced endothelial hyperpermeability in vitro. Therefore, PIK2 could be used as novel alternative to other cell‐permeant inhibitors of MLCK in cell culture‐based and in vivo studies where MLCK catalytic activity inhibition is required. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Phosphorylation and partial sequence of pregnant sheep myometrium myosin light chain kinase

The function of the uterine smooth muscle in gestation and parturition is affected by a variety of hormones and biomolecules, some of which alter the intracellular levels of cAMP and Ca²⁺. Since the activity of smooth muscle MLCK has been shown to be modulated by phosphorylation, the effect of this modification of pregnant sheep myometrium (psm) MLCK by the catalytic subunit of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was studied. In contrast to other smooth muscle MLCK reported, PKA incorporates 2.0–2.2 moles phosphate into a mole of psm MLCK both in the presence and absence of Ca²⁺-calmodulin. Modification of serine residues inhibited the activity of the enzyme. PKC also incorporated 2.0–2.1 moles of phosphate per mole psmMLCK under both conditions but had no effect on the MLCK activity. Sequential phosphorylation by PKC and PKA incorporated 3.8–4.1 moles phosphate suggesting that the amino acid residues modified by the two kinases are different. Phosphoamino acid analysis of the MLCK revealed that PKC phosphorylated serine and threonine residues. The double reciprocal plots of the enzyme activity and calmodulin concentrations showed that the V_max of the reaction is not altered by phosphorylation by PKA but the calmodulin concentration require for half-maximal activation is increased about 4-fold. Only 10 out of 17 monoclonal antibodies to various regions of the turkey gizzard MLCK cross-reacted with psmMLCK suggesting structural differences between these enzymes. Comparison of the deduced amino acid sequence of the cDNA encoding the C-terminal half of the psmMLCK molecule showed that while cgMLCK and psmMLCK are highly homologous, a number of nonconservative substitutions are present, particularly near the PKA phosphrylation site B (S⁸²⁸).     

Theoretical model of the interactions between Ca2+, calmodulin and myosin light chain kinase

Active Ca²⁺/calmodulin (CaM)-dependent myosin light chain kinase (MLCK) plays an important role in the process of MLC phosphorylation and consecutive smooth muscle contraction. Here, we propose a mathematical model of a detailed kinetic scheme describing interactions among Ca²⁺, CaM and MLCK and taking into account eight different aggregates. The main model result is the prediction of the Ca²⁺ dependent active form of MLCK, which is in the model taken as proportional to the concentration of Ca₄CaM · MLCK complex. Wegscheider’s condition is additionally applied as a constraint enabling the prediction of some parameter values that have not yet been obtained by experiments.     

Chicken smooth muscle myosin light chain kinase is acetylated on its NH2-terminal methionine

The reported cDNA structrre, of chicken smooth muscle myosin light chain kinase (smMLCK) encodes a protein of 972 residues (Olsonet al. Proc. Natl. Acad. Sci USA, 87: 2284–2288, 1990). The calculated Mr is 107, 534 whereas the estimate by SDS-PAGE is approximately 130, 000. Gibson and Higgins (DNA Sequence (in press)) have recently reported the possibility of errors, in the cDNA sequence for non-muscle MLCK and that the NH₂-terminus of both it and smMLCK may extend beyond the reported coding region. The native smMLCK is NH₂-terminally blocked. A CNBr peptide derived from smMLCK contains the NH₂-terminal sequence Asp-Phe-Arg-Ala corresponding to residues 2 to 4 in the smMLCK sequence indicating, that Met-1 is present. Using a limited thermolysin digest we isolated an NH₂-terminally blocked peptide by reversed-phase HPLC. This thermolytic peptide had a mass of approximately 797 by time of flight mass spectrometry. Amino acid analysis and Edman sequencing of a CNBr-subfragment of the thermolytic peptide indicated that it had the composition and sequence, (Met)-Asp-Phe-Arg-Ala-Asn, with a calculated mass of 753. The difference in mass corresponds to the NH₂-terminal Met being blocked by actylation. The results demonstrate that the NH₂-terminal sequence of smMLCK inferred from the reported cDNA sequence is correct and that the proposed initiating, Met is not removed, but modified by -NH₂ acetylation of the translation product.     

Effects of purified myosin light chain kinase on myosin light chain phosphorylation and catecholamine secretion in digitonin-permeabilized chromaffin cells

Many non-muscle cells including chromaffin cells contain actin and myosin. The 20,000 dalton light chain subunits of myosin can be phosphorylated by a Ca²⁺/calmodulin-dependent enzyme, myosin light chain kinase. In tissues other than striated muscle, light chain phosphorylation is required for actin-induced myosin ATPase activity. The possibility that actin and myosin are involved in catecholamine secretion was investigated by determining whether increased phosphorylation in the presence of [-³²P]ATP of myosin light chain by myosin light chain kinase enhances secretion from digitonin-treated chromaffin cells. In the absence of exogenous myosin light chain kinase, 1 M Ca²⁺ caused a 30–40% enhancement of the phosphorylation of a 20 kDa protein. This protein was identified on 2-dimensional gels as myosin light chain by its comigration with purified myosin light chain. Purified myosin light chain kinase (400 g/ml) in the presence of calmodulin (10 M) caused little or no enhancement of myosin light chain phosphorylation in the absence of Ca²⁺ in digitonin-treated cells. In the presence of 1 M Ca²⁺, myosin light chain kinase (400 g/ml) caused an approximately two-fold increase in myosin light chain phosphorylation in digitonin-treated cells in 5 min. The phosphorylation required permeabilization of the cells by digitonin and occurred within the cells rather than in the medium. Myosin light chain kinase-induced phosphorylation of myosin light chain was maximal at 1 M. Ca²⁺. Under identical conditions to those of the phosphorylation experiments, secretion was unaltered by myosin light chain kinase. The experiments indicate that the phosphorylation of myosin light chain by myosin light chain kinase is not a limiting factor in secretion in digitonin-treated chromaffin cells and suggest that the activation of myosin is not directly involved in secretion from the cells. The experiments also demonstrate the feasibility of investigation of effects of exogenously added proteins on secretion in digitonin-treated cells.Abbreviations EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - KGEPM solution containing potassium glutamate, EGTA, PIPES and MgCl₂ - NE norepinephrine - PIPES piperazine-N,-N-bis-(2-ethanesulfonic acid) - PSS physiological salt solution     

Intercommunications between brain capillary endothelial cells and glial cells increase the transcellular permeability of the blood-brain barrier during ischaemia

Increased cerebrovascular permeability is an important factor in the development of cerebral oedema after stroke, implicating the blood-brain barrier (BBB). To investigate the effect of hypoxia on the permeability changes, we used a cell culture model of the BBB consisting of a co-culture of brain capillary endothelial cells and glial cells. When endothelial cells from this co-culture model were submitted alone to hypoxic conditions, long exposures (48 h) were necessary to result in an increase in endothelial cell monolayer permeability to [3H]inulin. When endothelial cells were incubated in presence of glial cells, a huge increase in permeability occurred after 9 h of hypoxic conditions. Oxygen glucose deprivation (OGD) resulted in a much shorter time (i.e. 2 h) required for an increase in permeability. We have demonstrated that this OGD-induced permeability increase involves a transcellular rather than a paracellular pathway. Conditioned medium experiments showed that glial cells secrete soluble permeability factors during OGD. However, endothelial cells have to be made sensitive by OGD in order to respond to these glial soluble factors. This work shows that an early cross-talk between glial and endothelial cells occurs during ischaemic stroke and alters BBB transcellular transport by means of glial factor secretions.     

Localization of an actin binding domain in smooth muscle myosin light chain kinase

Phosphorylation of the regulatory light chain of myosin II by myosinlight chain kinase is important for regulating many contractile processes.Smooth muscle myosin light chain kinase has been shown to be associated withboth actin and myosin filaments in vitro and in vivo. In this report wedefine an actin binding region by using molecular deletions to generaterecombinant mutant proteins that were analyzed by co-sedimentation withF-actin. An actin binding region restricted to residues 2-42 in the animoterminus of the rabbit smooth muscle myosin light chain kinase wasidentified.     

A low molecular weight copper chelator crosses the blood-brain barrier and attenuates experimental autoimmune encephalomyelitis

Increasing evidence suggests that enhanced production of reactive oxygen species (ROS) activates the MAP kinases, c-Jun N-terminal protein kinase (JNK) and mitogen-activated protein kinase MAPK (p38). These phosphorylated intermediates at the stress-activated pathway induce expression of matrix metalloproteinases (MMPs), leading to inflammatory responses and pathological damages involved in the etiology of multiple sclerosis (MS). Here we report that N-acetylcysteine amide (AD4) crosses the blood-brain barrier (BBB), chelates Cu(2+), which catalyzes free radical formation, and prevents ROS-induced activation of JNK, p38 and MMP-9. In the myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, oral administration of AD4 drastically reduced the clinical signs, inflammation, MMP-9 activity, and protected axons from demylination damages. In agreement with the in vitro studies, we propose that ROS scavenging by AD4 in MOG-treated animals prevented MMP's induction and subsequent damages through inhibition of MAPK pathway. The low toxicity of AD4 coupled with BBB penetration makes this compound an excellent potential candidate for the therapy of MS and other neurodegenerative disorders.     

Structure and function of the essential light chain of myosin

The review summarizes the recent data on the structure and function of the essential light chain of myosin. It is known that the essential light chain of myosin stabilizes the lever arm. Consistent with the model of the shift of the dynamic population of conformations, the conformational flexibility of the essential light chain is emphasized, which opens the way to determining its new functions. It is proposed that the interaction between the C-terminal domain of the essential light chain and the N-terminal subdomain of the heavy chain of myosin may be involved in the coupling of ATP hydrolysis and rotation of the lever arm. The recent data indicate that the isoforms of the essential light chain with the additional N-terminal peptide are capable of interacting with actin and src-homologous domain 3 of myosin. The structural aspects of these interactions and the modulatory role of the isoforms of the essential light chain of myosin are discussed.     

Development and characterization of fluorescently-labeled myosin light chain kinase calmodulin-binding domain peptides

Calmodulin-dependent protein kinases such as myosin light chain kinase (MLCK), calmodulin kinase II, and phosphorylase kinase contain specific sequences responsible for binding calmodulin. These regions are known as calmodulin-binding domains and in many cases are contained within sequences that are short enough to be synthesized by solidphase techniques. The ability to chemically-synthesize target enzyme calmodulin-binding domains has permitted the use of a variety of biophysical techniques to study the interactions between calmodulin and calmodulin-binding domain peptides. The work reviewed here describes the development and characterization of peptides based on the sequence, of the calmodulin-binding domain of skeletal muscle myosin light chain kinase which were labeled with the fluorescent reagent, acrylodan. Data are presented demonstrating the use of fluorescently-labeled peptides to study various aspects of calmodulin-peptide interactions including binding affinity, stoichiometry, specificity, changes in peptide conformation, and thermal stability of the peptide-calmodulin complex. These data indicate the peptides exhibit many of the salient features seen with calmodulin-target enzyme interactions. The fluorescently-labeled peptides should thus serve as useful models for studying calmodulin-target enzyme interactions at the molecular level.     

The l-isomer-selective transport of aspartic acid is mediated by ASCT2 at the blood-brain barrier

Aspartic acid (Asp) undergoes l-isomer-selective efflux transport across the blood-brain barrier (BBB). This transport system appears to play an important role in regulating l- and d-Asp levels in the brain. The purpose of this study was to identify the responsible transporters and elucidate the mechanism for l-isomer-selective Asp transport at the BBB. The l-isomer-selective uptake of Asp by conditionally immortalized mouse brain capillary endothelial cells used as an in vitro model of the BBB took place in an Na+- and pH-dependent manner. This process was inhibited by system ASC substrates such as l-alanine and l-serine, suggesting that system ASC transporters, ASCT1 and ASCT2, are involved in the l-isomer selective transport. Indeed, l-Asp uptake by oocytes injected with either ASCT1 or ASCT2 cRNA took place in a similar manner to that in cultured BBB cells, whereas no significant d-Asp uptake occurred. Although both ASCT1 and ASCT2 mRNA were expressed in the cultured BBB cells, the expression of ASCT2 mRNA was 6.7-fold greater than that of ASCT1. Moreover, immunohistochemical analysis suggests that ASCT2 is localized at the abluminal side of the mouse BBB. These results suggest that ASCT2 plays a key role in l-isomer-selective Asp efflux transport at the BBB.     

Two-site phosphorylation of the phosphorylatable light chain (20-kDa light chain) of chicken gizzard myosin

When prepared under specified conditions chicken gizzard myosin was obtained which when incubated with ATP gave rise to a diphosphorylated as well as the monophosphorylated form of P light chain. Formation of the diphosphorylated light chain occurred more readily with these myosin preparations, but could also be obtained by prolonged incubation of the isolated whole light chain fraction with kinase preparations from rabbit skeletal and chicken gizzard muscles. Using isolated light chains as substrate the more readily formed monophosphorylated light chain contained serine phosphate while the diphosphorylated form contained serine and threonine phosphates.     

Oxidative stress activates protein tyrosine kinase and matrix metalloproteinases leading to blood-brain barrier dysfunction

The blood-brain barrier (BBB) formed by brain microvascular endothelial cells (BMVEC) regulates the passage of molecules and leukocytes in and out of the brain. Oxidative stress is a major underlying cause of neurodegenerative and neuroinflammatory disorders and BBB injury associated with them. Using human BMVEC grown on porous membranes covered with basement membrane (BM) matrix (BBB models), we demonstrated that reactive oxygen species (ROS) augmented permeability and monocyte migration across BBB. ROS activated matrix metalloproteinases (MMP-1, -2, and -9) and decreased tissue inhibitors of MMPs (TIMP-1 and -2) in a protein tyrosine kinase (PTK)-dependent manner. Increase in MMPs and PTK activities paralleled degradation of BM protein and enhanced tyrosine phosphorylation of tight junction (TJ) protein. These effects and enhanced permeability/monocyte migration were prevented by inhibitors of MMPs, PTKs, or antioxidant suggesting that oxidative stress caused BBB injury via degradation of BM protein by activated MMPs and by PTK-mediated TJ protein phosphorylation. These findings point to new therapeutic interventions ameliorating BBB dysfunction in neurological disorders such as stroke or neuroinflammation.