Acute lung injury (ALI) associated with sepsis and iatrogenic ventilator-induced lung injury resulting from mechanical ventilation are major medical problems with an unmet need for small molecule therapeutics. Prevailing hypotheses identify endothelial cell (EC) layer dysfunction as a cardinal event in the pathophysiology, with intracellular protein kinases as critical mediators of normal physiology and possible targets for drug discovery. The 210,000 molecular weight myosin light chain kinase (MLCK210, also called EC MLCK because of its abundance in EC) is hypothesized to be important for EC barrier function and might be a potential therapeutic target. To test these hypotheses directly, we made a selective MLCK210 knockout mouse that retains production of MLCK108 (also called smooth-muscle MLCK) from the same gene. The MLCK210 knockout mice are less susceptible to ALI induced by i.p. injection of the endotoxin lipopolysaccharide and show enhanced survival during subsequent mechanical ventilation. Using a complementary chemical biology approach, we developed a new class of small-molecule MLCK inhibitor based on the pharmacologically privileged aminopyridazine and found that a single i.p. injection of the inhibitor protected WT mice against ALI and death from mechanical ventilation complications. These convergent results from two independent approaches demonstrate a pivotal in vivo role for MLCK in susceptibility to lung injury and validate MLCK as a potential drug discovery target for lung injury.C urrent hypotheses (1, 2) identify dysfunction of the endothelial cell (EC) layer as a cardinal event in the pathophysiology of multiple medical conditions, including sepsis. The mortality associated with sepsis alone is similar (3) to that of acute myocardial infarction (MI). In contrast to MI, available therapies are limited for the treatment of sepsis and its associated tissue injuries (3, 4). Mechanical ventilation is often required for the support of patients with sepsis but is itself associated with additional, iatrogenic pulmonary injury that also appears to involve EC barrier dysfunction (5). Recent progress in the use of controlled ventilator strategies, such as positive end-expiratory pressure (PEEP), lessens the potential for ventilator-induced lung injury (VILI), but a need still exists for therapies that would prevent this clinical complication (5). Most recently, in vitro studies have linked a variety of EC signal transduction pathways to the physiological mechanisms of EC barrier function and identified several endothelial protein kinases as potential drug discovery targets (1). However, the integration of the knowledge regarding in vitro signal transduction pathways with in vivo pathophysiology related to compromised EC barrier function and the validation of potential EC therapeutic targets have not occurred.Homeostasis and resistance to tissue injury are maintained by a balance between intracellular EC cytoskeletal contractionrelaxation cycles and modulation of EC extracellular adhesion properties, whi...

Human umbilical vein endothelial cells (EC) were grown on elastic silicone membranes subjected to cyclic stretch, simulating arterial wall motion. Stretching conditions (20% amplitude, 52 cycle/min) stimulated stress fiber formation and their orientation transversely to the strain direction. Cell bodies aligned along the same axis after the actin cytoskeleton. EC orientation response was inhibited by the adenylate cyclase activator, forskolin (10(-5) M), which caused stress fiber disassembly and the redistribution of F-actin to the cortical cytoplasm. Preoriented EC depleted of stress fibers by forskolin treatment retained their aligned state. Thus, stress fibers are essential for the process of EC orientation induced by repeated strain, but not for the maintenance of EC orientation. The monolayer formed by EC grown to confluence in conditions of intermittent strain consisted of uniform elongated cells and was resistant to deformation. In contrast, the monolayer assembled in stationary conditions was less compliant and exposed local denudations on initiation of stretching. When stretched in the presence of 10(-5) M forskolin it rapidly (3-4 h) reestablished integrity but gained a heterogeneous appearance since denuded areas were covered by giant cells. The protective effect of forskolin was because of the stimulation of EC spreading. This feature of forskolin was demonstrated while studying its action on EC spreading and repair of a scratched EC monolayer in conventional culture. Thus mechanical deformation and adenylate cyclase activity may be important factors in the control of endothelium morphology in human arteries.

The exertion of periodic dynamic strain on the arterial wall is hypothesized to be relevant to smooth muscle cell morphology and function. This study has investigated the effect of cyclic mechanical stretching on rabbit aortic smooth muscle cell proliferation and expression of contractile phenotype protein markers. Cells were cultured on flexible-bottomed dishes and cyclic stretch was applied (frequency 30 cycles/min, 15% elongation) using a Flexercell Strain unit. Cyclic stretch potentiated smooth muscle cell proliferation in serum-activated cultures but not in cultures maintained in 0.5% fetal calf serum. Stretching induced a serum-independent increase of h-caldesmon expression and this effect was reversible following termination of mechanical stimulation. Strain was without effect on smooth muscle myosin or calponin expression. In cells grown on laminin stretch-induced h-caldesmon expression was more prominent than in cells cultured on collagen types I and IV, poly-L-lysine and gelatin. These data suggest that cyclic mechanical stimulation possesses dual effect on vascular smooth muscle cell phenotype characteristics since it: 1) potentiates proliferation, an attribute of a dedifferentiated phenotype; and 2) increases expression of h-caldesmon considered a marker of a differentiated smooth muscle cell state.

Kinase-related protein, also known as KRP or telokin, is an independently expressed protein product derived from a gene within the gene for myosin light chain kinase (MLCK). KRP binds to unphosphorylated smooth muscle myosin filaments and stabilizes them against ATP-induced depolymerization in vitro. KRP competes with MLCK for binding to myosin, suggesting that both proteins bind to myosin by the KRP domain (Shirinsky, V. P., Vorotnikov, A. V., Birukov, K. G., Nanaev, A. K., Collinge, M., Lukas, T. J., Sellers, J. R., and Watterson, D. M. (1993) J. Biol. Chem. 268, 16578 -16583). In this study, we investigated which regions of myosin and KRP interact in vitro. Using cosedimentation assays, we determined that KRP binds to unphosphorylated myosin with a stoichiometry of 1 mol of KRP/1 mol of myosin and an affinity of 5.5 M. KRP slows the rate of proteolytic cleavage of the head-tail junction of heavy meromyosin by papain and chymotrypsin, suggesting it is binding to this region of myosin. In addition, competition experiments, using soluble headless fragments of nonmuscle myosin, confirmed that KRP interacts with the regulatory light chain binding region of myosin. The regions important for KRP's binding to myosin were investigated using bacterially expressed KRP truncation mutants. We determined that the acid-rich sequence between Gly 138 and Asp 151 of KRP is required for high affinity myosin binding, and that the amino terminus and ␤-barrel regions weakly interact with myosin. All KRP truncations, at concentrations comparable to their K D values, exhibited some stabilization of myosin filaments against ATP depolymerization in vitro, suggesting that KRP's ability to stabilize myosin filaments is commensurate with its myosin binding affinity. KRP weakened the K m but not the V max of phosphorylation of myosin by MLCK, demonstrating that bound KRP does not prevent MLCK from activating myosin.

Abstract-Different forms of mechanical stimulation are among the physiological factors constantly acting on the vessel wall. We previously demonstrated that subjecting vascular smooth muscle cells (VSMCs) in culture to cyclic stretch increased the expression of high-molecular-weight caldesmon, a marker protein of a differentiated, contractile, VSMC phenotype. In the present work the effects of mechanical factors, in the form of circumferential stress and shear stress, on the characteristics of SM contractile phenotype were studied in an organ culture of rabbit aorta. Application of an intralumininal pressure of 80 mm Hg to aortic segments cultured in Dulbecco's modified Eagle's medium containing 20% fetal calf serum for 3 days prevented the decrease in high-molecular-weight caldesmon content (70Ϯ4% of initial level in nonpressurized vessel, 116Ϯ17% at 80 mm Hg) and filamin content (80Ϯ5% in nonpressurized vessel, 100Ϯ2% at 80 mm Hg). SM myosin and low-molecular-weight caldesmon contents showed no dependence on vessel pressurization. Neither endothelial denudation nor alteration of intraluminal flow rates affected marker protein content in 3-day vessel culture, thus excluding the possibility of any shear or endothelial effects. Maintenance of high high-molecular-weight caldesmon and filamin levels in the organ cultures of pressurized and stretched vessels demonstrates the positive role of mechanical factors in the control of the VSMC differentiated phenotype. (Arterioscler Thromb Vasc Biol. 1998;18:922-927.)Key Words: stretch Ⅲ aorta Ⅲ marker protein Ⅲ caldesmon Ⅲ filamin T he arterial wall is continuously subjected to the action of mechanical forces in the form of tensile stress and shear stress. In addition, the pulsatile nature of blood pressure imposes cyclic stretch on the wall. In vivo studies and clinical observations suggest that decreased values of tensile and shear stress in surgically injured vessels are correlated with activation of cell proliferation and extracellular matrix production, which may result in vessel occlusion.1 In contrast, reestablishment of baseline tensile stress and shear stress levels is correlated with inhibition of cell proliferation and restoration of vessel wall morphology and cellular ultrastructure characteristic of the differentiated VSMC phenotype. 1,2These features and morphology indicate that VSMCs undergo phenotypic changes in regions of altered hemodynamic patterns. Experiments on isolated vessels suggest that appropriate mechanical stimulation is essential for the maintenance of VSMC contractile function and sensitivity to vasoconstrictors, 3 as well as morphology of the vessel wall. 4,5 However, the role of mechanical stimulation in the maintenance of SM marker protein content is still unclear.Changes in the expression of VSMC marker proteins are usually coordinated during VSMC transition to the synthetic phenotype in primary culture or at the loci of vascular injury.6,7 However, under certain conditions, expression of some marker proteins may be regulated independent...

Calponin and caldesmon, constituents of smooth-muscle thin filaments, are considered to be potential modulators of smooth-muscle contraction. Both of them interact with actin and inhibit ATPase activity of smooth- and skeletal-muscle actomyosin. Here we show that calponin and caldesmon could bind simultaneously to F-actin when used in subsaturating amounts, whereas each one used in excess caused displacement of the other from the complex with F-actin. Calponin was more effective than caldesmon in this competition: when F-actin was saturated with calponin the binding of caldesmon was eliminated almost completely, whereas even at high molar excess of caldesmon one-third of calponin (relative to the saturation level) always remained bound to actin. The inhibitory effects of low concentrations of calponin and caldesmon on skeletal-muscle actomyosin ATPase were additive, whereas the maximum inhibition of the ATPase attained at high concentration of each of them was practically unaffected by the other one. These data suggest that calponin and caldesmon cannot operate on the same thin filaments. CA(2+)-calmodulin competed with actin for calponin binding, and at high molar excess dissociated the calponin-actin complex and reversed the calponin-induced inhibition of actomyosin ATPase activity.

The nonmuscle/smooth muscle myosin light chain kinase (MLCK) and the kinase related protein (KRP) that lacks protein kinase activity are myosin II binding proteins encoded in the vertebrate genome by a true gene within a gene relationship. The genomic organization and expression result in the same amino acid sequence in different molecular contexts from two different sizes of mRNA. We report here the identification and characterization of a third size class of gene products. The protein appears to be a higher molecular weight form of MLCK with additional amino terminal tail sequence which might provide differential subcellular targeting characteristics.

Myosin light chain kinase (MLCK) is the key regulator of cell motility and smooth muscle contraction in higher vertebrates. We searched for the features of the high molecular weight MLCK (MLCK-210) associated with its unique N-terminal sequence not found in a more ubiquitous lower molecular weight MLCK (MLCK-108). MLCK-210 demonstrates stronger association with the Triton-insoluble cytoskeletons than MLCK-108, suggesting the role for this sequence in subcellular targeting. Indeed, the expressed unique domain of MLCK-210 binds and bundles F-actin in vitro and colocalises with the microfilaments in transfected cells reproducing endogenous MLCK-210 distribution. Thus, MLCK-210 features an extensive actin binding interface and, perhaps, acts as an actin cytoskeleton stabiliser.z 1999 Federation of European Biochemical Societies.