Cadherin engagement regulates Rho family GTPases.

The formation of cell-cell adherens junctions is a cadherin-mediated process associated with reorganization of the actin cytoskeleton. Because Rho family GTPases regulate actin dynamics, we investigated whether cadherin-mediated adhesion regulates the activity of RhoA, Rac1, and Cdc42. Confluent epithelial cells were found to have elevated Rac1 and Cdc42 activity but decreased RhoA activity when compared with low density cultures. Using a calcium switch method to manipulate junction assembly, we found that induction of cell-cell junctions increased Rac1 activity, and this was inhibited by E-cadherin function-blocking antibodies. Using the same calcium switch procedure, we found little effect on RhoA activity during the first hour of junction assembly. However, over several hours, RhoA activity significantly decreased. To determine whether these effects are mediated directly through cadherins or indirectly through engagement of other surface proteins downstream from junction assembly, we used a model system in which cadherin engagement is induced without cell-cell contact. For these experiments, Chinese hamster ovary cells expressing C-cadherin were plated on the extracellular domain of C-cadherin immobilized on tissue culture plates. Whereas direct cadherin engagement did not stimulate Cdc42 activity, it strongly inhibited RhoA activity but increased Rac1 activity. Deletion of the C-cadherin cytoplasmic domain abolished these effects.     

The Rho GTPase effector ROCK regulates cyclin A, cyclin D1, and p27Kip1 levels by distinct mechanisms

The members of the Rho GTPase family are well known for their regulation of actin cytoskeletal structures. In addition, they influence progression through the cell cycle. The RhoA and RhoC proteins regulate numerous effector proteins, with a central and vital signaling role mediated by the ROCK I and ROCK II serine/threonine kinases. The requirement for ROCK function in the proliferation of numerous cell types has been revealed by studies utilizing ROCK-selective inhibitors such as Y-27632. However, the mechanisms by which ROCK signaling promotes cell cycle progression have not been thoroughly characterized. Using a conditionally activated ROCK-estrogen receptor fusion protein, we found that ROCK activation is sufficient to stimulate G1/S cell cycle progression in NIH 3T3 mouse fibroblasts. Further analysis revealed that ROCK acts via independent pathways to alter the levels of cell cycle regulatory proteins: cyclin D1 and p21(Cip1) elevation via Ras and the mitogen-activated protein kinase pathway, increased cyclin A via LIM kinase 2, and reduction of p27(Kip1) protein levels. Therefore, the influence of ROCK on cell cycle regulatory proteins occurs by multiple independent mechanisms.     

External mechanical strain regulates membrane targeting of Rho GTPases by controlling microtubule assembly

Transmission of externallyapplied mechanical forces to the interior of a cell requirescoordination of biochemical signaling pathways with changes incytoskeletal assembly and organization. In this study, we addressed onepotential mechanism for this signal integration by applying uniformsingle external mechanical strains to aortic smooth muscle cells (SMCs)via their adhesion substrate. A tensile strain applied to the substratefor 15 min significantly increased microtubule (MT) assembly by 32 ± 7%, with no apparent effect on the cells' focal adhesions asrevealed by immunofluorescence and quantitative analysis of TritonX-100-insoluble vinculin levels. A compressive strain decreased MT massby 24 ± 9% but did not influence the level of vinculin in focaladhesions. To understand the decoupling of these two cell responses tomechanical strain, we examined a redistribution of the small GTPasesRhoA and Rac. Tensile strain was found to decrease the amount ofmembrane-associated RhoA and Rac by 70 ± 9% and 45 ± 11%,respectively, compared with static controls. In contrast, compressivestrain increased membrane-associated RhoA and Rac levels by 74 ± 17% and 36 ± 13%, respectively. Disruption of the MT network byprolonged treatments with low doses of either nocodazole or paclitaxelbefore the application of strain abolished the redistribution of RhoAand Rac in response to the applied forces. Combined, these resultsindicate that the effects of externally applied mechanical strain onthe distribution and activation of the Rho family GTPases requirechanges in the state of MT polymerization.

     

Caveolin-1 regulates cell polarization and directional migration through Src kinase and Rho GTPases

Development, angiogenesis, wound healing, and metastasis all involve the movement of cells in response to changes in the extracellular environment. To determine whether caveolin-1 plays a role in cell migration, we have used fibroblasts from knockout mice. Caveolin-1–deficient cells lose normal cell polarity, exhibit impaired wound healing, and have decreased Rho and increased Rac and Cdc42 GTPase activities. Directional persistency of migration is lost, and the cells show an impaired response to external directional stimuli. Both Src inactivation and p190RhoGAP knockdown restore the wild-type phenotype to caveolin-1–deficient cells, suggesting that caveolin-1 stimulates normal Rho GTP loading through inactivation of the Src–p190RhoGAP pathway. These findings highlight the importance of caveolin-1 in the establishment of cell polarity during directional migration through coordination of the signaling of Src kinase and Rho GTPases.     

The integrin cytoplasmic domain-associated protein ICAP-1 binds and regulates Rho family GTPases during cell spreading.

Using two-hybrid screening, we isolated the integrin cytoplasmic domain-associated protein (ICAP-1), an interactor for the COOH terminal region of the beta1A integrin cytoplasmic domain. To investigate the role of ICAP-1 in integrin-mediated adhesive function, we expressed the full-length molecule in NIH3T3 cells. ICAP-1 expression strongly prevents NIH3T3 cell spreading on extracellular matrix. This inhibition is transient and can be counteracted by coexpression of a constitutively activated mutant of Cdc42, suggesting that ICAP-1 acts upstream of this GTPase. In addition, we found that ICAP-1 binds both to Cdc42 and Rac1 in vitro, and its expression markedly inhibits activation of these GTPases during integrin-mediated cell adhesion to fibronectin as detected by PAK binding assay. In the attempt to define the molecular mechanism of this inhibition, we show that ICAP-1 reduces both the intrinsic and the exchange factor-induced dissociation of GDP from Cdc42; moreover, purified ICAP-1 displaces this GTPase from cellular membranes. Together, these data show for the first time that ICAP-1 regulates Rho family GTPases during integrin-mediated cell matrix adhesion, acting as guanine dissociation inhibitor.     

p120 catenin regulates the actin cytoskeleton via Rho family GTPases

Cadherins are calcium-dependent adhesion molecules responsible for the establishment of tight cell-cell contacts. p120 catenin (p120ctn) binds to the cytoplasmic domain of cadherins in the juxtamembrane region, which has been implicated in regulating cell motility. It has previously been shown that overexpression of p120ctn induces a dendritic morphology in fibroblasts (Reynolds, A.B. , J. Daniel, Y. Mo, J. Wu, and Z. Zhang. 1996. Exp. Cell Res. 225:328-337.). We show here that this phenotype is suppressed by coexpression of cadherin constructs that contain the juxtamembrane region, but not by constructs lacking this domain. Overexpression of p120ctn disrupts stress fibers and focal adhesions and results in a decrease in RhoA activity. The p120ctn-induced phenotype is blocked by dominant negative Cdc42 and Rac1 and by constitutively active Rho-kinase, but is enhanced by dominant negative RhoA. p120ctn overexpression increased the activity of endogenous Cdc42 and Rac1. Exploring how p120ctn may regulate Rho family GTPases, we find that p120ctn binds the Rho family exchange factor Vav2. The behavior of p120ctn suggests that it is a vehicle for cross-talk between cell-cell junctions and the motile machinery of cells. We propose a model in which p120ctn can shuttle between a cadherin-bound state and a cytoplasmic pool in which it can interact with regulators of Rho family GTPases. Factors that perturb cell-cell junctions, such that the cytoplasmic pool of p120ctn is increased, are predicted to decrease RhoA activity but to elevate active Rac1 and Cdc42, thereby promoting cell migration.     

Rho GTPases and spermatogenesis

Rho GTPases, such as Rho, Rac and Cdc42, are known to regulate many cellular processes including cell movement and cell adhesion. While the cellular events of germ cell movement are crucial to spermatogenesis since developing germ cells must migrate progressively from the basal to the adluminal compartment but remain attached to the seminiferous epithelium, the physiological significance of Rho GTPases in spermatogenesis remains largely unexplored. This paper reviews some recent findings on Rho GTPases in the field with emphasis on the studies in the testis, upon which future studies can be designed to delineate the role of Rho GTPases in spermatogenesis.     

Control of vesicular trafficking by Rho GTPases

Although vesicular trafficking is essential for a large variety of cellular processes, the regulation of vesicular trafficking is still poorly understood. Members of the Rho family of small GTPases have recently emerged as important control elements of many stages of vesicular trafficking, providing new insight into the regulation of these events. We will discuss the diverse roles played by Rho proteins in membrane trafficking and focus on the biological implications of these functions.     

Regulation of innate immunity by Rho GTPases

Leukocytes are key cellular components of innate immunity. These phagocytic cells respond to bacteria at sites of infection through chemotactic sensing and directed motility regulated by Rho GTPases. The development of sensitive probes of Rho GTPase dynamics has provided insights into the temporal and spatial aspects of GTPase regulation during chemotaxis and subsequent microbial phagocytosis. The resulting destruction of ingested bacteria by means of reactive oxygen species (ROS) depends on a Rac-regulated "molecular switch" that is modulated by antagonistic crosstalk involving Cdc42. Recent studies of leukocytes derived from Rac1- and Rac2-knockout mice have shown that these highly homologous GTPases have unique biological roles. An understanding of the biochemical basis for such distinct activities should provide novel insights into the molecular details of Rho GTPase function and regulation in innate immunity.     

Regulation of PTEN by Rho small GTPases

PTEN (phosphatase and tensin homologue) is a phosphatase that dephosphorylates both protein and phosphoinositide substrates. It is mutated in a variety of human tumours and has important roles in a diverse range of biological processes, including cell migration and chemotaxis. PTEN's intracellular localization and presumably activity are regulated by chemoattractants in Dictyostelium and mouse neutrophils. However, the mechanisms for its regulation remain elusive. Here we show that RhoA and Cdc42, members of the Rho family of small GTPases, regulate the intracellular localization of PTEN in leukocytes and human transfected embryonic kidney cells. In addition, active RhoA is able to stimulate the phospholipid phosphatase activity of PTEN in human embryonic kidney cells and leukocytes, and this regulation seems to require RhoA's downstream effector, RhoA-associated kinase (Rock). Furthermore, we have identified key residues on PTEN that are required for its regulation by the small GTPase, and show that small GTPase-mediated regulation of PTEN has a significant role in the regulation of chemotaxis.     

Rho GTPases与肿瘤

Rho GTPases参与调控细胞的多种关键生物学行为,特别是细胞的生长、细胞骨架的形成、转录调节等生物学过程. 在肿瘤的发生发展中Rho GTPases也扮演了重要的角色．本文将回顾Rho GTPases的调控（包括经典及非经典调控方式）及其关键成员（RhoA、Cdc42及Rac1）与临床肿瘤的研究进展,特别是它们参与调控肿瘤的增殖、迁移、侵袭、凋亡等恶性生物学行为,从而为研发靶向Rho GTPases的小分子/基因药物了奠定基础．     

Lck regulates Vav activation of members of the Rho family of GTPases.

Vav is a member of a family of oncogene proteins that share an approximately 250-amino-acid motif called a Dbl homology domain. Paradoxically, Dbl itself and other proteins containing a Dbl domain catalyze GTP-GDP exchange for Rho family proteins, whereas Vav has been reported to catalyze GTP-GDP exchange for Ras proteins. We present Saccharomyces cerevisiae genetic data, in vitro biochemical data, and animal cell biological data indicating that Vav is a guanine nucleotide exchange factor for Rho-related proteins, but in similar genetic and biochemical experiments we fail to find evidence that Vav is a guanine nucleotide exchange factor for Ras. Further, we present data indicating that the Lck kinase activates the guanine nucleotide exchange factor and transforming activity of Vav.     

Podosome regulation by Rho GTPases in myeloid cells

Myeloid cells form a first line of defense against infections. They migrate from the circulation to the infected tissues by adhering to and subsequently crossing the vascular wall. This process requires precise control and proper regulation of these interactions with the environment is therefore crucial. Podosomes are the most prominent adhesion structures in myeloid cells. Podosomes control both the adhesive and migratory properties of myeloid cells and the regulation of podosomes is key to the proper functioning of these cells. Here we discuss the regulation of podosomes by Rho GTPases, well known regulators of adhesion and migration, focusing on myeloid cells. In addition, the regulation of podosomes by GTPase regulators such as GEFs and GAPs, as well as the effects of some Rho GTPase effector pathways, will be discussed.     

Rho GTPases in hepatocellular carcinoma

Rho GTPases are major regulators of signal transduction pathways and play key roles in processes including actin dynamics, cell cycle progression, cell survival and gene expression, whose deregulation may lead to tumorigenesis. A growing number of in vitro and in vivo studies using tumor-derived cell lines, primary tumors and animal cancer models strongly suggest that altered Rho GTPase signaling plays an important role in the initiation as well as in the progression of hepatocellular carcinoma (HCC), one of the deadliest human cancers in the world. These alterations can occur at the level of the GTPases themselves or of one of their regulators or effectors. The participation into the tumorigenic process can occur either through the over-expression of one of these components which presents an oncogenic activity as illustrated with RhoA and C or through the attenuation of the expression of a component presenting tumor suppressor activity as for Cdc42 or the RhoGAP, DLC-1. Consequently, these observations reflect the heterogeneity and the complexity of liver carcinogenesis. Recently, pharmacological approaches targeting Rho GTPase signaling have been used in HCC-derived models with relative success but remain to be validated in more physiologically relevant systems. Therefore, therapeutic approaches targeting Rho GTPase signaling may provide a novel alternative for anti-HCC therapy.     

The role of Rho GTPases in disease development

The functionality and efficacy of Rho GTPase signaling is pivotal for a plethora of biological processes. Due to the integral nature of these molecules, the dysregulation of their activities can result in diverse aberrant phenotypes. Dysregulation can, as will be described below, be based on an altered signaling strength on the level of a specific regulator or that of the respective GTPase itself. Alternatively, effector pathways emanating from a specific Rho GTPase may be under- or overactivated. In this review, we address the role of the Rho-type GTPases as a subfamily of the Ras-superfamily of small GTP-binding proteins in the development of various disease phenotypes. The steadily growing list of genetic alterations that specifically impinge on proper Rho GTPase function corresponds to pathological categories such as cancer progression, mental disabilities and a group of quite diverse and unrelated disorders. We will provide an overview of disease-rendering mutations in genes that have been positively correlated with Rho GTPase signaling and will discuss the cellular and molecular mechanisms that may be affected by them.     

The Rho GTPases in Macrophage Motility and Chemotaxis

The GTP-binding proteins, Rho, Rac and Cdc42 are known to regulate actin organisation. Rho induces the assembly of contractile actin-based microfilaments such as stress fibres, Rac regulates the formation of membrane ruffles and lamellipodia, and Cdc42 activation is necessary for the formation of filopodia. In addition, all three proteins can also regulate the assembly of integrin-containing focal adhesion complexes. The orchestration of these distinct cytoskeletal changes is thought to form the basis of the co-ordination of cell motility and we have investigated the roles of Rho family proteins in migration using a model system. We have found that in the macrophage cell line Bacl, the cytokine CSF-1 rapidly induces actin reorganisation: it stimulates the formation of filopodia, lamellipodia and membrane ruffles, as well as the appearance of fine actin cables within the cell. We have shown that Cdc42, Rac and Rho regulate the CSF-1 induced formation of these distinct actin filament-based structures. Using a cell tracking procedure we found that both Rho and Rac were required for CSF-1 stimulated cell translocation. In contrast, inhibition of Cdc42 does not prevent macrophages migrating in response to CSF-1, but does prevent recognition of a CSF-1 concentration gradient, so that cells now migrate randomly rather than up the gradient of this chemotactic cytokine. This implies that Cdc42, and thus probably filopodia, are required for gradient sensing and cell polarisation in macrophages.     

Integrin-mediated adhesion regulates cell polarity and membrane protrusion through the Rho family of GTPases

Integrin-mediated adhesion is a critical regulator of cell migration. Here we demonstrate that integrin-mediated adhesion to high fibronectin concentrations induces a stop signal for cell migration by inhibiting cell polarization and protrusion. On fibronectin, the stop signal is generated through alpha 5 beta 1 integrin-mediated signaling to the Rho family of GTPases. Specifically, Cdc42 and Rac1 activation exhibits a biphasic dependence on fibronectin concentration that parallels optimum cell polarization and protrusion. In contrast, RhoA activity increases with increasing substratum concentration. We find that cross talk between Cdc42 and Rac1 is required for substratum-stimulated protrusion, whereas RhoA activity is inhibitory. We also show that Cdc42 activity is inhibited by Rac1 activation, suggesting that Rac1 activity may down-regulate Cdc42 activity and promote the formation of stabilized rather than transient protrusion. Furthermore, expression of RhoA down-regulates Cdc42 and Rac1 activity, providing a mechanism whereby RhoA may inhibit cell polarization and protrusion. These findings implicate adhesion-dependent signaling as a mechanism to stop cell migration by regulating cell polarity and protrusion via the Rho family of GTPases.     

Regulation of Rho GTPases by p120-catenin.

Three recent reports indicate that p120-catenin can modulate the activities of RhoA, Rac and Cdc42, suggesting an elegant and previously unexpected mechanism for regulating the balance between adhesive and motile cellular phenotypes. The observations in these reports provide important new clues toward p120's mechanism of action and provide a potential explanation for the metastatic phenotype exhibited in carcinoma cells that have lost E cadherin expression.