TBOPP enhances the anticancer effect of cisplatin by inhibiting DOCK1 in renal cell carcinoma.

The treatment of renal cell carcinoma (RCC) with chemotherapy remains a challenge; therefore, improving the knowledge of the molecular mechanisms underlying RCC chemoresistance and developing novel therapeutic strategies is important. Dedicator of cytokinesis 1 (DOCK1), the first member of the DOCK family to be discovered, displays various roles during tumorigenesis; however, its role during RCC progression is not completely understood. Therefore, the present study aimed to clarify the function of DOCK1 and 1­[2­(3'­(trifluoromethyl)­(1,1'­biphenyl)­4­yl)­2­oxoethyl]­5­pyrrolidinylsulfonyl­2 (1H)­pyridone (TBOPP), a DOCK1­sensitive inhibitor, during RCC development and chemoresistance. The results of CCK­8 and EdU assay indicated that TBOPP decreased RCC cell viability and proliferation compared with the control group, and sensitized RCC cells to cisplatin. Moreover, RCC cells with high DOCK1 expression levels displayed increased resistance to cisplatin, whereas DOCK1 knockdown enhanced the lethal effects of cisplatin on RCC cells. Furthermore, the results determined by western blotting, CCK­8 and cell apoptosis assay indicated that TBOPP effectively reduced DOCK1 expression levels compared with the control group, and the TBOPP­mediated cisplatin sensitizing effect was mediated by DOCK1 inhibition. The present study suggests that DOCK1 plays a vital role in RCC cell chemoresistance to cisplatin; therefore, TBOPP may serve as a novel therapeutic agent for RCC chemoresistance.

Opposing roles of hematopoietic-specific small GTPase Rac2 and the guanine nucleotide exchange factor Vav1 in osteoclast differentiation.

Vav1 regulates Rac activation as a hematopoietic-specific Rho/Rac-family guanine nucleotide exchange factor. Rac is a subfamily of Rho GTPases that regulates the bone-resorbing capacity of osteoclasts (OCs). In this study, we show that hematopoietic-specific Rac2 and Vav1 play opposing roles by enhancing or attenuating OC differentiation, respectively. This was demonstrated by higher and lower bone density in the femurs from Rac2-deficient (Rac2-/-) and Vav1-deficient (Vav1-/-) mice, respectively, compared to the wild-type (WT) mice. Accordingly, Rac2-/- cells displayed low numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (41%) compared to WT cells, whereas, Vav1-/- cells showed high TRAP-positive cell numbers (150%), and the double-knockout Rac2-/-Vav1-/- mice nullified the effects on OC numbers achieved by the individual knockouts. These reciprocal roles of Rac2 and Vav1 in OC differentiation were confirmed by reduced and increased levels of OC-specific markers, such as TRAP, calcitonin receptor, cathepsin K, and DC-STAMP in the Rac2-/- and Vav1-/- OCs, respectively. Our findings of decrease and increase in actin ring formation and αvß3 integrin-mediated adhesion in Rac2-/- and Vav1-/- mice, respectively, suggest that Vav1 and its downstream GTPase, Rac2, may counteract to fine-tune OC differentiation and bone resorption.

Rac-GTPases and Rac-GEFs in neutrophil adhesion, migration and recruitment.

Rac-GTPases and their Rac-GEF activators play important roles in the recruitment and host defence functions of neutrophils. These proteins control the activation of adhesion molecules and the cytoskeletal dynamics that enable the adhesion, migration and tissue recruitment of neutrophils. They also regulate the effector functions that allow neutrophils to kill bacterial and fungal pathogens, and to clear debris. This review focuses on the roles of Rac-GTPases and Rac-GEFs in neutrophil adhesion, migration and recruitment.

Frazzled can act through distinct molecular pathways in epithelial cells to regulate motility, apical constriction, and localisation of E-Cadherin.

Netrin receptors of the DCC/NEO/UNC-40/Frazzled family have well established roles in cell migration and axon guidance but can also regulate epithelial features such as adhesion, polarity and adherens junction (AJ) stability. Previously, we have shown that overexpression of Drosophila Frazzled (Fra) in the peripodial epithelium (PE) inhibits wing disc eversion and also generates cellular protrusions typical of motile cells. Here, we tested whether the molecular pathways by which Fra inhibits eversion are distinct from those driving motility. We show that in disc proper (DP) epithelial cells Fra, in addition to inducing F-Actin rich protrusions, can affect localization of AJ components and columnar cell shape. We then show that these phenotypes have different requirements for the three conserved Fra cytoplasmic P-motifs and for downstream genes. The formation of protrusions required the P3 motif of Fra, as well as integrins (mys and mew), the Rac pathway (Rac1, wave and, arpc3) and myosin regulatory light chain (Sqh). In contrast, apico-basal cell shape change, which was accompanied by increased myosin phosphorylation, was critically dependent upon the P1 motif and was promoted by RhoGef2 but inhibited by Rac1. Fra also caused a loss of AJ proteins (DE-Cad and Arm) from basolateral regions of epithelial cells. This phenotype required all 3 P-motifs, and was dependent upon the polarity factor par6. par6 was not required for protrusions or cell shape change, but was required to block eversion suggesting that control of AJ components may underlie the ability of Fra to promote epithelial stability. The results imply that multiple molecular pathways act downstream of Fra in epithelial cells.

Rac3 regulates cell proliferation through cell cycle pathway and predicts prognosis in lung adenocarcinoma.

Lung cancer is still the leading cause of malignant deaths in the world. It is of great importance to find novel functional genes for the tumorigenesis of lung cancer. We demonstrated that Rac3 could promote cell proliferation and inhibit apoptosis in lung adenocarcinoma cell line A549 previously. The aim of this study was to investigate the function and mechanism of Rac3 in lung adenocarcinoma cell lines. Immunohistochemistry staining was performed in 107 lung adenocarcinoma tissues and matched non-tumor tissues. Multivariate analysis and Kaplan-Meier analysis were used to investigate the correlation between Rac3 expression and the clinical outcomes. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, and flow cytometry analysis were employed to determine the proliferative ability, cell cycle distribution, and apoptosis in H1299 and H1975 cell lines. Gene expression microarray and pathway analysis between the Rac3-siRNA group and the control group in A549 cells were performed to investigate the pathways and mechanism of Rac3 regulation. Rac3 was shown to be positively expressed in lung adenocarcinoma tissues, and the expression of Rac3 associates with longer survival in lung adenocarcinoma patients. Silencing of Rac3 significantly induced cell growth inhibition, colony formation decrease, cell cycle arrest, and apoptosis of lung adenocarcinoma cell lines, which accompanied by obvious downregulation of CCND1, MYC, and TFDP1 of cell cycle pathway involving in the tumorigenesis of lung adenocarcinoma based on the gene expression microarray. In conclusion, these findings suggest that Rac3 has the potential of being a therapeutic target for lung adenocarcinoma.

RAC3 more than a nuclear receptor coactivator: a key inhibitor of senescence that is downregulated in aging.

Receptor-associated coactivator 3 (RAC3) is a nuclear receptor coactivator usually overexpressed in tumors that exerts oncogenic functions in the cytoplasm and the nucleus. Although as part of its oncogenic actions it was previously identified as an inhibitor of apoptosis and autophagy, its expression is required in order to preserve the pluripotency and embryonic stem cell self-renewal. In this work we investigated its role in cellular senescence. We found that RAC3 overexpression in the nontumoral HEK293 cells inhibits the premature senescence induced by hydrogen peroxide or rapamycin. The mechanism involves not only the inhibition of autophagy early induced by these stimuli in the pathway to senescence, but also the increase in levels and nuclear localization of both the cell cycle suppressors p53/p21 and the longevity promoters FOXO1A, FOXO3A and SIRT1. Furthermore, we found that RAC3 overexpression is required in order to maintain the telomerase activity. In tumoral HeLa cells its activity was inhibited by depletion of RAC3 inducing replicative senescence. Moreover, we demonstrated that in vivo, levels of RAC3 are downregulated in the liver from aged as compared with young rats, whereas the levels of p21 are increased, correlating with the expected senescent cell contents in aged tissues. A similar downregulation of RAC3 was observed in the premature and replicative senescence of human fetal WI-38 cells and premature senescence of hepatocyte HepG2 cell line. Taken together, all these results demonstrate that RAC3 is an inhibitor of senescence whose downregulation in aged individuals could be probably a tumor suppressor mechanism, avoiding the clonal expansion of risky old cells having damaged DNA.

DRK/DOS/SOS converge with Crk/Mbc/dCed-12 to activate Rac1 during glial engulfment of axonal debris.

Nervous system injury or disease leads to activation of glia, which govern postinjury responses in the nervous system. Axonal injury in Drosophila results in transcriptional up-regulation of the glial engulfment receptor Draper; there is extension of glial membranes to the injury site (termed activation), and then axonal debris is internalized and degraded. Loss of the small GTPase Rac1 from glia completely suppresses glial responses to injury, but upstream activators remain poorly defined. Loss of the Rac guanine nucleotide exchange factor (GEF) Crk/myoblast city (Mbc)/dCed-12 has no effect on glial activation, but blocks internalization and degradation of debris. Here we show that the signaling molecules downstream of receptor kinase (DRK) and daughter of sevenless (DOS) (mammalian homologs, Grb2 and Gab2, respectively) and the GEF son of sevenless (SOS) (mammalian homolog, mSOS) are required for efficient activation of glia after axotomy and internalization/degradation of axonal debris. At the earliest steps of glial activation, DRK/DOS/SOS function in a partially redundant manner with Crk/Mbc/dCed-12, with blockade of both complexes strongly suppressing all glial responses, similar to loss of Rac1. This work identifies DRK/DOS/SOS as the upstream Rac GEF complex required for glial responses to axonal injury, and demonstrates a critical requirement for multiple GEFs in efficient glial activation after injury and internalization/degradation of axonal debris.

Rac2 controls tumor growth, metastasis and M1-M2 macrophage differentiation in vivo.

Although it is well-established that the macrophage M1 to M2 transition plays a role in tumor progression, the molecular basis for this process remains incompletely understood. Herein, we demonstrate that the small GTPase, Rac2 controls macrophage M1 to M2 differentiation and the metastatic phenotype in vivo. Using a genetic approach, combined with syngeneic and orthotopic tumor models we demonstrate that Rac2-/- mice display a marked defect in tumor growth, angiogenesis and metastasis. Microarray, RT-PCR and metabolomic analysis on bone marrow derived macrophages isolated from the Rac2-/- mice identify an important role for Rac2 in M2 macrophage differentiation. Furthermore, we define a novel molecular mechanism by which signals transmitted from the extracellular matrix via the α4ß1 integrin and MCSF receptor lead to the activation of Rac2 and potentially regulate macrophage M2 differentiation. Collectively, our findings demonstrate a macrophage autonomous process by which the Rac2 GTPase is activated downstream of the α4ß1 integrin and the MCSF receptor to control tumor growth, metastasis and macrophage differentiation into the M2 phenotype. Finally, using gene expression and metabolomic data from our Rac2-/- model, and information related to M1-M2 macrophage differentiation curated from the literature we executed a systems biologic analysis of hierarchical protein-protein interaction networks in an effort to develop an iterative interactome map which will predict additional mechanisms by which Rac2 may coordinately control macrophage M1 to M2 differentiation and metastasis.

Rho GTPases orient directional sensing in chemotaxis.

During chemotaxis, cells sense extracellular chemical gradients and position Ras GTPase activation and phosphatidylinositol (3,4,5)-triphosphate (PIP3) production toward chemoattractants. These two major signaling events are visualized by biosensors in a crescent-like zone at the plasma membrane. Here, we show that a Dictyostelium Rho GTPase, RacE, and a guanine nucleotide exchange factor, GxcT, stabilize the orientation of Ras activation and PIP3 production in response to chemoattractant gradients, and this regulation occurred independently of the actin cytoskeleton and cell polarity. Cells lacking RacE or GxcT fail to persistently direct Ras activation and PIP3 production toward chemoattractants, leading to lateral pseudopod extension and impaired chemotaxis. Constitutively active forms of RacE and human RhoA are located on the portion of the plasma membrane that faces lower concentrations of chemoattractants, opposite of PIP3 production. Mechanisms that control the localization of the constitutively active form of RacE require its effector domain, but not PIP3. Our findings reveal a critical role for Rho GTPases in positioning Ras activation and thereby establishing the accuracy of directional sensing.

Pak and Rac GTPases promote oncogenic KIT-induced neoplasms.

An acquired somatic mutation at codon 816 in the KIT receptor tyrosine kinase is associated with poor prognosis in patients with systemic mastocytosis and acute myeloid leukemia (AML). Treatment of leukemic cells bearing this mutation with an allosteric inhibitor of p21-activated kinase (Pak) or its genetic inactivation results in growth repression due to enhanced apoptosis. Inhibition of the upstream effector Rac abrogates the oncogene-induced growth and activity of Pak. Although both Rac1 and Rac2 are constitutively activated via the guanine nucleotide exchange factor (GEF) Vav1, loss of Rac1 or Rac2 alone moderately corrected the growth of KIT-bearing leukemic cells, whereas the combined loss resulted in 75% growth repression. In vivo, the inhibition of Vav or Rac or Pak delayed the onset of myeloproliferative neoplasms (MPNs) and corrected the associated pathology in mice. To assess the role of Rac GEFs in oncogene-induced transformation, we used an inhibitor of Rac, EHop-016, which specifically targets Vav1 and found that EHop-016 was a potent inhibitor of human and murine leukemic cell growth. These studies identify Pak and Rac GTPases, including Vav1, as potential therapeutic targets in MPN and AML involving an oncogenic form of KIT.

Neuronal Rac1 is required for learning-evoked neurogenesis.

Hippocampus-dependent learning and memory relies on synaptic plasticity as well as network adaptations provided by the addition of adult-born neurons. We have previously shown that activity-induced intracellular signaling through the Rho family small GTPase Rac1 is necessary in forebrain projection neurons for normal synaptic plasticity in vivo, and here we show that selective loss of neuronal Rac1 also impairs the learning-evoked increase in neurogenesis in the adult mouse hippocampus. Earlier work has indicated that experience elevates the abundance of adult-born neurons in the hippocampus primarily by enhancing the survival of neurons produced just before the learning event. Loss of Rac1 in mature projection neurons did reduce learning-evoked neurogenesis but, contrary to our expectations, these effects were not mediated by altering the survival of young neurons in the hippocampus. Instead, loss of neuronal Rac1 activation selectively impaired a learning-evoked increase in the proliferation and accumulation of neural precursors generated during the learning event itself. This indicates that experience-induced alterations in neurogenesis can be mechanistically resolved into two effects: (1) the well documented but Rac1-independent signaling cascade that enhances the survival of young postmitotic neurons; and (2) a previously unrecognized Rac1-dependent signaling cascade that stimulates the proliferative production and retention of new neurons generated during learning itself.

Macrophage migration arrest due to a winning balance of Rac2/Sp1 repression over ß-catenin-induced PLD expression.

Monocytes and neutrophils infiltrate into tissues during inflammation and stay for extended periods of time until the initial insult is resolved or sometimes remain even longer in the case of chronic inflammation. The mechanism as to why phagocytes become immobilized after the initial cell migration event is not understood completely. Here, we show that overexpression or hyperactivation of Rac2 decreases sustained chemotactic responses of macrophages to MCP-1/CCL2. The resulting leukocyte arrest is not caused by a diminished availability of the cytokine receptor CCR2 that remains intact during MCP-1 stimulation. We show a novel mechanism that links the Rac2-dependent arrest of chemotaxis to decreased expression of PLD2 through the transcription regulator Sp1. Prolonged Rac2 activity leads to nuclear overactivation of Sp1, which acts as a repressor for PLD2. Also, another signaling component plays a regulatory role: ß-catenin. Although early times of stimulation (≈ 20 min) with MCP-1/CCL2 resulted in activation of ß-catenin with a positive effect on PLD2, after ≈ 3 h of stimulation, the levels of ß-catenin were reduced and not able to prevent the negative effect of Rac2 on PLD2 activity. This is a novel molecular mechanism underlying immobilization of monocyte/macrophage migration that is important for the physiological maintenance of leukocytes at the site of inflammation. If this immobilization is prolonged enough, it could lead to chronic inflammation.

Sac-1004, a novel vascular leakage blocker, enhances endothelial barrier through the cAMP/Rac/cortactin pathway.

The maintenance of endothelial barrier is critical for the vascular homeostasis and is maintained by the interaction of adherens junction (AJ) and tight junction (TJ) proteins between adjacent cells. This interaction is stabilized by actin cytoskeleton forming cortical actin ring. Here, we developed a novel vascular leakage blocker, Sac-1004 and investigated its mechanism of action in endothelial cells (ECs). Sac-1004 inhibited endothelial hyperpermeability induced by vascular endothelial growth factor, histamine and thrombin via stabilization of cortical actin ring and AJ proteins at the cell-cell junction. Treatment of Sac-1004 in ECs increased cAMP levels and activated Rac, both of which are known to strengthen endothelial barrier. Furthermore, Sac-1004 induced phosphorylation of cortactin and its localization at cell membrane that is essential for the stabilization of cortical actin ring. These effects of Sac-1004 on ECs were significantly abrogated by dideoxyadenosine (adenylyl cyclase inhibitor) and NSC23766 (Rac inhibitor). Taken together, our findings indicate that Sac-1004 blocks vascular leakage by enhancing endothelial integrity via the cAMP/Rac/cortactin pathway and imply the potential usefulness of Sac-1004 in the development of therapeutic means for vascular leakage-related diseases.

Rac1 signaling regulates sepsis-induced pathologic inflammation in the lung via attenuation of Mac-1 expression and CXC chemokine formation.

Excessive neutrophil recruitment is a major feature in septic lung damage although the signaling mechanisms behind pulmonary infiltration of neutrophils in sepsis remain elusive. In the present study, we hypothesized that Rac1 might play an important role in pulmonary neutrophil accumulation and tissue injury in abdominal sepsis. Male C57BL/6 mice were treated with Rac1 inhibitor NSC23766 (5 mg/kg) before cecal ligation and puncture (CLP). Bronchoalveolar lavage fluid and lung tissue were collected for the quantification of neutrophil recruitment and edema and CXC chemokine formation. Blood was collected for the determination of Mac-1 on neutrophils and proinflammatory compounds in plasma. Gene expression of CXC chemokines and tumor necrosis factor alpha was determined by quantitative reverse transcription-polymerase chain reaction in alveolar macrophages. Rac1 activity was increased in lungs from septic animals, and NSC23766 significantly decreased pulmonary activity of Rac1 induced by CLP. Administration of NSC23766 markedly reduced CLP-triggered neutrophil infiltration, edema formation, and tissue damage in the lung. Inhibition of Rac1 decreased CLP-induced neutrophil expression of Mac-1 and pulmonary formation of CXC chemokines. Moreover, NSC23766 abolished the sepsis-evoked elevation of messenger RNA levels of CXC chemokines and tumor necrosis factor alpha in alveolar macrophages. Rac1 inhibition decreased the CLP-induced increase in plasma levels of high mobility group protein B1 and interleukin 6, indicating a role of Rac1 in systemic inflammation. In conclusion, our results demonstrate that Rac1 signaling plays a key role in regulating pulmonary infiltration of neutrophils and tissue injury via regulation of chemokine production in the lung and Mac-1 expression on neutrophils in abdominal sepsis. Thus, targeting Rac1 activity might be a useful strategy to protect the lung in abdominal sepsis.

Preventive and therapeutic effects of Smad7 on radiation-induced oral mucositis.

We report that K5.Smad7 mice, which express a Smad7 transgene under the control of a keratin 5 promoter, were resistant to radiation-induced oral mucositis, a painful oral ulceration. In addition to nuclear factor κB (NF-κB) activation, which is known to contribute to oral mucositis, we found activated transforming growth factor ß (TGF-ß) signaling in cells from this condition. Smad7 dampened both pathways to attenuate inflammation, growth inhibition and apoptosis. Additionally, Smad7 promoted oral epithelial migration to close the wound. Further analyses revealed that TGF-ß signaling Smads and their co-repressor C-terminal binding protein 1 (CtBP1) transcriptionally repressed Rac1, and that Smad7 abrogated this repression. Knocking down Rac1 expression in mouse keratinocytes abrogated Smad7-induced migration. Topical application of Smad7 protein conjugated with a cell-permeable Tat tag to oral mucosa showed prophylactic and therapeutic effects on radiation-induced oral mucositis in mice. Thus, we have identified new molecular mechanisms involved in oral mucositis pathogenesis, and our data suggest an alternative therapeutic strategy to block multiple pathological processes in this condition.

The Rac GTPase effector p21-activated kinase is essential for hematopoietic stem/progenitor cell migration and engraftment.

The p21-activated kinases (Paks) are serine/threonine kinases that are major effectors of the Rho guanosine 5'\x{2011}triphosphatase, Rac, and Cdc42. Rac and Cdc42 are known regulators of hematopoietic stem and progenitor cell (HSPC) function, however, a direct role for Paks in HSPCs has yet to be elucidated. Lin(-)Sca1(+)c-kit(+) (LSK) cells from wild-type mice were transduced with retrovirus expressing Pak inhibitory domain (PID), a well-characterized inhibitor of Pak activation. Defects in marrow homing and in vitro cell migration, assembly of the actin cytoskeleton, proliferation, and survival were associated with engraftment failure of PID-LSK. The PID-LSK demonstrated decreased phosphorylation of extracellular signal-regulated kinase (ERK), whereas constitutive activation of ERK in these cells led to rescue of hematopoietic progenitor cell proliferation in vitro and partial rescue of Pak-deficient HSPC homing and engraftment in vivo. Using conditional knock-out mice, we demonstrate that among group A Paks, Pak2(-/-) HSPC show reduced homing to the bone marrow and altered cell shape similar to PID-LSK cells in vitro and are completely defective in HSPC engraftment. These data demonstrate that Pak proteins are key components of multiple engraftment-associated HSPC functions and play a direct role in activation of ERK in HSPCs, and that Pak2 is specifically essential for HSPC engraftment.

Iloprost improves endothelial barrier function in lipopolysaccharide-induced lung injury.

The protective effects of prostacyclin and its stable analogue iloprost are mediated by elevation of intracellular cyclic AMP (cAMP) leading to enhancement of the peripheral actin cytoskeleton and cell-cell adhesive structures. This study tested the hypothesis that iloprost may exhibit protective effects against lung injury and endothelial barrier dysfunction induced by bacterial wall lipopolysaccharide (LPS). Endothelial barrier dysfunction was assessed by measurements of transendothelial permeability, morphologically and by analysis of LPS-activated inflammatory signalling. In vivo, C57BL/6J mice were challenged with LPS with or without iloprost or 8-bromoadenosine-3',5'-cyclic monophosphate (Br-cAMP) treatment. Lung injury was monitored by measurements of bronchoalveolar lavage protein content, cell count and Evans blue extravasation. Iloprost and Br-cAMP attenuated the disruption of the endothelial monolayer, and suppressed the activation of p38 mitogen-activated protein kinase (MAPK), the nuclear factor (NF)-κB pathway, Rho signalling, intercellular adhesion molecular (ICAM)-1 expression and neutrophil migration after LPS challenge. In vivo, iloprost was effective against LPS-induced protein and neutrophil accumulation in bronchoalveolar lavage fluid, and reduced myeloperoxidase activation, ICAM-1 expression and Evans blue extravasation in the lungs. Inhibition of Rac activity abolished the barrier-protective and anti-inflammatory effects of iloprost and Br-cAMP. Iloprost-induced elevation of intracellular cAMP triggers Rac signalling, which attenuates LPS-induced NF-κB and p38 MAPK inflammatory pathways and the Rho-dependent mechanism of endothelial permeability.

Rac1 is required for Prkar1a-mediated Nf2 suppression in Schwann cell tumors.

Schwannomas are peripheral nerve sheath tumors that often occur in the setting of an inherited tumor predisposition syndrome, including neurofibromatosis types 1 (NF1) and 2 (NF2), familial schwannomatosis and Carney complex. Loss of the NF2 tumor suppressor (encoding NF2, or Merlin) is associated with upregulation of the Rac1 small GTPase, which is thought to have a key role in mediating tumor formation. In prior studies, we generated a mouse model of schwannomas by performing tissue-specific knockout (KO) of the Carney complex gene Prkar1a, which encodes the type 1A regulatory subunit of protein kinase A. These tumors exhibited down-regulation of Nf2 protein and an increase in activated Rac1. To assess the requirement for Rac1 in schwannoma formation, we generated a double KO (DKO) of Prkar1a and Rac1 in Schwann cells and monitored tumor formation. Loss of Rac1 reduced tumor formation by reducing proliferation and enhancing apoptosis. Surprisingly, the reduction of tumor formation was accompanied by re-expression of the Nf2 protein. Furthermore, activated Rac1 was able to downregulate Nf2 in vitro in a Pak-dependent manner. These in vivo data indicate that activation of Rac1 is responsible for suppression of Nf2 protein production; deficiency of Nf2 in Schwann cells leads to loss of cellular growth control and tumor formation. Further, PKA activation through mutation in Prkar1a is sufficient to initiate Rac1 signaling, with subsequent reduction of Nf2 and schwannomagenesis. Although in vitro evidence has shown that loss of Nf2 activates Rac1, our data indicate that signaling between Nf2 and Rac1 occurs in a bidirectional fashion, and these interactions are modulated by PKA.

Function of Ahnak protein in aortic smooth muscle cell migration through Rac activation.

AIMS: Ahnak protein acts as a scaffold protein networking phospholipase C-γ and protein kinase C-α, which subsequently stimulate an extracellular signal-regulated kinase (Erk) pathway. In mouse aortic smooth muscle cells (ASMCs), the activation of the signalling cascade ultimately promotes the cell migration through an unknown mechanism. We aimed to dissect the Ahnak-mediated cell signalling network involved in the migration of ASMCs. METHODS AND RESULTS: Migration of ASMCs from wild-type mice was significantly increased by platelet-derived growth factor (PDGF) stimulation in transwell chamber and wound-healing assays, whereas migration of ASMCs from Ahnak knockout mice was reduced. Consistently, stimulation of wild-type ASMCs with PDGF resulted in Rac activation-mediated lamellipodial protrusion in migrating cells. In contrast, Ahnak knockout ASMCs displayed lower activation of Rac in response to PDGF and slow lamellipodial protrusion rate and cell migration. Ahnak signalling complex was analysed by immunoprecipitation with antibody to p21-activated protein kinase (PAK). Ahnak protein was shown to function as the signalling scaffold interacting with the multiple protein complex of Erk, PAK, and p21-activated kinase-interacting exchange factor ß. The proposed role of Ahnak in cell migration was examined using a restenosis model in which the carotid arteries of mice were subjected to post-ligation injury. We show neointimal formation and SMC migration after ligation injury in Ahnak knockout mice were significantly retarded compared with wild-type mice. CONCLUSION: Ahnak protein plays an important scaffolding function connecting Erk and Rac activation in PDGF-dependent migration of ASMC.

TIPE2 controls innate immunity to RNA by targeting the phosphatidylinositol 3-kinase-Rac pathway.

RNA receptors such as TLR3 and retinoid acid-inducible gene I/melanoma differentiation-associated gene 5 play essential roles in innate immunity to RNA viruses. However, how innate immunity to RNAs is controlled at the molecular level is not well understood. We describe in this study a new regulatory pathway of anti-RNA immunity that is composed of PI3K, its target GTPase Rac, and the newly described immune regulator TNF-α-induced protein 8 like-2 (TIPE2, or TNFAIP8L2). Polyinosinic-polycytidylic acid [Poly (I:C)], a dsRNA receptor ligand, activates Rac via its guanine nucleotide exchange factor Tiam; this leads to the activation of cytokine genes and, paradoxically, downregulation of the Tipe2 gene. TIPE2 is a negative regulator of immunity; its deficiency leads to hyperactivation of the PI3K-Rac pathway as exemplified by enhanced AKT, Rac, P21-activated kinase, and IFN regulatory factor 3 activities. As a consequence, TIPE2 knockout myeloid cells are hyperreactive to Poly (I:C) stimulation, and TIPE2 knockout mice are hypersensitive to Poly (I:C)-induced lethality. These results indicate that TIPE2 controls innate immunity to RNA by targeting the PI3K-Rac pathway. Therefore, manipulating TIPE2 or Rac functions can be effective for controlling RNA viral infections.