Modulation of epithelial tubule formation by Rho kinase

2004 ◽  
Vol 286 (4) ◽  
pp. C857-C866 ◽  
Author(s):  
Randi Eisen ◽  
Don R. Ratcliffe ◽  
George K. Ojakian
Keyword(s):  
Mdck Cells ◽  
Myosin Ii ◽  
Rho Kinase ◽  
Signal Transduction Pathway ◽  
Collagen Gel ◽  
Leading Edge ◽  
Transduction Pathway ◽  
Tubule Formation ◽  
Epithelial Remodeling ◽  
Lamellipodia Formation

We have developed a model system for studying integrin regulation of mammalian epithelial tubule formation. Application of collagen gel overlays to Madin-Darby canine kidney (MDCK) cells induced coordinated disassembly of junctional complexes that was accompanied by lamellipodia formation and cell rearrangement (termed epithelial remodeling). In this study, we present evidence that the Rho signal transduction pathway regulates epithelial remodeling and tubule formation. Incubation of MDCK cells with collagen gel overlays facilitated formation of migrating lamellipodia with membrane-associated actin. Inhibitors of myosin II and actin prevented lamellipodia formation, which suggests that actomyosin function was involved in regulation of epithelial remodeling. To determine this, changes in myosin II distribution, function, and phosphorylation were studied during epithelial tubule biogenesis. Myosin II colocalized with actin at the leading edge of lamellipodia thereby providing evidence that myosin is important in epithelial remodeling. This possibility is supported by observations that inhibition of Rho kinase, a regulator of myosin II function, alters formation of lamellipodia and results in attenuated epithelial tubule development. These data and those demonstrating myosin regulatory light-chain phosphorylation at the leading edge of lamellipodia strongly suggest that Rho kinase and myosin II are important modulators of epithelial remodeling. They support a hypothesis that the Rho signal transduction pathway plays a significant role in regulation of epithelial tubule formation.

Regulation of epithelial tubule formation by Rho family GTPases

2006 ◽  
Vol 290 (5) ◽  
pp. C1297-C1309 ◽  
Author(s):  
Randi Eisen ◽  
Shereaf Walid ◽  
Don R. Ratcliffe ◽  
George K. Ojakian
Keyword(s):  
Signaling Pathway ◽  
Tight Junctions ◽  
Mdck Cells ◽  
Rho Kinase ◽  
Collagen Gel ◽  
Dominant Negative ◽  
Tubule Formation ◽  
Rho Family Gtpases ◽  
Epithelial Remodeling ◽  
Rho Family

Previous work has established that the integrin signal transduction pathway plays an important role in the regulation of epithelial tubule formation. Furthermore, it has been demonstrated that Rho-kinase, an effector of the Rho signaling pathway, is an important downstream modulator of collagen-mediated renal and mammary epithelial tubule morphogenesis. In the present study, MDCK cells that expressed mutant dominant-negative, constitutively active Rho family GTPases were used to provide further insight into Rho-GTPase signaling and the regulation of epithelial tubule formation. Using collagen gel overlays on MDCK cells as a model system, we observed phosphorylated myosin light chain (pMLC) at the leading edge of migrating lamellipodia. This epithelial remodeling led to the formation of multicellular branching epithelial tubular structures with extensive tight junctions. However, in cells expressing dominant-negative RhoN19, MLC phosphorylation, epithelial remodeling, and tubule formation were inhibited. Instead, only small apical lumens with a solitary tight junctional ring were observed, providing further evidence that Rho signaling through Rho-kinase is important in the regulation of epithelial tubule formation. Because the present model for the Rho signaling pathway proposes that Rac plays a prominent but reciprocal role in cell regulation, experiments were conducted using cells that expressed constitutively active RacV12. When incubated with collagen gels, RacV12-expressing cells formed small apical lumens with simple tight junctions, suggesting that Rac1 signaling also has a prominent role in the regulation of epithelial morphogenesis. Complementary collagen gel overlay experiments with wild-type MDCK cells demonstrated that endogenous Rac1 activation levels decreased over a time course consistent with lamellipodia and tubule formation. Under these conditions, Rac1 was initially localized to the basolateral membrane. However, after epithelial remodeling, activated Rac1 was observed primarily in lamellipodia. These studies support a model in which Rac1 and RhoA are important modulators of epithelial tubule formation.

scholarly journals Cdc42 and noncanonical Wnt signal transduction pathways cooperate to promote cell polarity

2007 ◽  
Vol 178 (3) ◽  
pp. 355-361 ◽  
Author(s):  
Karni Schlessinger ◽  
Edward J. McManus ◽  
Alan Hall
Keyword(s):  
Signal Transduction ◽  
Wnt Signaling Pathway ◽  
Signal Transduction Pathway ◽  
Leading Edge ◽  
Transduction Pathway ◽  
Adenomatous Polyposis ◽  
Polyposis Coli ◽  
Gsk 3Β ◽  
Apc Protein ◽  
Wnt Signal

Scratch-induced disruption of cultured monolayers induces polarity in front row cells that can be visualized by spatially localized polymerization of actin at the front of the cell and reorientation of the centrosome/Golgi to face the leading edge. We previously reported that centrosomal reorientation and microtubule polarization depend on a Cdc42-regulated signal transduction pathway involving activation of the Par6/aPKC complex followed by inhibition of GSK-3β and accumulation of the adenomatous polyposis coli (APC) protein at the plus ends of leading-edge microtubules. Using monolayers of primary rodent embryo fibroblasts, we show here that dishevelled (Dvl) and axin, two major components of the Wnt signaling pathway are required for centrosome reorientation and that Wnt5a is required for activation of this pathway. We conclude that disruption of cell–cell contacts leads to the activation of a noncanonical Wnt/dishevelled signal transduction pathway that cooperates with Cdc42/Par6/aPKC to promote polarized reorganization of the microtubule cytoskeleton.

scholarly journals Role of Phosphatidylinositol 4,5-Bisphosphate in Ras/Rac-Induced Disruption of the Cortactin-Actomyosin II Complex and Malignant Transformation

1998 ◽  
Vol 18 (7) ◽  
pp. 3829-3837 ◽  
Author(s):  
Hong He ◽  
Takeshi Watanabe ◽  
Xi Zhan ◽  
Cai Huang ◽  
Ed Schuuring ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Malignant Transformation ◽  
Myosin Ii ◽  
Signal Transduction Pathway ◽  
Dependent Manner ◽  
Transduction Pathway ◽  
Membrane Ruffling ◽  
Oncogenic Ras ◽  
Cytoskeletal Change

ABSTRACT Oncogenic Ras mutants such as v-Ha-Ras cause a rapid rearrangement of actin cytoskeleton during malignant transformation of fibroblasts or epithelial cells. Both PI-3 kinase and Rac are required for Ras-induced malignant transformation and membrane ruffling. However, the signal transduction pathway(s) downstream of Rac that leads to membrane ruffling and other cytoskeletal change(s) as well as the exact biochemical nature of the cytoskeletal change remain unknown. Cortactin/EMS1 is the first identified molecule that is dissociated in a Rac–phosphatidylinositol 4,5-biphosphate (PIP2)-dependent manner from the actin-myosin II complex during Ras-induced malignant transformation; either the PIP2 binder HS1 or the Rac blocker SCH51344 restores the ability of EMS1 to bind the complex and suppresses the oncogenicity of Ras. Furthermore, while PIP2 inhibits the actin-EMS1 interaction, HS1 reverses the PIP2 effect. Thus, we propose that PIP2, an end-product of the oncogenic Ras/PI-3 kinase/Rac pathway, serves as a second messenger in the Ras/Rac-induced disruption of the actin cytoskeleton and discuss the anticancer drug potential of PIP2-binding molecules.

Drosophila myosin phosphatase and its role in dorsal closure

Development ◽  
2002 ◽  
Vol 129 (5) ◽  
pp. 1215-1223 ◽  
Author(s):  
Tomoaki Mizuno ◽  
Kyoko Tsutsui ◽  
Yasuyoshi Nishida
Keyword(s):  
Genetic Interaction ◽  
Shape Change ◽  
Myosin Ii ◽  
Rho Kinase ◽  
Leading Edge ◽  
Subcellular Location ◽  
Dorsal Closure ◽  
Nonmuscle Myosin ◽  
Myosin Phosphatase ◽  
Nonmuscle Myosin Ii

Myosin phosphatase negatively regulates nonmuscle myosin II through dephosphorylation of the myosin regulatory light chain (MRLC). Its regulatory myosin-binding subunit, MBS, is responsible for regulating the catalytic subunit in response to upstream signals and for determining the substrate specificity. DMBS, the Drosophila homolog of MBS, was identified to study the roles of myosin phosphatase in morphogenesis. The embryos defective for both maternal and zygotic DMBS demonstrated a failure in dorsal closure. In the mutant embryos, the defects were mainly confined to the leading edge cells which failed to fully elongate. Ectopic accumulation of phosphorylated MRLC was detected in lateral region of the leading edge cells, suggesting that the role of DMBS is to repress the activation of nonmuscle myosin II at the subcellular location for coordinated cell shape change. Aberrant accumulation of F-actin within the leading edge cells may correspond to the morphological aberrations of such cells. Similar defects were seen in embryos overexpressing Rho-kinase, suggesting that myosin phosphatase and Rho-kinase function antagonistically. The genetic interaction of DMBS with mutations in the components of the Rho signaling cascade also indicates that DMBS functions antagonistically to the Rho signal transduction pathway. The results indicate an important role for myosin phosphatase in morphogenesis.

scholarly journals CKA, a Novel Multidomain Protein, Regulates the JUN N-Terminal Kinase Signal Transduction Pathway in Drosophila

2002 ◽  
Vol 22 (6) ◽  
pp. 1792-1803 ◽  
Author(s):  
Hua-Wei Chen ◽  
Maria Julia Marinissen ◽  
Su-Wan Oh ◽  
Xiu Chen ◽  
Michael Melnick ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Cell Sheet ◽  
Signal Transduction Pathway ◽  
Leading Edge ◽  
Genetic Screen ◽  
Signal Transduction Pathways ◽  
Dorsal Closure ◽  
Transduction Pathway ◽  
Temporal Expression ◽  
Multidomain Protein

ABSTRACT The Drosophila melanogaster JUN N-terminal kinase (DJNK) and DPP (decapentaplegic) signal transduction pathways coordinately regulate epithelial cell sheet movement during the process of dorsal closure in the embryo. By a genetic screen of mutations affecting dorsal closure in Drosophila, we have now identified a multidomain protein, connector of kinase to AP-1 (cka), that functions in the DJNK pathway and controls the localized expression of dpp in the leading-edge cells. We have also investigated how CKA acts. This unique molecule forms a complex with HEP (DJNKK), BSK (DJNK), DJUN, and DFOS. Complex formation activates BSK kinase, which in turn phosphorylates and activates DJUN and DFOS. These data suggest that CKA represents a novel molecule regulating AP-1 activity by organizing a molecular complex of kinases and transcription factors, thus coordinating the spatial-temporal expression of AP-1-regulated genes.

scholarly journals Arp2/3 complex–dependent actin networks constrain myosin II function in driving retrograde actin flow

2012 ◽  
Vol 197 (7) ◽  
pp. 939-956 ◽  
Author(s):  
Qing Yang ◽  
Xiao-Feng Zhang ◽  
Thomas D. Pollard ◽  
Paul Forscher
Keyword(s):  
Growth Cone ◽  
Myosin Ii ◽  
Rho Kinase ◽  
Leading Edge ◽  
Actin Dynamics ◽  
Actin Networks ◽  
Motile Cells ◽  
Nonmuscle Myosin Ii ◽  
Neuronal Growth Cone ◽  
Contractile Forces

The Arp2/3 complex nucleates actin filaments to generate networks at the leading edge of motile cells. Nonmuscle myosin II produces contractile forces involved in driving actin network translocation. We inhibited the Arp2/3 complex and/or myosin II with small molecules to investigate their respective functions in neuronal growth cone actin dynamics. Inhibition of the Arp2/3 complex with CK666 reduced barbed end actin assembly site density at the leading edge, disrupted actin veils, and resulted in veil retraction. Strikingly, retrograde actin flow rates increased with Arp2/3 complex inhibition; however, when myosin II activity was blocked, Arp2/3 complex inhibition now resulted in slowing of retrograde actin flow and veils no longer retracted. Retrograde flow rate increases induced by Arp2/3 complex inhibition were independent of Rho kinase activity. These results provide evidence that, although the Arp2/3 complex and myosin II are spatially segregated, actin networks assembled by the Arp2/3 complex can restrict myosin II–dependent contractility with consequent effects on growth cone motility.

Rho kinase activation maintains high pulmonary vascular resistance in the ovine fetal lung

2006 ◽  
Vol 291 (5) ◽  
pp. L976-L982 ◽  
Author(s):  
Thomas A. Parker ◽  
Gates Roe ◽  
Theresa R. Grover ◽  
Steven H. Abman
Keyword(s):  
Kinase Inhibitors ◽  
Rho Kinase ◽  
Signal Transduction Pathway ◽  
Fetal Lung ◽  
No Synthase ◽  
Transduction Pathway ◽  
Pulmonary Vasodilation ◽  
Rho Kinase Inhibitors ◽  
Pulmonary Vasoconstriction ◽  
High Pulmonary Vascular Resistance

Mechanisms that maintain high pulmonary vascular resistance (PVR) in the fetal lung are poorly understood. Activation of the Rho kinase signal transduction pathway, which promotes actin-myosin interaction in vascular smooth muscle cells, is increased in the pulmonary circulation of adult animals with experimental pulmonary hypertension. However, the role of Rho kinase has not been studied in the fetal lung. We hypothesized that activation of Rho kinase contributes to elevated PVR in the fetus. To address this hypothesis, we studied the pulmonary hemodynamic effects of brief (10 min) intrapulmonary infusions of two specific Rho kinase inhibitors, Y-27632 (15–500 μg) and HA-1077 (500 μg), in chronically prepared late-gestation fetal lambs ( n = 9). Y-27632 caused potent, dose-dependent pulmonary vasodilation, lowering PVR from 0.67 ± 0.18 to 0.16 ± 0.02 mmHg·ml−1·min−1 ( P < 0.01) at the highest dose tested without lowering systemic arterial pressure. Despite brief infusions, Y-27632-induced pulmonary vasodilation was sustained for 50 min. HA-1077 caused a similar fall in PVR, from 0.39 ± 0.03 to 0.19 ± 0.03 ( P < 0.05). To study nitric oxide (NO)-Rho kinase interactions in the fetal lung, we tested the effect of Rho kinase inhibition on pulmonary vasoconstriction caused by inhibition of endogenous NO production with nitro-l-arginine (l-NA; 15–30 mg), a selective NO synthase antagonist. l-NA increased PVR by 127 ± 73% above baseline under control conditions, but this vasoconstrictor response was completely prevented by treatment with Y-27632 ( P < 0.05). We conclude that the Rho kinase signal transduction pathway maintains high PVR in the normal fetal lung and that activation of the Rho kinase pathway mediates pulmonary vasoconstriction after NO synthase inhibition. We speculate that Rho kinase plays an essential role in the normal fetal pulmonary circulation and that Rho kinase inhibitors may provide novel therapy for neonatal pulmonary hypertension.

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