Role of Rho, Rac, and Rho-kinase in phosphorylation of myosin light chain, development of polarity, and spontaneous migration of Walker 256 carcinosarcoma cells

As previously shown, constitutive activation of the small GTPase Rho and its downstream target Rho-kinase is crucial for spontaneous migration of Walker carcinosarcoma cells. We now show that after treatment of cells with either the Rho inhibitor C3 exoenzyme or the Rho-kinase inhibitor Y-27632, constitutive myosin light chain (MLC) phosphorylation is significantly decreased, correlating with inhibition of cell polarization and migration. Transfection with a dominant-negative Rho-kinase mutant similarly inhibits cell polarization and MLC phosphorylation. Transfection with a dominant-active Rho-kinase mutant leads to significantly increased MLC phosphorylation, membrane blebbing, and inhibition of cell polarization. This Rho-kinase-induced membrane blebbing can be inhibited by Y-27632, ML-7, and blebbistatin. Unexpectedly, overactivation of RhoA has similar effects as its inhibition. Introduction of a bacterially expressed constitutively activated mutant protein (but not of wild-type RhoA) into the cells or transfection of cells with a constitutively active RhoA mutant both inhibit polarization and decrease MLC phosphorylation. Transfection of cells with constitutively active or dominant-negative Rac both abrogate polarity, and the latter inhibits MLC phosphorylation. Our findings suggest an important role of Rac, Rho/Rho-kinase, and MLCK in controlling myosin activity in Walker carcinosarcoma cells and show that an appropriate level of RhoA, Rac, and Rho-kinase activity is required to regulate cell polarity and migration.     

Rac-induced increase of phosphorylation of myosin regulatory light chain in HeLa cells

The pathways by which activation of the small GTP-binding protein Rac causes cytoskeletal changes are not fully understood but are likely to involve both assembly of new actin filaments and reorganization of actin filaments driven by the actin-dependent ATPase activity of myosin II. Here we show that expression of active RacQ61 in growing HeLa cells, in addition to inducing ruffling, substantially enhances the level of phosphorylation of serine-19 of the myosin II regulatory light chain (MLC), which would increase actomyosin II ATPase and motor activities. Phosphorylated myosin was localized to RacQ61-induced ruffles and stress fibers. RacQ61-induced phosphorylation of MLC was reduced by a maximum of about 38% by an inhibitor (Tat-PAK) of p21-activated kinase (PAK), about 35% by an inhibitor (Y-27632) of Rho kinase, 51% by Tat-PAK plus Y-27632, and 10% by an inhibitor (ML7) of myosin light chain kinase. Staurosporine, a non-specific inhibitor of serine/threonine kinases, reduced RacQ61-induced phosphorylation of MLC by about 58%, at the maximum concentration that did not kill cells. Since Rac activates PAK and PAK can phosphorylate MLC, these data strongly suggest that PAK is responsible for a significant fraction of RacQ61-induced MLC phosphorylation. To our knowledge, this is the first evidence that active Rac causes phosphorylation of MLC in cells, thus implicating activation of the ATPase activity of actomyosin II as one of the ways by which Rac may induce cytoskeletal changes.     

Inhibition of RhoA but not ROCK induces chondrogenesis of chick limb mesenchymal cells

Cell shape change and cytoskeletal reorganization are known to be involved in the chondrogenesis. Negative role of RhoA, a cytoskeleton-regulating protein, and its downstream target, Rho-associated protein kinase (ROCK) in the chondrogenesis has been studied in many different culture systems including primary chondrocytes, chondrogenic cell lines, dedifferentiated chondrocytes, and micromass culture of mesenchymal cells. To further investigate the role of RhoA and ROCK in the chondrogenesis, we examined the RhoA-ROCK-myosin light chains (MLC) pathway in low density culture of chick limb bud mesenchymal cells. We observed for the first time that inhibition of RhoA by C3 cell-permeable transferase, CT04, induced chondrogenesis of undifferentiated mesenchymal single cells following dissolution of actin stress fibers. Inhibition of RhoA activity by CT04 was confirmed by pull down assay using the Rho-GTP binding domain of Rhotekin. CT04 also inhibited ROCK activity. In contrast, inhibition of ROCK by Y27632 neither altered the actin stress fibers nor induced chondrogenesis. In addition, inhibition of RhoA or ROCK did not affect the phosphorylation of MLC. Inhibition of myosin light chain kinase (MLCK) by ML-7 or inhibition of myosin ATPase with blebbistatin dissolved actin stress fibers and induced chondrogenesis. ML-7 reduced the MLC phosphorylation. Taken together, our current study suggests that RhoA uses other pathway than ROCK/MLC in the modulation of actin stress fibers and chondrogenesis. Our data also imply that, irrespective of mechanisms, dissolution of actin stress fibers is crucial for chondrogenesis.     

Differential roles of Rho-kinase and myosin light chain kinase in regulating shape, adhesion, and migration of HT1080 fibrosarcoma cells

We present evidence for differential roles of Rho-kinase and myosin light chain kinase (MLCK) in regulating shape, adhesion, migration, and chemotaxis of human fibrosarcoma HT1080 cells on laminin-coated surfaces. Pharmacological inhibition of Rho-kinase by Y-27632 or inhibition of MLCK by W-7 or ML-7 resulted in significant attenuation of constitutive myosin light chain phosphorylation. Rho-kinase inhibition resulted in sickle-shaped cells featuring long, thin F-actin-rich protrusions. These cells adhered more strongly to laminin and migrated faster. Inhibition of MLCK in contrast resulted in spherical cells and marked impairment of adhesion and migration. Inhibition of myosin II activation with blebbistatin resulted in a morphology similar to that induced by Y-27632 and enhanced migration and adhesion. Cells treated first with blebbistatin and then with ML-7 also rounded up, suggesting that effects of MLCK inhibition on HT1080 cell shape and motility are independent of inhibition of myosin activity.     

Rho kinase signaling pathways during stretch in primary alveolar epithelia

Alveolar epithelial cells (AECs) maintain integrity of the blood-gas barrier with actin-anchored intercellular tight junctions. Stretched type I-like AECs undergo magnitude- and frequency-dependent actin cytoskeletal remodeling into perijunctional actin rings. On the basis of published studies in human pulmonary artery endothelial cells (HPAECs), we hypothesize that RhoA activity, Rho kinase (ROCK) activity, and phosphorylation of myosin light chain II (MLC2) increase in stretched type I-like AECs in a manner that is dependent on stretch magnitude, and that RhoA, ROCK, or MLC2 activity inhibition will attenuate stretch-induced actin remodeling and preserve barrier properties. Primary type I-like AEC monolayers were stretched biaxially to create a change in surface area (ΔSA) of 12%, 25%, or 37% in a cyclic manner at 0.25 Hz for up to 60 min or left unstretched. Type I-like AECs were also treated with Rho pathway inhibitors (ML-7, Y-27632, or blebbistatin) and stained for F-actin or treated with the myosin phosphatase inhibitor calyculin-A and quantified for monolayer permeability. Counter to our hypothesis, ROCK activity and MLC2 phosphorylation decreased in type I-like AECs stretched to 25% and 37% ΔSA and did not change in monolayers stretched to 12% ΔSA. Furthermore, RhoA activity decreased in type I-like AECs stretched to 37% ΔSA. In contrast, MLC2 phosphorylation in HPAECs increased when HPAECs were stretched to 12% ΔSA but then decreased when they were stretched to 37% ΔSA, similar to type I-like AECs. Perijunctional actin rings were observed in unstretched type I-like AECs treated with the Rho pathway inhibitor blebbistatin. Myosin phosphatase inhibition increased MLC2 phosphorylation in stretched type I-like AECs but had no effect on monolayer permeability. In summary, stretch alters RhoA activity, ROCK activity, and MLC2 phosphorylation in a manner dependent on stretch magnitude and cell type.     

The suppression of myosin light chain (MLC) phosphorylation during the response to lipopolysaccharide (LPS): beneficial or detrimental to endothelial barrier?

Sepsis-induced vascular leakage is a major underlying cause of the respiratory dysfunction seen in severe sepsis. Here, we studied the role of MLC phosphorylation in LPS-induced endothelial hyperpermeability and assessed how the changes in phospho-MLC distribution affect LPS-induced barrier dysfunction. We demonstrated that the changes in human lung microvascular endothelial permeability are preceded by the increase in intracellular calcium level, and increase in MYPT and MLC phosphorylation. Using the siRNA approach, we showed that both LPS-induced barrier dysfunction and MLC phosphorylation are attenuated by the depletion of the smooth muscle isoform of MLC kinase (MLCK) and Rho kinase 2 (ROCK2). Surprisingly, pharmacological inhibition of both ROCK1 and 2 with Y-27632 exacerbated LPS-induced drop in transendothelial resistance, although significantly decreasing MLC phosphorylation level. We next studied the involvement of protein kinase A (PKA)-dependent pathways in LPS-induced barrier dysfunction. We showed that LPS decreased the level of PKA-dependent phosphorylation in endothelial cells; and the pretreatment with forskolin or PKA activator bnz-cAMP counteracted this effect. Forskolin and bnz-cAMP also attenuated LPS-induced increase in MLC phosphorylation level. As we have shown earlier (Bogatcheva et al., 2009), forskolin and bnz-cAMP provide protection from LPS-induced barrier dysfunction. We compared the effects of bnz-cAMP and Y-27632 on phospho-MLC distribution and observed that while bnz-cAMP increased the association of the phospho-MLC signal with the cortical structures, Y-27632 decreased this association. These data indicate that an overall decrease in MLC phosphorylation could be either beneficial or detrimental to endothelial barrier, depending on the intracellular locale of major phospho-MLC changes.     

Heat shock augments myosin phosphatase target-subunit phosphorylation

Our previous study demonstrated that heat shock augmented vascular contraction. In the present study, we hypothesized that heat shock augments myosin phosphatase target-subunit (MYPT1) phosphorylation resulting in augmented vascular contraction. Endothelium-denuded rat aortic rings were mounted in organ baths, exposed to heat shock (42 degrees C for 45 min), and subjected to contraction 4 h after the heat shock followed by Western blot analysis for MLC(20) (the 20 kDa light chains of myosin II) or MYPT1. The contractile responses in both control and heat shock-treated aorta were inhibited by Y27632, an inhibitor of Rho-kinase. The level of the MLC(20) and MYPT1(Thr855) phosphorylation in response to KCl was higher in heat shock-treated aorta than that in timed-control. The increased MYPT1(Thr855) phosphorylation was inhibited by Y27632 (1.0 microM) in parallel with inhibition of MLC(20) phosphorylation and vascular contraction. These results indicate that heat shock augments MYPT1 phosphorylation resulting in augmented vascular contraction.     

Rho kinase inhibitors stimulate the migration of human cultured osteoblastic cells by regulating actomyosin activity

We investigated the effects of Rho-associated kinase (ROCK) on migration and cytoskeletal organization in primary human osteoblasts and Saos-2 human osteosarcoma cells. Both cell types were exposed to two different ROCK inhibitors, Y-27632 and HA-1077. In the improved motility assay used in the present study, Y-27632 and HA-1077 significantly increased the migration of both osteoblasts and osteosarcoma cells on plastic in a dose-dependent and reversible manner. Fluorescent images showed that cells of both types cultured with Y-27632 or HA-1077 exhibited a stellate appearance, with poor assembly of stress fibers and focal contacts. Western blotting showed that ROCK inhibitors reduced myosin light chain (MLC) phosphorylation within 5 min without affecting overall myosin light-chain protein levels. Inhibition of ROCK activity is thought to enhance the migration of human osteoblasts through reorganization of the actin cytoskeleton and regulation of myosin activity. ROCK inhibitors may be potentially useful as anabolic agents to enhance the biocompatibility of bone and joint prostheses.     

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.     

LPA(1) -induced migration requires nonmuscle myosin II light chain phosphorylation in breast cancer cells

The enhanced migration found in tumor cells is often caused by external stimuli and the sequential participation of cytoskeleton‐related signaling molecules. However, until now, the molecular connection between the lysophosphatidic acid (LPA) receptor and nonmuscle myosin II (NM II) has not been analyzed in detail for LPA‐induced migration. Here, we demonstrate that LPA induces migration by activating the LPA₁ receptor which promotes phosphorylation of the 20 kDa NM II light chain through activation of Rho kinase (ROCK). We show that LPA‐induced migration is insensitive to pertussis toxin (PTX) but does require the LPA₁ receptor as determined by siRNA and receptor antagonists. LPA activates ROCK and also increases GTP‐bound RhoA activity, concomitant with the enhanced membrane recruitment of RhoA. LPA‐induced migration and invasion are attenuated by specific inhibitors including C3 cell‐permeable transferase and Y‐27632. We demonstrate that NM II plays an important role in LPA‐induced migration and invasion by inhibiting its cellular function with blebbistatin and shRNA lentivirus directed against NM II‐A or II‐B. Inhibition or loss of either NM II‐A or NM II‐B in 4T1 cells results in a decrease in migration and invasion. Restoration of the expression of NM II‐A or NM II‐B also rescued LPA‐induced migration. Taken together, these results suggest defined pathways for signaling through the LPA₁ receptor to promote LPA‐mediated NM II activation and subsequent cell migration in 4T1 breast cancer cells. J. Cell. Physiol. 226: 2881–2893, 2011. © 2011 Wiley‐Liss, Inc.     

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.     

Distinct roles for ROCK1 and ROCK2 in the regulation of cell detachment

This study, using mouse embryonic fibroblast (MEF) cells derived from ROCK1^−/− and ROCK2^−/− mice, is designed to dissect roles for ROCK1 and ROCK2 in regulating actin cytoskeleton reorganization induced by doxorubicin, a chemotherapeutic drug. ROCK1^−/− MEFs exhibited improved actin cytoskeleton stability characterized by attenuated periphery actomyosin ring formation and preserved central stress fibers, associated with decreased myosin light chain 2 (MLC2) phosphorylation but preserved cofilin phosphorylation. These effects resulted in a significant reduction in cell shrinkage, detachment, and predetachment apoptosis. In contrast, ROCK2^−/− MEFs showed increased periphery membrane folding and impaired cell adhesion, associated with reduced phosphorylation of both MLC2 and cofilin. Treatment with inhibitor of myosin (blebbistatin), inhibitor of actin polymerization (cytochalasin D), and ROCK pan-inhibitor (Y27632) confirmed the contributions of actomyosin contraction and stress fiber instability to stress-induced actin cytoskeleton reorganization. These results support a novel concept that ROCK1 is involved in destabilizing actin cytoskeleton through regulating MLC2 phosphorylation and peripheral actomyosin contraction, whereas ROCK2 is required for stabilizing actin cytoskeleton through regulating cofilin phosphorylation. Consequently, ROCK1 and ROCK2 can be functional different in regulating stress-induced stress fiber disassembly and cell detachment.     

Role of RhoA and its effectors ROCK and mDia1 in the modulation of deformation-induced FAK, ERK, p38, and MLC motogenic signals in human Caco-2 intestinal epithelial cells

Repetitive deformation enhances intestinal epithelial migration across tissue fibronectin. We evaluated the contribution of RhoA and its effectors Rho-associated kinase (ROK/ROCK) and mammalian diaphanous formins (mDia1) to deformation-induced intestinal epithelial motility across fibronectin and the responsible focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), p38, and myosin light chain (MLC) signaling. We reduced RhoA, ROCK1, ROCK2, and mDia1 by smart-pool double-stranded short-interfering RNAs (siRNA) and pharmacologically inhibited RhoA, ROCK, and FAK in human Caco-2 intestinal epithelial monolayers on fibronectin-coated membranes subjected to 10% repetitive deformation at 10 cycles/min. Migration was measured by wound closure. Stimulation of migration by deformation was prevented by exoenzyme C3, Y27632, or selective RhoA, ROCK1, and ROCK2 or mDia1 siRNAs. RhoA, ROCK inhibition, or RhoA, ROCK1, ROCK2, mDia1, and FAK reduction by siRNA blocked deformation-induced nuclear ERK phosphorylation without preventing ERK phosphorylation in the cytoplasmic protein fraction. Furthermore, RhoA, ROCK inhibition or RhoA, ROCK1, ROCK2, and mDia1 reduction by siRNA also blocked strain-induced FAK-Tyr(925), p38, and MLC phosphorylation. These results suggest that RhoA, ROCK, mDia1, FAK, ERK, p38, and MLC all mediate the stimulation of intestinal epithelial migration by repetitive deformation. This pathway may be an important target for interventions to promote mechanotransduced mucosal healing during inflammation.     

Distinct roles of MLCK and ROCK in the regulation of membrane protrusions and focal adhesion dynamics during cell migration of fibroblasts

We examined the role of regulatory myosin light chain (MLC) phosphorylation of myosin II in cell migration of fibroblasts. Myosin light chain kinase (MLCK) inhibition blocked MLC phosphorylation at the cell periphery, but not in the center. MLCK-inhibited cells did not assemble zyxin-containing adhesions at the periphery, but maintained focal adhesions in the center. They generated membrane protrusions all around the cell, turned more frequently, and migrated less effectively. In contrast, Rho-associated kinase (ROCK) inhibition blocked MLC phosphorylation in the center, but not at the periphery. ROCK-inhibited cells assembled zyxin-containing adhesions at the periphery, but not focal adhesions in the center. They moved faster and more straight. On the other hand, inhibition of myosin phosphatase increased MLC phosphorylation and blocked peripheral membrane ruffling, as well as turnover of focal adhesions and cell migration. Our results suggest that myosin II activated by MLCK at the cell periphery controls membrane ruffling, and that the spatial regulation of MLC phosphorylation plays critical roles in controlling cell migration of fibroblasts.     

Targeting by myosin phosphatase-RhoA interacting protein mediates RhoA/ROCK regulation of myosin phosphatase

Vascular smooth muscle cell contractile state is the primary determinant of blood vessel tone. Vascular smooth muscle cell contractility is directly related to the phosphorylation of myosin light chains (MLCs), which in turn is tightly regulated by the opposing activities of myosin light chain kinase (MLCK) and myosin phosphatase. Myosin phosphatase is the principal enzyme that dephosphorylates MLCs leading to relaxation. Myosin phosphatase is regulated by both vasoconstrictors that inhibit its activity to cause MLC phosphorylation and contraction, and vasodilators that activate its activity to cause MLC dephosphorylation and relaxation. The RhoA/ROCK pathway is activated by vasoconstrictors to inhibit myosin phosphatase activity. The mechanism by which RhoA and ROCK are localized to and interact with myosin light chain phosphatase (MLCP) is not well understood. We recently found a new member of the myosin phosphatase complex, myosin phosphatase-rho interacting protein, that directly binds to both RhoA and the myosin-binding subunit of myosin phosphatase in vitro, and targets myosin phosphatase to the actinomyosin contractile filament in smooth muscle cells. Because myosin phosphatase-rho interacting protein binds both RhoA and MLCP, we investigated whether myosin phosphatase-rho interacting protein was required for RhoA/ROCK-mediated myosin phosphatase regulation. Myosin phosphatase-rho interacting protein silencing prevented LPA-mediated myosin-binding subunit phosphorylation, and inhibition of myosin phosphatase activity. Myosin phosphatase-rho interacting protein did not regulate the activation of RhoA or ROCK in vascular smooth muscle cells. Silencing of M-RIP lead to loss of stress fiber-associated RhoA, suggesting that myosin phosphatase-rho interacting protein is a scaffold linking RhoA to regulate myosin phosphatase at the stress fiber.     

Organized migration of epithelial cells requires control of adhesion and protrusion through Rho kinase effectors

Migration of epithelial cell sheets, a process involving F-actin restructuring through Rho family GTPases, is both physiologically and pathophysiologically important. Our objective was to clarify the mechanisms whereby the downstream RhoA effector Rho-associated coil-coil-forming kinase (ROCK) influences coordinated epithelial cell motility. Although cells exposed to a pharmacological ROCK inhibitor (Y-27632) exhibited increased spreading in wound closure assays, they failed to migrate in a cohesive manner. Two main phenomena were implicated: the formation of aberrant protrusions at the migrating front and the basal accumulation of F-actin aggregates. Aggregates reflected increased membrane affiliation and detergent insolubility of the actin-binding protein ezrin and enhanced coassociation of ezrin with the membrane protein CD44. While F-actin aggregation following ROCK inhibition was recapitulated by inhibiting myosin light chain (MLC) phosphorylation with the MLC kinase inhibitor ML-7, the latter did not influence protrusiveness and, in fact, significantly decreased cell migration. Our results suggest that excessive protrusiveness downstream of ROCK inhibition reflects an influence of ROCK on F-actin stability via LIM kinase 1 (LIMK-1), which phosphorylates and inactivates cofilin. Y-27632 reduced the levels of both active LIMK-1 and inactive cofilin (phospho forms), and expression of a dominant negative LIMK-1 mutant stimulated leading edge protrusiveness. Furthermore, Y-27632-induced protrusions were partially reversed by overexpression of LIMK-1 to restore cofilin phosphorylation. In summary, our results provide new evidence suggesting that adhesive and protrusive events involved in organized epithelial motility downstream of ROCK are separately coordinated through the phosphorylation of (respectively) MLC and cofilin.     

非磷酸化肌球蛋白在血小板衍生生长因子诱导的血管平滑肌细胞迁移中的作用

为了阐明非磷酸化肌球蛋白在平滑肌细胞迁移中的作用,研究探讨了非磷酸化肌球蛋白是否介导了血小板衍生生长因子(PDGF)诱导豚鼠脑基底动脉平滑肌细胞(GbaSM-4)的迁移。研究结果显示,20ng/ml以下剂量的PDGF可诱导GbaSM-4细胞发生迁移,此时肌球蛋白轻链(MLC20)磷酸化水平无变化。该迁移作用可被肌球蛋白特异性抑制剂blebbistatin所拮抗。应用RNA干扰技术抑制肌球蛋白轻链激酶表达,经免疫印迹检测经果显示,MLC20的磷酸化水平发生了显著下降;但对PDGF诱导的迁移作用无影响;在RNA干扰后blebbistatin也可抑制其迁移作用。体外ATP酶活性测定结果显示,blebbistatin对从平滑肌中提取的非磷酸化肌球蛋白的ATP酶活性有明显的抑制作用,其主要作用位点位于肌球蛋白头的头部S1。上述结果提示,非磷酸化的肌球蛋白参与了PDGF诱导的平滑肌细胞迁移。     

RhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes

RhoA is a small guanosine-5'-triphosphatase (GTPase) suggested to be essential for cytokinesis, stress fiber formation, and epithelial cell-cell contacts. In skin, loss of RhoA was suggested to underlie pemphigus skin blistering. To analyze RhoA function in vivo, we generated mice with a keratinocyte-restricted deletion of the RhoA gene. Despite a severe reduction of cofilin and myosin light chain (MLC) phosphorylation, these mice showed normal skin development. Primary RhoA-null keratinocytes, however, displayed an increased percentage of multinucleated cells, defective maturation of cell-cell contacts. Furthermore we observed increased cell spreading due to impaired RhoA-ROCK (Rho-associated protein kinase)-MLC phosphatase-MLC-mediated cell contraction, independent of Rac1. Rho-inhibiting toxins further increased multinucleation of RhoA-null cells but had no significant effect on spreading, suggesting that RhoB and RhoC have partially overlapping functions with RhoA. Loss of RhoA decreased directed cell migration in vitro caused by reduced migration speed and directional persistence. These defects were not related to the decreased cell contraction and were independent of ROCK, as ROCK inhibition by Y27632 increased directed migration of both control and RhoA-null keratinocytes. Our data indicate a crucial role for RhoA and contraction in regulating cell spreading and a contraction-independent function of RhoA in keratinocyte migration. In addition, our data show that RhoA is dispensable for skin development.     

Citron kinase,a Rho-dependent kinase,induces di-phosphorylation of regulatory light chain of myosin II

Citron kinase is a Rho-effector protein kinase that is related to Rho-associated kinases of ROCK/ROK/Rho-kinase family. Both ROCK and citron kinase are suggested to play a role in cytokinesis. However, no substrates are known for citron kinase. We found that citron kinase phosphorylated regulatory light chain (MLC) of myosin II at both Ser-19 and Thr-18 in vitro. Unlike ROCK, however, citron kinase did not phosphorylate the myosin binding subunit of myosin phosphatase, indicating that it does not inhibit myosin phosphatase. We found that the expression of the kinase domain of citron kinase resulted in an increase in MLC di-phosphorylation. Furthermore, the kinase domain was able to increase di-phosphorylation and restore stress fiber assembly even when ROCK was inhibited with a specific inhibitor, Y-27632. The expression of full-length citron kinase also increased di-phosphorylation during cytokinesis. These observations suggest that citron kinase phosphorylates MLC to generate di-phosphorylated MLC in vivo. Although both mono- and di-phosphorylated MLC were found in cleavage furrows, di-phosphorylated MLC showed more constrained localization than did mono-phosphorylated MLC. Because citron kinase is localized in cleavage furrows, citron kinase may be involved in regulating di-phosphorylation of MLC during cytokinesis.     

Inhibition of pancreatic adenocarcinoma cellular invasiveness by blebbistatin: a novel myosin II inhibitor

Blebbistatin is a novel 1-phenyl-2-pyrrolidinone derivative capable of inhibiting non-muscle myosin II activity with a high degree of specificity. We examined the effects of blebbistatin on pancreatic adenocarcinoma cellular migration, invasion, adhesion, and spreading. Blebbistatin dose-dependently inhibited cellular migration and invasiveness, quantified by modified Boyden chamber assay. Matrix metalloproteinase 2 and 9 activities were unaffected by blebbistatin and cellular proliferation was inhibited only by concentrations of blebbistatin exceeding those required to inhibit myosin II activity and to interfere with migration and invasion. While blebbistatin treatment did not affect cell adhesion to the extracellular matrix component fibronectin, it markedly impaired cell spreading on this substrate. Cell surface expression of the archetypal fibronectin receptor (alpha(5)beta(1) integrin) was unaffected by blebbistatin. Our observations illustrate the critical role of non-muscle myosin II in pancreatic adenocarcinoma cellular invasiveness and extracellular matrix interaction and suggest that therapeutic strategies targeting myosin II warrant further investigation.