Neurite outgrowth from PC12 cells by basic fibroblast growth factor (bFGF) is mediated by RhoA inactivation through p190RhoGAP and ARAP3

The rat pheochromocytoma cell line PC12 has been widely used as a model to study neuronal differentiation. PC12 cells give rise to neurites in response to basic fibroblast growth factor (bFGF). However, it is unclear whether bFGF promotes neurite outgrowth by inducing RhoA inactivation, and a mechanism for RhoA inactivation in PC12 cells in response to bFGF has not been reported. Lysophosphatidic acid (LPA) treatment and the expression of constitutively active (CA)‐RhoA (RhoA V14) impaired neurite formation in response to bFGF, while Tat‐C3 exoenzyme and the expression of dominant negative (DN)‐RhoA (RhoA N19) stimulated neurite outgrowth. GTP‐bound RhoA levels were reduced in response to bFGF, which suggests that the inactivation of RhoA is essential to neurite outgrowth in response to bFGF. To investigate the mechanism of RhoA inactivation, this study examined the roles of p190RhoGAP and Rap‐dependent RhoGAP (ARAP3). DN‐p190RhoGAP prevented neurite outgrowth, while WT‐p190RhoGAP and Src synergistically stimulated neurite outgrowth; these findings suggest that bFGF promotes the inactivation of RhoA and subsequent neurite outgrowth through p190RhoGAP and Src. Furthermore, DN‐Rap1 and DN‐ARAP3 reduced neurite formation in PC12 cells. These results suggest that RhoA is likely to be inactivated by p190RhoGAP and ARAP3 during neurite outgrowth in response to bFGF. J. Cell. Physiol. 224: 786–794, 2010. © 2010 Wiley‐Liss, Inc.     

Serine phosphorylation differentially affects RhoA binding to effectors: implications to NGF-induced neurite outgrowth

Activation of RhoA prevents NGF-induced outgrowth and causes retraction of neurites in neuronal cells, including PC12 cells. Despite its inhibitory effect on neurite outgrowth, NGF activates GTP loading of and effector binding to RhoA, setting up an apparent contradiction. According to the molecular switch hypothesis of GTPase function GTP-loading of RhoA should be sufficient to activate its effectors uniformly. However, when monitoring NGF-induced binding of GTP-RhoA to multiple targets, we noted differential interactions with its effectors. We found that NGF elicits a protein kinase A-mediated phosphorylation of RhoA on serine(188), which renders it unable to bind to Rho-associated kinase (ROK), whereas it retains the ability to interact with other RhoA targets including rhotekin, mDia-1 and PKN. We show in vitro and in vivo that phosphorylation of serine(188) represents an additional switch, capable of directing signals among effector pathways. In the context of PC12 cell differentiation, NGF-induced phosphorylation of RhoA on serine(188) prevents it from interacting with ROK, which would otherwise block neurite outgrowth. Transfection of RhoA(S188A) mutant into PC12 cells prevents NGF-induced neurite outgrowth, just like constitutively activated RhoA(14V) does, indicating the requirement of this phosphorylation site. Replacement of serine(188) with the phosphomimetic glutamate residue in RhoA(V14/S188E) selectively impairs interaction with ROK and when transfected into PC12 cells restores NGF-induced neurite outgrowth. Therefore, phosphorylation of serine(188) may serve as a novel secondary switch of RhoA capable of overriding GTP-binding-elicited effector activation to a subset of targets such as ROK, which interact with the C-terminus of RhoA.     

Cadherin engagement inhibits RhoA via p190RhoGAP

Cadherins are transmembrane receptors that mediate cell-cell adhesion in epithelial cells. A number of changes occur during cadherin-mediated junction formation, one of which is a rearrangement of the actin cytoskeleton. Key regulators of actin cytoskeletal dynamics in cells are the Rho family of GTPases. We have demonstrated in previous studies that cadherin signaling suppresses RhoA activity and activates Rac1. The signaling events downstream of cadherins that modulate the activity of Rho family proteins remain unknown. Here we have identified a pathway by which RhoA becomes inactivated by cadherins. To determine whether cadherins regulate RhoA through activation of a GTPase-activating protein (GAP) for RhoA, we used constitutively active RhoA to isolate activated GAPs. Using this assay, we have identified the RhoA-specific GAP, p190RhoGAP, downstream from engaged cadherins. We found that cadherin engagement induced tyrosine phosphorylation of p190RhoGAP and increased its binding to p120RasGAP. The increased precipitation of p190RhoGAP with 63LRhoA was blocked by addition of PP2 suggesting that Src family kinases are required downstream from cadherin signaling. The inhibition of RhoA activity by cadherins was antagonized by expression of a dominant negative p190RhoGAP. Taken together, these data demonstrate that p190RhoGAP activity is critical for RhoA inactivation by cadherins.     

A possible role for p190RhoGAP in PKCepsilon-induced morphological effects

We have previously shown that protein kinase C (PKC) epsilon induces neurite outgrowth via its regulatory domain. This is accompanied by PKC-induced stress fibre loss. Here, we show that the regulatory domain (RD) of PKCepsilon induces processes also in NIH-3T3 fibroblasts, similar to what has been observed with p190RhoGAP. This study also shows that p190RhoGAP induces neurite outgrowth in SK-N-BE(2) neuroblastoma cells. We therefore investigated whether p190RhoGAP may be downstream of PKCepsilon. We could detect a co-localization of p190RhoGAP and PKCepsilon at membrane ruffles and an increased association between the proteins in fibroblasts treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). The association is also seen in neuroblastoma cells, and nerve growth factor (NGF) treatment of SH-SYSY/TrkA cells decreases the interaction. However, overexpressed PKCepsilon did not coprecipitate overexpressed p190RhoGAP in CHO cells, indicating that the proteins do not interact directly. This raises the possibility that p190RhoGAP is involved in mediating the morphological effects of PKCepsilon.     

p190 RhoGAP is the principal Src substrate in brain and regulates axon outgrowth, guidance and fasciculation

The Src tyrosine kinases have been implicated in several aspects of neural development and nervous system function; however, their relevant substrates in brain and their mechanism of action in neurons remain to be established clearly. Here we identify the potent Rho regulatory protein, p190 RhoGAP (GTPase-activating protein), as the principal Src substrate detected in the developing and mature nervous system. We also find that mice lacking functional p190 RhoGAP exhibit defects in axon guidance and fasciculation. p190 RhoGAP is co-enriched with F-actin in the distal tips of axons, and overexpressing p190 RhoGAP in neuroblastoma cells promotes extensive neurite outgrowth, indicating that p190 RhoGAP may be an important regulator of Rho-mediated actin reorganization in neuronal growth cones. p190 RhoGAP transduces signals downstream of cell-surface adhesion molecules, and we find that p190-RhoGAP-mediated neurite outgrowth is promoted by the extracellular matrix protein laminin. Together with the fact that mice lacking neural adhesion molecules or Src kinases also exhibit defects in axon outgrowth, guidance and fasciculation, our results suggest that p190 RhoGAP mediates a Src-dependent adhesion signal for neuritogenesis to the actin cytoskeleton through the Rho GTPase.     

A Blk–p190RhoGAP signaling module downstream of activated Gα13 functionally opposes CXCL12-stimulated RhoA activation and cell invasion

Activation of the GTPase RhoA linked to cell invasion can be tightly regulated following Gα₁₃ stimulation. We have used a cellular model displaying Gα₁₃-dependent inhibition of RhoA activation associated with defective cell invasion to the chemokine CXCL12 to characterize the molecular players regulating these processes. Using both RNAi transfection approaches and protein overexpression experiments here we show that the Src kinase Blk is involved in Gα₁₃-activated tyrosine phosphorylation of p190RhoGAP, which causes RhoA inactivation and ultimately leads to deficient cell invasion. Characterization of molecular interplays between Gα₁₃, Blk and p190RhoGAP revealed that Blk binds Gα₁₃, and that Blk-mediated p190RhoGAP phosphorylation upon Gα₁₃ activation correlates with weakening of Gα₁₃–Blk association connected to increased Blk–p190RhoGAP assembly. These results place Blk upstream of the p190RhoGAP–RhoA pathway in Gα₁₃-activated cells, overall representing an opposing signaling module during CXCL12-triggered invasion. In addition, analyses with Blk- or Gα₁₃-knockdown cells indicated that Blk can also mediate CXCL12-triggered phosphorylation of p190RhoGAP independently of Gα₁₃. However, even if CXCL12 induces the Blk-mediated GAP phosphorylation, the simultaneous stimulation of the guanine-nucleotide exchange factor Vav1 by the chemokine, as earlier reported, leads to a net increase in RhoA activation. Therefore, when Gα₁₃ is concurrently stimulated with CXCL12 there appears to be sufficient Blk activity to promote adequate levels of p190RhoGAP tyrosine phosphorylation to inactivate RhoA and to impair cell invasiveness.     

HIV-1 Nef disrupts the podocyte actin cytoskeleton by interacting with diaphanous interacting protein

The ability of the human immunodeficiency virus, type 1 (HIV-1) protein Nef to induce cytoskeleton changes in infected host cells is a key event in viral replication. In renal podocytes, we found that Nef induced loss of stress fibers and increased lamellipodia, pathological changes leading to proteinuria in HIV-associated nephropathy. These morphological changes were mediated by Nef-induced Rac1 activation and RhoA inhibition. We identified a new interaction between Nef and diaphanous interacting protein (DIP), a recently described regulator of Rho and Rac signaling. We found that the Src homology 3 binding domain of DIP and the Nef PXXP motif were required for this interaction. Nef also interacts with Vav2 in podocytes. DIP and Vav2 both interact directly with Nef in a competitive manner. DIP interacts with p190RhoGAP, and intact DIP was required for Nef-induced phosphorylation of p190RhoGAP. DIP also interacts with Vav2, and although DIP enhanced baseline phosphorylation of Vav2, it was not required for Nef-induced Vav2 activation. In Nef-infected podocytes, Src kinase induces phosphorylation of DIP, p190RhoGAP, and Vav2, leading to RhoA inhibition and Rac1 activation. Inhibition of the Nef-induced signaling pathway by using a dominant negative of either Src or DIP or siRNA for DIP or p190RhoAGAP restored RhoA activity and stress fiber formation in Nef-infected podocytes, whereas siRNA for Vav2 reduced Rac1 activity and formation of lamellipodia. We conclude that in HIV-infected podocytes, Nef, through the recruitment of DIP and p190RhoAGAP to Nef-Src complex, activates p190RhoAGAP and down-regulates RhoA activity.     

Role of p190RhoGAP in beta 2 integrin regulation of RhoA in human neutrophils

We found that engagement of beta(2) integrins on human neutrophils induced activation of RhoA, as indicated by the increased ratio of GTP:GTP + GDP recovered on RhoA and translocation of RhoA to a membrane fraction. The clustering of beta(2) integrins also induced a time-dependent increase in GDP bound to RhoA, which correlated with beta(2) integrin-induced activation of p190RHOGAP: The activation of p190RhoGAP was completely blocked by [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] (PP1), a selective inhibitor of Src family tyrosine kinases. However, clustering of beta(2) integrins did not increase the basal tyrosine phosphorylation of p190RhoGAP, nor did it affect the amount of p120RasGAP bound to p190RHOGAP: Instead, the beta(2) integrin-induced activation of p190RhoGAP was accompanied by increased tyrosine phosphorylation of a p190RhoGAP-associated protein, p120RasGAP, and accumulation of both p120RasGAP and p190RhoGAP in a membrane fraction. PP1 blocked the beta(2) integrin-induced phosphorylation of p120RasGAP, as well as the translocation of p190RhoGAP and p120RasGAP, but it did not affect the accumulation of RhoA in the membrane fraction. In agreement with the mentioned findings, PP1 also increased the GTP:GTP + GDP ratio recovered on RhoA immunoprecipitated from beta(2) integrin-stimulated cells. Thus, in neutrophils, beta(2) integrin-induced activation of p190RhoGAP requires a signal from a Src family tyrosine kinase, but it does not occur via the signaling pathway responsible for activation of RHOA:     

Protein kinase C induces actin reorganization via a Src- and Rho-dependent pathway

We have investigated the mechanism of PKC-induced actin reorganization in A7r5 vascular smooth muscle cells. PKC activation by 12-O-tetradecanoylphorbol-13-acetate induces the disassembly of actin stress fibers concomitant with the appearance of membrane ruffles. PKC also induces rapid tyrosine phosphorylation in these cells. As we could show, utilizing the Src-specific inhibitor PP2 and a kinase-deficient c-Src mutant, actin reorganization is dependent on PKC-induced Src activation. Subsequently, the activity of the small G-protein RhoA is decreased, whereas Rac and Cdc42 activities remain unchanged. Disassembly of actin stress fibers could also be observed using the Rho kinase-specific inhibitor Y-27632, indicating that the decrease in RhoA activity on its own is responsible for actin reorganization. In addition, we show that tyrosine phosphorylation of p190RhoGAP is increased upon 12-O-tetradecanoylphorbol-13-acetate stimulation, directly linking Src activation to a decrease in RhoA activity. Our data provide substantial evidence for a model elucidating the molecular mechanisms of PKC-induced actin rearrangements.     

Activation of Rac1 by phosphatidylinositol 3-kinase in vivo: role in activation of mitogen-activated protein kinase (MAPK) pathways and retinoic acid-induced neuronal differentiation of SH-SY5Y cells

Rho GTPases such as RhoA, Rac1 and Cdc42 are crucial players in the regulation of signal transduction pathways required for neuronal differentiation. Using an in vitro cell culture model of neuroblastoma SH-SY5Y cells, we demonstrated previously that RhoA is an in vivo substrate of tissue transglutaminase (TGase) and retinoic acid (RA) promoted activation of RhoA by transamidation. Although activation of RhoA promoted cytoskeletal rearrangement in SH-SY5Y cells, it was not involved in induction of neurite outgrowth. Here, we demonstrate that RA promotes activation of Rac1 in SH-SY5Y cells in a transamidation-independent manner. RA-induced activation of Rac1 is mediated by phosphatidylinositol 3-kinase (PI3K), probably because of phosphorylation of the p85 regulatory subunit by Src kinases. Over-expression of constitutively active PI3K or Rac1-V12 induces neurite outgrowth, activation of mitogen activated protein kinases (MAPKs), and expression of neuronal markers. The PI3K inhibitor LY294002, or over-expression of dominant negative Rac1-N17, blocks RA-induced neurite outgrowth, activation of MAPKs, and expression of neuronal markers, suggesting that activation of PI3K/Rac1 signaling represents a potential mechanism for regulation of neuronal differentiation in SH-SY5Y cells.     

Characterization of a brain-specific Rho GTPase-activating protein,p200RhoGAP

The Rho GTPase-activating proteins (RhoGAPs) are a family of multifunctional molecules that transduce diverse intracellular signals by regulating Rho GTPase activities. A novel RhoGAP family member, p200RhoGAP, is cloned in human and mouse. The murine p200RhoGAP shares 86% sequence identity with the human homolog. In addition to a conserved RhoGAP domain at the N terminus, multiple proline-rich motifs are found in the C-terminal region of the molecules. Northern blot analysis revealed a brain-specific expression pattern of p200RhoGAP. The RhoGAP domain of p200RhoGAP stimulated the GTPase activities of Rac1 and RhoA in vitro and in vivo, and the conserved catalytic arginine residue (Arg-58) contributed to the GAP activity. Expression of the RhoGAP domain of p200RhoGAP in Swiss 3T3 fibroblasts inhibited actin stress fiber formation stimulated by lysophosphatidic acid and platelet-derived growth factor-induced membrane ruffling but not Bradykinin-induced filopodia formation. Endogenous p200RhoGAP was localized to cortical actin in naive N1E-115 neuroblastoma cells and to the edges of extended neurites of differentiated N1E-115 cells. Transient expression of the RhoGAP domain and the full-length molecule, but not the catalytic arginine mutants, readily induced a differentiation phenotype in N1E-115 cells. Finally, p200RhoGAP was capable of binding to the Src homology 3 domains of Src, Crk, and phospholipase Cgamma in vitro and became tyrosine-phosphorylated upon association with activated Src in cells. These results suggest that p200RhoGAP is involved in the regulation of neurite outgrowth by exerting its RhoGAP activity and that its cellular activity may be regulated through interaction with Src-like tyrosine kinases.     

Depolarization-induced differentiation of PC12 cells is mediated by phospholipase D2 through the transcription factor CREB pathway

The present study examined the role of phospholipase D2 (PLD2) in the regulation of depolarization-induced neurite outgrowth and the expression of growth-associated protein-43 (GAP-43) and synapsin I in rat pheochromocytoma (PC12) cells. Depolarization of PC12 cells with 50 mmol/L KCl increased neurite outgrowth and elevated mRNA and protein expression of GAP-43 and synapsin I. These increases were suppressed by inhibition of Ca²⁺-calmodulin-dependent protein kinase II (CaMKII), PLD, or mitogen-activated protein kinase kinase (MEK). Knockdown of PLD2 by small interfering RNA (siRNA) suppressed the depolarization-induced neurite outgrowth, and the increase in GAP-43 and synapsin I expression. Depolarization evoked a Ca²⁺ rise that activated various signaling enzymes and the cAMP response element-binding protein (CREB). Silencing CaMKIIδ by siRNA blocked KCl-induced phosphorylation of proline-rich protein tyrosine kinase 2 (Pyk2), Src kinase, and extracellular signal-regulated kinase (ERK). Inhibition of Src or MEK abolished phosphorylation of ERK and CREB. Furthermore, phosphorylation of Pyk2, ERK, and CREB was suppressed by the PLD inhibitor, 1-butanol and transfection of PLD2 siRNA, whereas it was enhanced by over-expression of wild-type PLD2. Depolarization-induced PLD2 activation was suppressed by CaMKII and Src inhibitors, but not by MEK or protein kinase A inhibitors. These results suggest that the signaling pathway of depolarization-induced PLD2 activation was downstream of CaMKIIδ and Src, and upstream of Pyk2(Y881) and ERK/CREB, but independent of the protein kinase A. This is the first demonstration that PLD2 activation is involved in GAP-43 and synapsin I expression during depolarization-induced neuronal differentiation in PC12 cells.     

Cyclic AMP induces transactivation of the receptors for epidermal growth factor and nerve growth factor,thereby modulating activation of MAP kinase,Akt, and neurite outgrowth in PC12 cells

In PC12 cells, a well studied model for neuronal differentiation, an elevation in the intracellular cAMP level increases cell survival, stimulates neurite outgrowth, and causes activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2). Here we show that an increase in the intracellular cAMP concentration induces tyrosine phosphorylation of two receptor tyrosine kinases, i.e. the epidermal growth factor (EGF) receptor and the high affinity receptor for nerve growth factor (NGF), also termed Trk(A). cAMP-induced tyrosine phosphorylation of the EGF receptor is rapid and correlates with ERK1/2 activation. It occurs also in Panc-1, but not in human mesangial cells. cAMP-induced tyrosine phosphorylation of the NGF receptor is slower and correlates with Akt activation. Inhibition of EGF receptor tyrosine phosphorylation, but not of the NGF receptor, reduces cAMP-induced neurite outgrowth. Expression of dominant-negative Akt does not abolish cAMP-induced survival in serum-free media, but increases cAMP-induced ERK1/2 activation and neurite outgrowth. Together, our results demonstrate that cAMP induces dual signaling in PC12 cells: transactivation of the EGF receptor triggering the ERK1/2 pathway and neurite outgrowth; and transactivation of the NGF receptor promoting Akt activation and thereby modulating ERK1/2 activation and neurite outgrowth.     

A positive role of the PI3-K/Akt signaling pathway in PC12 cell differentiation

Activation of phosphatidylinositol 3-kinase (PI3-K) is considered to be a key event upon stimulation of cells with growth factors. Akt is known to be a downstream target of PI3-K when it is activated by nerve growth factor (NGF). NGF induces cell differentiation of PC12 cells as indicated by neurite outgrowth. In order to investigate the role of PI3-K/Akt in NGF-induced differentiation of PC12 cells, we generated cells ectopically expressing constitutively activated (CA), wild type (WT) and dominant negative (DN) forms of Akt. NGF-induced neurite outgrowth was greatly accelerated in the cells expressing CA-Akt, and dramatically inhibited in those expressing DN-Akt. Pre-treatment with an Akt inhibitor, ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H- hexahydro-1,4-diazepine], inhibited NGF-induced Akt phosphorylation as well as neurite outgrowth but did not markedly affect the activities of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). The PI3-K inhibitors wortmannin and LY294002 blocked NGF-induced Akt phosphorylation as well as neurite outgrowth. These results indicate that PI3-K/Akt is a positive regulator of NGF-induced neuronal differentiation in PC12 cells.     

β3 integrin-EGF receptor cross-talk activates p190RhoGAP in mouse mammary gland epithelial cells

Active RhoA localizes to plasma membrane, where it stimulates formation of focal adhesions and stress fibers. RhoA activity is inhibited by p190RhoGAP following integrin-mediated cell attachment to allow sampling of new adhesive environments. p190RhoGAP is itself activated by Src-dependent tyrosine phosphorylation, which facilitates complex formation with p120RasGAP. This complex then translocates to the cell surface, where p190RhoGAP down-regulates RhoA. Here we demonstrate that the epidermal growth factor receptor (EGFR) cooperates with β3 integrin to regulate p190RhoGAP activity in mouse mammary gland epithelial cells. Adhesion to fibronectin stimulates tyrosine phosphorylation of the EGFR in the absence of receptor ligands. Use of a dominant inhibitory EGFR mutant demonstrates that fibronectin-activated EGFR recruits p120RasGAP to the cell periphery. Expression of an inactive β3 integrin subunit abolishes p190RhoGAP tyrosine phosphorylation, demonstrating a mechanistic link between β3 integrin-activated Src and EGFR regulation of the RhoA inhibitor. The β3 integrin/EGFR pathway also has a positive role in formation of filopodia. Together our data suggest that EGFR constitutes an important intrinsic migratory cue since fibronectin is a key component of the microenvironment in normal mammary gland development and breast cancer. Our data also suggest that EGFR expressed at high levels has a role in eliciting cell shape changes associated with epithelial-to-mesenchymal transition.     

GTK, a Src-related tyrosine kinase, induces nerve growth factor-independent neurite outgrowth in PC12 cells through activation of the Rap1 pathway. Relationship to Shb tyrosine phosphorylation and elevated levels of focal adhesion kinase

The rat pheochromocytoma cell line PC12 is extensively used as a model for studies of neuronal cell differentiation. These cells develop a sympathetic neuron-like phenotype when cultured in the presence of nerve growth factor. The present study was performed in order to assess the role of mouse GTK (previously named BSK/IYK), a cytoplasmic tyrosine kinase belonging to the Src family, for neurite outgrowth in PC12 cells. We report that PC12 cells stably overexpressing GTK exhibit a larger fraction of cells with neurites as compared with control cells, and this response is not accompanied by an increased ERK activity. Treatment of the cells with the MEK inhibitor PD98059 did not reduce the GTK-dependent increased in neurite outgrowth. GTK expression induces a nerve growth factor-independent Rap1 activation, probably through altered CrkII signaling. We observe increased CrkII complex formation with p130(Cas), focal adhesion kinase (FAK), and Shb in PC12-GTK cells. The expression of GTK also correlates with a markedly increased content of FAK, phosphorylation of the adaptor protein Shb, and an association between these two proteins. Transient transfection of GTK-overexpressing cells with RalGDS-RBD or Rap1GAP, inhibitors of the Rap1 pathway, reduces the GTK-dependent neurite outgrowth. These data suggest that GTK participates in a signaling pathway, perhaps involving Shb, FAK and Rap1, that induces neurite outgrowth in PC12 cells.     

DIP (mDia interacting protein) is a key molecule regulating Rho and Rac in a Src-dependent manner

Cell movement is driven by the coordinated regulation of cytoskeletal reorganization through Rho GTPases downstream of integrin and growth-factor receptor signaling. We have reported that mDia, a target protein of Rho, interacts with Src and DIP. Here we show that DIP binds to p190RhoGAP and Vav2, and that DIP is phosphorylated by Src and mediates the phosphorylation of p190RhoGAP and Vav2 upon EGF stimulation. When endogenous DIP was inhibited by expressing dominant-negative mutants of DIP or siRNA, phosphorylation of p190RhoGAP and Vav2 upon EGF stimulation was diminished, and EGF-induced actin organization, distribution of p190RhoGAP and Vav2, and cell movement were affected. Therefore, DIP seems to transfer the complex of the three proteins from cytosol to beneath the membrane, and the three proteins, in turn, can be phosphorylated by Src. DIP inactivated Rho and activated Rac following EGF stimulation in the membrane fraction. Thus, DIP acts as a regulatory molecule causing Src kinase-dependent feedback modulation of Rho GTPases downstream of Rho-mDia upon EGF stimulation, and plays an important role in cell motility.     

Luteolin Induces microRNA-132 Expression and Modulates Neurite Outgrowth in PC12 Cells

Luteolin (3',4',5,7-tetrahydroxyflavone), a food-derived flavonoid, has been reported to exert neurotrophic properties that are associated with its capacity to promote neuronal survival and neurite outgrowth. In this study, we report for the first time that luteolin induces the persistent expression of microRNA-132 (miR-132) in PC12 cells. The correlation between miR-132 knockdown and a decrease in luteolin-mediated neurite outgrowth may indicate a mechanistic link by which miR-132 functions as a mediator for neuritogenesis. Furthermore, we find that luteolin led to the phosphorylation and activation of cAMP response element binding protein (CREB), which is associated with the up-regulation of miR-132 and neurite outgrowth. Moreover, luteolin-induced CREB activation, miR-132 expression and neurite outgrowth were inhibited by adenylate cyclase, protein kinase A (PKA) and MAPK/ERK kinase 1/2 (MEK1/2) inhibitors but not by protein kinase C (PKC) or calcium/calmodulin-dependent protein kinase II (CaMK II) inhibitors. Consistently, we find that luteolin treatment increases ERK phosphorylation and PKA activity in PC12 cells. These results show that luteolin induces the up-regulation of miR-132, which serves as an important regulator for neurotrophic actions, mainly acting through the activation of cAMP/PKA- and ERK-dependent CREB signaling pathways in PC12 cells.     

Rho‐associated kinase (ROCK) inhibitor,Y27632, promotes neurite outgrowth in PC12 cells in the absence of NGF

Neurite extension and retraction are very important processes in the formation of neuronal networks. A strategy for fostering axonal regrowth/regeneration of injured adult neurons is attractive therapeutically for various diseases such as traumatic brain injury, stroke and Alzheimer's disease. The Rho family of small GTPases, including Rac and Cdc42 have been shown to be involved in promoting neurite outgrowth. On the other hand, activation of RhoA induces collapse of growth cone and retraction of neurites. Rho‐associated kinase (ROCK) an effector molecule of RhoA, is downstream of a number of axonal outgrowth and growth cone collapse inhibition mechanisms. In the present study, we sought to identify the role of ROCK in neurite outgrowth in PC12 cells. Y27632, a specific inhibitor of ROCK, induced a robust increase in neurite outgrowth in these cells within 24–48 h as visualized by phase contrast microscopy. Staining with FITC‐tubulin or phalloidin show extended neurites in PC12 cells treated with Y27632, comparable to that with 100 ng/mL of NGF. Assessment of other biochemical markers of neurite outgrowth such as GAP43, neurofilament and tyrosine hydroxylase phosphorylation further indicates that inhibition of ROCK in PC12 cells causes differentiation of these cells to a neuronal phenotype.     

Suppression of RhoA activity by focal adhesion kinase-induced activation of p190RhoGAP: role in regulation of endothelial permeability

The interaction of endothelial cells with extracellular matrix proteins at focal adhesions sites contributes to the integrity of vascular endothelial barrier. Although focal adhesion kinase (FAK) activation is required for the recovery of the barrier function after increased endothelial junctional permeability, the basis for the recovery remains unclear. We tested the hypothesis that FAK activates p190RhoGAP and, thus, negatively regulates RhoA activity and promotes endothelial barrier restoration in response to the permeability-increasing mediator thrombin. We observed that thrombin caused a transient activation of RhoA but a more prolonged FAK activation temporally coupled to the recovery of barrier function. Thrombin also induced tyrosine phosphorylation of p190RhoGAP, which coincided with decrease in RhoA activity. We further showed that FAK was associated with p190RhoGAP, and importantly, recombinant FAK phosphorylated p190RhoGAP in vitro. Inhibition of FAK by adenoviral expression of FRNK (a dominant negative FAK construct) in monolayers prevented p190RhoGAP phosphorylation, increased RhoA activity, induced actin stress fiber formation, and produced an irreversible increase in endothelial permeability in response to thrombin. We also observed that p190RhoGAP was unable to attenuate RhoA activation in the absence of FAK activation induced by FRNK. The inhibition of RhoA by the C3 toxin (Clostridium botulinum toxin) restored endothelial barrier function in the FRNK-expressing cells. These findings in endothelial cells were recapitulated in the lung microcirculation in which FRNK expression in microvessel endothelia increased vascular permeability. Our studies demonstrate that FAK-induced down-modulation of RhoA activity via p190RhoGAP is a crucial step in signaling endothelial barrier restoration after increased endothelial permeability.