Na–H Exchange Acts Downstream of RhoA to Regulate Integrin-induced Cell Adhesion and Spreading

The ubiquitously expressed Na–H exchanger NHE1 functions in regulating intracellular pH and cell volume. NHE1 activity is stimulated by hormones, growth factors, and activation of integrin receptors. We recently determined that NHE1 activity is also stimulated by activation of the low molecular weight GTPase RhoA and that increases in NHE1 activity are necessary for RhoA-induced formation of actin stress fibers. We now show that NHE1 acts downstream of RhoA to modulate initial steps in integrin signaling for the assembly of focal adhesions. Adhesion of CCL39 fibroblasts on fibronectin was markedly delayed in the presence of the NHE inhibitor ethylisopropylamiloride. In mutant PS120 cells, derived from CCL39 fibroblasts but lacking NHE1, adhesion was also delayed but was rescued in PS120 cells stably expressing NHE1. In the absence of NHE1 activity, cell spreading was inhibited, and the accumulation of integrins, paxillin, and vinculin at focal contacts was impaired. Additionally, tyrosine phosphorylation of p125^FAK induced by integrin clustering was also impaired. Inactivation of RhoA with C3 transferase and inhibition of the Rho-kinase p160ROCK with the pyridine derivative Y-27632 completely abolished activation of NHE1 by integrins but not by platelet-derived growth factor. These findings indicate that NHE1 acts downstream of RhoA to contribute a previously unrecognized critical signal to proximal events in integrin-induced cytoskeletal reorganization.     

Specific activation of LIM kinase 2 via phosphorylation of threonine 505 by ROCK, a Rho-dependent protein kinase

LIM-kinase 1 (LIMK1) and LIM-kinase 2 (LIMK2) regulate actin cytoskeletal reorganization via cofilin phosphorylation downstream of distinct Rho family GTPases. We report our findings that ROCK, a downstream protein kinase of Rho, specifically activates LIMK2 but not LIMK1 downstream of RhoA. LIMK1 and LIMK2 activities toward cofilin phosphorylation were stimulated by co-expression with the active form of ROCK (ROCK-Delta3), whereas full-length ROCK selectively activates LIMK2 but not LIMK1. Activation of LIMK2 by RhoA was inhibited by Y-27632, a specific inhibitor of ROCK, but Rac1-mediated activation of LIMK1 was not. ROCK directly phosphorylated the threonine 505 residue within the activation segment of LIMK2 and markedly stimulated LIMK2 activity. A LIMK2 mutant with replacement of threonine 505 by valine abolished LIMK2 activities for cofilin phosphorylation and actin cytoskeletal changes, whereas replacement by glutamate enhanced the protein kinase activity and stress fiber formation by LIMK2. These results indicate that ROCK directly phosphorylates threonine 505 and activates LIMK2 downstream of RhoA and that this phosphorylation is essential for LIMK2 to induce actin cytoskeletal reorganization. Together with the finding that LIMK1 is regulated by Pak1, LIMK1 and LIMK2 are regulated by different protein kinases downstream of distinct Rho family GTPases.     

RhoA and rho kinase regulate the epithelial Na+/H+ exchanger NHE3. Role of myosin light chain phosphorylation

The activity of the Na(+)/H(+) exchanger NHE3 isoform, which is found primarily in epithelial cells, is sensitive to the state of actin polymerization. Actin assembly, in turn, is controlled by members of the small GTPase Rho family, namely Rac1, Cdc42, and RhoA. We therefore investigated the possible role of these GTPases in modulating NHE3 activity. Cells stably expressing NHE3 were transiently transfected with inhibitory forms of Rac1, Cdc42, or RhoA and transport activity was assessed using microfluorimetry. NHE3 activity was not adversely affected by either dominant-negative Rac1 or Cdc42. By contrast, the inhibitory form of RhoA greatly depressed NHE3 activity, without noticeably altering its subcellular distribution. NHE3 activity was equally reduced by inhibiting p160 Rho-associated kinase I (ROK), a downstream effector of RhoA, with the selective antagonist Y-27632 and a dominant-negative form of ROK. Furthermore, inhibition of ROK reduced the phosphorylation of myosin light chain. A comparable net dephosphorylation was achieved by the myosin light chain kinase inhibitor ML9, which similarly inhibited NHE3. These data suggest that optimal NHE3 activity requires a functional RhoA-ROK signaling pathway which acts, at least partly, by controlling the phosphorylation of myosin light chain and, ultimately, the organization of the actin cytoskeleton.     

ROCK and mDia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts

The small GTPase Rho acts on two effectors, ROCK and mDia1, and induces stress fibers and focal adhesions. However, how ROCK and mDia1 individually regulate signals and dynamics of these structures remains unknown. We stimulated serum-starved Swiss 3T3 fibroblasts with LPA and compared the effects of C3 exoenzyme, a Rho inhibitor, with those of Y-27632, a ROCK inhibitor. Y-27632 treatment suppressed LPA-induced formation of stress fibers and focal adhesions as did C3 exoenzyme but induced membrane ruffles and focal complexes, which were absent in the C3 exoenzyme-treated cells. This phenotype was suppressed by expression of N17Rac. Consistently, the amount of GTP-Rac increased significantly by Y-27632 in LPA-stimulated cells. Biochemically, Y-27632 suppressed tyrosine phosphorylation of paxillin and focal adhesion kinase and not that of Cas. Inhibition of Cas phosphorylation with PP1 or expression of a dominant negative Cas mutant inhibited Y-27632-induced membrane ruffle formation. Moreover, Crk-II mutants lacking in binding to either phosphorylated Cas or DOCK180 suppressed the Y-27632-induced membrane ruffle formation. Finally, expression of a dominant negative mDia1 mutant also inhibited the membrane ruffle formation by Y-27632. Thus, these results have revealed the Rho-dependent Rac activation signaling that is mediated by mDia1 through Cas phosphorylation and antagonized by the action of ROCK.     

The functional role of rho and rho-associated coiled-coil forming protein kinase in eotaxin signaling of eosinophils

The CC chemokine eotaxin plays a pivotal role in local accumulation of eosinophils. Very little is known about the eotaxin signaling in eosinophils except the activation of the mitogen-activated protein (MAP) kinase family. The p21 G protein Rho and its substrate Rho-associated coiled-coil forming protein kinase (ROCK) regulate the formation of stress fibers and focal adhesions. In the present study, we studied the functional relevance of Rho and ROCK in eosinophils using the ROCK inhibitor (Y-27632) and exoenzyme C3, a specific Rho inhibitor. Eotaxin stimulates activation of Rho A and ROCK II in eosinophils. Exoenzyme C3 almost completely inhibited the ROCK activity, indicating that ROCK is downstream of Rho. We then examined the role of Rho and ROCK in eosinophil chemotaxis. The eotaxin-induced eosinophil chemotaxis was significantly inhibited by exoenzyme C3 or Y-27632. Because extracellular signal-regulated kinase (ERK)1/2 and p38 MAP kinases are activated by eotaxin and are critical for eosinophil chemotaxis, we investigated whether Rho and ROCK are upstream of these MAP kinases. C3 partially inhibited eotaxin-induced phosphorylation of ERK1/2 but not p38. In contrast, neither ERK1/2 nor p38 phosphorylation was abrogated by Y-27632. Both C3 and Y-27632 reduced reactive oxygen species production from eosinophils. We conclude that both Rho and ROCK are important for eosinophil chemotaxis and reactive oxygen species production. There is a dichotomy of downstream signaling pathways of Rho, namely, Rho-ROCK and Rho-ERK pathways. Taken together, eosinophil chemotaxis is regulated by multiple signaling pathways that involve at least ROCK, ERK, and p38 MAP kinase.     

RhoA-Rho kinase pathway mediates thrombin- and U-46619-induced phosphorylation of a myosin phosphatase inhibitor, CPI-17, in vascular smooth muscle cells

Protein kinase C-potentiated phosphatase inhibitor of 17 kDa (CPI-17) mediates some agonist-induced smooth muscle contraction by suppressing the myosin phosphatase in a phosphorylation-dependent manner. The physiologically relevant kinases that phosphorylate CPI-17 remain to be identified. Several previous studies have shown that some agonist-induced CPI-17 phosphorylation in smooth muscle tissues was attenuated by the Rho kinase (ROCK) inhibitor Y-27632, suggesting that ROCK is involved in agonist-induced CPI-17 phosphorylation. However, Y-27632 has recently been found to inhibit protein kinase C (PKC)-, a well-recognized CPI-17 kinase. Thus the role of ROCK in agonist-induced CPI-17 phosphorylation remains uncertain. The present study was designed to address this important issue. We selectively activated the RhoA pathway using inducible adenovirus-mediated expression of a constitutively active mutant RhoA (V14RhoA) in primary cultured rabbit aortic vascular smooth muscle cells (VSMCs). V14RhoA caused expression level-dependent CPI-17 phosphorylation at Thr38 as well as myosin phosphatase phosphorylation at Thr853. Importantly, we have shown that V14RhoA-induced CPI-17 phosphorylation was not affected by the PKC inhibitor GF109203X but was abolished by Y-27632, suggesting that ROCK but not PKC was involved. Furthermore, we have shown that the contractile agonists thrombin and U-46619 induced CPI-17 phosphorylation in VSMCs. Similarly to V14RhoA-induced CPI-17 phosphorylation, thrombin-induced CPI-17 phosphorylation was not affected by inhibition of PKC with GF109203X, but it was blocked by inhibition of RhoA with adenovirus-mediated expression of exoenzyme C3 as well as by Y-27632. Taken together, our present data provide the first clear evidence indicating that ROCK is responsible for thrombin- and U-46619-induced CPI-17 phosphorylation in primary cultured VSMCs. protein kinase C; signal transduction; adenovirus     

Cofilin phosphorylation by protein kinase testicular protein kinase 1 and its role in integrin-mediated actin reorganization and focal adhesion formation

Testicular protein kinase 1 (TESK1) is a serine/threonine kinase with a structure composed of a kinase domain related to those of LIM-kinases and a unique C-terminal proline-rich domain. Like LIM-kinases, TESK1 phosphorylated cofilin specifically at Ser-3, both in vitro and in vivo. When expressed in HeLa cells, TESK1 stimulated the formation of actin stress fibers and focal adhesions. In contrast to LIM-kinases, the kinase activity of TESK1 was not enhanced by Rho-associated kinase (ROCK) or p21-activated kinase, indicating that TESK1 is not their downstream effector. Both the kinase activity of TESK1 and the level of cofilin phosphorylation increased by plating cells on fibronectin. Y-27632, a specific inhibitor of ROCK, inhibited LIM-kinase-induced cofilin phosphorylation but did not affect fibronectin-induced or TESK1-induced cofilin phosphorylation in HeLa cells. Expression of a kinase-negative TESK1 suppressed cofilin phosphorylation and formation of stress fibers and focal adhesions induced in cells plated on fibronectin. These results suggest that TESK1 functions downstream of integrins and plays a key role in integrin-mediated actin reorganization, presumably through phosphorylating and inactivating cofilin. We propose that TESK1 and LIM-kinases commonly phosphorylate cofilin but are regulated in different ways and play distinct roles in actin reorganization in living cells.     

ROCK mediates thrombin's endothelial barrier dysfunction

Thrombin-induced endothelial monolayer hyperpermeability is thought toresult from increased F-actin stress fiber-related contractile tension,a process regulated by the small GTP-binding protein Rho. We testedwhether this process was dependent on the Rho-associated proteinkinase, ROCK, using a specific ROCK inhibitor, Y-27632. The effects ofY-27632 on thrombin-induced myosin light chain phosphorylation (MLCP)and tyrosine phosphorylation of p125 focal adhesion kinase(p125^FAK) and paxillin were measured by Western blotting.F-actin organization and content were analyzed by digital imaging, andendothelial monolayer permeability was measured in bovine pulmonaryartery endothelial cell (EC) monolayers using a size-selectivepermeability assay. Y-27632 enhanced EC monolayer barrier function dueto a decline in small-pore number that was associated with increased ECsurface area, reduced F-actin content, and reorganization of F-actin to-catenin-containing cell-cell adherens junctions. Although Y-27632prevented thrombin-induced MLCP, stress fiber formation, and theincreased phosphotyrosine content of paxillin and p125^FAK,it attenuated but did not prevent the thrombin-induced formation oflarge paracellular holes. These data indicate that thrombin-induced stress fiber formation is ROCK dependent. In contrast, thrombin-induced paracellular hole formation occurs in a ROCK-independent manner, whereas thrombin-induced monolayer hyperpermeability appears to bepartially ROCK dependent.

     

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.     

Phosphorylation of profilin by ROCK1 regulates polyglutamine aggregation

Y-27632, an inhibitor of the Rho-associated kinase ROCK, is a therapeutic lead for Huntington disease (HD). The downstream targets that mediate its inhibitory effects on huntingtin (Htt) aggregation and toxicity are unknown. We have identified profilin, a small actin-binding factor that also interacts with Htt, as being a direct target of the ROCK1 isoform. The overexpression of profilin reduces the aggregation of polyglutamine-expanded Htt and androgen receptor (AR) peptides. This requires profilin's G-actin binding activity and its direct interaction with Htt, which are both inhibited by the ROCK1-mediated phosphorylation of profilin at Ser-137. Y-27632 blocks the phosphorylation of profilin in HEK293 cells and primary neurons, which maintains profilin in an active state. The knockdown of profilin blocks the inhibitory effect of Y-27632 on both AR and Htt aggregation. A signaling pathway from ROCK1 to profilin thus controls polyglutamine protein aggregation and is targeted by a promising therapeutic lead for HD.     

p38 MAP Kinase Inhibitor Suppresses Transforming Growth Factor-β2–Induced Type 1 Collagen Production in Trabecular Meshwork Cells

Glaucoma is an age-related neurodegenerative disease of retinal ganglion cells, and appropriate turnover of the extracellular matrix in the trabecular meshwork is important in its pathology. Here, we report the effects of Rho-associated kinase (ROCK) and p38 MAP kinase on transforming growth factor (TGF)-β2–induced type I collagen production in human trabecular meshwork cells. TGF-β2 increased RhoA activity, actin polymerization, and myosin light chain 2 phosphorylation. These effects were significantly inhibited by Y-27632, but not SB203580. TGF-β2 also increased promoter activity, mRNA synthesis, and protein expression of COL1A2. These effects were significantly inhibited by SB203580, but not Y-27632. Additionally, Y-27632 did not significantly inhibit TGF-β2–induced promoter activation, or phosphorylation or nuclear translocation of Smad2/3, whereas SB203580 partially suppressed these processes. Collectively, TGF-β2–induced production of type 1 collagen is suppressed by p38 inhibition and accompanied by partial inactivation of Smad2/3, in human trabecular meshwork cells.     

Y-27632, an Inhibitor of Rho-Associated Kinases, Prevents Tyrosine Phosphorylation of Focal Adhesion Kinase and Paxillin Induced by Bombesin: Dissociation from Tyrosine Phosphorylation of p130

A rapid increase in tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and Crk-associated substrate (CAS) are prominent early events triggered by many G protein-coupled receptors (GPCRs), but the mechanisms involved remain unclear. Here, we examined whether the Rho-associated protein serine/threonine kinase family (ROCK) is a critical Rho effector in the pathway that links GPCR activation to the tyrosine phosphorylation of FAK, CAS, and paxillin. Treatment of Swiss 3T3 cells with Y-27632, a preferential inhibitor of ROCK, dramatically inhibited the formation of actin stress fibers, the assembly of focal contacts, and the increase in tyrosine phosphorylation of FAK and paxillin induced by bombesin in these cells. Surprisingly, we found that treatment with Y-27632 did not produce any detectable effect on bombesin-elicited CAS tyrosine phosphorylation even at the highest concentrations of Y-27632 tested. HA-1077, a preferential inhibitor of ROCK activity structurally unrelated to Y-27632, also attenuated the increase in the tyrosine phosphorylation of FAK and paxillin but did not affect the tyrosine phosphorylation of CAS induced by bombesin in Swiss 3T3 cells. The results demonstrate that ROCK-dependent tyrosine phosphorylation of FAK and paxillin can be dissociated from a ROCK-independent pathway leading to tyrosine phosphorylation of CAS.     

Y-27632, an inhibitor of Rho-associated kinases, prevents tyrosine phosphorylation of focal adhesion kinase and paxillin induced by bombesin: dissociation from tyrosine phosphorylation of p130(CAS)

A rapid increase in tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and Crk-associated substrate (CAS) are prominent early events triggered by many G protein-coupled receptors (GPCRs), but the mechanisms involved remain unclear. Here, we examined whether the Rho-associated protein serine/threonine kinase family (ROCK) is a critical Rho effector in the pathway that links GPCR activation to the tyrosine phosphorylation of FAK, CAS, and paxillin. Treatment of Swiss 3T3 cells with Y-27632, a preferential inhibitor of ROCK, dramatically inhibited the formation of actin stress fibers, the assembly of focal contacts, and the increase in tyrosine phosphorylation of FAK and paxillin induced by bombesin in these cells. Surprisingly, we found that treatment with Y-27632 did not produce any detectable effect on bombesin-elicited CAS tyrosine phosphorylation even at the highest concentrations of Y-27632 tested. HA-1077, a preferential inhibitor of ROCK activity structurally unrelated to Y-27632, also attenuated the increase in the tyrosine phosphorylation of FAK and paxillin but did not affect the tyrosine phosphorylation of CAS induced by bombesin in Swiss 3T3 cells. The results demonstrate that ROCK-dependent tyrosine phosphorylation of FAK and paxillin can be dissociated from a ROCK-independent pathway leading to tyrosine phosphorylation of CAS.     

RhoA/ROCK signaling is critical to FAK activation by cyclic stretch in cardiac myocytes

Focal adhesion kinase (FAK) has been shown to be activated in cardiac myocytes exposed to mechanical stress. However, details of how mechanical stimuli induce FAK activation are unknown. We investigated whether signaling events mediated by the RhoA/Rho-associated coiled coil-containing kinase (ROCK) pathway are involved in regulation of stretch-induced FAK phosphorylation at Tyr(397) in neonatal rat ventricular myocytes (NRVMs). Immunostaining showed that RhoA localized to regions of myofilaments alternated with phalloidin (actin) staining. The results of coimmunoprecipitation assays indicated that FAK and RhoA are associated in nonstretched NRVMs, but cyclic stretch significantly reduced the amount of RhoA recovered from anti-FAK immunoprecipitates. Cyclic stretch induced rapid and sustained (up to 2 h) increases in phosphorylation of FAK at Tyr(397) and ERK1/2 at Thr(202)/Tyr(204). Blockade of RhoA/ROCK signaling by pharmacological inhibitors of RhoA (Clostridium botulinum C3 exoenzyme) or ROCK (Y-27632, 10 micromol/l, 1 h) markedly attenuated stretch-induced FAK and ERK1/2 phosphorylation. Similar effects were observed in cells treated with the inhibitor of actin polymerization cytochalasin D. Transfection of NRVMs with RhoA antisense oligonucleotide attenuated stretch-induced FAK and ERK1/2 phosphorylation and expression of beta-myosin heavy chain mRNA. Similar results were seen in cells transfected with FAK antisense oligonucleotide. These findings demonstrate that RhoA/ROCK signaling plays a crucial role in stretch-induced FAK phosphorylation, presumably by coordinating upstream events operationally linked to the actin cytoskeleton.     

Protein kinase A gating of a pseudopodial-located RhoA/ROCK/p38/NHE1 signal module regulates invasion in breast cancer cell lines

Metastasis results from a sequence of selective events often involving interactions with elements of the tumor-specific physiological microenvironment. The low-serum component of this microenvironment confers increased motility and invasion in breast cancer cells by activating the Na+/H+ exchanger isoform 1 (NHE1). The present study was undertaken to characterize the signal transduction mechanisms underlying this serum deprivation-dependent activation of both the NHE1 and the concomitant invasive characteristics such as leading edge pseudopodia development and penetration of matrigel in breast cancer cell lines representing different stages of metastatic progression. Using pharmacological and genetic manipulation together with transport and kinase activity assays, we observe that the activation of the NHE1 and subsequent invasion by serum deprivation in metastatic human breast cells is coordinated by a sequential RhoA/p160ROCK/p38MAPK signaling pathway gated by direct protein kinase A phosphorylation and inhibition of RhoA. Fluorescence resonance energy transfer imaging of RhoA activity and immunofluorescence analysis of phospho-RhoA and NHE1 show that serum deprivation dynamically remodels the cell, forming long, leading edge pseudopodia and that this signal module is preferentially compartmentalized in these leading edge pseudopodia, suggesting a tight topographic relation of the signaling module to an invasion-specific cell structure.     

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.     

RhoA and Rho-kinase dependent and independent signals mediate TGF-beta-induced pulmonary endothelial cytoskeletal reorganization and permeability

Transforming growth factor (TGF)-beta is a potent inflammatory mediator involved in acute lung injury. TGF-beta directly increases pulmonary endothelial myosin light chain (MLC) phosphorylation, which is associated with increased endothelial stress fiber formation, gap formation, and protein permeability, all hallmarks of pulmonary endothelial responses during acute lung injury. We performed the following experiments in pulmonary endothelial monolayers to determine whether RhoA and Rho-kinase mediate these TGF-beta-induced responses. TGF-beta caused the sustained activation of RhoA 2 h posttreatment associated with increased MLC phosphorylation. Inhibition of either RhoA or Rho-kinase with either C3 exoenzyme or Y-27632 blocked MLC phosphorylation. In addition, both C3 and Y-27632 partially attenuated the maximal TGF-beta-induced increase in permeability but did not affect the initial phase of compromised barrier integrity. Inhibition of Rho-kinase completely blocked the TGF-beta-induced increase in the content of filamentous actin (F-actin) but only partially inhibited TGF-beta-induced changes in actin reorganization. To assess the contribution of Rho-kinase in RhoA-mediated responses independent of additional TGF-beta-induced signals, cells were infected with a constitutively active RhoA adenovirus (RhoAQ63L) with or without Y-27632. RhoAQ63L increased MLC phosphorylation, F-actin content, and permeability. Treatment with Y-27632 blocked these responses, suggesting that Rho-kinase mediates these RhoA-induced effects. Collectively, these data suggest the following: 1) the RhoA/Rho-kinase pathway is an important component of TGF-beta-induced effects on endothelial MLC phosphorylation, cytoskeletal reorganization, and barrier integrity; and 2) additional signaling mechanisms independent of the RhoA/Rho-kinase signaling cascade contribute to TGF-beta-induced changes in cytoskeletal organization and permeability.     

Acetylcholine-induced phosphorylation of CPI-17 in rat bronchial smooth muscle: the roles of Rho-kinase and protein kinase C

It has been demonstrated that CPI-17 provokes an inhibition of myosin light chain phosphatase to increase myosin light chain phosphorylaton and Ca(2+) sensitivity during contraction of vascular smooth muscle. However, expression and agonist-mediated regulation of CPI-17 in bronchial smooth muscle have not been documented. Thus, expression and phosphorylation of CPI-17 mediated by PKC and ROCK were investigated using rat bronchial preparations. Acetylcholine (ACh)-induced contraction and Ca(2+) sensitization were both attenuated by 10(-6) mol Y-27632 /L, a ROCK inhibitor, 10(-6) mol calphostin C/L, a PKC inhibitor, and their combination. A PKC activator, PDBu, induced a Ca(2+) sensitization in alpha-toxin-permeabilized bronchial smooth muscle. In this case, the Ca(2+) sensitizing effect was significantly inhibited by caphostin C but not by Y-27632. An immunoblot study demonstrated CPI-17 expression in the rat bronchial smooth muscle. Acetylcholine induced a phosphorylation of CPI-17 in a concentration-dependent manner, which was significantly inhibited by Y-27632 and calphostin C. In conclusion, these data suggest that both PKC and ROCK are involved in force development, Ca(2+) sensitization, and CPI-17 phosphorylation induced by ACh stimulation in rat bronchial smooth muscle. As such, RhoA/ROCK, PKC/CPI-17, and RhoA/ROCK/CPI pathways may play important roles in the ACh-induced Ca(2+) sensitization of bronchial smooth muscle contraction.