Small GTPase Rho regulates R-cadherin through Dia1/profilin-1

R-cadherin is a member of the classical cadherins. Though much is known about E-cadherin in adherens junction formation in epithelial cells, the role of R-cadherin in epithelial cells remains elusive. This study examines regulation of R-cadherin adherens junctions by the small GTPase Rho and its downstream effectors in MDA-MB-231 breast cancer cells, MDA-MB-231 cells stably expressing the N-terminus of c-Cbl, and MCF10A normal breast epithelial cells. We find that the small GTPase Rho regulates R-cadherin adherens junction formation via Dia1 (also known as p140mDia) and profilin-1-mediated signaling pathway. The role played by Rho in regulating R-cadherin is underscored by the fact that constitutively active RhoA(Q63L) induces R-cadherin junction formation in MDA-MB-231 cells. Importantly, R-cadherin adherens junction formation facilitates a mesenchymal to epithelial-like transition in MDA-MB-231 cells. Additionally, our data suggest an inverse relationship between EGFR signaling and R-cadherin adherens junction formation. Taken together, results from this study indicate that R-cadherin is a critical regulator of epithelial phenotype.     

Differential expression of Rho1GTPase and Rho3GTPase during isotropic and polarized growth of Mucor circinelloides

Evidence has been obtained that indicates the presence of small 22 kDa GTP-binding Rho proteins through ADP-ribosylation by Clostridium botulinum C3 exotoxin in Mucor circinelloides. Rho protein was detected at all stages of growth studied. During polarized growth, both under aerobic conditions and during the yeast-mycelia transition, the radiolabeling of the [32P]ADP-ribosylated protein increased when tube formation occurred and decreased as the hyphae branched. However, when Mucor grew isotropically, the Rho protein band was thick and its intensity did not vary significantly even after bud formation and separation of daughter cells. Crude extracts of yeast and mycelial cells exhibited a broad 22 kDa band of the [32P]ADP-ribosylated Rho protein that was resolved into a protein with a pI of 6.0, after two-dimensional electrophoresis, corresponding to the Rho1p homolog. Furthermore, [32P]ADP-ribosylated Rho protein from soluble and particulate extracts of multipolarized mycelial cells obtained from the yeast-mycelia transition was separated into two proteins with pI of 6.0 and 6.4, respectively, after two-dimensional electrophoresis. These correspond to the Rho1p and Rho3p homologs, respectively. Therefore, our results show that an increase in Rho accumulation is associated with polarized growth.     

WNK2 modulates MEK1 activity through the Rho GTPase pathway

WNK protein kinases form a kinase subfamily expressed in multi-cellular organisms and the human genome encodes four distinct WNK genes. Human WNK2 has been recently identified as a cell growth regulator that modulates activation of the ERK1/2 protein kinase and is epigenetically silenced in gliomas. Here we provide mechanistic insight into how WNK2 affects ERK activation. We found that WNK2 depletion decreased RhoA activation and promoted GTP-loading of Rac1, leading to stimulation of the Rac1-effector PAK1, which is the kinase responsible for subsequent phosphorylation of MEK1 at serine 298, thereby increasing MEK affinity towards ERK1/2. We propose that WNK2 controls a RhoA-mediated cross-talk mechanism that regulates the efficiency with which MEK1 can activate ERK1/2 upon growth factor stimulation.     

Cyclic AMP blocks bacterial lipopolysaccharide-induced myosin light chain phosphorylation in endothelial cells through inhibition of Rho/Rho kinase signaling

During Gram-negative sepsis bacterial LPS induces endothelial cell contraction, actin reorganization, and loss of endothelial integrity by an unknown signal mechanism. In this study, we provide evidence that LPS-stimulation of endothelial cells (HUVEC) decreases myosin light chain (MLC) phosphatase, resulting in an increase in MLC phosphorylation followed by cell contraction. All of these LPS effects could be blocked by the Rho-GTPase inhibitor C3 transferase from Clostridium botulinum or the Rho kinase inhibitor Y-27632. These data suggest that LPS induces MLC phosphorylation via Rho/Rho kinase-mediated inhibition of MLC phosphatase in HUVEC. Furthermore, we observed that cAMP-elevating drugs, known to exert a vasoprotective function, mimicked the effects of C3 transferase and Y-27632, i.e., inhibited LPS-induced MLC phosphatase inactivation and MLC phosphorylation. cAMP elevation did not inhibit myosin phosphorylation induced by constitutively active V14Rho or the MLC phosphatase inhibitor calyculin and did not induce phosphorylation of RhoA in HUVEC, indicating inhibition of an upstream regulator of Rho/Rho kinase. Taken together, Rho/Rho kinase appears to be a central target for inflammatory mediators causing endothelial cell contraction such as bacterial toxins, but also for vasoprotective molecules elevating intracellular cAMP.     

GTPase regulation: getting aRnd Rock and Rho inhibition

Rnd proteins are atypical members of the Rho small G protein family that inhibit the formation of actomyosin contractile fibers via activation of RhoGAPs and inhibition of a Rho effector, the Ser/Thr kinase Rock. These mechanisms might be used to fine-tune Rho GTPase inhibition locally at sites where particular actin structures need to be made.     

Chrysin suppresses lipopolysaccharide-induced cyclooxygenase-2 expression through the inhibition of nuclear factor for IL-6 (NF-IL6) DNA-binding activity

Chrysin is a natural, biologically active compound extracted from many plants, honey and propolis. It possesses potent anti-inflammation, anti-cancer and anti-oxidation properties. The mechanism by which chrysin suppresses COX-2 expression remains poorly understood. In the present report, we investigated the effect of chrysin on the expression of COX-2 in lipopolysaccharide (LPS)-activated Raw 264.7 cells. Chrysin significantly suppressed the LPS-induced COX-2 protein and mRNA expression in a dose-dependent manner. The ability of chrysin to suppress the expression of the COX-2 was investigated using luciferase reporters controlled by various cis-elements in COX-2 promoter region. Mutational analysis and electrophoretic mobility shift assay verified that nuclear factor for IL-6 was identified as responsible for the chrysin-mediated COX-2 downregulation. These results will provide new insights into the anti-inflammatory and anti-carcinogenic properties of chrysin.     

Rho GTPase function in tumorigenesis

Malignant tumor cells display uncontrolled proliferation, loss of epithelial cell polarity, altered interactions with neighboring cells and the surrounding extracellular matrix, and enhanced migratory properties. Proteins of the Rho GTPase family regulate all these processes in cell culture and, for that reason, Rho GTPases, their regulators, and their effectors have been suggested to control tumor formation and progression in humans. However, while the tumor-relevant functions of Rho GTPases are very well documented in vitro, we are only now beginning to assess their contribution to cancer in human patients and in animal models. This review will give a very brief overview of Rho GTPase function in general and then focus on in vivo evidence for a role of Rho GTPases in malignant tumors, both in human patients and in genetically modified mice.     

The Rho family GTPase Rif induces filopodia through mDia2

Eukaryotic cells produce a variety of specialized actin-rich surface protrusions. These include filopodia-thin, highly dynamic projections that help cells to sense their external environment. Filopodia consist of parallel filaments of actin, bundled by actin crosslinking proteins. The filaments are oriented with their rapidly growing "barbed" ends at the protruding tip and their slowly growing "pointed" ends at the base. Extension occurs by polymerization at the tip and is controlled by regulation of filament capping. The Rho GTPase Cdc42 is a key mediator of filopodia formation, which it regulates through binding CRIB domain-containing effectors. Cdc42 binds and activates the WASP proteins, which in turn activate the actin-nucleating complex Arp2/3. It also binds and activates IRSp53, which recruits the Ena/WASP family protein Mena to the filopodial tip and protects elongating actin filaments from capping. Previously, we identified another Rho family GTPase, Rif, as a potent stimulator of filopodial protrusion through a mechanism that does not require Cdc42. Here we characterize the differences between filopodia induced by these two small GTPases and show that the Rif effector in this pathway is the Diaphanous-related formin mDia2. Thus, Rif and Cdc42 represent two distinct routes to the induction of filopodia-producing structures with both shared and unique properties.     

Rho GTPase/Rho kinase negatively regulates endothelial nitric oxide synthase phosphorylation through the inhibition of protein kinase B/Akt in human endothelial cells

Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiovascular homeostasis by production of nitric oxide (NO) from vascular endothelial cells. It can be activated by protein kinase B (PKB)/Akt via phosphorylation at Ser-1177. We are interested in the role of Rho GTPase/Rho kinase (ROCK) pathway in regulation of eNOS expression and activation. Using adenovirus-mediated gene transfer in human umbilical vein endothelial cells (HUVECs), we show here that both active RhoA and ROCK not only downregulate eNOS gene expression as reported previously but also inhibit eNOS phosphorylation at Ser-1177 and cellular NO production with concomitant suppression of PKB activation. Moreover, coexpression of a constitutive active form of PKB restores the phosphorylation but not gene expression of eNOS in the presence of active RhoA. Furthermore, we show that thrombin inhibits eNOS phosphorylation, as well as expression via Rho/ROCK pathway. Expression of the active PKB reverses eNOS phosphorylation but has no effect on downregulation of eNOS expression induced by thrombin. Taken together, these data demonstrate that Rho/ROCK pathway negatively regulates eNOS phosphorylation through inhibition of PKB, whereas it downregulates eNOS expression independent of PKB.     

Rho GTPase expression in tumourigenesis: evidence for a significant link

Rho proteins belong to the small GTPases superfamily. They function as molecular switches that, in response to diverse stimuli, control key signaling and structural aspects of the cell. Although early studies proposed a role for Rho GTPases in cellular transformation, this effect was underestimated due to the fact that no genetic mutations affecting Rho-encoding genes were found in tumors. Recently, it has become evident that Rho GTPases participate in the carcinogenic process by either overexpression of some of the members of the family with oncogenic activity, downmodulation of other members with suggested tumor suppressor activity, or by alteration of upstream modulators or downstream effectors. Thus, alteration of the levels of expression of different members of the family of Rho GTPases has been detected in many types of human tumors leading to a great interest in the cellular effects elicited by these oncoproteins. This essay reviews the current evidence of dysregulation of Rho signaling by overexpression in human tumors.     

Simvastatin enhances aquaporin-2 surface expression and urinary concentration in vasopressin-deficient Brattleboro rats through modulation of Rho GTPase

Statins are 3-hydroxyl-3-methyglutaryl-CoA reductase inhibitors that are commonly used to inhibit cholesterol biosynthesis. Emerging data have suggested that they also have "pleotropic effects," including modulating actin cytoskeleton reorganization. Here, we report an effect of simvastatin on the trafficking of aquaporin-2 (AQP2). Specifically, simvastatin induced the membrane accumulation of AQP2 in cell cultures and kidneys in situ. The effect of simvastatin was independent of protein kinase A activation and phosphorylation at AQP2-Ser(256), a critical event involved in vasopressin (VP)-regulated AQP2 trafficking. Further investigation showed that simvastatin inhibited endocytosis in parallel with downregulation of RhoA activity. Overexpression of active RhoA attenuated simvastatin's effect, suggesting the involvement of this small GTPase in simvastatin-mediated AQP2 trafficking. Finally, the effect of simvastatin on urinary concentration was investigated in VP-deficient Brattleboro rats. Simvastatin acutely (3-6 h) increased urinary concentration and decreased urine output in these animals. In summary, simvastatin regulates AQP2 trafficking in vitro and urinary concentration in vivo via events involving downregulation of Rho GTPase activity and inhibition of endocytosis. Our study provides an alternative mechanism to regulate AQP2 trafficking, bypassing the VP-vasopressin receptor signaling pathway.     

Bordetella dermonecrotic toxin exerting toxicity through activation of the small GTPase Rho

Bordetella dermonecrotic toxin (DNT) is a virulence factor produced by bacteria belonging to the genus Bordetella. The toxin possesses novel transglutaminase activity that catalyzes polyamination or deamidation of the small GTPases of the Rho family. The modified GTPases loose their GTP hydrolyzing activity, function as a constitutive active molecule, and continuously transduce signals to downstream effectors, which mediate the consequent phenotypes of cells intoxicated by DNT. A dynamin-dependent endocytosis is required for the toxin to be internalized into cells although it is unlikely transported to deep organelles such as the Golgi apparatus or the ER. Several lines of evidence show that the toxin undergoes proteolytic cleavage by furin or furin-like protease probably in the early endosome, and then escapes into the cytoplasm to reach the GTPase.     

Spatial regulation of the exocyst complex by Rho1 GTPase

Spatial regulation of membrane traffic is fundamental to many biological processes, including epithelial cell polarization and neuronal synaptogenesis. The multiprotein exocyst complex is localized to sites of polarized exocytosis, and is required for vesicle targeting and docking at specific domains of the plasma membrane. One component of the complex, Sec3, is thought to be a spatial landmark for polarized exocytosis. We have searched for proteins that regulate the polarized localization of the exocyst in the budding yeast Saccharomyces cerevisiae. Here we report that certain rho1 mutant alleles specifically affect the localization of the exocyst proteins. Sec3 interacts directly with Rho1 in its GTP-bound form, and functional Rho1 is needed both to establish and to maintain the polarized localization of Sec3. Sec3 is not the only mediator of the effect of Rho1 on the exocyst, because some members of the complex are correctly targeted independently of the interaction between Rho1 and Sec3. These results reveal the action of parallel pathways for the polarized localization of the exocytic machinery, both of which are under the control of Rho1, a master regulator of cell polarity.     

The TSC1 tumour suppressor hamartin regulates cell adhesion through ERM proteins and the GTPase Rho

Loss of the tumour-suppressor gene TSC1 is responsible for hamartoma development in tuberous sclerosis complex (TSC), which renders several organs susceptible to benign tumours. Hamartin, the protein encoded by TSC1, contains a coiled-coil domain and is expressed in most adult tissues, although its function is unknown. Here we show that hamartin interacts with the ezrin-radixin-moesin (ERM) family of actin-binding proteins. Inhibition of hamartin function in cells containing focal adhesions results in loss of adhesion to the cell substrate, whereas overexpression of hamartin in cells lacking focal adhesions results in activation of the small GTP-binding protein Rho, assembly of actin stress fibres and formation of focal adhesions. Interaction of endogenous hamartin with ERM-family proteins is required for activation of Rho by serum or by lysophosphatidic acid (LPA). Our data indicate that disruption of adhesion to the cell matrix through loss of hamartin may initiate the development of TSC hamartomas and that a Rho-mediated signalling pathway regulating cell adhesion may constitute a rate-limiting step in tumour formation.     

TOR under stress: Targeting TORC1 by Rho1 GTPase

In the yeast Saccharomyces cerevisiae, small GTPase Rho1 controls polarized actin distribution and cell wall expansion in response to many different environmental and intracellular stimuli. Its activity is essential for cell survival and adaptation under various stress conditions. A recent study identified the TOR complex 1 (TORC1), a central regulator in cell growth and metabolism, as a direct target of the small GTPase. This novel crosstalk extends the signaling network of Rho1 into many TORC1-dependent processes and sheds light on how yeast cells coordinate polarized spatial expansion with mass increase.