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Rho-associated Kinase (Rho-associat + kinase)
Selected AbstractsROCK inhibitor (Y27632) increases apoptosis and disrupts the actin cortical mat in embryonic avian corneal epitheliumDEVELOPMENTAL DYNAMICS, Issue 3 2004Kathy K.H. Svoboda Abstract The embryonic chicken corneal epithelium is a unique tissue that has been used as an in vitro epithelial sheet organ culture model for over 30 years (Hay and Revel [1969] Fine structure of the developing Avian cornea. Basel, Switzerland: S. Karger A.G.). This tissue was used to establish that epithelial cells could produce extracellular matrix (ECM) proteins such as collagen and proteoglycans (Dodson and Hay [1971] Exp Cell Res 65:215,220; Meier and Hay [1973] Dev Biol 35:318,331; Linsenmayer et al. [1977] Proc Natl Acad Sci U S A 74:39,43; Hendrix et al. [1982] Invest Ophthalmol Vis Sci 22:359,375). This historic model was also used to establish that ECM proteins could stimulate actin reorganization and increase collagen synthesis (Sugrue and Hay [1981] J Cell Biol 91:45,54; Sugrue and Hay [1982] Dev Biol 92:97,106; Sugrue and Hay [1986] J Cell Biol 102:1907,1916). Our laboratory has used the model to establish the signal transduction pathways involved in ECM-stimulated actin reorganization (Svoboda et al. [1999] Anat Rec 254:348,359; Chu et al. [2000] Invest Ophthalmol Vis Sci 41:3374,3382; Reenstra et al. [2002] Invest Ophthalmol Vis Sci 43:3181,3189). The goal of the current study was to investigate the role of ECM in epithelial cell survival and the role of Rho-associated kinase (p160 ROCK, ROCK-1, ROCK-2, referred to as ROCK), in ECM and lysophosphatidic acid (LPA) -mediated actin reorganization. Whole sheets of avian embryonic corneal epithelium were cultured in the presence of the ROCK inhibitor, Y27632 at 0, 0.03, 0.3, 3, or 10 ,M before stimulating the cells with either collagen (COL) or LPA. Apoptosis was assessed by Caspase-3 activity assays and visualized with annexin V binding. The ROCK inhibitor blocked actin cortical mat reformation and disrupted the basal cell lateral membranes in a dose-dependent manner and increased the apoptosis marker annexin V. In addition, an in vitro caspase-3 activity assay was used to determine that caspase-3 activity was higher in epithelia treated with 10 ,M Y-27632 than in those isolated without the basal lamina or epithelia stimulated with fibronectin, COL, or LPA. In conclusion, ECM molecules decreased apoptosis markers and inhibiting the ROCK pathway blocked ECM stimulated actin cortical mat reformation and increased apoptosis in embryonic corneal epithelial cells. Developmental Dynamics 229:579,590, 2004. © 2004 Wiley-Liss, Inc. [source] Activation of p21-activated kinase 1 is required for lysophosphatidic acid-induced focal adhesion kinase phosphorylation and cell motility in human melanoma A2058 cellsFEBS JOURNAL, Issue 8 2004In Duk Jung Lysophosphatidic acid (LPA), one of the naturally occurring phospholipids, stimulates cell motility through the activation of Rho family members, but the signaling mechanisms remain to be elucidated. In the present study, we investigated the roles of p21-activated kinase 1 (PAK1) on LPA-induced focal adhesion kinase (FAK) phosphorylation and cell motility. Treatment of human melanoma cells A2058 with LPA increased phosphorylation and activation of PAK1, which was blocked by treatment with pertussis toxin and by inhibition of phosphoinositide 3-kinase (PI3K) with an inhibitor LY294002 or by overexpression of catalytically inactive mutant of PI3K,, indicating that LPA-induced PAK1 activation was mediated via a Gi protein and the PI3K, signaling pathway. In addition, we demonstrated that Rac1/Cdc42 signals acted as upstream effector molecules of LPA-induced PAK activation. However, Rho-associated kinase, MAP kinase kinase 1/2 or phospholipase C might not be involved in LPA-induced PAK1 activation or cell motility stimulation. Furthermore, PAK1 was necessary for FAK phosphorylation by LPA, which might cause cell migration, as transfection of the kinase deficient mutant of PAK1 or PAK auto-inhibitory domain significantly abrogated LPA-induced FAK phosphorylation. Taken together, these findings strongly indicated that PAK1 activation was necessary for LPA-induced cell motility and FAK phosphorylation that might be mediated by sequential activation of Gi protein, PI3K, and Rac1/Cdc42. [source] Ca2+ -independent phospholipase A2-dependent sustained Rho-kinase activation exhibits all-or-none responseGENES TO CELLS, Issue 9 2006Akio Maeda Sustained contraction of cells depends on sustained Rho-associated kinase (Rho-kinase) activation. We developed a computational model of the Rho-kinase pathway to understand the systems characteristics. Thrombin-dependent in vivo transient responses of Rho activation and Ca2+ increase could be reproduced in silico. Low and high thrombin stimulation induced transient and sustained phosphorylation, respectively, of myosin light chain (MLC) and myosin phosphatase targeting subunit 1 (MYPT1) in vivo. The transient phosphorylation of MLC and MYPT1 could be reproduced in silico, but their sustained phosphorylation could not. This discrepancy between in vivo and in silico in the sustained responses downstream of Rho-kinase indicates that a missing pathway(s) may be responsible for the sustained Rho-kinase activation. We found, experimentally, that the sustained phosphorylation of MLC and MYPT1 exhibit all-or-none responses. Bromoenol lactone, a specific inhibitor of Ca2+ -independent phospholipase A2 (iPLA2), inhibited sustained phosphorylation of MLC and MYPT1, which indicates that sustained Rho-kinase activation requires iPLA2 activity. Thus, the systems analysis of the Rho-kinase pathway identified a novel iPLA2-dependent mechanism of the sustained Rho-kinase activation, which exhibits an all-or-none response. [source] Implication of Rho-associated kinase in the elevation of extracellular dopamine levels and its related behaviors induced by methamphetamine in ratsJOURNAL OF NEUROCHEMISTRY, Issue 2 2003Minoru Narita Abstract A growing body of evidence suggests that several protein kinases are involved in the expression of pharmacological actions induced by a psychostimulant methamphetamine. The present study was designed to investigate the role of the Rho/Rho-associated kinase (ROCK)-dependent pathway in the expression of the increase in extracellular levels of dopamine in the nucleus accumbens and its related behaviors induced by methamphetamine in rats. Methamphetamine (1 mg/kg, subcutaneously) produced a substantial increase in extracellular levels of dopamine in the nucleus accumbens, with a progressive augmentation of dopamine-related behaviors including rearing and sniffing. Methamphetamine also induced the decrease in levels of its major metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA). Both the increase in extracellular levels of dopamine and the induction of dopamine-related behaviors by methamphetamine were significantly suppressed by pretreatment with an intranucleus accumbens injection of a selective ROCK inhibitor Y-27632. In contrast, Y-27632 had no effect on the decrease in levels of DOPAC and HVA induced by methamphetamine. Under these conditions, there were no changes in protein levels of membrane-bound RhoA in the nucleus accumbens following methamphetamine treatment. It is of interest to note that the microinjection of Y-27632 into the nucleus accumbens failed to suppress the increases in extracellular levels of dopamine, DOPAC, and HVA in the nucleus accumbens induced by subcutaneous injection of a prototype of µ-opioid receptor agonist morphine (10 mg/kg). Furthermore, perfusion of a selective blocker of voltage-dependent Na+ channels, tetrodotoxin (TTx) into the rat nucleus accumbens did not affect the increase in extracellular levels of dopamine in the rat nucleus accumbens by methamphetamine, whereas the morphine-induced dopamine elevation was eliminated by this application of TTx. The extracellular level of dopamine in the nucleus accumbens was also increased by perfusion of a selective dopamine re-uptake inhibitor 1-[2-[bis(4-fluorophenyl)methoxy]-4-(3-phenylpropyl)piperazine (GBR-12909) in the nucleus accumbens. This effect was not affected by pretreatment with intranucleus accumbens injection of Y-27632. These findings provide first evidence that Rho/ROCK pathway in the nucleus accumbens may contribute to the increase in extracellular levels of dopamine in the nucleus accumbens evoked by a single subcutaneous injection of methamphetamine. In contrast, this pathway is not essential for the increased level of dopamine in this region induced by morphine, providing further evidence for the different mechanisms of dopamine release by methamphetamine and morphine in rats. [source] Melatonin induces neuritogenesis at early stages in N1E-115 cells through actin rearrangements via activation of protein kinase C and Rho-associated kinaseJOURNAL OF PINEAL RESEARCH, Issue 3 2007Alfredo Bellon Abstract:, Melatonin increases neurite formation in N1E-115 cells through microtubule enlargement elicited by calmodulin antagonism and vimentin intermediate filament reorganization caused by protein kinase C (PKC) activation. Microfilament rearrangement is also a necessary process in growth cone formation during neurite outgrowth. In this work, we studied the effect of melatonin on microfilament rearrangements present at early stages of neurite formation and the possible participation of PKC and the Rho-associated kinase (ROCK), which is a downstream kinase in the PKC signaling pathway. The results showed that 1 nm melatonin increased both the number of cells with filopodia and with long neurites. Similar results were obtained with the PKC activator phorbol 12-myristate 13-acetate (PMA). Both melatonin and PMA increased the quantity of filamentous actin. In contrast, the PKC inhibitor bisindolylmaleimide abolished microfilament organization elicited by either melatonin or PMA, while the Rho inhibitor C3, or the ROCK inhibitor Y27632, abolished the bipolar neurite morphology of N1E-115 cells. Instead, these inhibitors prompted neurite ramification. ROCK activity measured in whole cell extracts and in N1E-115 cells was increased in the presence of melatonin and PMA. The results indicate that melatonin increases the number of cells with immature neurites and suggest that these neurites can be susceptible to differentiation by incoming extracellular signals. Data also indicate that PKC and ROCK are involved at initial stages of neurite formation in the mechanism by which melatonin recruits cells for later differentiation. [source] Melatonin increases stress fibers and focal adhesions in MDCK cells: participation of Rho-associated kinase and protein kinase CJOURNAL OF PINEAL RESEARCH, Issue 2 2007Gerardo Ramírez-Rodríguez Abstract:, Melatonin cyclically modifies water transport measured as dome formation in MDCK cells. An optimal increase in water transport, concomitant with elevated stress fiber (SF) formation, occurs at nocturnal plasma melatonin concentrations (1 nm) after 6 hr of incubation. Blockage in melatonin-elicited dome formation was observed with protein kinase C (PKC) inhibitors. Despite, this information on the precise mechanism by which melatonin increases SF formation involved in water transport is not known. Focal adhesion contacts (FAC) are cytoskeletal structures, which participate in MDCK membrane polarization. SF organization and vinculin phosphorylation are involved in FAC assembly and both processes are mediated by PKC, an enzyme stimulated by melatonin; in these processes also involved is Rho-associated kinase (ROCK). Thus, we studied FAC formation and the ROCK/PKC pathway as the mechanism by which melatonin increases SF formation and water transport. The results showed that 1 nM melatonin and the PKC agonist phorbol-12-miristate-13-acetate increased FAC. The PKC inhibitor GF109203x, and the ROCK inhibitor Y27632, blocked increased FAC caused by melatonin. ROCK and PKC activities, vinculin phosphorylation and FAC formation were increased with melatonin. The PKC inhibitor, GF109203x, abolished both melatonin stimulated FAC in whole cells and ROCK activity, indicating that ROCK is a downstream kinase in the melatonin-stimulated PKC pathway in MDCK cultured cells that causes an increase in SF and FAC formation. Data also document that melatonin modulates water transport through modifications of the cytoskeletal structure. [source] Enhancement of cell recovery for dissociated human embryonic stem cells after cryopreservationBIOTECHNOLOGY PROGRESS, Issue 3 2010Xia Xu Abstract Due to widespread applications of human embryonic stem (hES) cells, it is essential to establish effective protocols for cryopreservation and subsequent culture of hES cells to improve cell recovery. We have developed a new protocol for cryopreservation of dissociated hES cells and subsequent culture. We examined the effects of new formula of freezing solution containing 7.5% dimethylsulfoxide (DMSO) (v/v %) and 2.5% polyethylene glycol (PEG) (w/v %) on cell survival and recovery of hES cells after cryopreservation, and further investigated the role of the combination of Rho-associated kinase (ROCK) inhibitor and p53 inhibitor on cell recovery during the subsequent culture. Compared with the conventional slow-freezing method which uses 10% DMSO as a freezing solution and then cultured in the presence of ROCK inhibitor at the first day of culture, we found out that hES cell recovery was significantly enhanced by around 30 % (P < 0.05) by the new freezing solution. Moreover, at the first day of post-thaw culture, the presence of 10 ,M ROCK inhibitor (Y-27632) and 1 ,M pifithrin-, together further significantly improved cell recovery by around 20% (P < 0.05) either for feeder-dependent or feeder-independent culture. hES cells remained their undifferentiated status after using this novel protocol for cryopreservation and subsequent culture. Furthermore, this protocol is a scalable cryopreservation method for handling large quantities of hES cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Characterization of G proteins involved in activation of nonselective cation channels by endothelinB receptorBRITISH JOURNAL OF PHARMACOLOGY, Issue 7 2002Yoshifumi Kawanabe We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca2+ -permeable nonselective cation channels (NSCC-1 and NSCC-2) in Chinese hamster ovary cells expressing endothelinB receptors (CHO-ETBR) that couple with Gq and Gi. The purpose of the present study was to identify the G proteins involved in the activation of these Ca2+ channels by ET-1. For this purpose, we constructed CHO cells expressing an unpalmitoylated (Cys402Cys403 Cys405,Ser402Ser403Ser405) ETBR (CHO-SerETBR) and ETBR truncated at the cytoplasmic tail downstream of Cys403 (CHO-ETBR,403). Based on the data obtained from actin stress fibre formation, CHO-ETBR couple with G13. Therefore, CHO-ETBR couple with Gq, Gi and G13. CHO-SerETBR and CHO-ETBR,403 couple with G13 and Gq, respectively. ET-1 activated NSCC-1 in CHO-ETBR preincubated with phospholipase C (PLC) inhibitor, U73122, and in CHO-SerETBR. On the other hand, ET-1 failed to activate Ca2+ channels in CHO-ETBR,403. Microinjection of dominant negative mutants of G13 (G13G225A) abolished activation of NSCC-1 and NSCC-2 in CHO-ETBR and that of NSCC-1 in CHO-SerETBR. Y-27632, a specific Rho-associated kinase (ROCK) inhibitor, did not affect the ET-1-induced transient and sustained increase in [Ca2+]i in CHO-ETBR. These results indicate that (1) the cytoplasmic tail downstream of the palmitoylation sites of ETBR, but not the palmitoylation site itself, is essential for coupling with G13, (2) the activation mechanism of each Ca2+ channel by ET-1 is different in CHO-ETBR. NSCC-1 activation depends on G13 -dependent cascade, and NSCC-2 activation depends on both Gq/PLC- and G13 -dependent cascades. Moreover, ROCK-dependent cascade is not involved in the activation of these channels. British Journal of Pharmacology (2002) 136, 1015,1022. doi:10.1038/sj.bjp.0704805 [source] | ||||||||||