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Fungal infections cause ~1.5 million deaths each year worldwide, and the mortality rate of disseminated candidiasis currently exceeds that of breast cancer and malaria. The major reasons for the high mortality of candidiasis are the limited number of antifungal drugs and the emergence of drug-resistant species. Therefore, a better understanding of antifungal host defense mechanisms is crucial for the development of effective preventive and therapeutic strategies. Here, we report that DOCK2 (dedicator of cytokinesis 2) promotes indispensable antifungal innate immune signaling and proinflammatory gene expression in macrophages. DOCK2-deficient macrophages exhibit decreased RAC GTPase (Rac family small GTPase) activation and ROS (reactive oxygen species) production, which in turn attenuates the killing of intracellular fungi and the activation of downstream signaling pathways. Mechanistically, after fungal stimulation, activated SYK (spleen-associated tyrosine kinase) phosphorylates DOCK2 at tyrosine 985 and 1405, which promotes the recruitment and activation of RAC GTPases and then increases ROS production and downstream signaling activation. Importantly, nanoparticle-mediated delivery of in vitro transcribed (IVT) Rac1 mRNA promotes the activity of Rac1 and helps to eliminate fungal infection in vivo. Taken together, this study not only identifies a critical role of DOCK2 in antifungal immunity via regulation of RAC GTPase activity but also provides proof of concept for the treatment of invasive fungal infections by using IVT mRNA.

Collective cell migration underlies morphogenesis, wound healing and cancer invasion

Ageing is driven by a loss of cellular integrity1. Given the major role of ubiquitin modifications in cell function2, here we assess the link between ubiquitination and ageing by quantifying whole-proteome ubiquitin signatures in Caenorhabditis elegans. We find a remodelling of the ubiquitinated proteome during ageing, which is ameliorated by longevity paradigms such as dietary restriction and reduced insulin signalling. Notably, ageing causes a global loss of ubiquitination that is triggered by increased deubiquitinase activity. Because ubiquitination can tag proteins for recognition by the proteasome3, a fundamental question is whether deficits in targeted degradation influence longevity. By integrating data from worms with a defective proteasome, we identify proteasomal targets that accumulate with age owing to decreased ubiquitination and subsequent degradation. Lowering the levels of age-dysregulated proteasome targets prolongs longevity, whereas preventing their degradation shortens lifespan. Among the proteasomal targets, we find the IFB-2 intermediate filament4 and the EPS-8 modulator of RAC signalling5. While increased levels of IFB-2 promote the loss of intestinal integrity and bacterial colonization, upregulation of EPS-8 hyperactivates RAC in muscle and neurons, and leads to alterations in the actin cytoskeleton and protein kinase JNK. In summary, age-related changes in targeted degradation of structural and regulatory proteins across tissues determine longevity.

The vascular network is established and maintained through the processes of vasculogenesis and angiogenesis, which are tightly regulated during embryonic and postnatal life. The formation of a functional vasculature requires critical cellular mechanisms, such as cell migration, proliferation and adhesion, which are dependent on the activity of small Rho GTPases, controlled in part by the dedicator of cytokinesis (DOCK) protein family. Whilst the majority of DOCK proteins are associated with neuronal development, a growing body of evidence has indicated that members of the DOCK family may have key functions in the control of vasculogenic and angiogenic processes. This is supported by the involvement of several angiogenic signalling pathways, including chemokine receptor type 4 (CXCR4), vascular endothelial growth factor (VEGF) and phosphatidylinositol 3-kinase (PI3K), in the regulation of specific DOCK proteins. This review summarises recent progress in understanding the respective roles of DOCK family proteins during vascular development. We focus on existing in vivo and in vitro models and known human disease phenotypes and highlight potential mechanisms of DOCK protein dysfunction in the pathogenesis of vascular disease.

The plant ROP/RAC protein belongs to a subfamily of Rho family GTPases that inimitably exists in plants. It is considered an all-powerful molecular switch that modulates various plant developmental processes and plant defense responses to multifarious stresses. However, their exact roles are currently not entirely clear. Herein, the role of GhRac6, a ROP/RAC member from Gossypium hirsutum, was explored in the plant defense response to aphid feeding using transgenic technology. The deduced sequence of the GhRac6 protein is highly homologous to that of ROP/RAC proteins from other plants. GhRac6 expression was significantly upregulated in cotton leaves after damage by cotton aphids or wounding. The results of choice and no-choice tests showed that the number of aphids on GhRac6-overexpressing transgenic Arabidopsis was more than that on wild-type plants, while the secreted quantity of aphid honeydew in transgenic Arabidopsis was less than that in wild-type plants. Furthermore, the soluble sugar content and the proline content were higher in transgenic than in wild-type Arabidopsis to a certain extent. The activity of PAL and CAT in transgenic Arabidopsis was also higher than that in wild-type plants. Callose deposits were more abundant in transgenic than in wild-type Arabidopsis. The expression of related defense genes in the salicylic acid signaling pathway was considerably higher in transgenic than in wild-type Arabidopsis. These results collectively demonstrated that the cotton ROP gene GhRac6 improved the plant defense response to aphid feeding by regulating some aphid resistance indexes.

Osteoclasts are multinucleated cells responsible for bone resorption. Osteoclasts adhere to the bone surface through integrins and polarize to form actin rings, which are formed by the assembly of podosomes. The area contained within actin rings (also called sealing zones) has an acidic pH, which causes dissolution of bone minerals including hydroxyapatite and the degradation of matrix proteins including type I collagen by the protease cathepsin K. Osteoclasts resorb bone matrices while moving on bone surfaces. Osteoclasts change their cell shapes and exhibit three modes for bone resorption: motile resorbing mode for digging trenches, static resorbing mode for digging pits, and motile non-resorbing mode. Therefore, the actin cytoskeleton is actively remodeled in osteoclasts. Recent studies have revealed that many molecules, such as Rac, Cdc42, Rho, and small GTPase regulators and effectors, are involved in actin cytoskeletal remodeling during the formation of actin rings and resorption cavities on bone slices. In this review, we introduce how these molecules and non-canonical Wnt signaling regulate the bone-resorbing activity of osteoclasts.

The vascular barrier that separates blood from tissues is actively regulated by the endothelium and is essential for transport, inflammation, and haemostasis. Haemodynamic shear stress plays a critical role in maintaining endothelial barrier function, but how this occurs remains unknown. Here we use an engineered organotypic model of perfused microvessels to show that activation of the transmembrane receptor NOTCH1 directly regulates vascular barrier function through a non-canonical, transcription-independent signalling mechanism that drives assembly of adherens junctions, and confirm these findings in mouse models. Shear stress triggers DLL4-dependent proteolytic activation of NOTCH1 to expose the transmembrane domain of NOTCH1. This domain mediates establishment of the endothelial barrier; expression of the transmembrane domain of NOTCH1 is sufficient to rescue defects in barrier function induced by knockout of NOTCH1. The transmembrane domain restores barrier function by catalysing the formation of a receptor complex in the plasma membrane consisting of vascular endothelial cadherin, the transmembrane protein tyrosine phosphatase LAR, and the RAC1 guanidine-exchange factor TRIO. This complex activates RAC1 to drive assembly of adherens junctions and establish barrier function. Canonical transcriptional signalling via Notch is highly conserved in metazoans and is required for many processes in vascular development, including arterial-venous differentiation, angiogenesis and remodelling. We establish the existence of a non-canonical cortical NOTCH1 signalling pathway that regulates vascular barrier function, and thus provide a mechanism by which a single receptor might link transcriptional programs with adhesive and cytoskeletal remodelling.

The function of RHOG, a RAC1 activator, was explored in the ovary during ovarian follicular development and pathological conditions. With the help of immunoblotting and immunolocalization, we determined the expression and localization of RHOG in normal (estrous cycle) and polycystic ovaries using Sprague Dawley (SD) rat model. Employing polymerase chain reaction and flow cytometry, we analyzed the transcript and expression levels of downstream molecules of RHOG, DOCK1, and RAC1 in the polycystic ovarian syndrome (PCOS) ovary along with normal antral follicular theca and granulosa cells after dehydroepiandrosterone (DHEA) supplementation. The effect of RHOG knockdown on DOCK1, VAV, and RAC1 expression was evaluated in the human ovarian cells (SKOV3), theca cells, and granulosa cells from SD rats with the help of flow cytometry. Oocyte at secondary follicles along with stromal cells showed optimal expression of RHOG. Immunoblotting of RHOG revealed its maximum expression at diestrus and proestrus, which was downregulated at estrus stage. Mild immunostaining of RHOG was also present in the theca and granulosa cells of the secondary and antral follicles. Polycystic ovary exhibited weak immunostaining for RHOG and that was corroborated by immunoblotting-based investigations. RHOG effectors DOCK1 and ELMO1 were found reduced in the ovary in PCOS condition/DHEA. RHOG silencing reduced the expression of DOCK1 and RAC1 in the theca and granulosa cells from SD rat antral follicles and that was mirrored in the human ovarian cells. Collectively, RHOG can mediate signaling through downstream effectors DOCK1 and RAC1 during ovarian follicular development (theca and granulosa cells and oocyte), but DHEA downregulated them in the PCOS ovary.

Throughout development, tissues undergo complex morphological changes, resulting from cellular mechanics that evolve over time and in three-dimensional space. During Drosophila germ-band extension (GBE), cell intercalation is the key mechanism for tissue extension, and the associated apical junction remodelling is driven by polarized myosin-II-dependent contraction. However, the contribution of the basolateral cellular mechanics to GBE remains poorly understood. Here, we characterize how cells coordinate their shape from the apical to the basal side during rosette formation, a hallmark of cell intercalation. Basolateral rosette formation is driven by cells mostly located at the dorsal/ventral part of the rosette (D/V cells). These cells exhibit actin-rich wedge-shaped basolateral protrusions and migrate towards each other. Surprisingly, the formation of basolateral rosettes precedes that of the apical rosettes. Basolateral rosette formation is independent of apical contractility, but requires Rac1-dependent protrusive motility. Furthermore, we identified Src42A as a regulator of basolateral rosette formation. Our data show that in addition to apical contraction, active cell migration driven by basolateral protrusions plays a pivotal role in rosette formation and contributes to GBE.

Cancer stem cells (CSCs) may be responsible for tumour dormancy, relapse and the eventual death of most cancer patients(1). In addition, these cells are usually resistant to cytotoxic conditions. However, very little is known about the biology behind this resistance to therapeutics. Here we investigated stem-cell death in the digestive system of adult Drosophila melanogaster. We found that knockdown of the coat protein complex I (COPI)–Arf79F (also known as Arf1) complex selectively killed normal and transformed stem cells through necrosis, by attenuating the lipolysis pathway, but spared differentiated cells. The dying stem cells were engulfed by neighbouring differentiated cells through a draper–myoblast city–Rac1–basket (also known as JNK)-dependent autophagy pathway. Furthermore, Arf1 inhibitors reduced CSCs in human cancer cell lines. Thus, normal or cancer stem cells may rely primarily on lipid reserves for energy, in such a way that blocking lipolysis starves them to death. This finding may lead to new therapies that could help to eliminate CSCs in human cancers.

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.

Mitochondria need to be juxtaposed to phagosomes for the synergistic production of ample reactive oxygen species (ROS) in phagocytes to kill pathogens. However, how phagosomes transmit signals to recruit mitochondria has remained unclear. Here we found that the kinases Mst1 and Mst2 functioned to control ROS production by regulating mitochondrial trafficking and mitochondrion-phagosome juxtaposition. Mst1 and Mst2 activated the GTPase Rac to promote Toll-like receptor (TLR)-triggered assembly of the TRAF6-ECSIT complex that is required for the recruitment of mitochondria to phagosomes. Inactive forms of Rac, including the human Rac2(D57N) mutant, disrupted the TRAF6-ECSIT complex by sequestering TRAF6 and substantially diminished ROS production and enhanced susceptibility to bacterial infection. Our findings demonstrate that the TLR-Mst1-Mst2-Rac signaling axis is critical for effective phagosome-mitochondrion function and bactericidal activity.

Background Our previous study demonstrated that an up-regulation of the Brain-Derived Neurotrophic Factor (BDNF) signaling pathway is involved the mechanism causing the recurrence of triple negative breast cancer. The aim of this study is to investigate the effects of commonly used Chinese medicinal herbs on MDA-MB-231 and HUVEC cells and how they interact with BDNF. Methods Human TNBC MDA-MB-231 cells and human endothelial HUVEC cells were used to explore the effect of commonly used Chinese herbal medicines on cancer cells alone, on endothelial cells alone and on cancer cell/endothelial cell interactions; this was done via functional studies, including migration and invasion assays. Furthermore, Western blot analysis and real-time PCR investigations were also used to investigate migration signal transduction, invasion signal transduction, and angiogenic signal transduction in these systems. Finally, the effect of the Chinese medicinal herbs on cancer cell/endothelial cell interactions was assessed using co-culture and ELISA. Results In terms of autoregulation, BDNF up-regulated TrkB gene expression in both MDA-MB-231 and HUVEC cells. Furthermore, BDNF enhanced migration by MDA-MB-231 cells via Rac, Cdc42 and MMP, while also increasing the migration of HUVEC cells via MMP and COX-2 expression. As measured by ELISA, the Chinese herbal medicinal herbs A. membranaceus, P. lactiflora, L. chuanxiong, P. suffruticosa and L. lucidum increased BDNF secretion by MDA-MB-231 cells. Similarly, using a co-culture system with MDA-MB-231 cells, A. membranaceus and L. lucidum modulated BDNF-TrkB signaling by HUVEC cells. Conclusion We conclude that BDNF plays an important role in the metastatic interaction between MDA-MB-231 and HUVEC cells. Some Chinese medicinal herbs are able to enhance the BDNF-related metastatic potential of the interaction between cancer cells and endothelial cells. These findings provide important information that should help with the development of integrated medical therapies for breast cancer patients. Electronic supplementary material The online version of this article (doi:10.1186/s12906-017-1909-7) contains supplementary material, which is available to authorized users.

The docking protein p140Cap negatively regulates tumour cell features. Its relevance on breast cancer patient survival, as well as its ability to counteract relevant cancer signalling pathways, are not fully understood. Here we report that in patients with ERBB2-amplified breast cancer, a p140Cap-positive status associates with a significantly lower probability of developing a distant event, and a clear difference in survival. p140Cap dampens ERBB2-positive tumour cell progression, impairing tumour onset and growth in the NeuT mouse model, and counteracting epithelial mesenchymal transition, resulting in decreased metastasis formation. One major mechanism is the ability of p140Cap to interfere with ERBB2-dependent activation of Rac GTPase-controlled circuitries. Our findings point to a specific role of p140Cap in curbing the aggressiveness of ERBB2-amplified breast cancers and suggest that, due to its ability to impinge on specific molecular pathways, p140Cap may represent a predictive biomarker of response to targeted anti-ERBB2 therapies., p140Cap adaptor proteins interfere with adhesion and growth factor-dependent signalling in cancer cells but the mechanisms are unclear. Here the authors show that p140Cap interferes with ERBB2-dependent activation of Rac GTPase-controlled circuitries reducing metastasis and cancer progression.

Phosphatidylinositol 3-kinase (PI3K) promotes cancer cell survival, migration, growth and proliferation by generating phosphatidylinositol 3,4,5-trisphosphate (PIP3) in the inner leaflet of the plasma membrane. PIP3 recruits pleckstrin homology domain-containing proteins to the membrane to activate oncogenic signaling cascades. Anticancer therapeutics targeting the PI3K/AKT/mTOR (mammalian target of rapamycin) pathway are in clinical development. In a mass spectrometric screen to identify PIP3-regulated proteins in breast cancer cells, levels of the Rac activator PIP3-dependent Rac exchange factor-1 (P-REX1) increased in response to PI3K inhibition, and decreased upon loss of the PI3K antagonist phosphatase and tensin homolog (PTEN). P-REX1 mRNA and protein levels were positively correlated with ER expression, and inversely correlated with PI3K pathway activation in breast tumors as assessed by gene expression and phosphoproteomic analyses. P-REX1 increased activation of Rac1, PI3K/AKT and MEK/ERK signaling in a PTEN-independent manner, and promoted cell and tumor viability. Loss of P-REX1 or inhibition of Rac suppressed PI3K/AKT and MEK/ERK, and decreased viability. P-REX1 also promoted insulin-like growth factor-1 receptor activation, suggesting that P-REX1 provides positive feedback to activators upstream of PI3K. In support of a model where PIP3-driven P-REX1 promotes both PI3K/AKT and MEK/ERK signaling, high levels of P-REX1 mRNA (but not phospho-AKT or a transcriptomic signature of PI3K activation) were predictive of sensitivity to PI3K inhibitors among breast cancer cell lines. P-REX1 expression was highest in estrogen receptor-positive breast tumors compared with many other cancer subtypes, suggesting that neutralizing the P-REX1/Rac axis may provide a novel therapeutic approach to selectively abrogate oncogenic signaling in breast cancer cells.

Chimaerins are a family of diacylglycerol- and phorbol ester-regulated GTPase activating proteins (GAPs) for the small G-protein Rac. Extensive evidence indicates that these proteins play important roles in development, axon guidance, metabolism, cell motility, and T cell activation. Four isoforms have been reported to-date, which are products of CHN1 (α1- and α2-chimaerins) and CHN2 (β1- and β2-chimaerins) genes. Although these gene products are assumed to be generated by alternative splicing, bioinformatics analysis of the CHN2 gene revealed that β1- and β2-chimaerins are the products of alternative transcription start sites (TSSs) in different promoter regions. Furthermore, we found an additional TSS in CHN2 gene that leads to a novel product, which we named β3-chimaerin. Expression profile analysis revealed predominantly low levels for the β3-chimaerin transcript, with higher expression levels in epididymis, plasma blood leucocytes, spleen, thymus, as well as various areas of the brain. In addition to the prototypical SH2, C1, and Rac-GAP domains, β3-chimaerin has a unique N-terminal domain. Studies in cells established that β3-chimaerin has Rac-GAP activity and is responsive to phorbol esters. The enhanced responsiveness of β3-chimaerin for phorbol ester-induced translocation relative to β2-chimaerin suggests differential ligand accessibility to the C1 domain. Fil: Zubeldia Brenner, Lautaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Gutierrez Uzquiza, Alvaro. University of Pennsylvania; Estados Unidos Fil: Barrio Real, Laura. University of Pennsylvania; Estados Unidos Fil: Wang, Hongbin. University of Pennsylvania; Estados Unidos Fil: Kazanietz, Marcelo Gabriel. University of Pennsylvania; Estados Unidos Fil: Coluccio Leskow, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad Nacional de Luján. Departamento de Ciencias Básicas; Argentina

Rho GTPases are a class of evolutionarily conserved proteins comprising 20 members, which are predominantly known for their role in regulating the actin cytoskeleton. They are primarily regulated by binding of GTP/GDP, which is again controlled by regulators like GEFs, GAPs, and RhoGDIs. Rho GTPases are thus far well known for their role in the regulation of actin cytoskeleton and migration. Here we present an overview on the role of Rho GTPases in regulating cell shape and plasticity of cell migration. Finally, we discuss the emerging roles of ubiquitination and sumoylation in regulating Rho GTPases and cell migration.

Glioblastomas (GBMs), the most common and malignant brain tumors, are highly resistant to current therapies. The failure of targeted therapies against aberrantly activated oncogenic signaling, such as that of the EGFR-PI3K/Akt pathway, underscores the urgent need to understand alternative downstream pathways and to identify new molecular targets for the development of more effective treatments for gliomas. Here, we report that EGFRvIII (ΔEGFR/de2-7EGFR), a constitutively active EGFR mutant that is frequently co-overexpressed with EGFR in clinical GBM tumors, promotes glioma growth and invasion through protein kinase A (PKA)-dependent phosphorylation of Dock180, a bipartite guanine nucleotide exchange Factor (GEF) for Rac1. We demonstrate that EGFRvIII induces serine phosphorylation of Dock180, stimulates Rac1 activation and glioma cell migration. Treatments of glioma cells using the PKA inhibitors H89 and KT5720, overexpression of a PKA inhibitor (PKI), and in vitro PKA kinase assays show that EGFRvIII induction of serine phosphorylation of Dock180 is PKA-dependent. Significantly, PKA induces phosphorylation of Dock180 at amino acid residue S1250 that resides within its Rac1-activating DHR-2 domain. Expression of the Dock180S1250L mutant, but not wild type Dock180WT, protein in EGFRvIII-expressing glioma cells inhibited receptor-stimulated cell proliferation, survival, migration in vitro and glioma tumor growth and invasion in vivo. Together, our findings describe a novel mechanism by which EGFRvIII drives glioma tumorigenesis and invasion through PKA-dependent phosphorylation of Dock180, thereby suggesting that targeting EGFRvIII-PKA-Dock180-Rac1 signaling axis could provide a novel pathway to target in developing potential therapeutic strategies for malignant gliomas.

Drug reinforcement and the reinstatement of drug seeking are associated with the pathological processing of drug-associated cue memories that can be disrupted by manipulating memory consolidation and reconsolidation. Ras-related C3 botulinum toxin substrate (Rac) is involved in memory processing by regulating actin dynamics and neural structure plasticity. The nucleus accumbens (NAc) and amygdala have been implicated in the consolidation and reconsolidation of emotional memories. Therefore, we hypothesized that Rac in the NAc and amygdala plays a role in the consolidation and reconsolidation of cocaine-associated cue memory.Conditioned place preference (CPP) and microinjection of Rac inhibitor NSC23766 were used to determine the role of Rac in the NAc and amygdala in the consolidation and reconsolidation of cocaine-associated cue memory in rats.Microinjections of NSC23766 into the NAc core but not shell, basolateral (BLA), or central amygdala (CeA) after each cocaine-conditioning session inhibited the consolidation of cocaine-induced CPP. A microinjection of NSC23766 into the BLA but not CeA, NAc core, or NAc shell immediately after memory reactivation induced by exposure to a previously cocaine-paired context disrupted the reconsolidation of cocaine-induced CPP. The effect of memory disruption on cocaine reconsolidation was specific to reactivated memory, persisted at least 2 weeks, and was not reinstated by a cocaine-priming injection.Our findings indicate that Rac in the NAc core and BLA are required for the consolidation and reconsolidation of cocaine-associated cue memory, respectively.

During brain development, the small GTPases Rac1/Rac3 play key roles in neuronal migration, neuritogenesis, synaptic formation and plasticity, via control of actin cytoskeleton dynamic. Their activity is positively and negatively regulated by GEFs and GAPs molecules, respectively. However their in vivo roles are poorly known. The ArhGAP15 gene, coding for a Rac-specific GAP protein, is expressed in both excitatory and inhibitory neurons of the adult hippocampus, and its loss results in the hyperactivation of Rac1/Rac3. In the CA3 and dentate gyrus (DG) regions of the ArhGAP15 mutant hippocampus the CR+, PV+ and SST+ inhibitory neurons are reduced in number, due to reduced efficiency and directionality of their migration, while pyramidal neurons are unaffected. Loss of ArhGAP15 alters neuritogenesis and the balance between excitatory and inhibitory synapses, with a net functional result consisting in increased spike frequency and bursts, accompanied by poor synchronization. Thus, the loss of ArhGAP15 mainly impacts on interneuron-dependent inhibition. Adult ArhGAP15−/− mice showed defective hippocampus-dependent functions such as working and associative memories. These findings indicate that a normal architecture and function of hippocampal inhibitory neurons is essential for higher hippocampal functions, and is exquisitely sensitive to ArhGAP15-dependent modulation of Rac1/Rac3.

Promoting endothelial cell (EC) migration is important not only for therapeutic angiogenesis, but also for accelerating re-endothelialization after vessel injury. Several recent studies have shown that inhibition of protein tyrosine phosphatase 1B (PTP1B) may promote EC migration and angiogenesis by enhancing the vascular endothelial growth factor receptor-2 (VEGFR2) signalling. In the present study, we demonstrated that PTP1B inhibitor could promote EC adhesion, spreading and migration, which were abolished by the inhibitor of Rac1 but not RhoA GTPase. PTP1B inhibitor significantly increased phosphorylation of p130Cas and the interactions among p130Cas, Crk and DOCK180; whereas the phosphorylation levels of focal adhesion kinase, Src, paxillin, or Vav2 were unchanged. Gene silencing of DOCK180, but not Vav2, abrogated the effects of PTP1B inhibitor on EC motility. The effects of PTP1B inhibitor on EC motility and p130Cas/DOCK180 activation persisted in the presence of the VEGFR2 antagonist. In conclusion, we suggest that stimulation of the DOCK180 pathway represents an alternative mechanism of PTP1B inhibitor-stimulated EC motility, which does not require concomitant VEGFR2 activation as a prerequisite. Therefore, PTP1B inhibitor may be a useful therapeutic strategy for promoting EC migration in cardiovascular patients in which the VEGF/VEGFR functions are compromised.

The extracellular matrix has a crucial role in determining the spatial orientation of epithelial polarity and the formation of lumens in glandular tissues; however, the underlying mechanisms remain elusive. By using Cre–Lox deletion we show that β1 integrins are required for normal mammary gland morphogenesis and lumen formation, both in vivo and in a three-dimensional primary culture model in which epithelial cells directly contact a basement membrane. Downstream of basement membrane β1 integrins, Rac1 is not involved; however, ILK is needed to polarize microtubule plus ends at the basolateral membrane and disrupting each of these components prevents lumen formation. The integrin–microtubule axis is necessary for the endocytic removal of apical proteins from the basement-membrane–cell interface and for internal Golgi positioning. We propose that this integrin signalling network controls the delivery of apical components to the correct surface and thereby governs the orientation of polarity and development of lumens.

The ability of cells to invade into the dermis is a critical event in the development of cutaneous melanoma and ultimately an indicator of poor prognosis. However, the molecular events surrounding the acquisition of this invasive phenotype remain incompletely understood. Mutations in B-RAF are frequent in melanoma and are known to regulate the invasive phenotype. In this study, we sought to determine the molecular mechanisms controlling melanoma invasion. We found that mutant B-RAF signaling regulates a cadherin switch. In melanoma cells expressing mutant B-RAF we observed high levels of N-cadherin and low levels of E-cadherin. Depletion of mutant B-RAF, by small interfering RNA, caused a decrease in the levels of N-cadherin and an increase in the levels of E-cadherin. Mechanistically, we found that this cadherin switch required the activity of Rac1 and its GEF, Tiam1, both of which show suppressed activity in the presence of mutant B-RAF. Consistent with the work of others, we found that depletion of mutant B-RAF decreased the invasive capacity of the melanoma cells. However, simultaneous depletion of B-RAF and Rac or Tiam1 resulted in invasive capacity similar to that of control cells. Taken together, our results suggest that mutant B-RAF signaling downregulates Tiam1/Rac activity resulting in an increase in N-cadherin levels and a decrease in E-cadherin levels and ultimately enhanced invasion.

We previously demonstrated that the guanine nucleotide exchange factor, RasGrf1, binds nerve growth factor (NGF)-activated TrkA and facilitates neurotrophin-induced neurite outgrowth in PC12 cells. RasGrf1 can activate both Ras and Rac, via intrinsic Cdc25 and DH domains, respectively, suggesting that the activation of both could contribute to this process. Previous studies have assayed constitutive neurite outgrowth following RasGrf1 over-expression in PC12 cells, in either the absence or presence of ectopic H-Ras, and have suggested an essential role for either Ras or Rac depending on the presence of H-Ras over-expression. In contrast, in this study, we have addressed the mechanism of how RasGrf1 facilitates neurite outgrowth in response to the neurotrophins, NGF and BDNF. Using Ras/Rac activation assays and site-directed RasGrf1 mutants, we find that both Ras and Rac are essential to neurotrophin-induced neurite outgrowth. Moreover, we find that H-Ras over-expression rescues the loss of neurite outgrowth observed with a Rac minus mutant and that RasGrf1 differentially stimulates NGF-dependent activation of Rac or Ras, depending on cell type. Collectively, these studies clarify the mechanism of how RasGrf1 expression facilitates neurotrophin-induced neurite outgrowth. Moreover, they suggest that H-Ras over-expression should be used with caution to measure phenotypic responses.

Hyaluronic acid (HA) has been shown to promote angiogenesis. However, the mechanism behind this effect remains largely unknown. Therefore, in this study, the mechanism of HA-induced angiogenesis was examined. CD44 and PKCδ were shown to be necessary for induction of the receptor for HA-mediated cell motility (RHAMM), a HA-binding protein. RHAMM was necessary for HA-promoted cellular invasion and endothelial cell tube formation. Cytokine arrays showed that HA induced the expression of plasminogen activator-inhibitor-1 (PAI), a downstream target of TGFβ receptor signaling. The induction of PAI-1 was dependent on CD44 and PKCδ. HA also induced an interaction between RHAMM and TGFβ receptor I, and induction of PAI-1 was dependent on RHAMM and TGFβ receptor I. Histone deacetylase 3 (HDAC3), which is decreased by HA via rac1, reduced induction of plasminogen activator inhibitor-1 (PAI-1) by HA. ERK, which interacts with RHAMM, was necessary for induction of PAI-1 by HA. Snail, a downstream target of TGFβ signaling, was also necessary for induction of PAI-1. The down regulation of PAI-1 prevented HA from enhancing endothelial cell tube formation and from inducing expression of angiogenic factors, such as ICAM-1, VCAM-1 and MMP-2. HDAC3 also exerted reduced expression of MMP-2. In this study, we provide a novel mechanism of HA-promoted angiogenesis, which involved RHAMM-TGFβRI signaling necessary for induction of PAI-1.

Neutrophil polarization is critical for the inflammatory response. AKT is a serine/threonine protein kinase and has been implicated in cell migration. However, it is not completely clear whether AKT affects neutrophil polarization. In this study, we tested the hypothesis that AKT regulates the polarization of neutrophil-like differentiated HL-60 cells (dHL-60) in response to fMLP. HL-60 cells were differentiated into dHL-60 by incubation in medium containing 1.3 % DMSO for up to 6 days. Polarization of dHL-60 cells and primary human neutrophils were measured by Zigmond chamber. Phospho-Akt was analyzed by immunofluorescence and Western blot analysis. F-actin polymerization was detected by Rhodamine-Phalloidine staining. Rac2 activation was evaluated using GST Pull-down assay. We found that changes in the rate of cell polarization were consistent with the changes in AKT phosphorylation levels during HL-60 cell differentiation in response to fMLP. Moreover, cell polarization and AKT phosphorylation were reduced in fMLP-stimulated dHL-60 cells pretreated with the PI3 kinase inhibitors or the AKT inhibitors, which was confirmed in the primary human neutrophils. The AKT inhibitors altered fMLP-induced F-actin polymerization. Rac2 GTPases was also decreased by the AKT inhibitors in fMLP-stimulated dHL-60 cells. This study demonstrates that AKT activation plays a crucial role in dHL-60 cell polarization.

Reactive oxygen species (ROS) generation is linked to dynamic actin cytoskeleton reorganization, which is involved in tumor cell motility and metastasis. Thus, inhibition of ROS generation and actin polymerization in tumor cells may represent an effective anticancer strategy. However, the molecular basis of this signaling pathway is currently unknown. Here, we show that the Ecklonia cava-derived antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells. Steady-state intracellular ROS levels were higher in malignant B16F10 cells than in parental, nonmetastatic B16F0 cells. Elevation of ROS by H(2)O(2) treatment increased migration and invasion ability of B16F0 cells to level similar to that of B16F10 cells, suggesting that intracellular ROS signaling mediates the prometastatic properties of B16 mouse melanoma cells. ROS levels and the cell migration and invasion ability of B16 melanoma cells correlated with Rac1 activation and WAVE2 expression. Overexpression of dominant negative Rac1 and depletion of WAVE2 by siRNA suppressed H(2)O(2)-induced cell invasion of B16F0 and B16F10 cells. Similarly, dieckol attenuates the ROS-mediated Rac1 activation and WAVE2 expression, resulting in decreased migration and invasion of B16 melanoma cells. In addition, we found that dieckol decreases association between WAVE2 and NADPH oxidase subunit p47(phox). Therefore, this finding suggests that WAVE2 acts to couple intracellular Rac1/ROS signaling to the invasive migration of B16 melanoma cells, which is inhibited by dieckol.

Nuclear factor erythroid 2-related factor 2 (Nrf2, NM 006164, 605 AA) is essential for the antioxidant responsive element (ARE)-mediated expression of a group of detoxifying antioxidant genes that detoxify carcinogens and protect against oxidative stress. Several proteins have been identified as Nrf2-interacting molecules. In this study, we found that the overexpression of receptor-associated coactivator 3 (RAC3)/AIB-1/steroid receptor coactivator-3, a nuclear coregulator and oncogene frequently amplified in human breast cancers, induced heme oxygenase-1 (HO-1) through Nrf2 transactivation in HeLa cells. Next, we determined the interaction between RAC3 and Nrf2 proteins using a co-immunoprecipitation assay and fluorescence resonance energy transfer analysis. The results showed that RAC3 bound directly to the Nrf2 protein in the nucleus. Subsequently, we identified the interacting domains of Nrf2 and RAC3 using a glutathione S-transferase pull-down assay. The results showed that both the N-terminal RAC3-pasB and C-terminal RAC3-R3B3 domains were tightly bound to the Neh4 and Neh5 transactivation domains. Furthermore, chromatin immunoprecipitation showed that RAC3 bound tightly to the ARE enhancer region of the HO-1 promoter via Nrf2 binding. These data suggest that Nrf2 activation is modulated and directly controlled through interactions with the RAC3 protein in HeLa cells.

Phagocytosis of apoptotic cells by macrophages, known as efferocytosis, is a critical process in the resolution of inflammation. High mobility group box 1 (HMGB1) protein was first described as a nuclear nonhistone DNA-binding protein, but is now known to be secreted by activated cells during inflammatory processes, where it participates in diminishing efferocytosis. Although HMGB1 is known to undergo modification when secreted, the effect of such modifications on the inhibitory actions of HMGB1 during efferocytosis have not been reported. In the present studies, we found that HMGB1 secreted by Toll-like receptor 4 (TLR4) stimulated cells is highly poly(ADP-ribosyl)ated (PARylated). Gene deletion of poly(ADP)-ribose polymerase (PARP)-1 or pharmacological inhibition of PARP-1 decreased the release of HMGB1 from the nucleus to the extracellular milieu after TLR4 engagement. Preincubation of macrophages or apoptotic cells with HMGB1 diminished efferocytosis through mechanisms involving binding of HMGB1 to phosphatidylserine on apoptotic cells and to the receptor for advanced glycation end products (RAGE) on macrophages. Preincubation of either macrophages or apoptotic cells with PARylated HMGB1 inhibited efferocytosis to a greater degree than exposure to unmodified HMGB1, and PARylated HMGB1 demonstrated higher affinity for phosphatidylserine and RAGE than unmodified HMGB1. PARylated HMGB1 had a greater inhibitory effect on Ras-related C3 botulinum toxin substrate 1 (Rac-1) activation in macrophages during the uptake of apoptotic cells than unmodified HMGB1. The present results, showing that PARylation of HMGB1 enhances its ability to inhibit efferocytosis, provide a novel mechanism by which PARP-1 may promote inflammation.

Despite advances in pharmacological treatments, diabetes with hypertension continues to be a major public health problem with high morbidity and mortality rates. We recently identified a circulating peptide coupling factor 6 (CF6), which binds to the plasma membrane ATP synthase (ecto-F(1)F(o) complex), resulting in intracellular acidosis. We investigated whether overexpression of CF6 contributes to diabetes and hypertension by intracellular acidosis.Transgenic mice overexpressing CF6 (also known as ATP5J) were generated, and physiological, biochemical and molecular biology studies were performed.CF6 overexpression elicited a sustained decrease in intracellular pH in tissues (aorta, kidney, skeletal muscle and liver, with the exception of adipose tissue) that express its receptor, the β-subunit of ecto-F(1)F(o) complex. Consistent with the receptor distribution, phospho-insulin receptor β, phosphoinositide 3-kinase activity and the phospho-Akt1:total Akt1 ratio were all decreased in the skeletal muscle and the liver in transgenic compared with wild-type mice, resulting in a decrease of plasma membrane-bound GLUT4 and an increase in hepatic glucose production. Under a high-sucrose diet, transgenic mice had insulin resistance and mild glucose intolerance; under a high-salt diet, they had elevated blood pressure with increased renal RAS-related C3 botulinum substrate 1 (RAC1)-GTP, which is an activator of mineralocorticoid receptor.Through its action on the β-subunit of ecto-F(1)F(o) complex, which results in intracellular acidosis, CF6 plays a crucial role in the development of insulin resistance and hypertension. This finding might advance our understanding of the mechanisms underlying diabetes and hypertension, possibly also providing a novel therapeutic target against cardiovascular disease.

The Stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) system is considered to be of great importance in diseases involving cardiogenesis and angiogenesis. The SDF-1α-RAC signaling pathway plays a pivotal role in a cell's polarity and serves to activate cell morphology variation and to control the direction of migration. We aimed to study whether the polarity of endothelial progenitor cells (EPCs) is changed by the induction of the SDF-1α-RAC signaling pathway, to investigate the mechanism of the effect of polarity on the homing action of EPCs, and to explore the gene and protein expression of Rac1/2 during endothelial repair. We measured the EPC characteristics of polarity induced by various final concentrations of SDF-1α; our observations included morphology variation, migration direction, and excursion. Of the dynamic variation and cytoskeleton rearrangement of EPCs induced by different final concentrations of SDF-1α, the most obvious variation was exhibited at the SDF-1α concentration of 200 ng/ml. Obvious polarity variations were also found in the EPCs and signal receptors induced by the SDF-1α concentration of 200 ng/ml. The Western blot analysis of Rac1 and Rac2 showed that the addition of AMD 3100 significantly inhibited the expression of Rac. The SDF-1α pathway potentially regulates the expression of Rac1/2. The actual excursion vector and the direction of the migration of EPCs induced by SDF-1α follows polarity, thus indicating the importance of further exploration regarding the homing induction of EPCs.

The adaptor ASC contributes to innate immunity through the assembly of inflammasome complexes that activate the cysteine protease caspase-1. Here we demonstrate that ASC has an inflammasome-independent, cell-intrinsic role in cells of the adaptive immune response. ASC-deficient mice showed defective antigen presentation by dendritic cells (DCs) and lymphocyte migration due to impaired actin polymerization mediated by the small GTPase Rac. Genome-wide analysis showed that ASC, but not the cytoplasmic receptor NLRP3 or caspase-1, controlled the mRNA stability and expression of Dock2, a guanine nucleotide-exchange factor that mediates Rac-dependent signaling in cells of the immune response. Dock2-deficient DCs showed defective antigen uptake similar to that of ASC-deficient cells. Ectopic expression of Dock2 in ASC-deficient cells restored Rac-mediated actin polymerization, antigen uptake and chemotaxis. Thus, ASC shapes adaptive immunity independently of inflammasomes by modulating Dock2-dependent Rac activation and actin polymerization in DCs and lymphocytes.

Estrogen receptor alpha (ERα) is a ligand-dependent nuclear receptor that is important in breast cancer genesis, behavior and response to hormone-based therapies. A T7 phage display screen against full-length human ERα, coupled with genome-wide exon arrays, was used to identify RAC3 as a putative ERα co-regulator. RAC3 is a Rho family small GTPase that is associated with cytoskeletal rearrangement. We demonstrate a novel role for nuclear RAC3 as an ERα transcriptional activator, with prognostic implications for metastatic disease. Through in vitro and cell-based studies, RAC3 was shown to exist in a GTP-bound state and act as a ligand specific ERα co-activator of E2-induced transcription. Overexpression of RAC3 induced pro-growth and pro-migratory genes that resulted in increased migration of ERα-positive breast cancer cells. Chemical inhibition and genetic knockdown of RAC3 antagonized E2-induced cell proliferation, cell migration and ERα mediated gene expression, indicating that RAC3 is necessary for full ERα transcriptional activity. In agreement with the molecular and cellular data, RAC3 overexpression in ERα-positive breast cancers correlated with a significant decrease in recurrence free survival and a significant increase in the odds ratio of metastasis. In conclusion, RAC3 is a novel ERα co-activator that promotes cell migration and has prognostic value for ERα-positive breast cancer metastasis. RAC3 may also be a useful therapeutic target for ERα-positive breast cancers.

The roles of Rac1 and Rac2 in regulating osteoclast-mediated bone quality in postmenopausal osteoporosis were evaluated using an ovariectomized murine model. Animals' bone composition and architecture were evaluated. Our results demonstrate that the deletion of Rac1 increases vertebral bone quality compared to wild-type bones in an ovariectomized model. To determine the roles of the Rho family small GTPases Rac1 and Rac2 in regulating osteoclast-mediated bone quality in a model of postmenopausal osteoporosis. Twelve-month-old female mice from three genotypes—wild type (WT), Rac1 null (LysM.Rac1 KO), and Rac2 null (Rac2KO)—were studied in control and ovariectomized groups (mice previously ovariectomized at 4 months of age). Animals were sacrificed at 12 months of age, and the femora and vertebrae were harvested for mechanical testing, bone densitometry, micro-computed tomography, and histomorphometric analyses to evaluate bone mineralization and architecture. The results were compared between groups using ANOVA and LSD post-hoc tests. We observed that LysM.Rac1 KO mice showed higher vertebral bone mineral density compared to WT in both control and ovariectomized groups. Consistent with this finding, LysM.Rac1 KO vertebrae showed increased resistance to fracture and increased trabecular connectivity compared to WT in both groups. Micro-CT analysis revealed that Rac2KO ovariectomized vertebrae have more trabecular bone compared to WT and LysM.Rac1 KO, but this did not translate into increased fracture resistance. Our results demonstrate that the deletion of Rac1 increases vertebral bone quality compared to WT bones in a postmenopausal osteoporosis model.

Thymic carcinoid is an important component of the tumor spectrum causing Ectopic ACTH Syndrome (EAS) and usually carries a poor prognosis. Efforts have been focused on exploring the mechanism of the excessive ACTH production in non-pituitary tumors, whereas few studies have reported the molecular events underlying the tumor progression. In this study, seven patients with ACTH producing thymic carcinoids were enrolled. Of note is that five of them showed either lymph node metastasis, local invasion or distant metastasis. By using cDNA profiling approach, we evaluated the expression of cell adhesion pathway genes and found a remarkable overexpression of p21-activated kinase 3 (PAK3) in all thymic carcinoids which was further confirmed at both transcriptional and translational level. RAC1, an upstream activator of PAK3, was also overexpressed in thymic carcinoids. Overexpression of PAK3 in NIH3T3 cell enhanced cell migration and invasion. Importantly, we observed c-Jun NH(2)-terminal kinase (JNK) was activated in PAK3 transfected cells, and inhibition of JNK activity by SP600125, a JNK pathway inhibitor, abolished PAK3 mediated cell migration. Activation of JNK pathway was also detected in thymic carcinoid with high level of PAK3 expression. Our findings suggested a potential role of PAK3 in the progression of ACTH-producing thymic carcinoid.

Rac1 has a role in proliferation and survival of tumor cells in vitro. The exact effects of Rac1 on growth, apoptosis and corresponding signaling pathways during tumorigenesis in vivo, however, have not been explored yet. Using mice with a keratinocyte-restricted deletion of the Rac1 gene, we found that Rac1 is essential for DMBA/TPA-induced skin tumor formation. This corresponded to a decreased keratinocyte hyperproliferation, although apoptosis was not detectably altered. Activated Rac1 promoted Erk-dependent hyperproliferation by Pak1-mediated Mek activation independent of Mek1 phosporylation at serine 298. Rac1 was furthermore required for Pak2-dependent hyperactivation of Akt, which under in vivo condition was restricted to the suprabasal cell layers corresponding to a suprabasal-specific expression of Pak2. It is surprising that none of these signaling pathways was altered in untreated Rac1-deficient skin, indicating a hyperproliferation-specific function of Rac1 in vivo. These data suggest that blocking of Rac1 function might allow tumor-specific growth repression, as Rac1 is not required for normal growth and growth signaling controlling pathways in skin in vivo.

The neurofibromatosis type 2 (NF2) tumor suppressor gene encodes merlin, a membrane/cytoskeleton protein necessary for maintenance of contact inhibition of growth in cells. Biallelic inactivation of NF2 is known to cause multiple cancers in both humans and mice. However, the mechanism through which merlin exerts its tumor suppressive function remains obscure. In this report we reveal that NF2 knockout mouse embryonic fibroblasts (MEFs) lost contact inhibition of cell proliferation and contained significantly increased canonical Wnt signaling. Inhibition of Rac1, whose activity is inversely regulated by NF2, through the use of a dominant negative mutant, small hairpin RNA, or a small molecule inhibitor in the NF2-deficient cells, was able to suppress the elevated Wnt signals as shown by reduced activity of the T-cell factor 4 (TCF4) transcription factor. Dominant negative TCF4 or Rac1 mutant, as well as a small molecule inhibition of Wnt, were able to curb the NF2 deficiency-elicited cell proliferation at the confluent state. Thus, Rac1-mediated canonical Wnt signaling is essential for the loss of contact inhibition in NF2-deficient cells.

Rotational movement of the node cilia generates a leftward fluid flow in the mouse embryo because the cilia are posteriorly tilted. However, it is not known how anterior-posterior information is translated into the posterior tilt of the node cilia. Here, we show that the basal body of node cilia is initially positioned centrally but then gradually shifts toward the posterior side of the node cells. Positioning of the basal body and unidirectional flow were found to be impaired in compound mutant mice lacking Dvl genes. Whereas the basal body was normally positioned in the node cells of Wnt3a(-/-) embryos, inhibition of Rac1, a component of the noncanonical Wnt signalling pathway, impaired the polarized localization of the basal body in wild-type embryos. Dvl2 and Dvl3 proteins were found to be localized to the apical side of the node cells, and their location was polarized to the posterior side of the cells before the posterior positioning of the basal body. These results suggest that posterior positioning of the basal body, which provides the posterior tilt to node cilia, is determined by planar polarization mediated by noncanonical Wnt signalling.

Small GTPases, particularly the Rho family, are key regulators of cell motility and migration. Dock180 was well known for the main target of signal adaptor protein Crk and acted as a guanine-nucleotide exchange factor for small GTPase Rac1. In the present study, Dock180 was found to combine primarily with CrkI other than CrkII, and its association with Elmo1 was also demonstrated in ovarian cancer cell SKOV3. To evaluate the role of Dock180 in human ovarian cancer cell, we performed RNAi-mediated knockdown of Dock180 in SKOV3 cells using small interfering RNA expression vector. In Dock180 knockdown cells, we found that Elmo1 expression and Rac1 activity were decreased simultaneously. By contrast, the expressions of both another Crk-combining molecule C3G and Rap1 activity were observed to increase obviously. Accordingly, all Dock180 knockdown cells present with evident change in cell morphology, reduced cell proliferation, and attenuated cell migration. Taken together, these results suggest that signal transfer of Crk/Dock180/Rac1 is implicated in actin cytoskeleton reorganization and thus in the cell proliferation, motility, invasion, and of human ovarian cancer cell line SKOV3.

Protein-protein interactions are essential for many cellular processes. We have developed a technology called light-activated dimerization (LAD) to artificially induce protein hetero- and homodimerization in live cells using light. Using the FKF1 and GIGANTEA (GI) proteins of Arabidopsis thaliana, we have generated protein tags whose interaction is controlled by blue light. We demonstrated the utility of this system with LAD constructs that can recruit the small G-protein Rac1 to the plasma membrane and induce the local formation of lamellipodia in response to focal illumination. We also generated a light-activated transcription factor by fusing domains of GI and FKF1 to the DNA binding domain of Gal4 and the transactivation domain of VP16, respectively, showing that this technology is easily adapted to other systems. These studies set the stage for the development of light-regulated signaling molecules for controlling receptor activation, synapse formation and other signaling events in organisms.

In response to invading microorganisms, macrophages engage in phagocytosis and rapidly release reactive oxygen species (ROS), which serve an important microbicidal function. However, how phagocytosis induces ROS production remains largely unknown. CARD9, a caspase-recruitment domain (CARD)-containing protein, is important for resistance to fungal and bacterial infection. The mechanism of CARD9-mediated bacterial clearance is still mostly unknown. Here we show that CARD9 is required for killing intracellular bacteria in macrophages. CARD9 associated with the GDP-dissociation inhibitor LyGDI in phagosomes after bacterial and fungal infection and binding of CARD9 suppressed LyGDI-mediated inhibition of the GTPase Rac1, thereby leading to ROS production and bacterial killing in macrophages. Thus, our studies identify a key pathway that leads to microbe-elicited ROS production.

Sphingosine 1-phosphate (S1P) is a bioactive lipid that acts through a family of G-protein-coupled receptors. Herein, we report evidence of a novel redox-based cross-talk between S1P and insulin signaling pathways. In skeletal muscle cells S1P, through engagement of its S1P(2) receptor, is found to produce a transient burst of reactive oxygen species through a calcium-dependent activation of the small GTPase Rac1. S1P-induced redox-signaling is sensed by protein tyrosine phosphatase-1B, the main negative regulator of insulin receptor phosphorylation, which undergoes oxidation and enzymatic inhibition. This redox-based inhibition of the phosphatase provokes a ligand-independent trans-phosphorylation of insulin receptor and a strong increase in glucose uptake. Our results propose a new role of S1P, recognizing the lipid as an insulin-mimetic cue and pointing at reactive oxygen species as critical regulators of the cross-talk between S1P and insulin pathways. Any possible implication of S1P-directed insulin signaling in diabetes and insulin resistance remains to be established.

Excessive activation of G-protein-coupled receptor (GPCR) and receptor tyrosine kinase (RTK) pathways has been linked to prostate cancer metastasis. Rac activation by guanine nucleotide exchange factors (GEFs) plays an important role in directional cell migration, a critical step of tumor metastasis cascades. We found that the upregulation of P-Rex1, a Rac-selective GEF synergistically activated by Gbetagamma freed during GPCR signaling, and PIP3, generated during either RTK or GPCR signaling, strongly correlates with metastatic phenotypes in both prostate cancer cell lines and human prostate cancer specimens. Silencing endogenous P-Rex1 in metastatic prostate cancer PC-3 cells selectively inhibited Rac activity and reduced cell migration and invasion in response to ligands of both epidermal growth factor receptor and G-protein-coupled CXC chemokine receptor 4. Conversely, expression of recombinant P-Rex1, but not its 'GEF-dead' mutant, in non-metastatic prostate cancer cells, such as CWR22Rv1, increased cell migration and invasion through Rac-dependent lamellipodia formation. More importantly, using a mouse xenograft model, we showed that the expression of P-Rex1, but not its mutant, induced lymph node metastasis of CWR22Rv1 cells without an effect on primary tumor growth. Thus, by functioning as a coincidence detector of chemotactic signals from both GPCRs and RTKs, P-Rex1-dependent activation of Rac promotes prostate cancer metastasis.

The motility of cancer cells in 3D matrices is of two types: mesenchymal motility, in which the cells are elongated and amoeboid motility, in which the cells are round. Amoeboid motility is driven by an actomyosin-based contractile force, which is regulated by the Rho/ROCK pathway. However, the molecular mechanisms underlying the motility of elongated cells remain unknown. Here, we show that the motility of elongated cells is regulated by Rac signaling through the WAVE2/Arp2/3-dependent formation of elongated pseudopodia and cell-substrate adhesion in 3D substrates. The involvement of Rac signaling in cell motility was different in cell lines that displayed an elongated morphology in 3D substrates. In U87MG glioblastoma cells, most of which exhibit mesenchymal motility, inhibition of Rac signaling blocked the invasion of these cells in 3D substrates. In HT1080 fibrosarcoma cells, which display mixed cell motility involving both elongated and rounded cells, inhibition of Rac1 signaling not only blocked mesenchymal motility but also caused a mesenchymal-amoeboid transition. Additionally, Rac1 and RhoA signaling regulated the mesenchymal and amoeboid motility in these cells, respectively, and the inhibition of both pathways dramatically decreased cell invasion. Hence, we could conclude that Rac1 and RhoA signaling simultaneously regulate cell invasion in 3D matrices.

Some of the characteristics of cancer cells are high rates of cell proliferation, cell survival, and the ability to invade surrounding tissue. The cytoskeleton has an essential role in these processes. Dynamic changes in the cytoskeleton are necessary for cell motility and cancer cells are dependent on motility for invasion and metastasis. The signaling pathways behind the reshaping and migrating properties of the cytoskeleton in cancer cells involve a group of Ras-related small GTPases and their effectors, including the p21-activated kinases (Paks). Paks are a family of serine/threonine protein kinases comprised of six isoforms (Pak 1-6), all of which are direct targets of the small GTPases Rac and Cdc42. Besides their role in cytoskeletal dynamics, Paks have recently been shown to regulate various other cellular activities, including cell survival, mitosis, and transcription. Paks are overexpressed and/or hyperactivated in several human tumors and their role in cell transformation makes them attractive therapeutic targets. Pak-targeted therapeutics may efficiently inhibit certain types of tumors and efforts to identify selective Pak-inhibitors are underway.

Rac1, a member of the Rho family of GTPases, is an intracellular transducer known to regulate multiple signaling pathways that control cytoskeleton organization, transcription, and cell proliferation. Deregulated expression or activation patterns of Rac1 can result in aberrant cell signaling and numerous pathological conditions. Here, we highlight the physiological functions and signaling mechanisms of Rac1 and their relevance to disease.

Fragile X syndrome is the most common genetic cause of mental disability. The mechanisms underlying the pathogenesis remain unclear and specific treatments are still under development. Previous studies have proposed an abnormal hypothalamic-pituitary-adrenal axis and high cortisol levels are demonstrated in the fragile X patients. Additionally, we have previously described that NADPH-oxidase activation leads to oxidative stress in the brain, representing a pathological mechanism in the fragile X mouse model. Fmr1-knockout mice develop an altered free radical production, abnormal glutathione homeostasis, high lipid and protein oxidation, accompanied by stress-dependent behavioral abnormalities and pathological changes in the first months of postnatal life. Chronic pharmacological treatment with alpha-tocopherol reversed pathophysiological hallmarks including free radical overproduction, oxidative stress, Rac1 and alpha-PKC activation, macroorchidism, and also behavior and learning deficits. The restoration of the oxidative status in the fragile X mouse emerges as a new and promising approach for further therapeutic research in fragile X syndrome.

Acute myeloid leukemia (AML) is intrinsically prone to resistance to conventional chemotherapeutic agents. Here we explore the Rac family of small GTPases as novel biologic targets for AML treatment. The Rac subfamily of Rho guanosine triphosphatases (GTPases) plays an essential role in regulating normal hematopoiesis (1). Rac proteins cycle between active GTP-bound and inactive GDP-bound states. Of the three known Rac family members, Rac1 and Rac3 are expressed ubiquitously, while Rac2 is restricted to the hematopoietic system. In hematopoietic cells, Rac proteins integrate signals from growth factor, chemokine, and adhesion receptors to mediate a variety of cellular responses, including cell growth and survival, gene transcription, adhesion, motility, and formation of the actin cytoskeleton (1). We recently identified the Rac GTPases as molecular targets in BCR-ABL-induced myeloproliferative disease (2, 3). However, the role of Rac in AML has thus far not been clearly defined. Elevated levels of GTP-bound Rac have been described in CD34+ cells isolated from patients with AML. In these samples, Rac signaling was identified as a critical mediator of stem/progenitor cell and stroma interaction (4). Recently, Wei et al. observed a critical role of Rac signaling in a disease model of human CD34+ cells transduced with the Mixed Lineage Leukemia (MLL)-AF9 fusion oncogene (5). Interestingly, while MLL-AF9 transduced cells were sensitive to Rac inhibition, cells transduced with the AML-ETO oncogene did not depend on Rac signaling for survival and proliferation. These discrepancies prompted us to further investigate the role of Rac signaling in a panel of human AML cell lines, including the MLL gene-rearranged ML-2 cell line and cell lines not harboring MLL rearrangements such as the histiocytic lymphoma U937 and the acute promyelocytic HL-60 cell line. We demonstrate the presence of GTP-Rac in all cell lines via p21-activated kinase (PAK)-binding domain (PBD) pull-down and immunoblot (data not shown). Compared to purified normal human CD34+ cells, ML-2 cells, which contain a MLL-AF6 translocation (6), showed the most profound inhibition of cell proliferation upon pharmacologic inhibition of Rac using the small molecule Rac inhibitor NSC23766 (7) (Figure 1A). To determine whether a correlation exists between Rac activation and the observed decrease in proliferation of ML-2 cells, we analyzed the effect of NSC23766 on GTP-Rac via PBD pull-down assay and observed abrogation of Rac activation with drug treatment (Figure 1B). We next wanted to determine whether NSC23766 treatment would impact apoptosis (Figure 1C) and/or cell cycle progression of ML-2 cells (Figure 1D). ML-2 cells displayed an increase of early and late apoptosis as measured by Annexin V/7AAD 72 hours after drug exposure (Figure 1C). In addition, Rac inhibition led to increased cell cycle arrest in G0/G1 (Figure 1D). Importantly, these effects were specific to ML-2 cells, as normal CD34+ cells were not significantly affected by NSC23766 exposure. Analogous effects were observed in the MLL-AF9 containing THP-1 cell line (Supplemental Figure 1). In contrast to these MLL gene rearranged cell lines, no significant effect of NSC23677 on cell cycle or apoptosis was observed in U937 cells and only marginal effects were observed in HL- 60 cells (data not shown). To further analyze the potential therapeutic efficacy of NSC23766 in a murine xenograft model, 2 × 107 ML-2 cells were transplanted into irradiated (350 Gy) NOD/SCID mice. Alzet osmotic pumps containing NSC23766 (2 pumps, 75 mM NSC23766 per pump) or PBS were implanted on day 21 post transplant. The pumps were exchanged for new pumps on day 35 and removed on day 49 post transplant. Animals were monitored for survival and bone marrow chimerism of ML-2 cells (human CD45+) was assessed by flow cytometry post-mortem. Animals with less than 15% human CD45+ chimerism were censored from the study (3 animals in the NSC23766- and four animals in the PBS-cohort). We noted a significant difference in the survival of PBS vs. NSC23677-treated animals (Figure 2). Death due to disease progression in the treatment group occurred predominantly following pump removal.

The clearance of apoptotic cells by phagocytes is an integral component of normal life, and defects in this process can have significant implications for self tolerance and autoimmunity. Recent studies have provided new insights into the engulfment process, including how phagocytes seek apoptotic cells, how they recognize and ingest these targets and how they maintain cellular homeostasis after the 'meal'. Several new factors that regulate engulfment have been identified, whereas the roles of some of the older players require revision. This Review focuses on these recent developments and attempts to highlight some of the important questions in this field.

Engulfment and subsequent degradation of apoptotic cells is an essential step that occurs throughout life in all multicellular organisms. ELMO/Dock180/Rac proteins are a conserved signalling module for promoting the internalization of apoptotic cell corpses; ELMO and Dock180 function together as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac, and thereby regulate the phagocyte actin cytoskeleton during engulfment. However, the receptor(s) upstream of the ELMO/Dock180/Rac module are still unknown. Here we identify brain-specific angiogenesis inhibitor 1 (BAI1) as a receptor upstream of ELMO and as a receptor that can bind phosphatidylserine on apoptotic cells. BAI1 is a seven-transmembrane protein belonging to the adhesion-type G-protein-coupled receptor family, with an extended extracellular region and no known ligands. We show that BAI1 functions as an engulfment receptor in both the recognition and subsequent internalization of apoptotic cells. Through multiple lines of investigation, we identify phosphatidylserine, a key 'eat-me' signal exposed on apoptotic cells, as a ligand for BAI1. The thrombospondin type 1 repeats within the extracellular region of BAI1 mediate direct binding to phosphatidylserine. As with intracellular signalling, BAI1 forms a trimeric complex with ELMO and Dock180, and functional studies suggest that BAI1 cooperates with ELMO/Dock180/Rac to promote maximal engulfment of apoptotic cells. Last, decreased BAI1 expression or interference with BAI1 function inhibits the engulfment of apoptotic targets ex vivo and in vivo. Thus, BAI1 is a phosphatidylserine recognition receptor that can directly recruit a Rac-GEF complex to mediate the uptake of apoptotic cells.