Rho-mediated cytoskeletal rearrangement in response to LPA is functionally antagonized by Rac1 and PIP2

G-protein-coupled receptors signal through Rho to induce actin cytoskeletal rearrangement. We previously demonstrated that thrombin stimulates Rho-dependent process retraction and rounding of 1321N1 astrocytoma cells. Surprisingly, while lysophosphatidic acid (LPA) activated RhoA in 1321N1 cells, it failed to produce cell rounding. Thrombin, unlike LPA, decreased Rac1 activity, and activated (GTPase-deficient) Rac1 inhibited thrombin-stimulated cell rounding, while expression of dominant-negative Rac1 promoted LPA-induced rounding. LPA and thrombin receptors appear to differ in coupling to Gi, as LPA but not thrombin-stimulated 1321N1 cell proliferation was pertussis toxin-sensitive. Blocking Gi with pertussis toxin enabled LPA to induce cell rounding and to decrease activated Rac1. These data support the hypothesis that Rac1 and Gi activation antagonize cell rounding. Thrombin and LPA receptors also differentially activated Gq pathways as thrombin but not LPA increased InsP3 formation and reduced phosphatidylinositol 4,5-bisphosphate (PIP2) levels. Microinjection of the plekstrin homology domain of phospholipase C (PLC)delta1, which binds PIP2, enabled LPA to elicit cell rounding, consistent with a requirement for PIP2 reduction. We suggest that Rho-mediated cytoskeletal responses are enhanced by concomitant reductions in cellular levels of PIP2 and Rac1 activation and thus effected only by G-protein-coupled receptors with appropriate subsets of G protein activation.     

Ezrin-related Phosphoinositide pathway modifies RhoA and Rac1 in human osteosarcoma cell lines

Selected Phosphoinositide-specific Phospholipase C (PI-PLC) enzymes occupy the convergence point of the broad range of pathways that promote Rho and Ras GTPase mediated signalling, which also regulate the activation of ezrin, a member of the ezrin-radixin-moesin (ERM) proteins family involved in the metastatic osteosarcoma spread. Previous studies described that in distinct human osteosarcoma cell lines ezrin networks the PI-PLC with complex interplay controlling the expression of the PLC genes, which codify for PI-PLC enzymes. In the present study, we analyzed the expression and the sub-cellular distribution of RhoA and Rac1 respectively after ezrin silencing and after PI-PLC ε silencing, in order to investigate whether ezrin-RhoGTPAses signalling might involve one or more specific PI-PLC isoforms in cultured 143B and Hs888 human osteosarcoma cell lines. In the present experiments, both ezrin and PLCE gene silencing had different effects upon RhoA and Rac1 expression and sub-cellular localization. Displacements of Ezrin and of RhoA localization were observed, probably playing functional roles.     

Rac1 activity is required for cardiac myocyte alignment in response to mechanical stress

Mechanical stretch is essential for the cardiac growth. The exposure of cardiac myocytes to the mechanical stretch leads to the cell alignment in parallel to the stretch direction, determining the cell polarity, though it remains to be addressed how mechanical stretch regulates cell orientation. In the present study, we investigated the signal transduction pathways responsible for the cell orientation response to mechanical stretch, focusing on Rho family proteins. Neonatal rat cardiomyocytes were cultured on silicon chambers and exposed to artificial uniaxial cyclic stretch. The pull-down assays revealed that Rac1 was rapidly activated by stretch, but not RhoA. To analyze the roles of Rho family proteins in cardiomyocyte orientation, adenoviral vectors expressing dominant-negative (dn) RhoA and Rac1 were generated. The transfection with adenovirus vector expressing dnRac1, but not dnRhoA, inhibited stretch-induced cell alignment. In conclusion, Rac1 activity is necessary for cardiomyocyte alignment in response to directional stretch.     

Valvular dystrophy associated filamin A mutations reveal a new role of its first repeats in small-GTPase regulation

Filamin A (FlnA) is a ubiquitous actin binding protein which anchors various transmembrane proteins to the cell cytoskeleton and provides a scaffold to many cytoplasmic signaling proteins involved in actin cytoskeleton remodeling in response to mechanical stress and cytokines stimulation. Although the vast majority of FlnA binding partners interact with the carboxy-terminal immunoglobulin like (Igl) repeats of FlnA, little is known on the role of the amino-N-terminal repeats. Here, using cardiac mitral valvular dystrophy associated FlnA–G288R and P637Q mutations located in the N-terminal Igl repeat 1 and 4 respectively as a model, we identified a new role of FlnA N-terminal repeats in small Rho-GTPases regulation. Using FlnA-deficient melanoma and HT1080 cell lines as expression systems we showed that FlnA mutations reduce cell spreading and migration capacities. Furthermore, we defined a signaling network in which FlnA mutations alter the balance between RhoA and Rac1 GTPases activities in favor of RhoA and provided evidences for a role of the Rac1 specific GTPase activating protein FilGAP in this process. Together our work ascribed a new role to the N-terminal repeats of FlnA in Small GTPases regulation and supports a conceptual framework for the role of FlnA mutations in cardiac valve diseases centered around signaling molecules regulating cellular actin cytoskeleton in response to mechanical stress.     

Cdc42 plays a critical role in assembly of sarcomere units in series of cardiac myocytes

Cardiomyocyte hypertrophy is observed in various cardiovascular diseases and causes heart failure. We here examined the role of small GTP-binding proteins of Rho family in phenylephrine (PE)-or leukocyte inhibitory factor (LIF)-induced hypertrophic morphogenesis of cultured neonatal rat cardiomyocytes. Both LIF and PE increased cell size of cardiomyocytes. LIF induced an increase in the length/width ratio of cardiomyocytes, while PE did not change the ratio. Adenoviral gene transfer of constitutively active mutants of Cdc42 increased the length/width ratio of cardiomyocytes and dominant negative mutants of Cdc42 conversely inhibited LIF-induced cell-elongation, while mutants of RhoA and Rac1 did not affect the length/width ratio of cardiomyocytes. These results suggest that Cdc42, but not RhoA and Rac1, is involved in LIF-induced sarcomere assembly in series in cardiomyocytes.     

Timing is everything

Cell adhesion to the extracellular matrix elicits a temporal reorganization of the actin cytoskeleton that is regulated first by Rac1 and later by RhoA. The signaling mechanisms controlling late stage RhoA activation are incompletely understood. Net1A is a RhoA/RhoB-specific guanine nucleotide exchange factor that is required for cancer cell motility. The ability of Net1A to stimulate RhoA activation is negatively regulated by nuclear sequestration. However, mechanisms controlling the plasma membrane localization of Net1A had not previously been reported. Recently we have shown that Rac1 activation stimulates plasma membrane relocalization and activation of Net1A. Net1A relocalization is independent of its catalytic activity and does not require its C-terminal pleckstrin homology or PDZ interacting domains. Rac1 activation during cell adhesion stimulates a transient relocalization of Net1A that is terminated by proteasomal degradation of Net1A. Importantly, plasma membrane localization of Net1A is required for efficient myosin light chain phosphorylation, focal adhesion maturation, and cell spreading. These data show for the first time a physiological mechanism controlling Net1A relocalization from the nucleus. They also demonstrate a previously unrecognized role for Net1A in controlling actomyosin contractility and focal adhesion dynamics during cell adhesion.     

RhoA/ROCK-mediated switching between Cdc42- and Rac1-dependent protrusion in MTLn3 carcinoma cells

Rho GTPases are versatile regulators of cell shape that act on the actin cytoskeleton. Studies using Rho GTPase mutants have shown that, in some cells, Rac1 and Cdc42 regulate the formation of lamellipodia and filopodia, respectively at the leading edge, whereas RhoA mediates contraction at the rear of moving cells. However, recent reports have described a zone of RhoA/ROCK activation at the front of cells undergoing motility. In this study, we use a FRET-based RhoA biosensor to show that RhoA activation localizes to the leading edge of EGF-stimulated cells. Inhibition of Rho or ROCK enhanced protrusion, yet markedly inhibited cell motility; these changes correlated with a marked activation of Rac-1 at the cell edge. Surprisingly, whereas EGF-stimulated protrusion in control MTLn3 cells is Rac-independent and Cdc42-dependent, the opposite pattern is observed in MTLn3 cells after inhibition of ROCK. Thus, Rho and ROCK suppress Rac-1 activation at the leading edge, and inhibition of ROCK causes a switch between Cdc42 and Rac-1 as the dominant Rho GTPase driving protrusion in carcinoma cells. These data describe a novel role for Rho in coordinating signaling by Rac and Cdc42.     

The regulation of RhoA at focal adhesions by StarD13 is important for astrocytoma cell motility

Malignant astrocytomas are highly invasive into adjacent and distant regions of the normal brain. Rho GTPases are small monomeric G proteins that play important roles in cytoskeleton rearrangement, cell motility, and tumor invasion. In the present study, we show that the knock down of StarD13, a GTPase activating protein (GAP) for RhoA and Cdc42, inhibits astrocytoma cell migration through modulating focal adhesion dynamics and cell adhesion. This effect is mediated by the resulting constitutive activation of RhoA and the subsequent indirect inhibition of Rac. Using Total Internal Reflection Fluorescence (TIRF)-based Förster Resonance Energy Transfer (FRET), we show that RhoA activity localizes with focal adhesions at the basal surface of astrocytoma cells. Moreover, the knock down of StarD13 inhibits the cycling of RhoA activation at the rear edge of cells, which makes them defective in retracting their tail. This study highlights the importance of the regulation of RhoA activity in focal adhesions of astrocytoma cells and establishes StarD13 as a GAP playing a major role in this process.