Members Of Rho GTPase Family Research Articles

A Novel Method of Mobilizing Leukemia Initiating Cells by a Small Molecule Cdc42 Activity-Specific Inhibitor (CASIN) for Acute Myeloid Leukemia Therapy.

Abstract 13A large body of studies has proposed that leukemia initiating cells (LICs) are the culprit of leukemia relapse from conventional therapies. Like normal hematopoietic stem/progenitor cells (HSPCs), LICs are thought to reside in the bone marrow (BM) endothelial and/or endosteal niche. Although the full nature of LIC-niche interaction remains elusive, it has been postulated as a useful target for leukemia therapy based on a dual rationale: on one hand, the survival of LICs may depend upon interactions with specific niche, while on the other hand, chasing LICs out of the BM niche may drive quiescent LICs into active cell cycle, sensitizing them for conventional chemotherapy. Recent progresses in studying the PML tumor suppressor and the CXCR4-antagonist AMD3100 have provided strong support for such a rationale (Blood 113, 6215; Nature 453, 1072). The Rho GTPase family member Cdc42 is a central regulator of cell proliferation, adhesion, and migration by integrating signals from multiple cell surface receptors. Ablation of Cdc42 in HSPCs in a conditional knockout mouse model leads to massive egress of HSPCs from BM to the peripheral blood (PB), a phenotype attributable to deficiencies in HSPC adhesion, migration, and F-actin polymerization. We recently demonstrated that pharmacological targeting of Cdc42 by a small molecule Cdc42 activity-specific inhibitor (CASIN) transiently and specifically inhibits Cdc42 activity and mimics the Cdc42 knockout mobilization phenotype by suppressing HSPC adhesion, migration, and F-actin polymerization. CASIN appears to be active in this regard on both murine and human blood progenitors in vitro and in xenografted mice (Blood 112: 68a, Nature Biotechnology under revision). In the present studies, we hypothesize that pharmacological targeting of Cdc42 by CASIN is effective in mobilizing LICs from the BM niche, thus providing a new method for combinatory therapy against LICs. To test this hypothesis, we used MLL-AF9/N-Ras human AML cells (MA9/N-Ras), whereby the N-Ras G12D oncogene was introduced into MLL-AF9 transduced human CD34+ umbilical cord blood (HCB) cells. These cells grow vigorously in vitro independent of cytokine supplementation and induce AML readily in humanized NOD/SCID-SGM3 (SGM3) mice. Xenotransplant experiments confirmed that MA9/N-Ras cells are clonal inducers of leukemia with the property of LICs, as animals transplanted with either bulk cultures or with single cell derived cultures succumbed to AML with similar latencies. In MA9/N-Ras cells CASIN effectively inhibited downstream effectors of Cdc42 such as p-PAK, p-MLC and p-FAK in a dose-dependent manner. In the SGM3 mouse xenograft, we found CASIN administration (1.2 mg/kg, IV) transiently elicited mobilization of LICs from BM to PB by 20 minutes post injection. This was correlated with in vitro suppression of SDF-1α induced F-actin polymerization detected by FACS analysis and directional migration detected by a transwell assay upon CASIN treatment of the LICs (5-10 μM). Similar observations were made using Cdc42-specific shRNA knockdown of endogenous Cdc42 in the LICs. Continuous CASIN infusion into the xenografted mice for 5 days (1.2 mg/kg, IV, once daily) led to a potent induction of apoptosis of LICs detected by AnnexinV/7AAD staining. Significantly, the CASIN infusion showed no effects on the survival of HCB cells in xenografted SGM3 mice. In addition to a potential niche-dependent survival mechanism, the LICs, not normal HCB cells, appear to directly depend on Cdc42 for survival signals as further in vitro culture studies found that a 24-hour CASIN treatment resulted in a dose-dependent apoptosis of MA9/N-Ras cells, but not of normal HCBs. Finally, mouse genetic studies using MA9 transduced Mx-cre;Cdc42lox/lox BM cells transplanted into congenic BoyJ recipients showed that none of the mice with deleted Cdc42 upon pIpC injection developed AML while all mock-injected mice die from leukemia with less than 4 weeks latency, providing genetic evidence that Cdc42 is required for MA9-induced initiation of AML. Whether CASIN is effective in sensitizing the LICs to conventional chemotherapy in a combinatory regiment is currently under investigation. Our studies present a novel concept that pharmacological targeting of the intracellular signal transducer Cdc42 may have therapeutic value in eradicating LICs. Disclosures:No relevant conflicts of interest to declare.

RhoG Promotes Neural Progenitor Cell Proliferation in Mouse Cerebral Cortex

In early cortical development, neural progenitor cells (NPCs) expand their population in the ventricular zone (VZ), and produce neurons. Although a series of studies have revealed the process of neurogenesis, the molecular mechanisms regulating NPC proliferation are still largely unknown. Here we found that RhoG, a member of Rho family GTPases, was expressed in the VZ at early stages of cortical development. Expression of constitutively active RhoG promoted NPC proliferation and incorporation of bromodeoxyuridine (BrdU) in vitro, and the proportion of Ki67-positive cells in vivo. In contrast, knockdown of RhoG by RNA interference suppressed the proliferation, BrdU incorporation, and the proportion of Ki67-positive cells in NPCs. However, knockdown of RhoG did not affect differentiation and survival of NPC. The RhoG-induced promotion of BrdU incorporation required phosphatidylinositol 3-kinase (PI3K) activity but not the interaction with ELMO. Taken together, these results indicate that RhoG promotes NPC proliferation through PI3K in cortical development.

Human Neutrophils Coordinate Chemotaxis by Differential Activation of Rac1 and Rac2

Rac1 and Rac2, members of the small Rho GTPase family, play essential roles in coordinating directional migration and superoxide production during neutrophil responses to chemoattractants. Although earlier studies in Rac1 and Rac2 knockout mice have demonstrated unique roles for each Rac isoform in chemotaxis and NADPH oxidase activation, it is still unclear how human neutrophils use Rac1 and Rac2 to achieve their immunological responses to foreign agent stimulation. In the current study, we used TAT dominant-negative Rac1-T17N and Rac2-T17N fusion proteins to acutely alter the activity of Rac1 and Rac2 individually in human neutrophils. We demonstrate distinct activation kinetics and different roles for Rac1 and Rac2 in response to low vs high concentrations of fMLP. These observations were verified using neutrophils from mice in which Rac1 or Rac2 was genetically absent. Based on these results, we propose a model to explain how human neutrophils kill invading microbes while limiting oxidative damage to the adjacent surrounding healthy tissue through the differential activation of Rac1 and Rac2 in response to different concentrations of chemoattractant.

Targeting Rho GTPases by Peptidic Structures

Rho GTPases represent a family of small GTP-binding proteins that are involved in many important cellular functions relevant to cancer including cell cytoskeleton organization, migration, transcription, and proliferation. Since deregulation of members of Rho GTPase family is often found associated with many disease states, targeting of Rho GTPases and related signaling pathways for potential therapeutic benefits has been extensively pursued. Recent progress in this field of studies by peptide and peptidomemic inhibitors has provided important validations to this principle. The possibility to design and synthesize specific peptides that can bind to specific surface of the targeting proteins to elicit transient and specific blockade of the signal flows that require defined protein-protein interactions makes peptide inhibitors an attractive approach. In this review we summarize the recent advances in the design and application of a number of polypeptide and peptidomimetic structures that specifically target individual members of Rho GTPases and their up- or down-stream signaling regulators/effectors with an emphasis on cancer, inflammation and neurodegenerative diseases. The principle derived from the peptidic inhibitors has led to discoveries of the first generation of small molecule inhibitors of Rac GTPase of the Rho family. The implication of these studies in the pathobiology of various human diseases makes targeting Rho GTPases a valid strategy for future therapies.

RhoBTB3: A Rho GTPase-Family ATPase Required for Endosome to Golgi Transport

Rho GTPases are key regulators of the actin-based cytoskeleton; Rab GTPases are key regulators of membrane traffic. We report here that the atypical Rho GTPase family member, RhoBTB3, binds directly to Rab9 GTPase and functions with Rab9 in protein transport from endosomes to the trans Golgi network. Gene replacement experiments show that RhoBTB3 function in cultured cells requires both RhoBTB3's N-terminal, Rho-related domain and C-terminal sequences that are important for Rab9 interaction. Biochemical analysis reveals that RhoBTB3 binds and hydrolyzes ATP rather than GTP. Rab9 binding opens the autoinhibited RhoBTB3 protein to permit maximal ATP hydrolysis. Because RhoBTB3 interacts with TIP47 on membranes, we propose that it may function to release this cargo selection protein from vesicles to permit their efficient docking and fusion at the Golgi.

The Actin Polymerization Regulator WAVE2 Is Required for Early Bone Marrow Repopulation by Hematopoietic Stem Cells

The Rho GTPase family members play essential roles in hematopoiesis. Of these, Rac1 is thought to be required for the appropriate spatial localization of hematopoietic stem and/or progenitor cells (HSPCs) within the bone marrow (BM), whereas Rac2 likely plays a role in BM retention of HSPCs. To elucidate the molecular mechanisms underlying Rac-mediated functions in hematopoietic stem cells (HSCs), we studied Wiskott-Aldrich syndrome protein family verprolin-homologous proteins (WAVEs), the specific effectors downstream of the Rac GTPases in actin polymerization. We here showed that CD34(-/low)c-Kit(+)Sca-1(+)lineage(-) HSCs (CD34(-)KSL HSCs) express WAVE2 but neither WAVE1 nor WAVE3. Because WAVE2 knockout mice are embryonic-lethal, we utilized HSCs in which the expression of WAVE2 was reduced by small interfering RNA. We found that knockdown (KD) of WAVE2 in HSCs affected neither in vitro colony formation nor cell proliferation but did impair in vivo long-term reconstitution. Interestingly, WAVE2 KD HSCs exhibited unaltered homing but showed poor BM repopulation detected as early as day 5 after transplantation. The mechanistic studies on WAVE2 KD HSCs revealed modest but significant impairment in both cobblestone-like area-forming on stromal layers and actin polymerization upon integrin ligation by fibronectin. These results suggested that WAVE2-mediated actin polymerization, potentially downstream of Rac1, plays an important role in intramarrow mobilization and proliferation of HSCs, which are believed to be crucial steps for long-term marrow reconstitution after transplantation.

Regulation of epithelial apical junctional complex by Rho family GTPases.

The apical junctional complex (AJC), encompassing the tight junction (TJ) and adherens junction (AJ) plays a vital role in regulating epithelial cell differentiation and barrier function of simple epithelia. Both AJ and TJ are comprised of multiprotein complexes consisting of transmembrane proteins, which interact with the underlying cytoskeleton via cytoplasmic scaffold proteins. These interactions are tightly controlled by a number of signaling proteins that are critical for the regulation of the AJC function. Among these signaling molecules Rho family of small GTPases have been demonstrated to regulate the AJC function in diverse physiological and pathological states. In this review we will focus on experimental data addressing the role of Rho GTPase family members, Rho, Rac and Cdc42 in the regulation of epithelial AJC, and analyze Rho GTPase-mediated signaling pathways that control maintenance, disassembly and assembly of the AJC in epithelial cells.

Cdc42 is highly expressed in colorectal adenocarcinoma and downregulates ID4 through an epigenetic mechanism

Cdc42, a member of Rho GTPases family, is involved in the regulation of several cellular functions, such as rearrangement of actin cytoskeleton, membrane trafficking, cell-cycle progression, and transcriptional regulation. Aberrant expression or activity of Cdc42 has been reported in several tumours. Here, the specific role of Cdc42 in development and progression of colorectal cancer was analyzed through microarrays technology. A comparative analysis of Cdc42 overexpressing cells versus cells with decreased Cdc42 levels through siRNA revealed that Cdc42 overexpression down-regulated the potential tumour suppressor gene ID4. Results were validated by quantitative RT-PCR and the methylation status of the specific promoter, analyzed. Methylation-specific PCR and bisulfite sequencing PCR analysis revealed that Cdc42 induced the methylation of the CpG island of the ID4 promoter. Colorectal adenocarcinoma samples were compared with the corresponding adjacent normal tissue of the same patient in order to determine specific gene expression levels. The downregulation of ID4 by Cdc42 was also found of relevance in colorectal adenocarcinoma biopsies. Cdc42 was found to be overexpressed with high incidence (60%) in colorectal cancer samples, and this expression was associated with silencing of ID4 with statistical significance (p<0.05). Cdc42 may have a role in the development of colon cancer. Furthermore, inhibition of Cdc42 activity may have a direct impact in the management of colorectal cancer.

Rac1 in cortical projection neurons is selectively required for midline crossing of commissural axonal formation

Rac1 is a member of Rho family GTPases and regulates multiple cellular functions through actin cytoskeleton reorganization. During cerebral corticogenesis, Rac1 has been assumed to be involved in neuronal migration, neurite formation, polarization and axonal guidance. Here we show the specific role of Rac1, regulating midline crossing of commissural axons during cortical development by using cortex-restricted Rac1-knockout mice. In the knockout mice, Rac1 was eliminated from the beginning of corticogenesis exclusively in the dorsal telencephalon where progenitors of cortical projection neurons are located. Cortical lamination was distorted only mildly in the knockout mice, being preserved with six layers of neurons. However, cortex-restricted Rac1 deletion exhibited striking agenesis of commissural axons including the corpus callosum and anterior commissure without affecting other corticofugal axons including corticospinal and corticothalamic projections. Of note, the commissural axons of the knockout mice were potent in extending their process, but failed to cross the midline. Therefore, these findings indicate that Rac1 specifically controls the midline crossing of the commissural fibers, but not axonal formation of corticospinal or corticothalamic fibers during cortical development.

CD81 Is a Central Regulator of Cellular Events Required for Hepatitis C Virus Infection of Human Hepatocytes

Infection with hepatitis C virus (HCV) is still a major public health problem, and the events leading to hepatocyte infection are not yet fully understood. Combining confocal microscopy with biochemical analysis and studies of infection requirements using pharmacological inhibitors and small interfering RNAs, we show here that engagement of CD81 activates the Rho GTPase family members Rac, Rho, and Cdc42 and that the block of these signaling pathways drastically reduces HCV infectivity. Activation of Rho GTPases mediates actin-dependent relocalization of the HCV E2/CD81 complex to cell-cell contact areas where CD81 comes into contact with the tight-junction proteins occludin, ZO-1, and claudin-1, which was recently described as an HCV coreceptor. Finally, we show that CD81 engagement activates the Raf/MEK/ERK signaling cascade and that this pathway affects postentry events of the virus life cycle. In conclusion, we describe a range of cellular events that are manipulated by HCV to coordinate interactions with its multiple coreceptors and to establish productive infections and find that CD81 is a central regulator of these events.

Nm23‐H1 modulates the activity of the guanine exchange factor Dbl‐1

Cytoskeleton rearrangement is necessary for tumor invasion and metastasis. Cellular molecules whose role is to regulate components of the cytoskeletal structure can dictate changes in cellular morphology. One of these molecules is the suppressor of tumor metastasis Nm23-H1. The level of Nm23-H1 expression has been linked to the invasiveness and metastatic potential of human cancers including melanoma and breast cancer. In this report, we demonstrate an interaction between the suppressor of tumor metastasis Nm23-H1, and Dbl-1, an oncoprotein which is associated with guanine exchange and belongs to a family of Guanine Exchange Factors (GEF). Nm23-H1 also was shown to bind pDbl which is the proto-oncoprotein of Dbl. Interestingly, the interaction between Nm23-H1 and Dbl-1 rescues the suppression of the cell motility activity Nm23-H1. Moreover, this interaction results in loss of the ability of the Dbl-1 oncoprotein to function as a GEF for the critical Rho-GTPase family member Cdc42. The loss of GTP loading onto Cdc42 resulted in a dramatic reduction in adhesion stimulated ruffles and suggests that Nm23-H1 can negatively regulate cell migration and tumor metastasis by modulating the activity of Cdc42 through direct interaction with Dbl-1.

The wanderings of hematopoietic stem cells

The Rho GTPase family member Rac1 functions in the migration of hematopoietic stem/progenitor cells in the embryo and is required for the emergence of intraembryonic hematopoiesis. Although it is generally agreed that migrating hematopoietic stem cells (HSCs) seed the developing fetal liver, where

Smurf1 directly targets hPEM-2, a GEF for Cdc42, via a novel combination of protein interaction modules in the ubiquitin-proteasome pathway

Smurf1, a member of HECT-type E3 ubiquitin ligases, regulates cell polarity and protrusive activity by inducing ubiquitination and subsequent proteasomal degradation of the small GTPase RhoA. We report here that hPEM-2, a guanine nucleotide exchange factor for the small GTPase Cdc42, is a novel target of Smurf1. Pulse-chase labeling and a ubiquitination experiment using MG132, a proteasomal inhibitor, indicate that Smurf1 induces proteasomal degradation of hPEM-2 in cells. GST pull-down assays with heterologously expressed firefly luciferase-fusion proteins that include partial sequences of hPEM-2 reveal that part of the PH domain (residues 318-343) of hPEM-2 is sufficient for binding to Smurf1. In contrast, the hPEM-2 binding domain in Smurf1 was mapped to the C2 domain. Although it has been reported that the binding activities of some C2 domains to target proteins are regulated by Ca2+, Smurf1 interacts with hPEM-2 in a Ca2+-independent manner. Our discovery that hPEM-2 is, in addition to RhoA, a target protein of Smurf1 suggests that Smurf1 plays a crucial role in the spatiotemporal regulation of Rho GTPase family members.

Cdc42 increases activation of MMP‐2 in endothelial cells

Angiogenesis is stimulated by VEGF and requires basement membrane degradation by the matrix metalloproteinases (MMPs). VEGF induces increased membrane type 1‐MMP on the cell surface (required for MMP‐2 activation) and signals through the RhoGTPase family members. The RhoGTPases and the MAPK p38 are known for their roles upstream of actin cytoskeleton reorganization and are purported to regulate angiogenesis. We hypothesize that Cdc42 mediates the effects of VEGF on activation of MMP‐2 via p38 and actin cytoskeleton reorganization. To investigate this we manipulated Cdc42 signaling with a constitutively active Cdc42 (CA‐Cdc42) TAT‐fusion protein and observed the effects in rat skeletal muscle endothelial cells. Confocal microscopy demonstrated that CA‐Cdc42 increased cortical actin and the proportion of retracted or protruded cells similar to VEGF‐induced actin stress fibre depolymerization. CA‐Cdc42 treatments resulted in increased phospho‐p38 levels as seen with VEGF via western blotting. Furthermore, MMP‐2 activation increased in cells treated with CA‐Cdc42 or VEGF independent of new protein synthesis, as tested by gelatin zymography. This evidence suggests that Cdc42 may mediate the effects of VEGF on actin cytoskeleton reorganization, p38 phosphorylation and the levels of active MMP‐2. Funded by NSERC.

Regulation of Human Lung Adenocarcinoma Cell Migration and Invasion by Macrophage Migration Inhibitory Factor

Macrophage migration inhibitory factor (MIF) is expressed and secreted in response to mitogens and integrin-dependent cell adhesion. Once released, autocrine MIF promotes the activation of RhoA GTPase leading to cell cycle progression in rodent fibroblasts. We now report that small interfering RNA-mediated knockdown of MIF and MIF small molecule antagonism results in a greater than 90% loss of both the migratory and invasive potential of human lung adenocarcinoma cells. Correlating with these phenotypes is a substantial reduction in steady state as well as serum-induced effector binding activity of the Rho GTPase family member, Rac1, in MIF-deficient cells. Conversely, MIF overexpression by adenovirus in human lung adenocarcinoma cells induces a dramatic enhancement of cell migration, and co-expression of a dominant interfering mutant of Rac1 (Rac1(N17)) completely abrogates this effect. Finally, our results indicate that MIF depletion results in defective partitioning of Rac1 to caveolin-containing membrane microdomains, raising the possibility that MIF promotes Rac1 activity and subsequent tumor cell motility through lipid raft stabilization.

Rac1 Signaling Stimulates N-cadherin Expression, Mesenchymal Condensation, and Chondrogenesis

The molecular mechanisms controlling differentiation of mesenchymal precursor cells into chondrocytes (chondrogenesis) are not completely understood. We have recently shown that the small GTPase RhoA inhibits this process. Here we demonstrate that a different Rho GTPase family member, Rac1, promotes chondrogenesis. Pharmacological inhibition of Rac1 expression in micromass culture resulted in reduced mRNA levels of the chondrogenic markers collagen II and aggrecan, and decreased accumulation of glycosaminoglycans. Expression of the essential chondrogenic transcription factors Sox9, Sox5, and Sox6 was also reduced upon inhibition of Rac1 signaling. In contrast, overexpression of Rac1 in the chondrogenic ATDC5 cell line increased mRNA transcripts of Sox9, 5, and 6, collagen II, and aggrecan. Inhibition of Rac1 resulted in a reduction in the number, size, and organization of cellular condensations and decreased expression of N-cadherin. Overexpression of Rac1 resulted in an increase in N-cadherin expression levels. Furthermore, genetic ablation of Rac1 in primary micromass cultures resulted in reduced expression of chondrogenic markers. Additionally, we provide evidence that Cdc42 also promotes chondrogenesis. Overexpression of Cdc42 in ATDC5 cells resulted in increased expression of Sox5, Sox9, and collagen II but not Sox6, aggrecan, or N-cadherin. Therefore, we demonstrate that Rac1 and Cdc42 are positive regulators of chondrogenesis, but act at least in part through different cellular and molecular mechanisms.

Dendrites Are Only a CLICK Away

STKE From one side of the neuronal cell body an axon emerges; from the other, a branched dendritic tree. These processes are crucial for the ability of a neuron to receive and transmit electrochemical signals via synapses. Neuronal activity is important in driving dendrite outgrowth, but the intermediary players are not well understood. Because neuronal activity increases intracellular Ca2+ concentration, roles for members of the family of Ca2+/calmodulin- dependent protein kinases (CaMKs) have been investigated. Takemoto-Kimura et al. have looked at CLICKIII (also known as CaMKIγ or CL3). They found that CL3 undergoes sequential lipid modifications of its C-terminal tail: prenylation, which anchors CL3 to the plasma membrane, followed by palmitoylation. Lipid fractionation experiments then showed that prenylated and palmitoylated CL3 was predominantly associated with lipid raft microdomains in the plasma membrane, and most of the lipid raft-localized CL3 was found in the proximal dendrites. Studies of rat embryonic neurons revealed that total dendrite length was enhanced by overexpression of wild-type but not kinase-deficient CL3, and knockdown of CL3 resulted in fewer and shorter dendrites. Lipid raft-localized CL3 in dendrites activated the Rho GTPase family member Rac, leading to rearrangement of the actin cytoskeleton of the growing dendrite. Together these data suggest that CL3 is a key factor in transducing Ca2+ transients into signals responsible for dendrite outgrowth. — JFF Neuron 54 , 755 (2007).

Dendrites Are Only a CLICK Away

Neurons are composed of a cell body and two terminal processes: the axon at one end and a branched dendritic tree at the other. These processes are crucial for the ability of one neuron to respond to another and pass on electrochemical signals through synapses. Neuronal activity is very important in driving dendrite outgrowth, but the precise mechanisms involved are not well understood. Because neuronal activity results in increased intracellular Ca 2+ concentrations, possible roles for members of the family of Ca 2+ /calmodulin (CaM)-dependent protein kinases (CaMKs) in translating this rise in Ca 2+ into signals driving dendrite outgrowth have been investigated. Takemoto-Kimura et al . examined the role of the CaMKI family member, CLICKIII (also known as CaMKIγ and CL3). From initial studies on wild-type and mutated forms of CL3 in transfected COS-7 cells cultured in medium containing radiolabeled lipid precursors, the authors demonstrated that CL3 undergoes sequential lipid modifications of its C-terminal tail. CL3 was first prenylated, which was responsible for anchoring CL3 to the plasma membrane [as demonstrated by visualization of a green fluorescent protein (GFP)-tagged form of CL3], and then palmitoylated at specific Cys residues. Palmitoylation of a kinase-deficient mutant of CL3 (which was prenylated normally) was greatly impaired compared with that of wild-type CL3, suggesting that kinase activity induced a conformational change that was necessary for this lipid modification. Lipid fractionation experiments performed on cultured cortical neurons that were transfected with GFP-tagged CL3 demonstrated that prenylated, palmitoylated CL3 was predominantly associated with lipid raft microdomains in the plasma membrane, and fluorescence microscopy revealed that the majority of the lipid raft-localized CL3 was found in the proximal dendrites. Further studies in neurons that were removed from day 19 embryonic rats, transfected with CL3 constructs, and then cultured for 48 hours revealed that total dendrite length was enhanced by overexpression of wild-type but not kinase-deficient CL3. Knockdown of CL3 in cultured neurons by short hairpin RNA (shRNA) techniques resulted in fewer and shorter dendrites compared with control neurons. Brain-derived neurotrophic factor (BDNF) activates neurons by stimulating intracellular Ca 2+ , resulting in dendrite outgrowth. The authors found that siRNA-mediated knockdown of CL3 blocked BDNF-mediated dendrite formation in cultured cortical neurons. Finally, lipid raft-localized CL3 in dendrites activated the Rho GTPase family member Rac, leading to rearrangement of the actin cytoskeleton of the growing dendrite. Together these data suggest that CL3 is a key factor in transducing Ca 2+ signals stimulated by extracellular factors into lipid-raft localized signals responsible for dendrite outgrowth. S. Takemoto-Kimura, N. Ageta-Ishihara, M. Nonaka, A. Adachi-Morishima, T. Mano, M. Okamura, H. Fujii, T. Fuse, M. Hoshino, S. Suzuki, M. Kojima, M. Mishina, H. Okuno, H. Bito, Regulation of dendritogenesis via a lipid-raft-associated Ca 2+ /calmodulin-dependent protein kinase CLICK-III/CaΜΚΙγ. Neuron 54 , 755-770 (2007). [PubMed]

Small Rho GTPases Mediate Tumor-Induced Inhibition of Endocytic Activity of Dendritic Cells

The generation, maturation, and function of dendritic cells (DC) have been shown to be markedly compromised in the tumor microenvironment in animals and humans. However, the molecular mechanisms and intracellular pathways involved in the regulation of the DC system in cancer are not yet fully understood. Recently, we have reported on the role of the small Rho GTPase family members Cdc42, Rac1, and RhoA in regulating DC adherence, motility, and Ag presentation. To investigate involvement of small Rho GTPases in dysregulation of DC function by tumors, we next evaluated how Cdc42, Rac1, and RhoA regulated endocytic activity of DC in the tumor microenvironment. We revealed a decreased uptake of dextran 40 and polystyrene beads by DC generated in the presence of different tumor cell lines, including RM1 prostate, MC38 colon, 3LL lung, and B7E3 oral squamous cell carcinomas in vitro and by DC prepared from tumor-bearing mice ex vivo. Impaired endocytic activity of DC cocultured with tumor cells was associated with decreased levels of active Cdc42 and Rac1. Transduction of DC with the dominant negative Cdc42 and Rac1 genes also led to reduced phagocytosis and receptor-mediated endocytosis. Furthermore, transduction of DC with the constitutively active Cdc42 and Rac1 genes restored endocytic activity of DC that was inhibited by the tumors. Thus, our results suggest that tumor-induced dysregulation of endocytic activity of DC is mediated by reduced activity of several members of the small Rho GTPase family, which might serve as new targets for improving the efficacy of DC vaccines.

Genetic and pharmacologic evidence that Rac1 GTPase is involved in regulation of platelet secretion and aggregation.

Rac1 GTPase, a member of the Ras-related Rho GTPase family, is the major Rac isoform present in platelets and has been shown to be involved in cell actin cytoskeleton reorganization and adhesion. Agonists that induce platelet secretion and aggregation also activate Rac1 GTPase, raising the possibility that Rac1 GTPase may be involved in regulation of platelet function. To rigorously define the role of Rac1 in platelet regulation. We have used a dual approach of gene targeting in mice and pharmacologic inhibition of Rac1 by NSC23766, a rationally designed specific small molecule inhibitor, to study the role of Rac1 in platelet function. Platelets from mice as well as human platelets treated with NSC23766 exhibited a significant decrease in: (i) active Rac1 species and phosphorylation of the Rac effector, p21-activated kinase; (ii) expression of P-selectin and secretion of adenosine triphosphate induced by thrombin or U46619; and (iii) aggregation induced by adenosine 5'-diphosphate, collagen, thrombin and U46619, a stable analog of thromboxane A(2). NSC23766 did not alter the cAMP or cGMP levels in platelets. Consistent with the requirement of Rac1 for normal platelet function, the bleeding times in Rac1(-/-) mice or mice given NSC23766 were significantly prolonged. Our data show that deficiency or inhibition of Rac1 GTPase blocks platelet secretion. The inhibition of secretion, at least in part, is responsible for diminished platelet aggregation and prolonged bleeding times observed in Rac1 knockout or Rac1 inhibitor-treated mice.