Rho GTPases in PC-3 prostate cancer cell morphology,invasion and tumor cell diapedesis

BACKGROUND: The Rho GTPases comprise one of the eight subfamilies of the Ras superfamily of monomeric GTP-binding proteins and are involved in cytoskeletal organization. Previously, using a dominant negative construct, we demonstrated a role for RhoC GTPase in conferring invasive capabilities to PC-3 human prostate cancer cells. Further, we demonstrated that inactivation of RhoC led to morphological changes commensurate with epithelial to mesenchymal transition (EMT) and was accompanied by increased random, linear motility and decreased directed migration and invasion. EMT was related positively to sustained expression and activity of Rac GTPase. In the current study we analyze the individual roles of RhoA, RhoC and Rac1 GTPases in PC-3 cell directed migration, invasion and tumor cell diapedesis across a human bone marrow endothelial cell layer in vitro. RESULTS: Use of specific shRNA directed against RhoA, RhoC or Rac1 GTPases demonstrated a role for each protein in maintaining cell morphology. Furthermore, we demonstrate that RhoC expression and activation is required for directed migration and invasion, while Rac1 expression and activation is required for tumor cell diapedesis. Inhibition of RhoA expression produced a slight increase in invasion and tumor cell diapedesis. CONCLUSIONS: Individual Rho GTPases are required for critical aspects of migration, invasion and tumor cell diapedesis. These data suggest that coordinated activation of individual Rho proteins is required for cells to successfully complete the extravasation process; a key step in distant metastasis.     

Role of small GTPases in Trypanosoma cruzi invasion in MDCK cell lines

Trypanosoma cruzi can modulate a large number of host intracellular responses during its invasion. GTPases such as RhoA, Rac1 and Cdc42 are examples of molecules that could be activated at this moment and trigger changes in the pattern of F-actin cytoskeleton leading to the formation of structures like stress fibers, lamellipodium and fillopodium, respectively. Here we investigate the role of these GTPases in the cytoskeletal rearrangement of MDCK cell transfectants expressing variants of RhoA, Rac1 and Cdc42 during T. cruzi infection. The adhesion, internalization and the survival rate were determined. Rac1 mutants showed the higher adhesion and internalization indexes but the lower survival index after 48 h of infection. Confocal laser scanning microscopy showed changes in the pattern of F-actin distribution and reorganization at the site of trypomastigote invasion. These observations suggest that these GTPases act in the signaling mechanisms that affect the F-actin cytoskeleton during T. cruzi invasion.     

Sequential activation of Rap1 and Rac1 small G proteins by PDGF locally at leading edges of NIH3T3 cells

Moving cells form protrusions, such as filopodia and lamellipodia, and focal complexes at leading edges, which eventually enhance cell movement. The Rho family small G proteins, Rac1, Cdc42 and RhoA, are involved in the formation of these leading edge structures. We investigated the role of another small G protein Rap1 in the platelet-derived growth factor (PDGF)-induced formation of leading edge structures and cell movement. Upon stimulation of NIH3T3 cells by PDGF, leading edge structures were formed and Necl-5, integrin α_Vβ₃, and PDGF receptor were accumulated at leading edges. Rap1, upstream regulators of Rap1 such as Crk and C3G, and a downstream effector RalGDS, were accumulated at peripheral ruffles over lamellipodia. Over-expression of Rap1GAP, which inactivates Rap1, and knockdown of Rap1 inhibited the PDGF-induced formation of leading edge structures, accumulation of these molecules, and cell movement. In addition, Rap1 activation subsequently induced accumulation of Rac1, Vav2 and PAK at peripheral ruffles, which was inhibited by Rap1GAP and knockdown of Rap1. These results indicate that Rap1, activated by PDGF, is recruited to leading edges and that Rac1 is thereby activated locally at peripheral ruffles. This process is pivotal for the PDGF-induced formation of leading edge structures and cell movement.     

Differential activation of Rac1 and RhoA in neuroblastoma cell fractions

The Rho guanine nucleotide triphosphatases (GTPases) Rac1 and RhoA are important regulators of axon growth. However, the specific roles each plays are complicated by implications that each is involved in promoting and inhibiting neurite outgrowth. Differential regulation of Rac1 and RhoA activation in cell bodies and growth cones may be important in directing axon growth. To test this, we separated neuroblastoma cells into growth cone and cell body fractions and assessed Rac1 and RhoA activation in response to outgrowth promoters, serum withdrawal and 8-bromoadeosine-5′,3′-cyclic monophosphate (8-Br-cAMP), and outgrowth inhibitors, chondroitin sulfate proteoglycans (CSPGs) or semaphorin 3A (Sema 3A). In whole cell lysates, serum withdrawal decreased and CSPGs or Sema 3A increased RhoA activity, but no treatments affected Rac1 activity. In growth cones, serum withdrawal or 8-Br-cAMP increased Rac1 activation and serum withdrawal decreased RhoA activation. Conversely, outgrowth inhibitors decreased Rac1 activity. Additionally, 8-Br-cAMP reversed increases in RhoA activity induced by Sema 3A in whole cell lysates and CSPGs in growth cones. These data suggest that activation of RhoA and Rac1 is differentially regulated in specific cellular regions, perhaps contributing to the complexity of Rho GTPase-mediated axon growth.     

Cdc42 GTPase and Rac1 GTPase act downstream of p120 catenin and require GTP exchange during gastrulation of zebrafish mesoderm

Background : We investigated the roles of p120 catenin, Cdc42, Rac1, and RhoA GTPases in regulating migration of presomitic mesoderm cells in zebrafish embryos. p120 catenin has dual roles: It binds the intracellular and juxtamembrane region of cadherins to stabilize cadherin‐mediated adhesion with the aid of RhoA GTPase, and it activates Cdc42 GTPase and Rac1 GTPase in the cytosol to initiate cell motility. Results : During gastrulation of zebrafish embryos, knockdown of the synthesis of zygotic p120 catenin δ1 mRNAs with a splice‐site morpholino caused lateral widening and anterior‐posterior shortening of the presomitic mesoderm and somites and a shortened anterior‐posterior axis. These phenotypes indicate a cell‐migration effect. Co‐injection of low amounts of wild‐type Cdc42 or wild‐type Rac1 or dominant‐negative RhoA mRNAs, but not constitutively‐active Cdc42 mRNA, rescued these p120 catenin δ1‐depleted embryos. Conclusions : These downstream small GTPases require appropriate spatiotemporal stimulation or cycling of GTP to guide mesodermal cell migration. A delicate balance of Rho GTPases and p120 catenin underlies normal development. Developmental Dynamics 241:1545–1561, 2012. © 2012 Wiley Periodicals Inc.     

RhoA activation promotes transendothelial migration of monocytes via ROCK

Monocyte infiltration into inflamed tissue requires the initial arrest of the cells on the endothelium followed by firm adhesion and their subsequent migration. Migration of monocytes and other leukocytes is believed to involve a coordinated remodeling of the actin cytoskeleton. The small GTPases RhoA, Rac1, and Cdc42 are critical regulators of actin reorganization. In this study, we have investigated the role of Rho-like GTPases RhoA, Rac1, and Cdc42 in the adhesion and migration of monocytes across brain endothelial cells by expressing their constitutively active or dominant-negative constructs in NR8383 rat monocytic cells. Monocytes expressing the active form of Cdc42 show a reduced migration, whereas Rac1 expression did not affect adhesion or migration. In contrast, expression of the active form of RhoA in monocytes leads to a dramatic increase in their adhesion and migration across endothelial cells. The effect of RhoA was found to be mediated by its down-stream effector Rho kinase (ROCK), as pretreatment with the selective ROCK inhibitor Y-27632 prevented this enhanced adhesion and migration. These results demonstrate that RhoA activation in monocytes is sufficient to enhance adhesion and migration across monolayers of endothelial cells.     

Rho GTPases control migration and polarization of adhesion molecules and cytoskeletal ERM components in T lymphocytes

Motile lymphocytes adopt a polarized morphology with different adhesion molecules (ICAM, CD43 and CD44) and ERM actin-binding proteins concentrated on the uropod, a slender posterior appendage with important functions in cell-cell interactions and lymphocyte recruitment. We have studied the role of Rho family of GTPases (Rho, Rac and Cdc42) in the control of lymphocyte polarity and migration by analyzing the effects of exogenously introduced Rho GTPase mutants. Transfection of T cell lines that constitutively display a polarized motile morphology with activated mutants of RhoA, Rac1 and Cdc42 impaired cell polarization. A guanosine nucleotide exchange factor for Rac, Tiam-1, induced the same effect as activated Rac1. Conversely, dominant negative forms of the three GTP-binding proteins induced a polarized phenotype in constitutively round-shaped T cells with redistribution of ICAM-3 and moesin to the uropod in an integrin-dependent manner. On the other hand, overexpression of dominant negative Cdc42 and activated mutants of all three Rho GTPases significantly inhibited SDF-1alpha-induced T cell chemotaxis. Together, these data demonstrate that Rho GTPases regulate lymphocyte polarization and chemokine-induced migration, and underscore the key role of Cdc42 in lymphocyte directional migration.     

Rho家族小GTP酶在黑素细胞树突形成和黏附中的作用

黑素细胞通过其树突将合成的黑素转运至角质形成细胞,进而发挥生理功能。黑素细胞树突形成是黑素转运过程中的重要环节,黑素转运必须在黑素细胞和角质形成细胞紧密接触后才能实现。黑素细胞形态学改变包括胞体大小和树突变化等细胞骨架的改变,细胞骨架变化主要与肌动蛋白和微管结构的重排有关。Rho家族小GTP酶包括20种成员,其中RhoA,Rac1和Cdc42对黑素细胞细胞骨架的变化和细胞黏附的调节起重要作用。     

Facilitation of Ca2+-dependent exocytosis by Rac1-GTPase in bovine chromaffin cells

Rho family GTPases are primary mediators of cytoskeletal reorganization, although they have also been reported to regulate cell secretion. Yet, the extent to which Rho family GTPases are activated by secretory stimuli in neural and neuroendocrine cells remains unknown. In bovine adrenal chromaffin cells, we found Rac1, but not Cdc42, to be rapidly and selectively activated by secretory stimuli using an assay selective for the activated GTPases. To examine effects of activated Rac1 on secretion, constitutively active mutants of Rac1 (Rac1-V12, Rac1-L61) were transiently expressed in adrenal chromaffin cells. These mutants facilitated secretory responses elicited from populations of intact and digitonin-permeabilized cells as well as from cells under whole cell patch clamp. A dominant negative Rac1 mutant (Rac1-N17) produced no effect on secretion. Expression of RhoGDI, a negative regulator of Rac1, inhibited secretory responses while overexpression of effectors of Rac1, notably, p21-activated kinase (Pak1) and actin depolymerization factor (ADF) promoted evoked secretion. In addition, expression of effector domain mutants of Rac1-V12 that exhibit reduced activation of the cytoskeletal regulators Pak1 and Partner of Rac1 (POR1) resulted in a loss of Rac1-V12-mediated enhancement of evoked secretion. These findings suggest that Rac1, in part, functions to modulate secretion through actions on the cytoskeleton. Consistent with this hypothesis, the actin modifying drugs phalloidin and jasplakinolide enhanced secretion, while latrunculin-A inhibited secretion and eliminated the secretory effects of Rac1-V12. In summary, Rac1 was activated by secretory stimuli and modulated the secretory pathway downstream of Ca²⁺ influx, partly through regulation of cytoskeletal organization.     

力-化学耦合作用在血管内皮细胞迁移中的作用及其力学生物学机制

目的 研究力学与化学因素的耦合在内皮细胞迁移过程中的作用以及其中的力学生物学机制。方法 在不同大小剪应力下分别用RTPCR、Western blot以及免疫荧光的方法检测内皮细胞CXCR1和CXCR2的表达变化;用antiIL8RA和antiIL8RB拮抗CXCR1和CXCR2,在剪应力作用下观察内皮细胞迁移情况;采用脂质体包绕法分别将Rac1及RhoA的野生型、活化型和抑制型3种质粒转染入内皮细胞,将转染了Rac1的3种质粒的细胞分别施加力学（剪应力）和化学（IL-8）刺激,对转染了RhoA的3种质粒的细胞施加化学刺激,检测以上条件下内皮细胞迁移情况。结果 CXCR1和CXCR2作为新型力学感受器参与调节内皮细胞迁移;Rac1与RhoA的高表达能促进内皮细胞迁移,反之,内皮细胞迁移被抑制。结论 IL-8Rs (CXCR1、CXCR2)、Rac1、RhoA是将力学、化学信号进行“耦合”的关键信号分子。     

Calcium-dependent interaction of Lis1 with IQGAP1 and Cdc42 promotes neuronal motility

Lis1 gene defects impair neuronal migration, causing the severe human brain malformation lissencephaly. Although much is known about its interactions with microtubules, microtubule-binding proteins such as CLIP-170, and with the dynein motor complex, the response of Lis1 to neuronal motility signals has not been elucidated. Lis1 deficiency is associated with deregulation of the Rho-family GTPases Cdc42, Rac1 and RhoA, and ensuing actin cytoskeletal defects, but the link between Lis1 and Rho GTPases remains unclear. We report here that calcium influx enhances neuronal motility through Lis1-dependent regulation of Rho GTPases. Lis1 promotes Cdc42 activation through interaction with the calcium sensitive GTPase scaffolding protein IQGAP1, maintaining the perimembrane localization of IQGAP1 and CLIP170 and thereby tethering microtubule ends to the cortical actin cytoskeleton. Lis1 thus is a key component of neuronal motility signal transduction that regulates the cytoskeleton by complexing with IQGAP1, active Cdc42 and CLIP-170 upon calcium influx.     

A gain-of-function mutation in the PDGFR-beta alters the kinetics of injury response in liver and skin

Platelet-derived growth factor (PDGF) isoforms stimulate cell proliferation, migration and survival. We recently generated mice carrying a gain-of-function mutation within the activation loop of PDGF beta-receptor (PDGFR-beta D849N). Embryonic fibroblasts derived from these mice show elevated basal phosphorylation and altered kinetics for ligand-induced activation of PDGFR-beta, as well as enhanced proliferation and migration. To investigate the effect of this mutation in vivo, we used carbon tetrachloride-induced liver injury as a model system. We observed a higher basal activation of mutant PDGFR-beta in unchallenged livers; however, the difference in activation upon carbon tetrachloride stimulation was lower than expected, an effect that might be explained by a delayed response of the mutated receptor toward reactive oxygen species. Mutant mice showed enhanced proliferation of nonparenchymal liver cells and activation of hepatic stellate cells, leading to a small increase in early fibrosis formation. Another mouse strain lacking the binding site for phosphatidylinositol-3' kinase in PDGFR-beta showed the reverse phenotype. These results suggest an important role for PDGFR-beta signaling in the early injury-response. We confirmed this hypothesis with a second injury model, cutaneous wound healing, where we observed earlier proliferation and formation of granulation tissue in D849N-mutant mice.     

Role of the Rho GTPase-activating protein RICS in neurite outgrowth

The Rho family of small GTPases, including RhoA, Rac1 and Cdc42, are critical regulators of the actin cytoskeleton. In neuronal systems, Rho GTPase-activating proteins (RhoGAPs) and their substrates, Rho GTPases, have been implicated in regulating multiple processes in the morphological development of neurons, including axonal growth and guidance, dendritic elaboration and formation of synapses. RICS is mainly expressed in the brain and functions as a RhoGAP protein for Cdc42 and Rac1 in vitro. To examine the biological function of RICS, we disrupted the RICS gene in mice. RICS knockout mice developed normally and were fertile. However, when cultured in vitro, Cdc42 activity in RICS(-/-) neurons was higher than that in wild-type neurons. Consistent with this finding, hippocampal and cerebellar granule neurons derived from RICS(-/-) mice bore longer neurites than those from wild-type mice. These findings suggest that RICS plays an important role in neurite extension by regulating Cdc42 in vivo.     

GTPases and T cell activation

Summary: Guanine nucleotide binding proteins rapidly cycle between a guanosine diphosphate (GDP)‐bound and guanosine triphosphate (GTP)‐bound state, and they operate as binary switches that control cell activation in response to environmental cues. GTPases adopt different conformations when binding GTP vs. GDP. The GTP‐bound state is generally considered to be the active conformation that allows GTPases to interact with downstream effectors and thereby initiate downstream signaling pathways, which regulate many important biological processes. Many members of the Ras family of GTPases, notably Ras and Rap1A, and the Rho family GTPases, Cdc42Hs, Rac1, Rac2 and RhoA, are important components of signal transduction pathways used by antigen receptors, costimulatory, cytokine and chemokine receptors to regulate the immune response. This review discusses current knowledge and ideas about the regulation and function of these GTPases in lymphocytes.     

Dominant-negative soluble PDGF-beta receptor inhibits hepatic stellate cell activation and attenuates liver fibrosis

Hepatic fibrogenesis is a consequence of hepatic stellate cells that become activated and transdifferentiate into a myofibroblastic phenotype with the ability to proliferate and synthesize large quantities of extracellular matrix components. In this process, platelet-derived growth factor (PDGF) is the most potent stimulus for hepatic stellate cell proliferation and migration, and is overexpressed during active hepatic fibrogenesis. This cytokine binds to the PDGF receptor type beta, activates Ras and sequentially propagates the stimulatory signal sequentially via phosphorylation of Raf-1, MEK and the extracellular-signal regulated kinases ERK1/ERK2. Hepatic injury is associated with both increased autocrine PDGF signaling and upregulation of PDGF receptor. In this study, we report that a dominant-negative soluble PDGF-beta receptor consisting of a chimeric IgG containing the extracellular portion of the PDGF receptor type beta blocks HSC activation and attenuates fibrogenesis induced by ligation of the common bile duct in rats. In culture-activated hepatic stellate cells, the soluble receptor blocks phosphorylation of endogenous PDGF receptor, phosphorylation of the ERK1/EKR2 signal and reduces proliferative activities of HSC. In vivo, both the delivery of the purified soluble PDGF antagonist and the administration of adenoviruses expressing the artificial transgene were able to reduce significantly the expression of collagen and alpha-smooth muscle actin. Our results demonstrate that PDGF plays a critical role in the progression and initiation of experimental liver fibrogenesis, and suggest that early anti-PDGF intervention should have a therapeutical impact on the treatment of liver fibrogenesis.     

Cadherin-11 regulates protrusive activity in Xenopus cranial neural crest cells upstream of Trio and the small GTPases

Xenopus Cadherin-11 (Xcad-11) is expressed when cranial neural crest cells (CNC) acquire motility. However, its function in stimulating cell migration is poorly understood. Here, we demonstrate that Xcad-11 initiates filopodia and lamellipodia formation, which is essential for CNC to populate pharyngeal pouches. We identified the cytoplasmic tail of Xcad-11 as both necessary and sufficient for proper CNC migration as long as it was linked to the plasma membrane. Our results showing that guanine nucleotide exchange factor (GEF)-Trio binds to Xcad-11 and can functionally substitute for it like constitutively active forms of RhoA, Rac, and cdc42 unravel a novel cadherin function.     

ERK activation of p21 activated kinase-1 (Pak1) is critical for medulloblastoma cell migration

We previously identified that overexpression of the platelet-derived growth factor receptor (PDGFR) is associated with metastatic medulloblastoma (MB) and showed that PDGF treatment increases ERK activity and promotes MB cell migration. In this study, we investigated whether ERK regulates Rac1/Pak1 signaling and is critically linked to MB cell migration. Herein we demonstrate that PDGF-BB treatment of MB cells induces concomitant activation of PDGFRβ, MEK1/ERK, Rac1 and Pak1, but suppresses Rho activity, which together significantly promotes cell migration. Conversely, cells transfected with either PDGFRβ or Pak1 siRNA or treated with an inhibitor of Rac1 (NSC23766) or N-myristoyltransferase-1 (Tris-dipalladium) are unable to activate Rac1 or Pak1 in response to PDGF, and consequently, are unable to undergo PDGF-mediated cell migration. Furthermore, we also demonstrate that either chemical inhibition of MEK/ERK (U0126) or stable downregulation of PDGFRβ by shRNA similarly results in the loss of PDGF-induced ERK phosphorylation and abolishes Rac1/Pak1 activation and cell migration in response to PDGF. However, specific depletion of Pak1 by siRNA has no effect on PDGF-induced ERK phosphorylation, indicating that in MB cells ERK signaling is Pak1-independent, but PDGF-induced migration is dependent on ERK-mediated activation of Pak1. Finally, using tissue microarrays, we detect phosphorylated Pak1 in 53% of medulloblastomas and show that immunopositivity is associated with unfavorable outcome. We conclude that Rac1/Pak1 signaling is critical to MB cell migration and is functionally dependent on PDGFRβ/ERK activity.     

Dynamic expression patterns of RhoV/Chp and RhoU/Wrch during chicken embryonic development.

Rho GTPases play central roles in the control of cell adhesion and migration, cell cycle progression, growth, and differentiation. However, although most of our knowledge of Rho GTPase function comes from the study of the three classic Rho GTPases RhoA, Rac1, and Cdc42, recent studies have begun to explore the expression, regulation, and function of some of the lesser-known members of the Rho GTPase family. In the present study, we cloned the avian orthologues of RhoV (or Chp for Cdc42 homologous protein) and RhoU (or Wrch-1 for Wnt-regulated Cdc42 homolog-1) and examined their expression patterns by in situ hybridization analysis both during early chick embryogenesis and later on, during gastrointestinal tract development. Our data show that both GTPases are detected in the primitive streak, the somites, the neural crest cells, and the gastrointestinal tract with distinct territories and/or temporal expression windows. Although both proteins are 90% identical, our results indicate that cRhoV and cRhoU are distinctly expressed during chicken embryonic development.     

Annexin 2 regulates intestinal epithelial cell spreading and wound closure through Rho-related signaling

Epithelial cell migration is a critical event in gastrointestinal mucosal wound healing and is dependent on actin cytoskeletal reorganization. We observed increased expression of an actin regulatory protein, annexin 2, in migrating intestinal epithelial cells. Small interfering RNA (siRNA)-mediated knockdown of annexin 2 expression in Caco-2 epithelial cells resulted in significant reductions in cell spreading and wound closure associated with decreased formation of filamentous actin bundles along the base of migrating cells. Because annexin 2 has been shown to influences actin cytoskeletal remodeling through targeting signaling molecules to membrane domains, we examined the membrane association and activation status of Rho GTPases after annexin 2 knockdown. We observed Rho dissociation from membranes and decreased Rho activity following annexin 2 siRNA transfection. Inhibition of cell spreading and wound closure in annexin 2 siRNA-transfected cells was prevented by expression of constitutively active RhoA. Rho colocalized with annexin 2 in lamellipodia and along the cytoplasmic face of the plasma membrane. In addition, annexin 2 was observed to co-immunoprecipitate with endogenous Rho and constitutively active RhoA. These findings suggest that annexin 2 plays a role in targeting Rho to cellular membranes, thereby modulating Rho-related signaling events regulating cytoskeletal reorganization during epithelial cell migration.     

Statins inhibit T-acute lymphoblastic leukemia cell adhesion and migration through Rap1b

Statins are known to inhibit signaling of Ras superfamily GTPases and reduce T cell adhesion to ICAM-1. Here, we address the hypothesis that statins affect T cell adhesion and migration by modulating the function of specific GTPases. Statins inhibit the synthesis of mevalonic acid, which is required for farnesyl and geranylgeranyl isoprenoid synthesis. Ras superfamily GTPases are post-translationally isoprenylated to facilitate their anchorage to membranes, where they function to stimulate signal transduction processes. We demonstrate that 1 μM statin inhibits the adhesion, migration, and chemotaxis of the T-ALL cell line CCRF-CEM and TEM of CCRF-CEM and PEER T-ALL cells, but higher statin concentrations are needed to inhibit adhesion of primary T cells. Similar effects are observed following treatment with GGTI-298 or RNA interference-mediated knockdown of Rap1b but not Rap1a, Rac1, Rac2, RhoA, or Cdc42. Statins also alter Rap1 activity and Rap1b localization. Rap1 levels are higher in primary T cells than T-ALL cells, which could explain their reduced sensitivity to statins. These results demonstrate for the first time that the closely related Rap1a and Rap1b isoforms have different functions and suggest that statins or Rap1b depletion could be used to reduce tissue invasion in T-ALL.