Transactivation of sphingosine 1-phosphate receptors is essential for vascular barrier regulation. Novel role for hyaluronan and CD44 receptor family

The role for hyaluronan (HA) and CD44 in vascular barrier regulation is unknown. We examined high and low molecular weight HA (HMW-HA, approximately 1,000 kDa; LMW-HA, approximately 2.5 kDa) effects on human transendothelial monolayer electrical resistance (TER). HMW-HA increased TER, whereas LMW-HA induced biphasic TER changes ultimately resulting in EC barrier disruption. HMW-HA induced the association of the CD44s isoform with, and AKT-mediated phosphorylation of, the barrier-promoting sphingosine 1-phosphate receptor (S1P1) within caveolin-enriched lipid raft microdomains, whereas LMW-HA induced brief CD44s association with S1P1 followed by sustained association of the CD44v10 isoform with, and Src and ROCK 1/2-mediated phosphorylation of, the barrier-disrupting S1P3 receptor. HA-induced EC cytoskeletal reorganization and TER alterations were abolished by either disruption of lipid raft formation, CD44 blocking antibody or siRNA-mediated reductions in expression of CD44 isoforms. Silencing S1P1, AKT1, or Rac1 blocked the barrier enhancing effects of HA whereas silencing S1P3, Src, ROCK1/2, or RhoA blocked the barrier disruption induced by LMW-HA. In summary, HA regulates EC barrier function through novel differential CD44 isoform interaction with S1P receptors, S1P receptor transactivation, and RhoA/Rac1 signaling to the EC cytoskeleton.     

Ccpg1, a novel scaffold protein that regulates the activity of the Rho guanine nucleotide exchange factor Dbs

Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) with in vitro exchange activity specific for RhoA and Cdc42. Like many RhoGEF family members, the in vivo exchange activity of Dbs is restricted in a cell-specific manner. Here we report the characterization of a novel scaffold protein (designated cell cycle progression protein 1 [Ccpg1]) that interacts with Dbs and modulates its in vivo exchange specificity. When coexpressed in mammalian cells, Ccpg1 binds to the Dbl homology/pleckstrin homology domain tandem motif of Dbs and inhibits its exchange activity toward RhoA, but not Cdc42. Expression of Ccpg1 correlates with the ability of Dbs to activate endogenous RhoA in cultured cells, and suppression of endogenous Ccpg1 expression potentiates Dbs exchange activity toward RhoA. The isolated Dbs binding domain of Ccpg1 is not sufficient to suppress Dbs exchange activity on RhoA, thus suggesting a regulatory interaction. Ccpg1 mediates recruitment of endogenous Src kinase into Dbs-containing complexes and interacts with the Rho family member Cdc42. Collectively, our studies suggest that Ccpg1 represents a new class of regulatory scaffold protein that can function as both an assembly platform for Rho protein signaling complexes and a regulatory protein which can restrict the substrate utilization of a promiscuous RhoGEF family member.     

CD44 regulates hepatocyte growth factor-mediated vascular integrity. Role of c-Met, Tiam1/Rac1, dynamin 2, and cortactin

The preservation of vascular endothelial cell (EC) barrier integrity is critical to normal vessel homeostasis, with barrier dysfunction being a feature of inflammation, tumor angiogenesis, atherosclerosis, and acute lung injury. Therefore, agents that preserve or restore vascular integrity have important therapeutic implications. In this study, we explored the regulation of hepatocyte growth factor (HGF)-mediated enhancement of EC barrier function via CD44 isoforms. We observed that HGF promoted c-Met association with CD44v10 and recruitment of c-Met into caveolin-enriched microdomains (CEM) containing CD44s (standard form). Treatment of EC with CD44v10-blocking antibodies inhibited HGF-mediated c-Met phosphorylation and c-Met recruitment to CEM. Silencing CD44 expression (small interfering RNA) attenuated HGF-induced recruitment of c-Met, Tiam1 (a Rac1 exchange factor), cortactin (an actin cytoskeletal regulator), and dynamin 2 (a vesicular regulator) to CEM as well as HGF-induced trans-EC electrical resistance. In addition, silencing Tiam1 or dynamin 2 reduced HGF-induced Rac1 activation, cortactin recruitment to CEM, and EC barrier regulation. We observed that both HGF- and high molecular weight hyaluronan (CD44 ligand)-mediated protection from lipopolysaccharide-induced pulmonary vascular hyperpermeability was significantly reduced in CD44 knock-out mice, thus validating these in vitro findings in an in vivo murine model of inflammatory lung injury. Taken together, these results suggest that CD44 is an important regulator of HGF/c-Met-mediated in vitro and in vivo barrier enhancement, a process with essential involvement of Tiam1, Rac1, dynamin 2, and cortactin.     

Modulation of functional responses of endothelial cells linked to angiogenesis and inflammation by shear stress: differential effects of the mechanotransducer CD31

We investigated the roles of the "mechanotransducer" CD31 in the effects of shear stress on endothelial gene expression and functional responses relevant to angiogenesis and inflammation. Human or murine endothelial cells (hEC or mEC) were exposed to different levels of shear stress, while expression of CD31 was modified using siRNA in the hEC, or mEC from CD31(-/-) mice. Quantitation of expression of genes linked to inflammation or angiogenesis showed several were sensitive to shear. In a "wound" assay, exposure of endothelial cells (EC) to shear stress tended to align migration with the direction of flow and decrease the rate of closure compared to static cultures. When EC were cultured on filters, shear stress promoted migration away from the luminal surface. EC conditioned by shear stress recruited fewer flowing neutrophils, and showed reduced up-regulation of E-selectin after stimulation with tumor necrosis factor-α (TNF). Use of siRNA against CD31 in the hEC, or testing of mEC from mice lacking CD31, indicated that expression of CD31 was not required for the shear-induced modification of wound closure. However, shear modulation of response to TNF was less effective in the absence of CD31, while reduction of CD31 reduced shear-sensitivity in some genes (e.g., eNOS), but not others (e.g., KLF-2). Thus, CD31 played a role in shear-sensitivity of some genes and of neutrophil recruitment, but not in modulation of endothelial migration. Different mechanotransducers may mediate different functional effects of shear stress. Hence, identification of the specific pathways may provide targets for therapeutic manipulation of angiogenesis or inflammation.     

Coelomocyte locomotion in the sipunculan Themiste petricola induced by exogenous and endogenous chemoattractants: role of a CD44-like antigen--HA interaction

Cell migration is a key event in the invertebrate immuno-defense system. Microbial products like lipopolysacharide (LPS) and formyl-methyl-leucyl-phenylalanine (fMLP) promote cell recruitment to sites of infection. In mammals, complement activation by factors such as zymosan induces C5a production, which influences leukocyte migration. The endogenous factor hyaluronic acid (HA), an extracellular matrix component, also promotes cell migration through its receptor CD44. We evaluated whether coelomocytes from the sipunculan worm T. petricola migrated towards LPS, fMLP, or zymosan treated plasma (ZTP) and if HA was involved in coelomocyte migration and adhesion. We also evaluated if antibodies specific for mouse HA receptor CD44 inhibited any of the effects induced by HA. Using microchemotaxis chambers we found that coelomocytes migrated towards exogenously and endogenously derived chemoattractants. We also observed that HA was a potent chemotactic signal and that coelomocytes adhered strongly to plates coated with LMW-HA but not with HMW-HA. In addition we found that these HA mediated effects were blocked by the monoclonal antibody IM7 directed to mouse CD44, suggesting that a CD44-like cross-reactive antigen might play a role in HA mediated coelomocyte locomotion.     

Involvement of caveolin‐1 in fibronectin‐induced mouse embryonic stem cell proliferation: Role of FAK,RhoA, PI3K/Akt,and ERK 1/2 pathways

Fibronectin (FN) is the foremost proliferation‐associated extracellular matrix component promoting cell adhesion, migration, and survival. We examined the effect of FN on cell proliferation and the related signaling pathways in mouse embryonic stem (ES) cells. FN increased integrin β1, Src, focal adhesion kinase (FAK), and caveolin‐1 phosphorylation levels in a time‐dependent manner. Phosphorylation of Src, FAK, and caveolin‐1 was attenuated by integrin β1 neutralizing antibody. Integrin β1, Src, and FAK coimmunoprecipitated with caveolin‐1 in the presence of FN. In addition, FN increased RhoA and Rho kinase activation, which were completely blocked by PP2, FAK small interfering RNA (siRNA), caveolin‐1 siRNA, or the caveolar disruptor methyl‐β‐cyclodextrin (MβCD). FN also increased phosphorylation of Akt and ERK 1/2, which were significantly blocked by either FAK siRNA, caveolin‐1 siRNA, MβCD, GGTI‐286 (RhoA inhibitor), or Y‐27632 (Rho kinase inhibitor). FN‐induced increase of protooncogenes (c‐fos, c‐myc, and c‐Jun) and cell‐cycle regulatory proteins (cyclin D1/CDK4 and cyclin E/CDK2) expression levels were attenuated by FAK siRNA or caveolin‐1 siRNA. Furthermore, inhibition of each pathway such as integrin β1, Src, FAK, caveolin‐1, RhoA, Akt, and ERK 1/2 blocked FN‐induced [³H]‐thymidine incorporation. We conclude that FN stimulates mouse ES cell proliferation via RhoA‐PI3K/Akt‐ERK 1/2 pathway through caveolin‐1 phosphorylation. J. Cell. Physiol. 226: 267–275, 2010. © 2010 Wiley‐Liss, Inc.     

Differential activation of ERK and Rac mediates the proliferative and anti-proliferative effects of hyaluronan and CD44

Hyaluronan, a widely distributed component of the extracellular matrix, exists in a high molecular weight (native) form and lower molecular weight form (HMW- and LMW-HA, respectively). These different forms of hyaluronan bind to CD44 but elicit distinct effects on cellular function. A striking example is the opposing effects of HMW- and LMW-HA on the proliferation of vascular smooth muscle cells; the binding of HMW-HA to CD44 inhibits cell cycle progression, whereas the binding of LMW-HA to CD44 stimulates cell cycle progression. We now report that cyclin D1 is the primary target of LMW-HA in human vascular smooth muscle cells, as it is for HMW-HA, and that the opposing cell cycle effects of these CD44 ligands result from differential regulation of signaling pathways to cyclin D1. HMW-HA binding to CD44 selectively inhibits the GTP loading of Rac and Rac-dependent signaling to the cyclin D1 gene, whereas LMW-HA binding to CD44 selectively stimulates ERK activation and ERK-dependent cyclin D1 gene expression. These data describe a novel mechanism of growth control in which a ligand-receptor system generates opposing effects on mitogenesis by differentially regulating signaling pathways to a common cell cycle target. They also emphasize how a seemingly subtle change in matrix composition can have a profound effect on cell proliferation.     

Differential involvement of the hyaluronan (HA) receptors CD44 and receptor for HA-mediated motility in endothelial cell function and angiogenesis.

Hyaluronan (HA), an important glycosaminoglycan constituent of the extracellular matrix, has been implicated in angiogenesis. It appears to exert its biological effects through binding interactions with at least two cell surface receptors: CD44 and receptor for HA-mediated motility (RHAMM). Recent in vitro studies have suggested potential roles for these two molecules in various aspects of endothelial function. However, the relative contribution of each receptor to endothelial functions critical to angiogenesis and their roles in vivo have not been established. We therefore investigated the endothelial expression of these proteins and determined the effects of antibodies against RHAMM and CD44 on endothelial cell (EC) function and in vivo angiogenesis. Both receptors were detected on vascular endothelium in situ, and on the surface of cultured EC. Further studies with active blocking antibodies revealed that anti-CD44 but not anti-RHAMM antibody inhibited EC adhesion to HA and EC proliferation, whereas anti-RHAMM but not CD44 antibody blocked EC migration through the basement membrane substrate, Matrigel. Although antibodies against both receptor inhibited in vitro endothelial tube formation, only the anti-RHAMM antibody blocked basic fibroblast growth factor-induced neovascularization in mice. These data suggest that RHAMM and CD44, through interactions with their ligands, are both important to processes required for the formation of new blood vessels.     

Targeted deletion of CD44v7 exon leads to decreased endothelial cell injury but not tumor cell killing mediated by interleukin-2-activated cytolytic lymphocytes

In the current study, we investigated the nature and role of CD44 variant isoforms involved in endothelial cell (EC) injury and tumor cell cytotoxicity mediated by IL-2-activated killer (LAK) cells. Treatment of CD44 wild-type lymphocytes with IL-2 led to increased gene expression of CD44 v6 and v7 variant isoforms and to significant induction of vascular leak syndrome (VLS). CD44v6-v7 knockout (KO) and CD44v7 KO mice showed markedly reduced levels of IL-2-induced VLS. The decreased VLS in CD44v6-v7 KO and CD44v7 KO mice did not result from differential activation and expansion of CD8+ T cells, NK, and NK-T cells or from altered degree of perivascular lymphocytic infiltration in the lungs. LAK cells from CD44v7 KO mice showed a significant decrease in their ability to adhere to and mediate lysis of EC but not lysis of P815 tumor cells in vitro. CD44v7-mediated lysis of EC by LAK cells was dependent on the activity of phosphatidylinositol 3-kinase and tyrosine kinases. Interestingly, IL-2-activated LAK cells expressing CD44hi but not CD44lo were responsible for EC lysis. Furthermore, lysis of EC targets could be blocked by addition of soluble or enzymatic cleavage of CD44v6-v7-binding glycosaminoglycans. Finally, anti-CD44v7 mAbs caused a significant reduction in the adherence to and killing of EC and led to suppression of IL-2-induced VLS. Together, this study suggests that the expression of CD44v7 on LAK cells plays a specific role in EC injury and that it may be possible to reduce EC injury but not tumor cell killing by specifically targeting CD44v7.     

Critical Role of S1PR1 and Integrin β4 in HGF/c-Met-mediated Increases in Vascular Integrity

Vascular endothelial cell (EC) barrier integrity is critical to vessel homeostasis whereas barrier dysfunction is a key feature of inflammatory disorders and tumor angiogenesis. We previously reported that hepatocyte growth factor (HGF)-mediated increases in EC barrier integrity are signaled through a dynamic complex present in lipid rafts involving its receptor, c-Met (1). We extended these observations to confirm that S1PR1 (sphingosine 1-phosphate receptor 1) and integrin β4 (ITGB4) are essential participants in HGF-induced EC barrier enhancement. Immunoprecipitation experiments demonstrated HGF-mediated recruitment of c-Met, ITGB4 and S1PR1 to caveolin-enriched lipid rafts in human lung EC with direct interactions of c-Met with both S1PR1 and ITGB4 accompanied by c-Met-dependent S1PR1 and ITGB4 transactivation. Reduced S1PR1 expression (siRNA) attenuated both ITGB4 and Rac1 activation as well as c-Met/ITGB4 interaction and resulted in decreased transendothelial electrical resistance. Furthermore, reduced ITGB4 expression attenuated HGF-induced c-Met activation, c-Met/S1PR1 interaction, and effected decreases in S1P- and HGF-induced EC barrier enhancement. Finally, the c-Met inhibitor, XL880, suppressed HGF-induced c-Met activation as well as S1PR1 and ITGB4 transactivation. These results support a critical role for S1PR1 and ITGB4 transactivation as rate-limiting events in the transduction of HGF signals via a dynamic c-Met complex resulting in enhanced EC barrier integrity.     

EphA4 signaling regulates blastomere adhesion in the Xenopus embryo by recruiting Pak1 to suppress Cdc42 function

The control of cell adhesion is an important mechanism by which Eph receptors regulate cell sorting during development. Activation of EphA4 in Xenopus blastulae induces a reversible, cell autonomous loss-of-adhesion and disruption of the blastocoel roof. We show this phenotype is rescued by Nckbeta (Grb4) dependent on its interaction with EphA4. Xenopus p21(Cdc42/Rac)-activated kinase xPAK1 interacts with Nck, is activated in embryo by EphA4 in an Nck-dependent manner, and is required for EphA4-induced loss-of-adhesion. Ectopic expression of xPAK1 phenocopies EphA4 activation. This does not require the catalytic activity of xPAK1, but it does require its GTPase binding domain and is enhanced by membrane targeting. Indeed, membrane targeting of the GTPase binding domain (GBD) of xPAK1 alone is sufficient to phenocopy EphA4 loss-of-adhesion. Both EphA4 and the xPAK1-GBD down-regulate RhoA-GTP levels, and consistent with this, loss-of-adhesion can be rescued by activated Cdc42, Rac, and RhoA and can be epistatically induced by dominant-negative RhoA. Despite this, neither Cdc42 nor Rac activities are down-regulated by EphA4 activation or by the xPAK1-GBD. Together, the data suggest that EphA4 activation sequesters active Cdc42 and in this way down-regulates cell-cell adhesion. This novel signaling pathway suggests a mechanism for EphA4-guided migration.     

RhoGEF specificity mutants implicate RhoA as a target for Dbs transforming activity

Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) that exhibits transforming activity when overexpressed in NIH 3T3 mouse fibroblasts. Like many RhoGEFs, the in vitro catalytic activity of Dbs is not limited to a single substrate. It can catalyze the exchange of GDP for GTP on RhoA and Cdc42, both of which are expressed in most cell types. This lack of substrate specificity, which is relatively common among members of the RhoGEF family, complicates efforts to determine the molecular basis of their transforming activity. We have recently determined crystal structures of several RhoGEFs bound to their cognate GTPases and have used these complexes to predict structural determinants dictating the specificities of coupling between RhoGEFs and GTPases. Guided by this information, we mutated Dbs to alter significantly its relative exchange activity for RhoA versus Cdc42 and show that the transformation potential of Dbs correlates with exchange on RhoA but not Cdc42. Supporting this conclusion, oncogenic Dbs activates endogenous RhoA but not endogenous Cdc42 in NIH 3T3 cells. Similarly, a competitive inhibitor that blocks RhoA activation also blocks Dbs-mediated transformation. In conclusion, this study highlights the usefulness of specificity mutants of RhoGEFs as tools to genetically dissect the multiple signaling pathways potentially activated by overexpressed or oncogenic RhoGEFs. These ideas are exemplified for Dbs, which is strongly implicated in the transformation of NIH 3T3 cells via RhoA and not Cdc42.     

Transformation by the Rho-specific guanine nucleotide exchange factor Dbs requires ROCK I-mediated phosphorylation of myosin light chain

Dbs was identified in a cDNA-based expression screen for sequences that can cause malignant growth when expressed in murine fibroblasts. In previous studies we have shown that Dbs is a Rho-specific guanine nucleotide exchange factor that can activate RhoA and/or Cdc42 in a cell-specific manner. In this current study we have used a combination of genetic and pharmacological approaches to examine the relative contributions of RhoA x PRK and RhoA x ROCK signaling to Dbs transformation. Our analysis indicates that ROCK is activated in Dbs-transformed cells and that Dbs transformation is dependent upon ROCK I activity. In contrast, there appears to be no requirement for PRK activation in Dbs transformation. Dbs transformation is also associated with increased phosphorylation of myosin light chain and stress fiber formation, both of which occur in a ROCK-dependent manner. Suppression of myosin light chain expression by small interfering RNAs impairs Dbs focus formation, thus establishing a direct link between actinomyosin contraction and Rho-specific guanine nucleotide exchange factor transformation.     

Mechanical stretch-induced RhoA activation is mediated by the RhoGEF Vav2 in mesangial cells

Increased intraglomerular pressure is an important hemodynamic determinant of glomerulosclerosis, and can be modelled in vitro by exposing mesangial cells (MC) to cyclic mechanical stretch. We have previously shown that the GTPase RhoA mediates stretch-induced fibronectin production. Here we investigate the role of the RhoGEF Vav2 in the activation of RhoA by stretch. Primary rat MC were exposed to 1 Hz cyclic stretch, previously shown to induce maximal RhoA activation at 1 min. Total Vav2 tyrosine phosphorylation and specific phosphorylation on Y172, required for activation, were increased by 1 min of stretch. Overexpression of dominant-negative Vav2 Y172/159F in COS-1 cells or downregulation of Vav2 by siRNA in MC prevented stretch-induced RhoA activation. Vav2 is known to be activated in response to growth factors, and we have previously shown the epidermal growth factor receptor (EGFR) to be transactivated by stretch in MC. Both Vav2 Y172 phosphorylation and RhoA activation were blocked by the EGFR inhibitor AG1478 and prevented in MC overexpressing kinase inactive EGFR. Stretch led to physical association between the EGFR and Vav2, and this was dependent on EGFR activation. EGFR Y992 phosphorylation, required for growth factor-induced Vav2 phosphorylation, was also induced by stretch. Activation of both Src and PI3K were necessary upstream mediators of stretch-induced Vav2 Y172 phosphorylation and RhoA activation. In summary, stretch-induced RhoA activation is dependent on transactivation of the EGFR and activation of the RhoGEF Vav2. Src and PI3K are both required upstream of Vav2 and RhoA activation.     

Role of RhoA and its effectors ROCK and mDia1 in the modulation of deformation-induced FAK, ERK, p38, and MLC motogenic signals in human Caco-2 intestinal epithelial cells

Repetitive deformation enhances intestinal epithelial migration across tissue fibronectin. We evaluated the contribution of RhoA and its effectors Rho-associated kinase (ROK/ROCK) and mammalian diaphanous formins (mDia1) to deformation-induced intestinal epithelial motility across fibronectin and the responsible focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), p38, and myosin light chain (MLC) signaling. We reduced RhoA, ROCK1, ROCK2, and mDia1 by smart-pool double-stranded short-interfering RNAs (siRNA) and pharmacologically inhibited RhoA, ROCK, and FAK in human Caco-2 intestinal epithelial monolayers on fibronectin-coated membranes subjected to 10% repetitive deformation at 10 cycles/min. Migration was measured by wound closure. Stimulation of migration by deformation was prevented by exoenzyme C3, Y27632, or selective RhoA, ROCK1, and ROCK2 or mDia1 siRNAs. RhoA, ROCK inhibition, or RhoA, ROCK1, ROCK2, mDia1, and FAK reduction by siRNA blocked deformation-induced nuclear ERK phosphorylation without preventing ERK phosphorylation in the cytoplasmic protein fraction. Furthermore, RhoA, ROCK inhibition or RhoA, ROCK1, ROCK2, and mDia1 reduction by siRNA also blocked strain-induced FAK-Tyr(925), p38, and MLC phosphorylation. These results suggest that RhoA, ROCK, mDia1, FAK, ERK, p38, and MLC all mediate the stimulation of intestinal epithelial migration by repetitive deformation. This pathway may be an important target for interventions to promote mechanotransduced mucosal healing during inflammation.     

Thy-1-Induced Migration Inhibition in Vascular Endothelial Cells through Reducing the RhoA Activity

Our previous study indicated that Thy-1, which is expressed on blood vessel endothelium in settings of pathological and a specific of physiological, but not during embryonic, angiogenesis, may be used as a marker for angiogenesis. However, the function of Thy-1 during angiogenesis is still not clear. Here, we demonstrate that knock-down of the endogenous Thy-1 expression by Thy-1 siRNA transfection promoted the migration of human umbilical vein endothelial cells (HUVEC). In contrast, treatment with interleukin-1β (IL-1β) or phorbol-12-myristate-13-acetate (PMA) increased the level of Thy-1 protein and reduced the migration of HUVEC. These effects were abolished by pre-transfection of HUVEC with Thy-1 siRNA to knock-down the expression of Thy-1. Moreover, over-expression of Thy-1 by transfection of HUVEC with Thy-1 pcDNA3.1 decreased the activity of RhoA and Rac-1 and inhibited the adhesion, migration and capillary-like tube formation of these cells. These effects were prevented by co-transfection of the cell with constitutively active RhoA construct (RhoA V14). On the other hand, pre-treatment with a ROCK (a kinase associated with RhoA for transducing RhoA signaling) inhibitor, Y27632, abolished the RhoA V14-induced prevention effect on the Thy-1-induced inhibition of endothelial cell migration and tube formation. Taken together, these results indicate that suppression of the RhoA-mediated pathway might participate in the Thy-1-induced migration inhibition in HUVEC. In the present study, we uncover a completely novel role of Thy-1 in endothelial cell behaviors.     

Fibronectin type I repeat is a nonactivating ligand for EphA1 and inhibits ATF3-dependent angiogenesis

ATF3 stimulated promoter activity of EphA1 by 3.4-fold in ATF3-dependent angiogenesis in vitro. Although tyrosine kinase activation of EphA1 was dispensable, binding of EphA1 to fibronectin through its type I repeat played an essential role in the angiogenesis. Recombinant proteins containing fibronectin 10th to 12th type I repeat (I 10-12) but not I 12 could inhibit the angiogenesis in vitro by competitively targeting EphA1 with the full-length fibronectin. However, I 12 acquired a higher affinity toward EphA2 with K(d) 18 nm and inhibited vascular endothelial growth factor-dependent angiogenic invasion in a Matrigel plug assay.     

Multifunctional roles for the PH domain of Dbs in regulating Rho GTPase activation

Dbl family members are guanine nucleotide exchange factors specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. Dbs, a Dbl family member specific for Cdc42 and RhoA, exhibits transforming activity when overexpressed in NIH 3T3 mouse fibroblasts. In this study, the PH domain of Dbs was mutated to impair selectively either guanine nucleotide exchange or phosphoinositide binding in vitro and resulting physiological alterations were assessed. As anticipated, substitution of residues within the PH domain of Dbs integral to the interface with GTPases reduced nucleotide exchange and eliminated the ability of Dbs to transform NIH 3T3 cells. More interestingly, substitutions within the PH domain that prevent interaction with phosphoinositides yet do not alter in vitro activation of GTPases also do not transform NIH 3T3 cell and fail to activate RhoA in vivo despite proper subcellular localization. Therefore, the PH domain of Dbs serves multiple roles in the activation of GTPases and cannot be viewed as a simple membrane-anchoring device. In particular, the data suggest that binding of phosphoinositides to the PH domain within the context of membrane surfaces may direct orientations or conformations of the linked DH and PH domains to regulate GTPases activation.     

The Small GTPase RhoA Is Required for Proper Locomotor Circuit Assembly

The assembly of neuronal circuits during development requires the precise navigation of axons, which is controlled by attractive and repulsive guidance cues. In the developing spinal cord, ephrinB3 functions as a short-range repulsive cue that prevents EphA4 receptor-expressing corticospinal tract and spinal interneuron axons from crossing the midline, ensuring proper formation of locomotor circuits. Here we report that the small GTPase RhoA, a key regulator of cytoskeletal dynamics, is also required for ephrinB3/EphA4-dependent locomotor circuit formation. Deletion of RhoA from neural progenitor cells results in mice that exhibit a rabbit-like hopping gait, which phenocopies mice lacking ephrinB3 or EphA4. Consistent with this locomotor defect, we found that corticospinal tract axons and spinal interneuron projections from RhoA-deficient mice aberrantly cross the spinal cord midline. Furthermore, we determined that loss of RhoA blocks ephrinB3-induced growth cone collapse of cortical axons and disrupts ephrinB3 expression at the spinal cord midline. Collectively, our results demonstrate that RhoA is essential for the ephrinB3/EphA4-dependent assembly of cortical and spinal motor circuits that control normal locomotor behavior.