Rac1 Mutant Research Articles

Abstract 4698: Mechanism of glucose deprivation-induced sensitization of tumor cells to receptor-mediated apoptosis: Downregulation of intracellular superoxide anion and cFLIP

Abstract One hallmark of cancer cells is their ability to switch their metabolism from oxidative phosphorylation to glycolysis. This is accomplished via an increase in glucose uptake and high rate of glycolysis. Therefore, one attractive strategy for a favorable anti-cancer response would be to deprive cancer cells of this essential nutrient. Here we investigated the effect of glucose withdrawal on death receptor-induced apoptosis in human leukemia cells overexpressing Bcl-2. Interestingly, subjecting Bcl-2 overexpressing cancer cells to a 30-60 minutes of glucose deprivation significantly increased sensitivity to CD95 (Fas/Apo1)-induced apoptosis. We have previously reported that death signaling in cancer cells is regulated by the cellular redox status in that a slight increase in superoxide anion (O2-) inhibited apoptotic signaling1. Of note, we also reported the ability of Bcl-2 and other oncogenes such as Rac1 to inhibit apoptosis via generating a pro-oxidant intracellular milieu. In line with those findings, we report here that glucose withdrawal resulted in a significant decrease in intracellular O2- and triggered strong activation of caspases 8, 9, and 3, translocation of Bax to the mitochondria and subsequent release of cytochrome C, independent of Bcl-2 expression. More importantly, we identified the death inhibitory protein cFLIP as a target of glucose withdrawal induced sensitization to death receptor induced apoptosis; glucose withdrawal decreased the expression and promoter activity of cFLIP, which correlated with increased caspase 8 activation. In order to provide evidence to link O2- to death inhibition, intracellular O2- levels were manipulated by incubation with PMA (62.5ng/ml), paraquat (50uM), or upon transient transfection with the constutively active form of the GTP binding protein Rac1 (RacV12) or a variety of functional mutants of Rac1, namely H103A, K166E, H40 and L37, and the effect on glucose withdrawal induced apoptosis was assessed. Indeed, increasing O2- strongly inhibited death signaling in cancer cells via an effect on the promoter activity and transcription of cFLIP, whereas blocking NADHP oxidase repressed cFLIP expression and facilitated death execution. This finding suggest that the glycolytic pathway may be important in sensitizing tumor cells to death receptor ligand by inhibiting cFLIP and facilitating the activation of initiator caspase-8 via a drop in intracellular O2-. 1 Clement, M-V., Hirpara, J.L., and Pervaiz, S. Decrease in intracellular superoxide sensitizes Bcl-2 overexpressing tumor cells to receptor- and drug-induced apoptosis independent of the mitochondria. Cell Death Diff. 10(11):1273-85, 2003. This work is supported by research grants to S.P. from the NMRC, Singapore. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4698. doi:10.1158/1538-7445.AM2011-4698

Immunoinhibitory Adapter Protein Src Homology Domain 3 Lymphocyte Protein 2 (SLy2) Regulates Actin Dynamics and B Cell Spreading

Appropriate B cell activation is essential for adaptive immunity. In contrast to the molecular mechanisms that regulate positive signaling in immune responses, the counterbalancing negative regulatory pathways remain insufficiently understood. The Src homology domain 3 (SH3)-containing adapter protein SH3 lymphocyte protein 2 (SLy2, also known as hematopoietic adapter-containing SH3 and sterile α-motif (SAM) domains 1; HACS1) is strongly up-regulated upon B cell activation and functions as an endogenous immunoinhibitor in vivo, but the underlying molecular mechanisms of SLy2 function have been elusive. We have generated transgenic mice overexpressing SLy2 in B and T cells and have studied the biological effects of elevated SLy2 levels in Jurkat and HeLa cells. Our results demonstrate that SLy2 induces Rac1-dependent membrane ruffle formation and regulates cell spreading and polarization and that the SLy2 SH3 domain is essential for these effects. Using immunoprecipitation and confocal microscopy, we provide evidence that the actin nucleation-promoting factor cortactin is an SH3 domain-directed interaction partner of SLy2. Consistent with an important role of SLy2 for actin cytoskeletal reorganization, we further show that SLy2-transgenic B cells are severely defective in cell spreading. Together, our findings extend our mechanistic understanding of the immunoinhibitory roles of SLy2 in vivo and suggest that the physiological up-regulation of SLy2 observed upon B cell activation functions to counteract excessive B cell spreading.

Rac-1 GTPase controls the capacity of human leukaemic lymphoblasts to migrate on fibronectin in response to SDF-1α (CXCL12)

Acute lymphoblastic leukaemia (ALL) is characterized by malignant cell infiltration of bone marrow, requiring chemotactic response to SDF-1α. Using time-lapse video, we measured the velocity of ALL cells on fibronectin, and found that SDF-1α increased their migration activity for 2h, but had no effect after 4h, following internalization of its receptor CXCR4. Transfection of ALL cells with dominant-negative Rac1 mutant significantly prolonged their chemotactic response to SDF-1α, and this effect was associated with an alteration of CXCR4 internalization. These data suggest a regulatory role for Rac1 in the chemotactic response of ALL cells to SDF-1α via receptor processing.

Protein kinase G signaling disrupts Rac1-dependent focal adhesion assembly in liver specific pericytes

Nitric oxide (NO) regulates the function of perivascular cells (pericytes), including hepatic stellate cells (HSC), mainly by activating cGMP and cGMP-dependent kinase (PKG) via NO/cGMP paracrine signaling. Although PKG is implicated in integrin-mediated cell adhesion to extracellular matrix, whether or how PKG signaling regulates the assembly of focal adhesion complexes (FA) and migration of HSC is not known. With the help of complementary molecular and cell biological approaches, we demonstrate here that activation of PKG signaling in HSC inhibits vascular tubulogenesis, migration/chemotaxis, and assembly of mature FA plaques, as assessed by vascular tubulogenesis assays and immunofluorescence localization of FA markers such as vinculin and vasodilator-stimulated phosphoprotein (VASP). To determine whether PKG inhibits FA assembly by phosphorylation of VASP at Ser-157, Ser-239, and Thr-278, we mutated these putative phosphorylation sites to alanine (VASP3A, phosphoresistant mutant) or aspartic acid (VASP3D, phosphomimetic), respectively. Data generated from these two mutants suggest that the effect of PKG on FA is independent of these three phosphorylation sites. In contrast, activation of PKG inhibits the activity of small GTPase Rac1 and its association with the effector protein IQGAP1. Moreover, PKG activation inhibits the formation of a trimeric protein complex containing Rac1, IQGAP1, and VASP. Finally, we found that expression of a constitutively active Rac1 mutant abolishes the inhibitory effects of PKG on FA formation. In summary, our data suggest that activation of PKG signaling in pericytes inhibits FA formation by inhibiting Rac1.

Thrombin Induces NF-κB Activation and IL-8/CXCL8 Expression in Lung Epithelial Cells by a Rac1-dependent PI3K/Akt Pathway

We previously showed that thrombin induces interleukin (IL)-8/CXCL8 expression via the protein kinase C (PKC)α/c-Src-dependent IκB kinase α/β (IKKα/β)/NF-κB signaling pathway in human lung epithelial cells. In this study, we further investigated the roles of Rac1, phosphoinositide 3-kinase (PI3K), and Akt in thrombin-induced NF-κB activation and IL-8/CXCL8 expression. Thrombin-induced IL-8/CXCL8 release and IL-8/CXCL8-luciferase activity were attenuated by a PI3K inhibitor (LY294002), an Akt inhibitor (1-L-6-hydroxymethyl-chiro-inositol-2-((R)-2-O-methyl-3-O-octadecylcarbonate)), and the dominant negative mutants of Rac1 (RacN17) and Akt (AktDN). Treatment of cells with thrombin caused activation of Rac and Akt. The thrombin-induced increase in Akt activation was inhibited by RacN17 and LY294002. Stimulation of cells with thrombin resulted in increases in IKKα/β activation and κB-luciferase activity; these effects were inhibited by RacN17, LY294002, an Akt inhibitor, and AktDN. Treatment of cells with thrombin induced Gβγ, p85α, and Rac1 complex formation in a time-dependent manner. These results imply that thrombin activates the Rac1/PI3K/Akt pathway through formation of the Gβγ, Rac1, and p85α complex to induce IKKα/β activation, NF-κB transactivation, and IL-8/CXCL8 expression in human lung epithelial cells.

Anti-inflammatory cytokine interleukin-19 inhibits smooth muscle cell migration and activation of cytoskeletal regulators of VSMC motility

Vascular smooth muscle cell (VSMC) migration is an important cellular event in multiple vascular diseases, including atherosclerosis, restenosis, and transplant vasculopathy. Little is known regarding the effects of anti-inflammatory interleukins on VSMC migration. This study tested the hypothesis that an anti-inflammatory Th2 interleukin, interleukin-19 (IL-19), could decrease VSMC motility. IL-19 significantly decreased platelet-derived growth factor (PDGF)-stimulated VSMC chemotaxis in Boyden chambers and migration in scratch wound assays. IL-19 significantly decreased VSMC spreading in response to PDGF. To determine the molecular mechanism(s) for these cellular effects, we examined the effect of IL-19 on activation of proteins that regulate VSMC cytoskeletal dynamics and locomotion. IL-19 decreased PDGF-driven activation of several cytoskeletal regulatory proteins that play an important role in smooth muscle cell motility, including heat shock protein-27 (HSP27), myosin light chain (MLC), and cofilin. IL-19 decreased PDGF activation of the Rac1 and RhoA GTPases, important integrators of migratory signals. IL-19 was unable to inhibit VSMC migration nor was able to inhibit activation of cytoskeletal regulatory proteins in VSMC transduced with a constitutively active Rac1 mutant (RacV14), suggesting that IL-19 inhibits events proximal to Rac1 activation. Together, these data are the first to indicate that IL-19 can have important inhibitory effects on VSMC motility and activation of cytoskeletal regulatory proteins. This has important implications for the use of anti-inflammatory cytokines in the treatment of vascular occlusive disease.

Rac GTPase-activating Protein (Rac GAP) α1-Chimaerin Undergoes Proteasomal Degradation and Is Stabilized by Diacylglycerol Signaling in Neurons

α1-Chimaerin is a neuron-specific member of the Rho GTPase-activating protein family that selectively inactivates the small GTPase Rac. It is known to regulate the structure of dendrites and dendritic spines. We describe here that under basal conditions α1-chimaerin becomes polyubiquitinated and undergoes rapid proteasomal degradation. This degradation is partly dependent on the N-terminal region that is unique to this isoform. Mimicking diacylglycerol (DAG) signaling with a phorbol ester stabilizes endogenous α1-chimaerin against degradation and causes accumulation of the protein. The stabilization requires phorbol ester binding via the C1 domain of the protein and is independent of PKC activity. In addition, overexpression of a constitutively active Rac1 mutant is sufficient to cause an accumulation of α1-chimaerin through a phospholipase C-dependent mechanism, showing that endogenous DAG signaling can also stabilize the protein. These results suggest that signaling via DAG may regulate the abundance of α1-chimaerin under physiological conditions, providing a new model for understanding how its activity could be controlled.

Differential roles of Smad2 and Smad3 in the regulation of TGF-β1-mediated growth inhibition and cell migration in pancreatic ductal adenocarcinoma cells: control by Rac1.

BackgroundProgression of pancreatic ductal adenocarcinoma (PDAC) is largely the result of genetic and/or epigenetic alterations in the transforming growth factor-beta (TGF-β)/Smad signalling pathway, eventually resulting in loss of TGF-β-mediated growth arrest and an increase in cellular migration, invasion, and metastasis. These cellular responses to TGF-β are mediated solely or partially through the canonical Smad signalling pathway which commences with activation of receptor-regulated Smads (R-Smads) Smad2 and Smad3 by the TGF-β type I receptor. However, little is known on the relative contribution of each R-Smad, the possible existence of functional antagonism, or the crosstalk with other signalling pathways in the control of TGF-β1-induced growth inhibition and cell migration. Using genetic and pharmacologic approaches we have inhibited in PDAC cells endogenous Smad2 and Smad3, as well as a potential regulator, the small GTPase Rac1, and have analysed the consequences for TGF-β1-mediated growth inhibition and cell migration (chemokinesis).ResultsSiRNA-mediated silencing of Smad3 in the TGF-β responsive PDAC cell line PANC-1 reduced TGF-β1-induced growth inhibition but increased the migratory response, while silencing of Smad2 enhanced growth inhibition but decreased chemokinesis. Interestingly, siRNA-mediated silencing of the small GTPase Rac1, or ectopic expression of a dominant-negative Rac1 mutant largely mimicked the effect of Smad2 silencing on both TGF-β1-induced growth inhibition, via upregulation of the cdk inhibitor p21WAF1, and cell migration. Inhibition of Rac1 activation reduced both TGF-β1-induction of a Smad2-specific transcriptional reporter and Smad2 C-terminal phosphorylation in PDAC cells while Smad3-specific transcriptional activity and Smad3 C-terminal phosphorylation appeared increased. Disruption of autocrine TGF-β signalling in PANC-1 cells rendered cells less susceptible to the growth-suppressive effect of Rac1 inhibition, suggesting that the decrease in "basal" proliferation upon Rac1 inhibition was caused by potentiation of autocrine TGF-β growth inhibition.ConclusionsIn malignant cells with a functional TGF-β signalling pathway Rac1 antagonizes the TGF-β1 growth inhibitory response and enhances cell migration by antagonistically regulating Smad2 and Smad3 activation. This study reveals that Rac1 is prooncogenic in that it can alter TGF-β signalling at the R-Smad level from a tumour-suppressive towards a tumour-promoting outcome. Hence, Rac1 might represent a viable target for therapeutic intervention to inhibit PDAC progression.

SUMO wrestling in cell movement

SUMO wrestling in cell movement

Semaphorin 5A and Plexin-B3 Inhibit Human Glioma Cell Motility through RhoGDIα-mediated Inactivation of Rac1 GTPase

Semaphorins and plexins are implicated in the progression of various types of cancer, although the molecular basis has not been fully elucidated. Here, we report the expression of plexin-B3 in glioma cells, which upon stimulation by its ligand Sema5A results in significant inhibition of cell migration and invasion. A search for the underlying mechanism revealed direct interaction of plexin-B3 with RhoGDP dissociation inhibitor α (RhoGDIα), a negative regulator of RhoGTPases that blocks guanine nucleotide exchange and sequesters them away from the plasma membrane. Glioma cells challenged with Sema5A indeed showed a marked reduction in Rac1-GTP levels by 60%, with a concomitant disruption of lamellipodia. The inactivation of Rac1 was corroborated to contribute to the impediment of glioma cell invasion by Sema5A, as supported by the abolishment of effect upon forced expression of a constitutively active Rac1 mutant. Furthermore, silencing the endogenous expression of RhoGDIα in glioma cells was found to be sufficient in abrogating the down-regulation of Rac1-GTP and the ensuing suppression of glioma cell motility induced by Sema5A. Mechanistically, we provide evidence that Sema5A promotes Rac1 recruitment to RhoGDIα and reduces its membrane localization in a plexin-B3-dependent manner, thereby preventing Rac1 activation. This represents a novel signaling of semaphorin and plexin in the control of cell motility by indirect inactivation of Rac1 through RhoGDIα.

Rac1 activity changes are associated with neuronal pathology and spatial memory long-term recovery after global cerebral ischemia

Excitotoxicity is the main event during neurological disorders producing drastic morphological and functional changes. Rac-GTPase is involved in cytoskeletal remodeling and survival. However, the role of Rac1 after cerebral ischemia has not been completely understood yet. In this study, we evaluated the activity of Rac1 and its immunoreactivity associated to neuropathological hallmarks and behavioral task analyses after global cerebral ischemia in an acute and long-term post-ischemia period. Our findings showed that during the acute phase (24 h) after global cerebral ischemia, a decrease of the active state of Rac1 was detected in the hippocampus, together with a down-regulation of survival signaling. In this same post-ischemia time, Rac1 immunoreactivity was redistributed to cytoplasm and to aberrant neurites, accompanied by dendritic and actin cytoskeletal retraction both in vivo and in vitro in neuronal primary cultures treated with glutamate. Neurons transfected with the constitutively active mutant of Rac1 were recovered from the glutamate-induced affection in vitro. However, in the in vivo model an inactive state of Rac1, and its cellular localization remained one month after ischemia, with still decreased survival signaling, significant tauopathy, and learning and memory alterations. These neuropathological hallmarks were reversed two months post-ischemia,related with a Rac1 activity state similar to control, as well as a “normalization” of the learning and memory tasks in the ischemic rats. In summary, our data suggests that changes in Rac1 activity are involved in the neurodegenerative processes after cerebral ischemia, and also in its long-term recovery.

Rac1-Dependent Collective Cell Migration Is Required for Specification of the Anterior-Posterior Body Axis of the Mouse

Author SummaryThe specification of the anterior-posterior body axis of the mouse embryo depends on migration of the anterior visceral endoderm (AVE) to a position that overlies the future head. By high-resolution imaging of intact embryos we show that movement of the AVE is a form of collective cell migration, as the migrating cells retain tight and adherens junctions while they migrate and exchange neighbors within the plane of the visceral endoderm epithelium. Using conditional knockouts, we find that the small GTPase Rac1 is absolutely required for specification of the anterior-posterior axis and acts cell-autonomously within the AVE to allow cells to extend long, dynamic lamellar projections that are required for movement. Rac1-mediated epithelial migration of the AVE is a crucial step in the establishment of the mammalian body plan, and Rac1 may be important for collective migration in general in mammalian tissues, including invading tumor cells.

The rho GTPase Rac1 is required for proliferation and survival of progenitors in the developing forebrain

Progenitor cells in the ventricular zone (VZ) and subventricular zone (SVZ) of the developing forebrain give rise to neurons and glial cells, and are characterized by distinct morphologies and proliferative behaviors. The mechanisms that distinguish VZ and SVZ progenitors are not well understood, although the homeodomain transcription factor Cux2 and Cyclin D2, a core component of the cell cycle machinery, are specifically involved in controlling SVZ cell proliferation. Rho GTPases have been implicated in regulating the proliferation, differentiation, and migration of many cell types, and one family member, Cdc42, affects the polarity and proliferation of radial glial cells in the VZ. Here, we show that another family member, Rac1, is required for the normal proliferation and differentiation of SVZ progenitors and for survival of both VZ and SVZ progenitors. A forebrain-specific loss of Rac1 leads to an SVZ-specific reduction in proliferation, a concomitant increase in cell cycle exit, and premature differentiation. In Rac1 mutants, the SVZ and VZ can no longer be delineated, but rather fuse to become a single compact zone of intermingled cells. Cyclin D2 expression, which is normally expressed by both VZ and SVZ progenitors, is reduced in Rac1 mutants, suggesting that the mutant cells differentiate precociously. Rac1-deficient mice can still generate SVZ-derived upper layer neurons, indicating that Rac1 is not required for the acquisition of upper layer neuronal fates, but instead is needed for the normal regulation of proliferation by progenitor cells in the SVZ.

Rac1 Recruits the Adapter Protein CMS/CD2AP to Cell-Cell Contacts

Rac1 is a member of the Rho family of small GTPases, which regulate cell adhesion and migration through their control of the actin cytoskeleton. Rho-GTPases are structurally very similar, with the exception of a hypervariable domain in the C terminus. Using peptide-based pulldown assays in combination with mass spectrometry, we previously showed that the hypervariable domain in Rac1 mediates specific protein-protein interactions. Most recently, we found that the Rac1 C terminus associates to the ubiquitously expressed adapter protein CMS/CD2AP. CD2AP is critical for the formation and maintenance of a specialized cell-cell contact between kidney podocyte foot processes, the slit diaphragm. Here, CD2AP links the cell adhesion protein nephrin to the actin cytoskeleton. In addition, CMS/CD2AP binds actin-regulating proteins, such as CAPZ and cortactin, and has been implicated in the internalization of growth factor receptors. We found that CD2AP specifically interacts with the C-terminal domain of Rac1 but not with that of other Rho family members. Efficient interaction between Rac1 and CD2AP requires both the proline-rich domain and the poly-basic region in the Rac1 C terminus, and at least two of the three N-terminal SH3 domains of CD2AP. CD2AP co-localizes with Rac1 to membrane ruffles, and small interfering RNA-based experiments showed that CD2AP links Rac1 to CAPZ and cortactin. Finally, expression of constitutive active Rac1 recruits CD2AP to cell-cell contacts in epithelial cells, where we found CD2AP to participate in the control of the epithelial barrier function. These data identify CD2AP as a novel Rac1-associated adapter protein that participates in the regulation of epithelial cell-cell contact.

The 19-Amino Acid Insertion in the Tumor-associated Splice Isoform Rac1b Confers Specific Binding to p120 Catenin

The Rac1b splice isoform contains a 19-amino acid insertion not found in Rac1; this insertion leads to decreased GTPase activity and reduced affinity for GDP, resulting in the intracellular predominance of GTP-bound Rac1b. Here, using co-precipitation and proteomic methods, we find that Rac1b does not bind to many common regulators of Rho family GTPases but that it does display enhanced binding to SmgGDS, RACK1, and p120 catenin (p120(ctn)), proteins involved in cell-cell adhesion, motility, and transcriptional regulation. We use molecular modeling and structure analysis approaches to determine that the interaction between Rac1b and p120(ctn) is dependent upon protein regions that are predicted to be unstructured in the absence of molecular complex formation, suggesting that the interaction between these two proteins involves coupled folding and binding. We also find that directed cell movement initiated by Rac1b is dependent upon p120. These results define a distinct binding functionality of Rac1b and provide insight into how the distinct phenotypic program activated by this protein may be implemented through molecular recognition of effectors distinct from those of Rac1.

CADM1 Interacts with Tiam1 and Promotes Invasive Phenotype of Human T-cell Leukemia Virus Type I-transformed Cells and Adult T-cell Leukemia Cells

CADM1 encodes a multifunctional immunoglobulin-like cell adhesion molecule whose cytoplasmic domain contains a type II PSD95/Dlg/ZO-1 (PDZ)-binding motif (BM) for associating with other intracellular proteins. Although CADM1 lacks expression in T lymphocytes of healthy individuals, it is overexpressed in adult T-cell leukemia-lymphoma (ATL) cells. It has been suggested that the expression of CADM1 protein promotes infiltration of leukemic cells into various organs and tissues, which is one of the frequent clinical manifestations of ATL. Amino acid sequence alignment revealed that Tiam1 (T-lymphoma invasion and metastasis 1), a Rac-specific guanine nucleotide exchange factor, has a type II PDZ domain similar to those of membrane-associated guanylate kinase homologs (MAGUKs) that are known to bind to the PDZ-BM of CADM1. In this study, we demonstrated that the cytoplasmic domain of CADM1 directly interacted with the PDZ domain of Tiam1 and induced formation of lamellipodia through Rac activation in HTLV-I-transformed cell lines as well as ATL cell lines. Our results indicate that Tiam1 integrates signals from CADM1 to regulate the actin cytoskeleton through Rac activation, which may lead to tissue infiltration of leukemic cells in ATL patients.

The Candida albicans ELMO homologue functions together with Rac1 and Dck1, upstream of the MAP Kinase Cek1, in invasive filamentous growth

Regulation of Rho G-proteins is critical for cytoskeletal organization and cell morphology in all eukaryotes. In the human opportunistic pathogen Candida albicans, Rac1 and its activator Dck1, a member of the CED5, Dock180, myoblast city family of guanine nucleotide exchange factors, are required for the budding to filamentous transition during invasive growth. We show that Lmo1, a protein with similarity to human ELMO1, is necessary for invasive filamentous growth, similar to Rac1 and Dck1. Furthermore, Rac1, Dck1 and Lmo1 are required for cell wall integrity, as the deletion mutants are sensitive to cell wall perturbing agents, but not to oxidative or osmotic stresses. The region of Lmo1 encompassing the ELMO and PH-like domains is sufficient for its function. Both Rac1 and Dck1 can bind Lmo1. Overexpression of a number of protein kinases in the rac1, dck1 and lmo1 deletion mutants indicates that Rac1, Dck1 and Lmo1 function upstream of the mitogen-activated protein kinases Cek1 and Mkc1, linking invasive filamentous growth to cell wall integrity. We conclude that the requirement of ELMO/CED12 family members for Rac1 function is conserved from fungi to humans.

NADPH oxidase 4 mediates reactive oxygen species induction of CD146 dimerization in VEGF signal transduction

CD146 dimerization plays an important role in tumor-induced angiogenesis. Stimulation of target cells with vascular endothelial growth factor (VEGF), a major angiogenic factor produced by tumor cells, elicits a burst of reactive oxygen species (ROS) that enhances angiogenesis. However, the molecular mechanism coupling CD146 dimerization with the VEGF-related oxidant-generating apparatus has not been elucidated. Here, we show that CD146 dimerization is induced by VEGF and is significantly diminished by pretreatment with diphenylene iodonium, an inhibitor of NADPH oxidase, suggesting a potential role for NADPH oxidase (NOX) in VEGF-induced CD146 dimerization. Importantly, we found that overexpression of NADPH oxidase 4 (NOX4), which is the predominant NOX expressed in endothelial cells, significantly enhances VEGF-induced ROS generation and CD146 dimerization. By contrast, these VEGF effects were dramatically attenuated after transfection with siRNA to reduce NOX4 expression. Furthermore, expression of Rac1 N17, a dominant negative mutant of Rac1, a member of the Rho family of small GTPases, suppressed VEGF-induced ROS generation and CD146 dimerization. These studies show for the first time that VEGF alteration of CD146 dimerization is mediated via a NOX4-dependent pathway and provide novel insight into the significant role of NOX in redox regulation of the dimerization of cell adhesion molecules.

Abstract 1025: Rac1 is a novel binding partner of the anti-apoptotic protein Bcl-2

Abstract We have previously reported that Bcl-2 expression resulted in an increase in intracellular superoxide anion and that a dominant negative mutant of the small GTPase Rac1 sensitized Bcl-2 expressing cells to apoptosis. Here we report that silencing and functional inhibition of Rac1 blocks Bcl-2 mediated increases in intracellular and mitochondrial superoxide levels in tumor cells. We provide evidence that this effect is mediated via specific interaction between the two proteins using co-immunoprecipitation, confocal and electron microscopy, as well as GST-fusion proteins. Analysis of the sub-cellular localization of these proteins revealed increased association of Bcl-2 and mitochondrial Rac1 in Bcl-2 overexpressing cells. This interaction can be blocked in vitro and in vivo by BH3 mimetics such as HA14-I, BH3-I as well as synthetic Bcl-2 BH3 domain peptides. That this interaction is functionally relevant is supported by the ability of the Bcl-2 BH3 peptide to inhibit intracellular superoxide production as well as overcome drug resistance in Bcl-2 overexpressing cells. Lastly, using patient-derived primary tissues, we observed the interaction only in cancerous tissues with marked overexpression of Bcl-2 and not in peripheral blood leukocytes or samples from non-cancerous tissue. These data provide a novel facet in the biology of Bcl-2 with potential implications for targeted anti-cancer drug design. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1025.

Small Rho GTPase Rac1 determines human epidermal stem cell fate in vitro

Epidermal stem cells (ESCs) are essential not only for tissue homeostasis but also for skin to respond to insults, but the mechanisms of stem cell regulation are unknown. To investigate the function of Rac1 in ESC development, we introduced either the dominant negative isoform or constitutively the active mutant of Rac1 in cultured human ESCs by using a retroviral vector and then analyzed the consequences. Upon activation, Rac1 increased surface alpha6/beta1 integrin levels and promoted colony forming efficiency of ESCs. Conversely, dominant negative Rac1 caused a progressive reduction in growth rate, an inhibition of adhesiveness and a marked stimulation of terminal differentiation, without any effect on the cell cycle. These results were consistent with the role of Rac1 in determining the fate of ESCs by controlling their exit from the stem cell compartment. Our results reveal a novel biological role for Rac1 and provide new insights into the mechanism regulating ESCs.