Rho-associated Protein Kinase Signaling Research Articles

Microenvironmental Stiffness Directs Chondrogenic Lineages of Stem Cells from the Human Apical Papilla via Cooperation between ROCK and Smad3 Signaling.

Cell-based cartilage tissue engineering faces a great challenge in the repair process, partly due to the special physical microenvironment. Human stem cell from apical papilla (hSCAP) shows great potential as seed cells because of its versatile differentiation capacity. However, whether hSCAP has potent chondrogenic differentiation ability in the physical microenvironment of chondroid remains unknown. In this study, we fabricated poly(dimethylsiloxane) (PDMS) substrates with different stiffnesses and investigated the chondrogenic differentiation potential of hSCAPs. First, we found that hSCAPs cultured on soft substrates spread more narrowly accompanied by cortical actin organization, a hallmark of differentiated chondrocytes. On the contrary, stiff substrates were favorable for cell spreading and stress fiber formation. More importantly, the increased chondrogenic differentiation of hSCAPs seeded on soft substrates was confirmed by characterizing increased extracellular proteoglycan aggregation through Alcian blue staining and Safranin O staining and enhanced markers toward chondrogenic differentiation including SRY-box transcription factor 9 (Sox9), type II collagen (Col2), and aggrecan in both normal α-minimum essential medium (αMEM) and specific chondrogenic medium (CM) culture conditions. Then, we investigated the mechanosensing/mechanotransduction governing the chondrogenic differentiation of hSCAPs in response to different stiffnesses and found that stiffness-sensitive integrin β1 and focal adhesion kinase (FAK) were essential for mechanical signal perception and were oriented at the start of mechanotransduction induced by matrix stiffness. We next showed that the increased nuclear accumulation of Smad3 signaling and target Sox9 facilitated the chondrogenic differentiation of hSCAPs on the soft substrates and further verified the importance of Rho-associated protein kinase (ROCK) signaling in regulating chondrogenic differentiation and its driving factors, Smad3 and Sox9. By using SIS3, the specific inhibitor of p-Smad3, and miRNA targeting Rho-associated protein kinase 1 (ROCK-1), we finally confirmed the importance of ROCK/Smad3/Sox9 axis in the chondrogenic differentiation of hSCAPs in response to substrate stiffness. These results help us to increase the understanding of how microenvironmental stiffness directs chondrogenic differentiation from the aspects of mechanosensing, mechanotransduction, and cell fate decision, which will be of great value in the application of hSCAPs in cartilage tissue engineering.

Vanillic Acid Alleviates Right Ventricular Function in Rats With MCT-Induced Pulmonary Arterial Hypertension

This study examined the molecular processes behind the effects of vanillic acid (VA) on right ventricular (RV) hypertrophy and function in rats with monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH). There were 40 male Sprague‒Dawley (SD) rats that were separated into 4 groups: Control, PAH, MCT + VA (50 mg/kg/d), and MCT + VA (100 mg/kg/d). Male SD rats were injected with MCT once under the skin to create the PAH model (40 mg/kg). RV morphological properties were evaluated using Masson and hematoxylin and eosin (H&E) staining. Echocardiography was used to evaluate RV functioning and right ventricle–pulmonary artery (RV-PA) coupling. In addition, Rho-associated protein kinase (ROCK) pathway-related factors were evaluated using Western blotting. Enzyme-linked immunosorbent assay (ELISA) was used to detect inflammatory markers as well as atrial natriuretic peptide (ANP) and brain-type natriuretic peptide (BNP) in the blood of PAH rats. As a result, VA effectively reduced the development of RV cardiomyocyte hypertrophy and fibrosis in PAH rats; levels of ANP, BNP, and inflammatory markers in the blood of PAH rats were also significantly decreased by VA intervention. Additionally, VA enhanced RV functioning and RV-PA coupling in PAH rats. In response to VA, the expression of proteins related to the ROCK pathway (ROCK1, ROCK2, NFATc3, P-STAT3, and Bax) was downregulated, whereas Bcl-2 expression was elevated. This study found that VA could attenuate RV remodeling and improve RV-PA coupling in PAH rats. RV remodeling and dysfunction may be linked to the dysregulation of the ROCK pathway, and the protective action of VA on RV function may be due to a block in the ROCK signaling pathway or its downstream signaling molecules.

Reducing Proteoglycan Synthesis and NOX Activity by ROCK Inhibitors: Therapeutic Targets in Atherosclerosis.

Atherosclerosis is a chronic inflammatory disease of the arteries characterized by the accumulation of inflammatory cells in the arterial wall. Hypertension, dyslipidemia, and hyperglycemia are major risk factors of atherosclerosis. Rho-associated protein kinase (ROCK), a serine/threonine kinase, is a downstream effector of the small GTPase RhoA. ROCK is involved in different stages of atherosclerosis. Accumulating evidence has demonstrated that ROCK signaling plays vital roles in various cellular functions, such as contraction, migration, and proliferation of smooth muscle cells. Dysregulation of the ROCK pathway is associated with atherosclerosis and hypertension. Experimental studies have shown that ROCK inhibitors may have favorable effects in ameliorating atherosclerosis. ROCK signaling has a role in proteoglycan synthesis through transactivation of the TGF-β receptor Type I (TβRI) mediated by G-protein-coupled receptor (GPCR) agonists (endothelin-1, angiotensin II and …), and ROCK inhibitors could decrease proteoglycan synthesis and atherosclerotic plaque formation. Based on the hypothesis that targeting ROCK pathway may be effective in ameliorating atherosclerosis, we suggest that ROCK inhibitors may have a potential therapeutic role in inhibition or slowing atherogenesis. However, for this hypothesis more research is needed.

Cadmium exposure enhances VE-cadherin expression in endothelial cells via suppression of ROCK signaling.

Vascular endothelium is a target of cadmium (Cd), which is a global pollutant of the environment. However, the detailed effects and underlying mechanisms remain to be elucidated. In the present study, human umbilical vein endothelial cells (HUVECs) were treated with 0.1, 1, 5, 10, 50 µM cadmium chloride (CdCl2) for 12 h. It was found that vascular endothelial (VE)-cadherin mRNA and protein expression was upregulated by Cd in HUVECs in a dose-dependent manner. Higher levels of VE-cadherin were detected at cell-to-cell junctions in HUVECs treated with 10 µM CdCl2 compared with normal condition. The phosphorylation level of myosin-binding subunit of myosin phosphatase, a downstream substrate of Rho-associated protein kinase (ROCK), was reduced by 10 µM CdCl2, suggesting that Cd inhibited the Rho/ROCK pathway. Activation of ROCK by narciclasine reversed the Cd-induced increase of VE-cadherin expression. By contrast, ROCK pathway inhibitor Y27632 increased VE-cadherin expression in HUVECs. Following inhibition of the ROCK pathway, Cd did not significantly alter the level of VE-cadherin. Taken together, the results suggested that Cd exposure enhanced VE-cadherin expression in endothelial cells via suppression of ROCK signaling.

Short‐Duration High Frequency MegaHertz‐Order Nanomechanostimulation Drives Early and Persistent Osteogenic Differentiation in Mesenchymal Stem Cells

Stem cell fate can be directed through the application of various external physical stimuli, enabling a controlled approach to targeted differentiation. Studies involving the use of dynamic mechanical cues driven by vibrational excitation to date have, however, been limited to low frequency (Hz to kHz) forcing over extended durations (typically continuous treatment for >7 days). Contrary to previous assertions that there is little benefit in applying frequencies beyond 1 kHz, weshow here that high frequency MHz-order mechanostimulation in the form of nanoscale amplitude surface reflected bulk waves are capable of triggering differentiation of human mesenchymal stem cells from various donor sources toward an osteoblast lineage, with early, short time stimuli inducing long-term osteogenic commitment. More specifically, rapid treatments (10 min daily over 5 days) of the high frequency (10 MHz) mechanostimulation are shown to trigger significant upregulation in early osteogenic markers (RUNX2, COL1A1) and sustained increase in late markers (osteocalcin, osteopontin) through a mechanistic pathway involving piezo channel activation and Rho-associated protein kinase signaling. Given the miniaturizability and low cost of the devices, the possibility for upscaling the platform toward practical bioreactors, to address a pressing need for more efficient stem cell differentiation technologies in the pursuit of translatable regenerative medicine strategies, isensivaged.

Genes That Escape X Chromosome Inactivation Modulate Sex Differences in Valve Myofibroblasts.

Aortic valve stenosis is a sexually dimorphic disease, with women often presenting with sustained fibrosis and men with more extensive calcification. However, the intracellular molecular mechanisms that drive these clinically important sex differences remain underexplored. Hydrogel biomaterials were designed to recapitulate key aspects of the valve tissue microenvironment and to serve as a culture platform for sex-specific valvular interstitial cells (VICs; precursors to profibrotic myofibroblasts). The hydrogel culture system was used to interrogate intracellular pathways involved in sex-dependent VIC-to-myofibroblast activation and deactivation. RNA sequencing was used to define pathways involved in driving sex-dependent activation. Interventions with small molecule inhibitors and siRNA transfections were performed to provide mechanistic insight into sex-specific cellular responses to microenvironmental cues, including matrix stiffness and exogenously delivered biochemical factors. In both healthy porcine and human aortic valves, female leaflets had higher baseline activation of the myofibroblast marker α-smooth muscle actin compared with male leaflets. When isolated and cultured, female porcine and human VICs had higher levels of basal α-smooth muscle actin stress fibers that further increased in response to the hydrogel matrix stiffness, both of which were higher than in male VICs. A transcriptomic analysis of male and female porcine VICs revealed Rho-associated protein kinase signaling as a potential driver of this sex-dependent myofibroblast activation. Furthermore, we found that genes that escape X-chromosome inactivation such as BMX and STS (encoding for Bmx nonreceptor tyrosine kinase and steroid sulfatase, respectively) partially regulate the elevated female myofibroblast activation through Rho-associated protein kinase signaling. This finding was confirmed by treating male and female VICs with endothelin-1 and plasminogen activator inhibitor-1, factors that are secreted by endothelial cells and known to drive myofibroblast activation through Rho-associated protein kinase signaling. Together, in vivo and in vitro results confirm sex dependencies in myofibroblast activation pathways and implicate genes that escape X-chromosome inactivation in regulating sex differences in myofibroblast activation and subsequent aortic valve stenosis progression. Our results underscore the importance of considering sex as a biological variable to understand the molecular mechanisms of aortic valve stenosis and to help guide sex-based precision therapies.

Expression patterns of Rho-associated protein kinase signaling pathway-related genes in mouse submandibular glands

Expression patterns of Rho-associated protein kinase signaling pathway-related genes in mouse submandibular glands

Activation of yes-associated protein mediates sphingosine-1-phosphate-induced proliferation and migration of pulmonary artery smooth muscle cells and its potential mechanisms.

The aims of the present study were to examine the molecular mechanisms underlying sphingosine-1-phosphate (S1P)-induced rat pulmonary artery smooth muscle cells (PASMCs) proliferation/migration and to determine the effect of yes-associated protein (YAP) activation on S1P-induced PASMCs proliferation/migration and its potential mechanisms. S1P induced YAP dephosphorylation and nuclear translocation, upregulated microRNA-130a/b (miR-130a/b) expression, reduced bone morphogenetic protein receptor 2 (BMPR2), and inhibitor of DNA binding 1(Id1) expression, and promoted PASMCs proliferation and migration. Pretreatment of cells with Rho-associated protein kinase (ROCK) inhibitor Y27632 suppressed S1P-induced YAP activation, miR-130a/b upregulation, BMPR2/Id1 downregulation, and PASMCs proliferation/migration. Knockdown of YAP using small interfering RNA also suppressed S1P-induced alterations of miR-130a/b, BMPR2, Id1, and PASMCs behavior. In addition, luciferase reporter assay indicated that miR-130a/b directly regulated BMPR2 expression in PASMCs. Inhibition of miR-130a/b functions by anti-miRNA oligonucleotides attenuated S1P-induced BMPR2/Id1 downregulation and the proliferation and migration of PASMCs. Taken together, our study indicates that S1P induces activation of YAP through ROCK signaling and subsequently increases miR-130a/b expression, which, in turn, downregulates BMPR2 and Id1 leading to PASMCs proliferation and migration.

Sphingosine-1-Phosphate Enhances α1-Adrenergic Vasoconstriction via S1P2-G12/13-ROCK Mediated Signaling.

Sphingosine-1-phosphate (S1P) has been implicated recently in the physiology and pathology of the cardiovascular system including regulation of vascular tone. Pilot experiments showed that the vasoconstrictor effect of S1P was enhanced markedly in the presence of phenylephrine (PE). Based on this observation, we hypothesized that S1P might modulate α1-adrenergic vasoactivity. In murine aortas, a 20-minute exposure to S1P but not to its vehicle increased the Emax and decreased the EC50 of PE-induced contractions indicating a hyperreactivity to α1-adrenergic stimulation. The potentiating effect of S1P disappeared in S1P2 but not in S1P3 receptor-deficient vessels. In addition, smooth muscle specific conditional deletion of G12/13 proteins or pharmacological inhibition of the Rho-associated protein kinase (ROCK) by Y-27632 or fasudil abolished the effect of S1P on α1-adrenergic vasoconstriction. Unexpectedly, PE-induced contractions remained enhanced markedly as late as three hours after S1P-exposure in wild-type (WT) and S1P3 KO but not in S1P2 KO vessels. In conclusion, the S1P–S1P2–G12/13–ROCK signaling pathway appears to have a major influence on α1-adrenergic vasoactivity. This cooperativity might lead to sustained vasoconstriction when increased sympathetic tone is accompanied by increased S1P production as it occurs during acute coronary syndrome and stroke.

The Effects of ROCK Inhibition on Mesenchymal Stem Cell Chondrogenesis Are Culture Model Dependent.

Rho-associated protein kinase (ROCK) signaling correlates with cell shape, with decreased cell spreading accompanied by decreased ROCK activity. However, how cell shape and ROCK activity impact the chondrogenesis of mesenchymal stem cells (MSCs) remains inconclusive. Here we examine the effects of ROCK inhibition on human MSC chondrogenesis in four different culture models, including three-dimensional (3D) microribbon (μRB) scaffolds, two-dimensional hydrogel (2D-HG) substrates, 3D hydrogels (3D-HGs), and pellet. For each culture model involving biomaterials, four polymers were compared, including gelatin, chondroitin sulfate, hyaluronic acid, and polyethylene glycol. ROCK inhibition decreased MSC chondrogenesis in μRB model, enhanced chondrogenesis in pellet, and had minimal effect in 2D-HG or 3D-HG models. Furthermore, we demonstrate that MSC chondrogenesis cannot be predicted using ROCK signaling alone. While varying biomaterial compositions can impact the amount or phenotype of resulting cartilage, varying biomaterials did not change the chondrogenic response to ROCK inhibition within each culture model. Regardless of culture model or ROCK expression, increased cartilage formation was always accompanied by enhanced N-cadherin expression and production, suggesting that N-cadherin is a robust marker to select culture conditions that promote chondrogenesis. Together, the results from this study may be used to enhance MSC-based cartilage regeneration in different culture models. Impact Statement Here we assessed the effects of Rho-associated protein kinase (ROCK) inhibition on mesenchymal stem cell (MSC) chondrogenesis in different culture models, including three-dimensional (3D) microribbon scaffolds, two-dimensional hydrogel substrates, 3D hydrogels, and pellet culture. Our results demonstrate that effects of ROCK inhibition on MSC chondrogenesis differ substantially depending on culture models. Furthermore, MSC chondrogenesis cannot be predicted using ROCK signaling alone. The results from this study fill in a gap of knowledge in the correlation between ROCK signaling and MSC chondrogenesis, which may be used to enhance MSC-based cartilage regeneration in different culture models.

ROCK inhibitor enhances the growth and migration of BRAF-mutant skin melanoma cells.

Rho‐associated protein kinase (ROCK) plays crucial roles in the proliferation and migration of different types of cells. ROCK inhibitor Y‐27632 was previously reported to inhibit melanoma cell growth, and ROCK signaling was suggested to be a therapeutic target for treating melanoma. However, the negative effect of Y‐27632 on melanoma cells was mainly seen in studies on murine B16 melanoma cells. Here, we reported that ROCK inhibitor actually promoted human melanoma cell growth and migration in vitro. Y‐27632 increased the growth and migration of BRAF‐mutated melanoma cells but had a negative effect on wild‐type melanoma cells or primary melanocytes. We discovered that Y‐27632 enhanced the growth of BRAF‐mutated melanoma cells through increased ATK and ERK activity. The in vivo study further confirmed the in vitro finding. These data suggested that the effect of ROCK inhibitor on melanoma cells is cell‐context dependent, and the application of ROCK inhibitor in the treatment of melanoma requires further study.

Manipulation of the response of human endothelial colony-forming cells by focal adhesion assembly using gradient nanopattern plates

Nano feature surfaces are of growing interest as materials for a controlled response of various cells. In this study, we successfully fabricated gradient nanopattern plates to manipulate the response of blood-derived hECFCs without any chemical stimulation. Interestingly, we find that the sensitive nanopillar size for manipulation of hECFCs is range between 120 nm and 200 nm, which decreased the area and increased the filopodial outgrowth of hECFCs. Furthermore, we only modulate the nanopillar size to increase ROCK expression can be an attractive method for modulating the cytoskeletal integrity and focal adhesion of hECFCs.

Y-27632, a Rho-Associated Protein Kinase Inhibitor, Inhibits Voltage-Dependent K+ Channels in Rabbit Coronary Arterial Smooth Muscle Cells

We examined the effects of the Rho-associated protein kinase (ROCK) inhibitor Y-27632 on voltage-dependent K⁺ (Kv) channels in rabbit coronary arterial smooth muscle cells using the whole-cell patch clamp technique. Y-27632 reduced the amplitude of the Kv current in a concentration-dependent manner, with an IC₅₀ of 0.87 ± 0.06 μmol/l and a Hill coefficient of 1.48 ± 0.06. Y-27632 did not affect the steady-state activation or inactivation curves, suggesting that the drug does not affect the voltage sensitivity of Kv channels. Another ROCK inhibitor, H-1152, did not affect the Kv current and had no significant effect on the Y-27632-induced inhibition of Kv channels, indicating that the inhibitory effect of Y-27632 on the Kv current is independent of ROCK signaling. From these results, we conclude that Y-27632 inhibits the Kv channel current in a dose-dependent and ROCK signaling-independent manner.

Involvement of Rho-associated protein kinase (ROCK) and bone morphogenetic protein-binding endothelial cell precursor-derived regulator (BMPER) in high glucose-increased alkaline phosphatase expression and activity in human coronary artery smooth muscle cells.

BackgroundVascular calcification is an independent risk factor for cardiovascular disease. Diabetes mellitus increases the incidence of vascular calcification; however, detailed molecular mechanisms of vascular calcification in diabetes mellitus remain unknown. We recently reported that bone morphogenetic protein-binding endothelial cell precursor-derived regulator (BMPER) regulates osteoblast-like trans-differentiation of human coronary artery smooth muscle cells (HCASMCs).MethodsWe investigated the effect of a hydroxymethylglutaryl-coenzyme A reductase inhibitor (statin), commonly used in patients with atherosclerotic diseases and diabetes mellitus, on alkaline phosphatase (ALP) mRNA expression in aortas of streptozotocin-induced diabetic mice. We also investigated the effects of the statin, Rho-associated protein kinase (ROCK) inhibitors and BMPER knockdown on ALP mRNA expression and activity in HCASMCs cultured in high glucose-containing media.ResultsAlkaline phosphatase mRNA expression was increased in aortas of streptozotocin-induced diabetic mice, and the increase was inhibited by rosuvastatin. ALP mRNA expression and activity were increased in HCASMCs cultured in high glucose-containing media, and the increases were suppressed by rosuvastatin. This suppression was reversed by the addition of mevalonate or geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate. High glucose-increased ALP mRNA expression and activity were suppressed by ROCK inhibitors. Moreover, BMPER mRNA expression was increased in diabetic mouse aortas and in HCASMCs cultured in high glucose-containing media, but was not inhibited by rosuvastatin or ROCK inhibitors. Knockdown of BMPER suppressed high glucose-increased ALP activity, but not ROCK activity in HCASMCs.ConclusionsThere are at least two independent pathways in high glucose-induced ALP activation in HCASMCs: the Rho–ROCK signaling pathway and the BMPER-dependent pathway.

Design of novel rho kinase inhibitors using energy based pharmacophore modeling, shape-based screening, in silico virtual screening, and biological evaluation.

Rho-associated protein kinase (ROCK) plays a key role in regulating a variety of cellular processes, and dysregulation of ROCK signaling or expression is implicated in numerous diseases and infections. ROCK proteins have therefore emerged as validated targets for therapeutic intervention in various pathophysiological conditions such as diabetes-related complications or hepatitis C-associated pathogenesis. In this study, we report on the design and identification of novel ROCK inhibitors utilizing energy based pharmacophores and shape-based approaches. The most potent compound 8 exhibited an IC50 value of 1.5 μM against ROCK kinase activity and inhibited methymercury-induced neurotoxicity of IMR-32 cells at GI50 value of 0.27 μM. Notably, differential scanning fluorometric analysis revealed that ROCK protein complexed with compound 8 with enhanced stability relative to Fasudil, a validated nanomolar range ROCK inhibitor. Furthermore, all compounds exhibited ≥96 μM CC50 (50% cytotoxicity) in Huh7 hepatoma cells, while 6 compounds displayed anti-HCV activity in HCV replicon cells. The identified lead thus constitutes a prototypical molecule for further optimization and development as anti-ROCK inhibitor.

Abstract 3899: ROCK inhibitor as a differentiation-inducing agent to target glioma stem cells

Abstract Only 3% of patients diagnosed each year with Glioblastoma Multiforme (GBM) survive longer than 5 years, making GBM one of the most lethal forms of human cancer. It has been proposed that GBMs are driven by a rare subset of tumor cells referred to as cancer stem cells (CSCs), which have the ability to resist conventional radio- and chemotherapy and initiate disease recurrence. Thus, the development of novel and effective therapeutic strategies targeting CSCs is required and should significantly improve the dismal prognosis for patients with GBM. A possible viable strategy to effectively target glioma stem cells (GSCs) is the use of bone morphogenetic proteins (BMPs) as differentiation inducers. Our results suggest that Rho-associated protein kinase (ROCK) could work as a pharmacological negative regulator of BMPs. Our preliminary data indicate that: i) Fasudil, a ROCK1 inhibitor, induces BMP2 and BMP4 expression both in vitro and in vivo in different human-derived glioma xenograft models; ii) Fasudil-induced BMPs can increase the expression of differentiation markers and decrease stemness in these glioma models; iii) temozolomide, a chemotherapeutic agent, converts glioma cells to a GSC phenotype, while Fasudil blocks this interconversion; iv) co-administration of Fasudil with temozolomide inhibits the invasive characteristics of GBM in an orthotropic glioma xenograft model; and v) the analysis of BMP expression in human-derived GBM samples from the Repository of Molecular Brain Neoplasia Database (REMBRABDT) reveals that patients with higher BMP2 expression survived four times longer than those with low or intermediate BMP2 expression (p<0.001). Based on this, we propose that, by blocking ROCK signaling with the FDA approved pharmacological inhibitor Fasudil, one can modulate BMP expression in human-derived gliomas. Fasudil, therefore, may redirect cellular signals regulating GSCs' stemness, fate, and initiate their differentiation. Such an agent will allow us to develop a more effective anti-glioma therapy through its combination with other conventional anti-cancer drugs. Citation Format: Brenda Auffinger, Alex L. Tobias, Yu Han, Maciej S. Lesniak, Atique U. Ahmed. ROCK inhibitor as a differentiation-inducing agent to target glioma stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3899. doi:10.1158/1538-7445.AM2014-3899

Phosphorylation and mRNA Splicing of Collapsin Response Mediator Protein-2 Determine Inhibition of Rho-associated Protein Kinase (ROCK) II Function in Carcinoma Cell Migration and Invasion

The Rho-associated protein kinases (ROCK I and II) are central regulators of important cellular processes such as migration and invasion downstream of the GTP-Rho. Recently, we reported collapsin response mediator protein (CRMP)-2 as an endogenous ROCK II inhibitor. To reveal how the CRMP-2-ROCK II interaction is controlled, we further mapped the ROCK II interaction site of CRMP-2 and examined whether phosphorylation states of CRMP-2 affected the interaction. Here, we show that an N-terminal fragment of the long CRMP-2 splice variant (CRMP-2L) alone binds ROCK II and inhibits colon carcinoma cell migration and invasion. Furthermore, the interaction of CRMP-2 and ROCK II is partially regulated by glycogen synthase kinase (GSK)-3 phosphorylation of CRMP-2, downstream of PI3K. Inhibition of PI3K reduced interaction of CRMP-2 with ROCK II, an effect rescued by simultaneous inhibition of GSK3. Inhibition of PI3K also reduced colocalization of ROCK II and CRMP-2 at the cell periphery in human breast carcinoma cells. Mimicking GSK3 phosphorylation of CRMP-2 significantly reduced CRMP-2 binding of recombinant full-length and catalytic domain of ROCK II. These data implicate GSK3 in the regulation of ROCK II-CRMP-2 interactions. Using phosphorylation-mimetic and -resistant CRMP-2L constructs, it was revealed that phosphorylation of CRMP-2L negatively regulates its inhibitory function in ROCK-dependent haptotactic cell migration, as well as invasion of human colon carcinoma cells. Collectively, the presented data show that CRMP-2-dependent regulation of ROCK II activity is mediated through interaction of the CRMP-2L N terminus with the ROCK II catalytic domain as well as by GSK3-dependent phosphorylation of CRMP-2.

Cholinergic regulation of epithelial sodium channels in rat alveolar type 2 epithelial cells

We and others have shown that epithelial Na(+) channels (ENaC) in alveolar type 2 (AT2) cells are activated by β2 agonists, steroid hormones, elevated oxygen tension, and by dopamine. Although acetylcholine receptors (AChRs) have been previously described in the lung, there are few reports of whether cholinergic agonists alter sodium transport in the alveolar epithelium. Therefore, we investigated how cholinergic receptors regulate ENaC activity in primary cultures of rat AT2 cells using cell-attached patch-clamp recordings to assess ENaC activity. We found that the muscarinic agonists, carbachol (CCh) and oxotremorine, activated ENaC in a dose-dependent manner but that nicotine did not. CCh-induced activation of ENaC was blocked by atropine. Western blotting and immunohistochemistry suggested that muscarinic M2 and M3 receptors (mAChRs) but not nicotinic receptors were present in AT2 cells. Endogenous RhoA and GTP-RhoA increased in response to CCh and the increase was reduced by pretreatment with atropine. We showed that Y-27632, an inhibitor of Rho-associated protein kinase (ROCK), abolished endogenous ENaC activity and inhibited the activation of ENaC by CCh. We also showed that ROCK signaling was necessary for ENaC stability in 2F3 cells, a model for AT2 cells. Our results showed that muscarinic agonists activated ENaC in rat AT2 cells through M2 and/or M3 mAChRs probably via a RhoA/ROCK signaling pathway.

Effects of ROCK inhibitor, Y-27632, on adhesion and mobility in esophageal squamous cell cancer cells

Rho-associated protein kinase (ROCK), a molecular switch, modulates cellular functions in many cancers, such as hepatocellular, breast, colon cancers, etc. However, little is known the effect of ROCK on cell adhesion and mobility in esophageal squamous cell cancer (ESCC), one of the most diagnosed cancers in China. In this study, Y-27632 was used to specifically block ROCK activity in ESCC cells. Adhesion of ESCC cells was detected by homotypic and heterotypic adhesion assay together with examination of E-cadherin expression. Motility of ESCC cells changes were examined by detection of phosphorylated cofilin and observed under confocal microscopy, respectively. We found that Y-27632 increased both heterotypic and homotypic adhesion, and the expression of E-cadherin; decreased phosphorylated cofilin resulting in actin rearrangement in ESCC cells. All these findings indicate that ROCK signaling pathway plays an important role in cell adhesion and mobility, suggesting that it may be used as a potential target for therapy of ESCC.