A fluorescent resonant energy transfer-based biosensor reveals transient and regional myosin light chain kinase activation in lamella and cleavage furrows

Approaches with high spatial and temporal resolution are required to understand the regulation of nonmuscle myosin II in vivo. Using fluorescence resonance energy transfer we have produced a novel biosensor allowing simultaneous determination of myosin light chain kinase (MLCK) localization and its [Ca2+]4/calmodulin-binding state in living cells. We observe transient recruitment of diffuse MLCK to stress fibers and its in situ activation before contraction. MLCK is highly active in the lamella of migrating cells, but not at the retracting tail. This unexpected result highlights a potential role for MLCK-mediated myosin contractility in the lamella as a driving force for migration. During cytokinesis, MLCK was enriched at the spindle equator during late metaphase, and was maximally activated just before cleavage furrow constriction. As furrow contraction was completed, active MLCK was redistributed to the poles of the daughter cells. These results show MLCK is a myosin regulator in the lamella and contractile ring, and pinpoints sites where myosin function may be mediated by other kinases.     

Distinct roles of ROCK (Rho-kinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fibers and focal adhesions in 3T3 fibroblasts

ROCK (Rho-kinase), an effector molecule of RhoA, phosphorylates the myosin binding subunit (MBS) of myosin phosphatase and inhibits the phosphatase activity. This inhibition increases phosphorylation of myosin light chain (MLC) of myosin II, which is suggested to induce RhoA-mediated assembly of stress fibers and focal adhesions. ROCK is also known to directly phosphorylate MLC in vitro; however, the physiological significance of this MLC kinase activity is unknown. It is also not clear whether MLC phosphorylation alone is sufficient for the assembly of stress fibers and focal adhesions.We have developed two reagents with opposing effects on myosin phosphatase. One is an antibody against MBS that is able to inhibit myosin phosphatase activity. The other is a truncation mutant of MBS that constitutively activates myosin phosphatase. Through microinjection of these two reagents followed by immunofluorescence with a specific antibody against phosphorylated MLC, we have found that MLC phosphorylation is both necessary and sufficient for the assembly of stress fibers and focal adhesions in 3T3 fibroblasts. The assembly of stress fibers in the center of cells requires ROCK activity in addition to the inhibition of myosin phosphatase, suggesting that ROCK not only inhibits myosin phosphatase but also phosphorylates MLC directly in the center of cells. At the cell periphery, on the other hand, MLCK but not ROCK appears to be the kinase responsible for phosphorylating MLC. These results suggest that ROCK and MLCK play distinct roles in spatial regulation of MLC phosphorylation.     

Myosin light chain kinase activation and calcium sensitization in smooth muscle in vivo

Ca(2+)/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) in smooth muscle by myosin light chain kinase (MLCK) and dephosphorylation by myosin light chain phosphatase (MLCP) are subject to modulatory cascades that influence the sensitivity of RLC phosphorylation and hence contraction to intracellular Ca(2+) concentration ([Ca(2+)](i)). We designed a CaM-sensor MLCK containing smooth muscle MLCK fused to two fluorescent proteins linked by the MLCK CaM-binding sequence to measure kinase activation in vivo and expressed it specifically in mouse smooth muscle. In phasic bladder muscle, there was greater RLC phosphorylation and force relative to MLCK activation and [Ca(2+)](i) with carbachol (CCh) compared with KCl treatment, consistent with agonist-dependent inhibition of MLCP. The dependence of force on MLCK activity was nonlinear such that at higher concentrations of CCh, force increased with no change in the net 20% activation of MLCK. A significant but smaller amount of MLCK activation was found during the sustained contractile phase. MLCP inhibition may occur through RhoA/Rho-kinase and/or PKC with phosphorylation of myosin phosphatase targeting subunit-1 (MYPT1) and PKC-potentiated phosphatase inhibitor (CPI-17), respectively. CCh treatment, but not KCl, resulted in MYPT1 and CPI-17 phosphorylation. Both Y27632 (Rho-kinase inhibitor) and calphostin C (PKC inhibitor) reduced CCh-dependent force, RLC phosphorylation, and phosphorylation of MYPT1 (Thr694) without changing MLCK activation. Calphostin C, but not Y27632, also reduced CCh-induced phosphorylation of CPI-17. CCh concentration responses showed that phosphorylation of CPI-17 was more sensitive than MYPT1. Thus the onset of agonist-induced contraction in phasic smooth muscle results from the rapid and coordinated activation of MLCK with hierarchical inhibition of MLCP by CPI-17 and MYPT1 phosphorylation.     

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.     

Stress fiber organization regulated by MLCK and Rho-kinase in cultured human fibroblasts

To understand the roles of Rho-kinase and myosin light chain kinase (MLCK) for the contraction and organization of stress fibers, we treated cultured human foreskin fibroblasts with several MLCK, Rho-kinase, or calmodulin inhibitors and analyzed F-actin organization in the cells. Some cells were transfected with green fluorescent protein (GFP)-labeled actin, and the effects of inhibitors were also studied in these living cells. The Rho-kinase inhibitors Y-27632 and HA1077 caused disassembly of stress fibers and focal adhesions in the central portion of the cell within 1 h. However, stress fibers located in the periphery of the cell were not severely affected by the Rho-kinase inhibitors. When these cells were washed with fresh medium, the central stress fibers and focal adhesions gradually reformed, and within 3 h the cells were completely recovered. ML-7 and KT5926 are specific MLCK inhibitors and caused disruption and/or shortening of peripheral stress fibers, leaving the central fibers relatively intact even though their number was reduced. The calmodulin inhibitors W-5 and W-7 gave essentially the same results as the MLCK inhibitors. The MLCK and calmodulin inhibitors, but not the Rho-kinase inhibitors, caused cells to lose the spread morphology, indicating that the peripheral fibers play a major role in keeping the flattened state of the cell. When stress fiber models were reactivated, the peripheral fibers contracted before the central fibers. Thus our study shows that there are at least two different stress fiber systems in the cell. The central stress fiber system is dependent more on the activity of Rho-kinase than on that of MLCK, while the peripheral stress fiber system depends on MLCK.     

Ca2+ signaling in hypoxic pulmonary vasoconstriction: effects of myosin light chain and Rho kinase antagonists

Antagonists of myosin light chain (MLC) kinase (MLCK) and Rho kinase (ROK) are thought to inhibit hypoxic pulmonary vasoconstriction (HPV) by decreasing the concentration of phosphorylated MLC at any intracellular Ca(2+) concentration ([Ca(2+)](i)) in pulmonary arterial smooth muscle cells (PASMC); however, these antagonists can also decrease [Ca(2+)](i). To determine whether MLCK and ROK antagonists alter Ca(2+) signaling in HPV, we measured the effects of ML-9, ML-7, Y-27632, and HA-1077 on [Ca(2+)](i), Ca(2+) entry, and Ca(2+) release in rat distal PASMC exposed to hypoxia or depolarizing concentrations of KCl. We performed parallel experiments in isolated rat lungs to confirm the inhibitory effects of these agents on pulmonary vasoconstriction. Our results demonstrate that MLCK and ROK antagonists caused concentration-dependent inhibition of hypoxia-induced increases in [Ca(2+)](i) in PASMC and HPV in isolated lungs and suggest that this inhibition was due to blockade of Ca(2+) release from the sarcoplasmic reticulum and Ca(2+) entry through store- and voltage-operated Ca(2+) channels in PASMC. Thus MLCK and ROK antagonists might block HPV by inhibiting Ca(2+) signaling, as well as the actin-myosin interaction, in PASMC. If effects on Ca(2+) signaling were due to decreased phosphorylated myosin light chain concentration, their diversity suggests that MLCK and ROK antagonists may have acted by inhibiting myosin motors and/or altering the cytoskeleton in a manner that prevented achievement of required spatial relationships among the cellular components of the response.     

Sustained muscle contraction induced by agonists, growth factors, and Ca(2+) mediated by distinct PKC isozymes

The role of protein kinase C (PKC) in sustained contraction was examined in intestinal circular and longitudinal muscle cells. Initial contraction induced by agonists (CCK-8 and neuromedin C) was abolished by 1) inhibitors of Ca(2+) mobilization (neomycin and dimethyleicosadienoic acid), 2) calmidazolium, and 3) myosin light chain (MLC) kinase (MLCK) inhibitor KT-5926. In contrast, sustained contraction was not affected by these inhibitors but was abolished by 1) the PKC inhibitors chelerythrine and calphostin C, 2) PKC-epsilon antibody, and 3) a pseudosubstrate PKC-epsilon inhibitor. GDPbetaS abolished both initial and sustained contraction, whereas a Galpha(q/11) antibody inhibited only initial contraction, implying that sustained contraction was dependent on activation of a distinct G protein. Sustained contraction induced by epidermal growth factor was inhibited by calphostin C, PKC-alpha,beta,gamma antibody, and a pseudosubstrate PKC-alpha inhibitor. Ca(2+) (0.4 microM) induced an initial contraction in permeabilized muscle cells that was blocked by calmodulin and MLCK inhibitors and a sustained contraction that was blocked by calphostin C and a PKC-alpha,beta,gamma antibody. Thus initial contraction induced by Ca(2+), agonists, and growth factors is mediated by MLCK, whereas sustained contraction is mediated by specific Ca(2+)-dependent and -independent PKC isozymes. G protein-coupled receptors are linked to PKC activation via distinct G proteins.     

Ca2+ activation of smooth muscle contraction: evidence for the involvement of calmodulin that is bound to the triton insoluble fraction even in the absence of Ca2+.

Smooth muscle contraction is activated by phosphorylation of the 20-kDa light chains of myosin catalyzed by Ca(2+)/calmodulin (CaM)-dependent myosin light chain kinase (MLCK). According to popular current theory, the CaM involved in MLCK regulation is Ca(2+)-free and dissociated from the kinase at resting cytosolic free Ca(2+) concentration ([Ca(2+)](i)). An increase in [Ca(2+)](i) saturates the four Ca(2+)-binding sites of CaM, which then binds to and activates actin-bound MLCK. The results of this study indicate that this theory requires revision. Sufficient CaM was retained after skinning (demembranation) of rat tail arterial smooth muscle in the presence of EGTA to support Ca(2+)-evoked contraction, as observed previously with other smooth muscle tissues. This tightly bound CaM was released by the CaM antagonist trifluoperazine (TFP) in the presence of Ca(2+). Following removal of the (Ca(2+))(4)-CaM-TFP(2) complex, Ca(2+) no longer induced contraction. The addition of exogenous CaM to TFP-treated tissue at a [Ca(2+)] subthreshold for contraction or even in the absence of Ca(2+) (presence of 5 mm EGTA), followed by washout of unbound CaM, restored Ca(2+)-induced contraction; this required MLCK activation, since it was blocked by the MLCK inhibitor ML-9. The data suggest, therefore, that a specific pool of cellular CaM, tightly bound to myofilaments at resting [Ca(2+)](i), or even in the absence of Ca(2+), is responsible for activation of contraction following a local increase in [Ca(2+)]. This mechanism would allow for localized changes in [Ca(2+)] in regions of the cell distant from the myofilaments to regulate distinct Ca(2+)-dependent processes without triggering a contractile response. Immobilized CaM, therefore, resembles troponin C, the Ca(2+)-binding regulatory protein of striated muscle, which is also bound to the thin filament in a Ca(2+)-independent manner.     

Myosin light-chain kinase regulates endothelial calcium entry and endothelium-dependent vasodilation.

Activation of smooth muscle myosin light-chain kinase (MLCK) causes contraction. Here we have proven that MLCK controls Ca2+ entry (CE) in endothelial cells (ECs): MLCK antisense oligonucleotides strongly prevented bradykinin (BK)- and thapsigargin (TG)-induced endothelial Ca2+ response, while MLCK sense did not. We also show that the relevant mechanism is not phosphorylation of myosin light-chain (MLC): MLC phosphorylation by BK required CE, but MLC phosphorylation caused by the phosphatase inhibitor calyculin A did not trigger Ca2+ response. Most important, we provide for the first time strong evidence that, in contrast to its role in smooth muscle cells, activation of MLCK in ECs stimulates the production of important endothelium-derived vascular relaxing factors: MLCK antisense and MLCK inhibitors abolished BK- and TG-induced nitric oxide production, and MLCK inhibitors substantially inhibited acetylcholine-stimulated hyperpolarization of smooth muscle cell membrane in rat mesenteric artery. These results indicate that MLCK controls endothelial CE, but not through MLC phosphorylation, and unveils a hitherto unknown physiological function of the enzyme: vasodilation through its action in endothelial cells. The study discovers a counter-balancing role of MLCK in the regulation of vascular tone.     

Caldesmon inhibits nonmuscle cell contractility and interferes with the formation of focal adhesions.

Caldesmon is known to inhibit the ATPase activity of actomyosin in a Ca(2+)-calmodulin-regulated manner. Although a nonmuscle isoform of caldesmon is widely expressed, its functional role has not yet been elucidated. We studied the effects of nonmuscle caldesmon on cellular contractility, actin cytoskeletal organization, and the formation of focal adhesions in fibroblasts. Transient transfection of nonmuscle caldesmon prevents myosin II-dependent cell contractility and induces a decrease in the number and size of tyrosine-phosphorylated focal adhesions. Expression of caldesmon interferes with Rho A-V14-mediated formation of focal adhesions and stress fibers as well as with formation of focal adhesions induced by microtubule disruption. This inhibitory effect depends on the actin- and myosin-binding regions of caldesmon, because a truncated variant lacking both of these regions is inactive. The effects of caldesmon are blocked by the ionophore A23187, thapsigargin, and membrane depolarization, presumably because of the ability of Ca(2+)-calmodulin or Ca(2+)-S100 proteins to antagonize the inhibitory function of caldesmon on actomyosin contraction. These results indicate a role for nonmuscle caldesmon in the physiological regulation of actomyosin contractility and adhesion-dependent signaling and further demonstrate the involvement of contractility in focal adhesion formation.     

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.     

Cell rounding in cultured human astrocytes and vascular endothelial cells upon inhibition of CK2 is mediated by actomyosin cytoskeleton alterations

Protein kinase CK2 participates in a wide range of cellular events, including the regulation of cellular morphology and migration, and may be an important mediator of angiogenesis. We previously showed that in the retina, CK2 immunolocalizes mostly to vascular endothelium and astrocytes in association with the cytoskeleton. Additionally, CK2 inhibitors significantly reduced retinal neovascularization and stem cell recruitment in the mouse model of oxygen-induced proliferative retinopathy. We have also shown that CK2 and F-actin co-localized in actin stress fibers in microvascular endothelial cells, and that highly specific CK2 inhibitors caused cell rounding in astrocytes and microvascular endothelial cells, which was alleviated by serum that promotes spreading by Rho/Rho-kinase (RhoK) activation of myosin II. Therefore, we examined a possible role of CK2 in the regulation of actin-myosin II-based contractility. Treatment with CK2 inhibitors correlated with disassembly of actomyosin stress fibers and cell shape changes, including cytoplasmic retraction and process formation that were similar to those occurring during astrocyte stellation. Low doses of specific inhibitors of kinases (RhoK and MLCK) that phosphorylate myosin light chain (MLC) enhanced the effect of suboptimal CK2 inhibition on cell shape. Such striking stellation-like alteration was accompanied by decreased level of phospho-MLC, thus implying a CK2 role in regulation of actomyosin cytoskeleton. Our results suggest an important role of CK2 in the control of cell contractility and motility, which may account for suppressing effect of CK2 inhibition on retinal neovascularization. Together, our data implicate protein kinase CK2 for the first time in stellation-like morphological transformation.     

Mutation analysis of the non-muscle myosin light chain kinase (MLCK) deletion constructs on CV1 fibroblast contractile activity and proliferation

Smooth muscle myosin light chain kinase (MLCK) is a multifunctional molecule composed of an N-terminal actin binding domain, a central kinase domain, and C-terminal calmodulin- and myosin-binding domains. We previously cloned and characterized a novel MLCK isoform from endothelial cells (EC MLCK) consisting of 1,914 amino acids displaying a higher molecular weight (210 kDa) and a novel-amino-terminal stretch of 922 amino acids not shared by the smooth muscle isoform (smMLCK, 150 kDa). To further define the role of specific EC MLCK motifs in endothelial and non-muscle cells, we constructed two epitope-tagged EC MLCK deletion mutants in mammalian expression vectors lacking either the C-terminal auto-inhibitory and calmodulin-binding domain (EC MLCK1745) or the ATP-binding site (EC MLCKATPdel). Expression of EC MLCK1745 in CV1 fibroblasts showed increased basal actin stress fiber formation, which was markedly enhanced after tumor necrosis factor (TNF-alpha) or thrombin treatment. Distribution of EC MLCK1745 was largely confined to stress fibers, cortical actin filaments, and focal adhesion contacts, and co-localized with myosin light chains (MLCs) diphosphorylated on Ser(19) and Thr(18). In contrast, immunofluorescence staining demonstrated that EC MLCKATPdel abolished thrombin- and TNFalpha-induced stress fiber formation and MLC phosphorylation, suggesting this kinase-dead mutant functions as a dominant-negative MLCK construct, thereby confirming the role of EC MLCK in stress fiber formation. Finally, we compared the serum-stimulated growth rate of mutant MLCK-transfected fibroblasts to sham controls, and found EC MLCK1745 to augment thymidine incorporation whereas EC MLCKATPdel reduced CV1 growth rates. These data demonstrate the necessary role for MLCK in driving the contractile apparatus via MLC phosphorylation, which can alter fibroblast growth and contractility.     

Cross talk between cyclic nucleotides and polyphosphoinositide hydrolysis, protein kinases, and contraction in smooth muscle

This article provides an update of a minireview published in 1996 (Abdel-Latif AA. Proc Soc Exp Biol Med 211:163-177, 1996), the purpose of which was to examine in nonvascular smooth muscle the biochemical and functional cross talk between the sympathetic nervous system, which governs the formation of cAMP and muscle relaxation, and the parasympathetic nervous system, which governs the generation of IP3 and diacylglycerol, from the polyphosphoinositides, Ca2+ mobilization, and contraction. This review examines further evidence, both from nonvascular and vascular smooth muscle, for cross talk between the cyclic nucleotides, cAMP and cGMP via their respective protein kinases, and the Ca2+-dependent- and Ca2+-independent-signaling pathways involved in agonist-induced contraction. These include the IP3-Ca2+-CaM- myosin light chain kinase (MLCK) pathway and the Ca2+-independent pathways, including protein kinase C-, MAP kinase-, and Rho-kinase. In addition, MLC phosphorylation and contraction can also be increased by a decrease in myosin phosphatase activity. A summary of the cross talk between the cyclic nucleotides and these signaling pathways was presented. In smooth muscle, there are several targets for cyclic nucleotide inhibition and consequent relaxation, including the receptor, G proteins, phospholipase C-beta1-4 isoforms, IP3 receptor, Ca2+ mobilization, MLCK, MAP kinase, Rho-kinase, and myosin phosphatase. While significant progress has been made in the past four years on this cross talk, the precise mechanisms underlying the biochemical basis for the cyclic nucleotide inhibition of Ca2+ mobilization and consequently muscle contraction remain to be established. Although it is well established that second-messenger cross talk plays an important role in smooth muscle relaxation, the many sources which exist in smooth muscle for Ca2+ mobilization, coupled with the multiple signaling pathways involved in agonist-induced contraction, contribute appreciably to the difficulties found by many investigators in identifying the targets for cyclic nucleotide inhibition and consequent relaxation. Better methodology and more novel interdisciplinary approaches are required for elucidating the mechanism(s) of cAMP- and cGMP-inhibition of smooth muscle contraction.     

Polyamines regulate Rho-kinase and myosin phosphorylation during intestinal epithelial restitution

Polyamines are required for the early phase of mucosal restitution that occurs as a consequence of epithelial cell migration. Our previous studies have shown that polyamines increase RhoA activity by elevating cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) through controlling voltage-gated K(+) channel expression and membrane potential (E(m)) during intestinal epithelial restitution. The current study went further to determine whether increased RhoA following elevated [Ca(2+)](cyt) activates Rho-kinase (ROK/ROCK) resulting in myosin light chain (MLC) phosphorylation. Studies were conducted in stable Cdx2-transfected intestinal epithelial cells (IEC-Cdx2L1), which were associated with a highly differentiated phenotype. Reduced [Ca(2+)](cyt), by either polyamine depletion or exposure to the Ca(2+)-free medium, decreased RhoA protein expression, which was paralleled by significant decreases in GTP-bound RhoA, ROCK-1, and ROKalpha proteins, Rho-kinase activity, and MLC phosphorylation. The reduction of [Ca(2+)](cyt) also inhibited cell migration after wounding. Elevation of [Ca(2+)](cyt) induced by the Ca(2+) ionophore ionomycin increased GTP-bound RhoA, ROCK-1, and ROKalpha proteins, Rho-kinase activity, and MLC phosphorylation. Inhibition of RhoA function by a dominant negative mutant RhoA decreased the Rho-kinase activity and resulted in cytoskeletal reorganization. Inhibition of ROK/ROCK activity by the specific inhibitor Y-27632 not only decreased MLC phosphorylation but also suppressed cell migration. These results indicate that increase in GTP-bound RhoA by polyamines via [Ca(2+)](cyt) can interact with and activate Rho-kinase during intestinal epithelial restitution. Activation of Rho-kinase results in increased MLC phosphorylation, leading to the stimulation of myosin stress fiber formation and cell migration.     

Sphingosine 1-phosphate induces cell contraction via calcium-independent/Rho-dependent pathways in undifferentiated skeletal muscle cells

We have previously shown that sphingosine 1-phosphate (S1P) can induce intracellular Ca(2+) mobilization and cell contraction in C2C12 myoblasts and that the two phenomena are temporally unrelated. Although Ca(2+)-independent mechanisms of cell contraction have been the focus of numerous studies on Ca(2+) sensitization of smooth muscle, comparatively less studies have focused on the role that these mechanisms play in the regulation of skeletal muscle contractility. Phosphorylation and activation of myosin by Rho-dependent kinase mediate most of Ca(2+)-independent contractile responses. In the present study, we examined the potential role of Rho/Rho-kinase cascade activation in S1P-induced C2C12 cell contraction. First, we showed that depletion of Ca(2+), by pre-treatment with BAPTA, did not affect S1P-induced myoblastic contractility, whereas it abolished S1P-induced Ca(2+) transients. These results correlated with the absence of troponin C and with the immature cytoskeletal organization of these cells. Experimental evidence demonstrating the involvement of Rho pathway in S1P-stimulated myoblast contraction included: the activation/translocation of RhoA to the membrane in response to agonist-stimulation in cells depleted of Ca(2+) and the inhibition of dynamic changes of the actin cytoskeleton in cells where Rho functions had been inhibited either by overexpression of RhoGDI, a physiological inhibitor of GDP dissociation from Rho proteins, or by pretreatment with Y-27632, a specific Rho kinase inhibitor. Contribution of protein kinase C in this cytoskeletal rearrangement was also evaluated. However, the pretreatment with G?6976 or rottlerin, specific inhibitors of PKC alpha and PKC delta, respectively, failed to inhibit the agonist-induced myoblastic contraction. Single particle tracking of G-actin fluorescent probe was performed to statistically evaluate actin cytoskeletal dynamics in response to S1P. Stimulation with S1P was also able to increase the phosphorylation level of myosin light chain II. In conclusion, our results strongly suggest that Ca(2+)-independent/Rho-Rho kinase-dependent pathways may exert an important role in S1P-induced myoblastic cell contraction.     

5,6-EET-induced contraction of intralobar pulmonary arteries depends on the activation of Rho-kinase.

The mechanism mediating epoxyeicosatrienoic acid (EET)-induced contraction of intralobar pulmonary arteries (PA) is currently unknown. EET-induced contraction of PA has been reported to require intact endothelium and activation of the thromboxane/endoperoxide (TP) receptor. Because TP receptor occupation with the thromboxane mimetic U-46619 contracts pulmonary artery via Rho-kinase activation, we examined the hypothesis that 5,6-EET-induced contraction of intralobar rabbit pulmonary arteries is mediated by a Rho-kinase-dependent signaling pathway. In isolated rings of second-order intralobar PA (1-2 mm OD) at basal tension, 5,6-EET (0.3-10 microM) induced increases in active tension that were inhibited by Y-27632 (1 microM) and HA-1077 (10 microM), selective inhibitors of Rho-kinase activity. In PA in which smooth muscle intracellular Ca(2+) concentration ([Ca(2+)](i)) was increased with KCl (25 mM) to produce a submaximal contraction, 5,6-EET (1 microM) induced a contraction that was 7.0 +/- 1.6 times greater than without KCl. 5,6-EET (10 microM) also contracted beta-escin permeabilized PA in which [Ca(2+)](i) was clamped at a concentration resulting in a submaximal contraction. Y-27632 inhibited the 5,6-EET-induced contraction in permeabilized PA. 5,6-EET (10 microM) increased phosphorylation of myosin light chain (MLC), increasing the ratio of phosphorylated MLC/total MLC from 0.10 +/- 0.03 to 0.30 +/- 0.02. Y-27632 prevented this increase in MLC phosphorylation. These data suggest that 5,6-EET induces contraction in intralobar PA by increasing Rho-kinase activity, phosphorylating MLC, and increasing the Ca(2+) sensitivity of the contractile apparatus.     

Mildly oxidized low density lipoprotein induces contraction of human endothelial cells through activation of Rho/Rho kinase and inhibition of myosin light chain phosphatase.

Mildly oxidized low density lipoprotein (mox-LDL) is critically involved in the early atherogenic responses of the endothelium and increases endothelial permeability through an unknown signal pathway. Here we show that (i) exposure of confluent human endothelial cells (HUVEC) to mox-LDL but not to native LDL induces the formation of actin stress fibers and intercellular gaps within minutes, leading to an increase in endothelial permeability; (ii) mox-LDL induces a transient decrease in myosin light chain (MLC) phosphatase that is paralleled by an increase in MLC phosphorylation; (iii) phosphorylated MLC stimulated by mox-LDL is incorporated into stress fibers; (iv) cytoskeletal rearrangements and MLC phosphorylation are inhibited by C3 transferase from Clostridium botulinum, a specific Rho inhibitor, and Y-27632, an inhibitor of Rho kinase; and (v) mox-LDL does not increase intracellular Ca(2+) concentration. Our data indicate that mox-LDL induces endothelial cell contraction through activation of Rho and its effector Rho kinase which inhibits MLC phosphatase and phosphorylates MLC. We suggest that inhibition of this novel cell signaling pathway of mox-LDL could be relevant for the prevention of atherosclerosis.     

Ca2+-independent smooth muscle contraction. a novel function for integrin-linked kinase

Smooth muscle contraction follows an increase in cytosolic Ca(2+) concentration, activation of myosin light chain kinase, and phosphorylation of the 20-kDa light chain of myosin at Ser(19). Several agonists acting via G protein-coupled receptors elicit a contraction without a change in [Ca(2+)](i) via inhibition of myosin light chain phosphatase and increased myosin phosphorylation. We showed that microcystin (phosphatase inhibitor)-induced contraction of skinned smooth muscle occurred in the absence of Ca(2+) and correlated with phosphorylation of myosin light chain at Ser(19) and Thr(18) by a kinase distinct from myosin light chain kinase. In this study, we identify this kinase as integrin-linked kinase. Chicken gizzard integrin-linked kinase cDNA was cloned, sequenced, expressed in E. coli, and shown to phosphorylate myosin light chain in the absence of Ca(2+) at Ser(19) and Thr(18). Subcellular fractionation revealed two distinct populations of integrin-linked kinase, including a Triton X-100-insoluble component that phosphorylates myosin in a Ca(2+)-independent manner. These results suggest a novel function for integrin-linked kinase in the regulation of smooth muscle contraction via Ca(2+)-independent phosphorylation of myosin, raise the possibility that integrin-linked kinase may also play a role in regulation of nonmuscle motility, and confirm that integrin-linked kinase is indeed a functional protein-serine/threonine kinase.     

Phorbol esters increase MLC phosphorylation and actin remodeling in bovine lung endothelium without increased contraction

Direct protein kinase C (PKC) activation with phorbol myristate acetate (PMA) results in the loss of endothelial monolayer integrity in bovine lung endothelial cells (EC) but produces barrier enhancement in human lung endothelium. To extend these findings, we studied EC contractile events and observed a 40% increase in myosin light chain (MLC) phosphorylation in bovine endothelium following PMA challenge. The increase in PMA-mediated MLC phosphorylation occurred at sites distinct from Ser19/Thr18, sites catalyzed by MLC kinase (MLCK), and immunoblotting with antibodies specific to phosphorylated Ser19/Thr18 demonstrated profound time-dependent Ser19/Thr18 dephosphorylation. These events occurred in conjunction with rearrangement of stress fibers into a grid-like network, but without an increase in cellular contraction as measured by silicone membrane wrinkling assay. The PMA-induced MLC dephosphorylation was not due to kinase inhibition but, rather, correlated with rapid increases in myosin-associated phosphatase 1 (PPase 1) activity. These data suggest that PMA-mediated EC barrier regulation may involve dual mechanisms that alter MLC phosphorylation. The increase in bovine MLC phosphorylation likely occurs via direct PKC-dependent MLC phosphorylation in conjunction with decreases in Ser19/Thr18 phosphorylation catalyzed by MLCK due to PMA-induced increases in PPase 1 activity. Together, these events result in stress fiber destabilization and profound actin rearrangement in bovine endothelium, which may result in the physiological alterations observed in these models.