Activation Of GTPase Rac1 Research Articles

A Novel Synthetic Derivative of Phloroglucinol Inhibits Neuroinflammatory Responses Through Attenuating Kalirin Signaling Pathway in Murine BV2 Microglial Cells.

Neuroinflammation has been implicated as an important factor in the neurodegenerative diseases, and multiple candidates with anti-inflammatory effects have been shown to be beneficial for the treatment of neurodegenerative diseases. Our previous study demonstrated that a novel synthetic phloroglucinol derivative from Lysidice rhodostegia roots (code name: Compound 21) exerted neuroprotective effect through suppressing neuroinflammation. The aim of this study was to reveal the underlying molecular mechanism. The results indicated that the anti-inflammatory effects of Compound 21 were mediated through suppression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and the production of reactive oxygen species (ROS). Further study showed that this suppression on NADPH oxidase was mediated by inhibiting the translocation and activity of its subunit Rac1. It is well established that Rac1 activation is regulated by a variety of guanine nucleotide exchange factors (GEFs), so we tried to find out whether GEFs were involved in the anti-inflammatory effects of Compound 21. The results showed that Compound 21 treatment down-regulated the expression and activity of GEF Kalirin, thus modulating the activity of Rac1 GTPase. Altogether, our data suggested that Compound 21 exerted the anti-neuroinflammatory effect through suppressing Kalirin signaling pathways, decreasing Rac1-NADPH oxidase activation and the subsequent pro-inflammatory cytokine production. The present study provided solid evidence to support Compound 21 as a potential candidate of neuroinflammatory inhibitor. Moreover, our findings have shed new light on the role of Rac1 and GEF Kalirin in neuroinflammation, which provides potential targets for neuroinflammation-related diseases, such as neurodegenerative diseases.

Distinct Roles of Small GTPases Rac1 and Rac2 in Histamine H4 Receptor-Mediated Chemotaxis of Mast Cells.

Histamine induces chemotaxis of mast cells through the H4 receptor. However, little is known about the precise intracellular signaling pathway that mediates this process. In this study, we identified small GTPases Rac1 and Rac2 as intracellular binding partners of the H4 receptor and characterized their roles in H4 receptor signaling. We showed that histamine induced Rac GTPase activation via the H4 receptor. A Rac inhibitor NSC23766 attenuated chemotaxis of mast cells toward histamine, as well as histamine-induced calcium mobilization and extracellular signal-regulated kinase (ERK) activation. Histamine-induced migration of mast cells was also sensitive to PD98059, an inhibitor of the mitogen-activated protein kinase kinase, indicating that the Rac-ERK pathway was involved in chemotaxis through the H4 receptor. Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) by LY294002 suppressed the histamine-induced chemotaxis and activation of Rac GTPases, suggesting that PI3K regulates chemotaxis upstream of Rac activation. Specific knockdown of Rac1 and Rac2 by short-hairpin RNA revealed that both Rac GTPases are necessary for histamine-induced migration. Downregulation of Rac1 and Rac2 led to attenuated response in calcium mobilization and ERK activation, respectively. These observations suggested that Rac1 and Rac2 have distinct and essential roles in intracellular signaling downstream of H4 receptor-PI3K in histamine-induced chemotaxis of mast cells.

SMARCB1 Deficiency Integrates Epigenetic Signals to Oncogenic Gene Expression Program Maintenance in Human Acute Myeloid Leukemia.

SWI/SNF is an evolutionarily conserved multi-subunit chromatin remodeling complex that regulates epigenetic architecture and cellular identity. Although SWI/SNF genes are altered in approximately 25% of human malignancies, evidences showing their involvement in tumor cell-autonomous chromatin regulation and transcriptional plasticity are limiting. This study demonstrates that human primary acute myeloid leukemia (AML) cells exhibit near complete loss of SMARCB1 (BAF47 or SNF5/INI1) and SMARCD2 (BAF60B) associated with nucleation of SWI/SNFΔ SMARCC1 (BAF155), an intact core component of SWI/SNFΔ, colocalized with H3K27Ac to target oncogenic loci in primary AML cells. Interestingly, gene ontology (GO) term and pathway analysis suggested that SMARCC1 occupancy was enriched on genes regulating Rac GTPase activation, cell trafficking, and AML-associated transcriptional dysregulation. Transcriptome profiling revealed that expression of these genes is upregulated in primary AML blasts, and loss-of-function studies confirmed transcriptional regulation of Rac GTPase guanine nucleotide exchange factors (GEF) by SMARCB1. Mechanistically, loss of SMARCB1 increased recruitment of SWI/SNFΔ and associated histone acetyltransferases (HAT) to target loci, thereby promoting H3K27Ac and gene expression. Together, SMARCB1 deficiency induced GEFs for Rac GTPase activation and augmented AML cell migration and survival. Collectively, these findings highlight tumor suppressor role of SMARCB1 and illustrate SWI/SNFΔ function in maintaining an oncogenic gene expression program in AML.Implications: Loss of SMARCB1 in AML associates with SWI/SNFΔ nucleation, which in turn promotes Rac GTPase GEF expression, Rac activation, migration, and survival of AML cells, highlighting SWI/SNFΔ downstream signaling as important molecular regulator in AML. Mol Cancer Res; 16(5); 791-804. ©2018 AACR.

The Role of Trio, a Rho Guanine Nucleotide Exchange Factor, in Glomerular Podocytes.

Nephrotic syndrome is a kidney disease featured by heavy proteinuria. It is caused by injury to the specialized epithelial cells called “podocytes” within the filtration unit of the kidney, glomerulus. Previous studies showed that hyperactivation of the RhoGTPase, Rac1, in podocytes causes podocyte injury and glomerulosclerosis (accumulation of extracellular matrix in the glomerulus). However, the mechanism by which Rac1 is activated during podocyte injury is unknown. Trio is a guanine nucleotide exchange factor (GEF) known to activate Rac1. By RNA-sequencing, we found that Trio mRNA is abundantly expressed in cultured human podocytes. Trio mRNA was also significantly upregulated in humans with minimal change disease and focal segmental glomerulosclerosis, two representative causes of nephrotic syndrome. Reduced expression of Trio in cultured human podocytes decreased basal Rac1 activity, cell size, attachment to laminin, and motility. Furthermore, while the pro-fibrotic cytokine, transforming growth factor β1 increased Rac1 activity in control cells, it decreases Rac1 activity in cells with reduced Trio expression. This was likely due to simultaneous activation of the Rac1-GTPase activation protein, CdGAP. Thus, Trio is important in the basal functions of podocytes and may also contribute to glomerular pathology, such as sclerosis, via Rac1 activation.

Affecting long-term fear memory formation through optical control of Rac1 GTPase and PAK activity in lateral amygdala

Fear conditioning, a behavioral model for studying fear-related disorders, is believed to be formed by alterations of synaptic efficacy mediated by changes in synaptic transmission and neuronal morphology in lateral amygdala (LA). Rac GTPase and its downstream effector p21-activated kinase (PAK) are involved in such key neuronal functions. Here we show that optical activation of Rac1 GTPase using photoactivatable form of Rac1 (PA-Rac1) in amygdala led to phosphorylation of PAK and inhibition of long-term but not short-term auditory fear conditioning memory formation. Activation of PA-Rac1 in LA one day after fear conditioning had no effect on long-term fear memory tested 24 hrs after PA-Rac1 activation. Inhibition of PAK in LA by microinjection of the PAK inhibitor IPA-3 30 minutes before fear conditioning enhanced long-term but not short-term fear memory formation. Our results demonstrate that photoactivation of Rac1 GTPase in lateral amygdala impairs fear memory formation. Moreover, Rac1 effector PAK activity during fear conditioning constrains the formation of fear memory in LA. Thus, Rac GTPase and PAK proteins may serve as targets for treatment of fear and anxiety disorders.

Prostaglandin E receptor-4 receptor mediates endothelial barrier-enhancing and anti-inflammatory effects of oxidized phospholipids.

Unlike other agonists that cause transient endothelial cell (EC) response, the products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) oxidation that contain cyclopenthenone groups, which recapitulate prostaglandin-like structure, cause sustained enhancement of the pulmonary EC barrier. The mechanisms that drive the sustained effects by oxidized PAPC (OxPAPC) remain unexplored. On the basis of the structural similarity of isoprostanoid moieties that are present in full-length oxygenated PAPC species, we used an inhibitory approach to perform the screening of prostanoid receptors as potential candidates that mediate OxPAPC effects. Results show that only prostaglandin E receptor-4 (EP4) was involved and mediated the sustained phase of the barrier-enhancing effects of OxPAPC that are associated with the activation of Rac GTPase and its cytoskeletal targets. EC incubation with OxPAPC also induced EP4 mRNA expression in pulmonary ECs and lung tissue. EP4 knockdown using gene-specific small interfering RNA did not affect the rapid phase of OxPAPC-induced EC barrier enhancement or the protective effects against thrombin-induced EC permeability, but abolished the advanced barrier enhancement phase and suppressed the protective effects of OxPAPC against more sustained EC barrier dysfunction and cell inflammatory response caused by TNF-α. Endothelial-specific knockout of the EP4 receptor in mice attenuated the protective effect of intravenous OxPAPC administration in the model of acute lung injury caused by intratracheal injection of LPS. Taken together, these results demonstrate a novel role for prostaglandin receptor EP4 in the mediation of barrier-enhancing and anti-inflammatory effects caused by oxidized phospholipids.-Oskolkova, O., Gawlak, G., Tian, Y., Ke, Y., Sarich, N., Son, S., Andreasson, K., Bochkov, V. N., Birukova, A. A., Birukov, K. G. Prostaglandin E receptor-4 receptor mediates endothelial barrier-enhancing and anti-inflammatory effects of oxidized phospholipids.

P115 RhoGEF activates the Rac1 GTPase signaling cascade in MCP1 chemokine–induced vascular smooth muscle cell migration and proliferation

Although the involvement of Rho proteins in the pathogenesis of vascular diseases is well studied, little is known about the role of their upstream regulators, the Rho guanine nucleotide exchange factors (RhoGEFs). Here, we sought to identify the RhoGEFs involved in monocyte chemotactic protein 1 (MCP1)-induced vascular wall remodeling. We found that, among the RhoGEFs tested, MCP1 induced tyrosine phosphorylation of p115 RhoGEF but not of PDZ RhoGEF or leukemia-associated RhoGEF in human aortic smooth muscle cells (HASMCs). Moreover, p115 RhoGEF inhibition suppressed MCP1-induced HASMC migration and proliferation. Consistent with these observations, balloon injury (BI) induced p115 RhoGEF tyrosine phosphorylation in rat common carotid arteries, and siRNA-mediated down-regulation of its levels substantially attenuated BI-induced smooth muscle cell migration and proliferation, resulting in reduced neointima formation. Furthermore, depletion of p115 RhoGEF levels also abrogated MCP1- or BI-induced Rac1-NFATc1-cyclin D1-CDK6-PKN1-CDK4-PAK1 signaling, which, as we reported previously, is involved in vascular wall remodeling. Our findings also show that protein kinase N1 (PKN1) downstream of Rac1-cyclin D1/CDK6 and upstream of CDK4-PAK1 in the p115 RhoGEF-Rac1-NFATc1-cyclin D1-CDK6-PKN1-CDK4-PAK1 signaling axis is involved in the modulation of vascular wall remodeling. Of note, we also observed that CCR2-Gi/o-Fyn signaling mediates MCP1-induced p115 RhoGEF and Rac1 GTPase activation. These findings suggest that p115 RhoGEF is critical for MCP1-induced HASMC migration and proliferation in vitro and for injury-induced neointima formation in vivo by modulating Rac1-NFATc1-cyclin D1-CDK6-PKN1-CDK4-PAK1 signaling.

Abstract 894: Heparanase-induced shedding of syndecan-1 promotes cancer cell invasion : prevention by inhibitory synstatin peptide

Abstract Syndecan-1 (Sdc1, CD138) is highly expressed in multiple myeloma (MM). Heparanase (HPSE), an enzyme that cleaves heparan sulfate (HS) chains on Sdc1, induces shedding of the Sdc1 ectodomain; this is associated with poor prognosis in this disease. However, the link between Sdc1 shedding and poor prognosis remains unclear. We now show that HPSE-induced shed Sdc1 (sSdc1) engages α4β1 integrin (VLA-4) and vascular endothelial cell growth factor receptor-2 (VEGFR2) to form a ternary receptor complex on the cell surface. This coupling of VEGFR2 to clusters of VLA-4 via sSdc1 leads to autoactivation of VEGFR2 and the subsequent activation of protein kinase A (PKA) bound to the VEGFR2 cytoplasmic domain. PKA activation proceeds via VEGFR2-mediated stimulation of the G-protein coupled cytokine receptor CXCR4 and the subsequent generation of cAMP by its activation of adenylate cyclase 7. This mechanism is prevented by SSTNVEGFR2, a peptide that competes for the VEGFR2 docking site in the Sdc1 ectodomain, thereby disrupting VEGFR2 binding to sSdc1. VLA-4 has been shown to mediate the polarized invasion of a variety of cells due to α4 integrin phosphorylation on Ser988 by PKA, followed by Rac GTPase activation, a process that is restricted to the leading edge of the cell. But how this is confined at the leading edge is wholly unknown. We find that HPSE-mediated shedding of Sdc1 promotes an invasive phenotype by re-localizing VLA-4 and VEGFR2 to the leading edge of migrating myeloma cells as well as human T-acute lymphoblastic leukemia, melanoma, and endothelial cells where VEGFR2-dependent PKA activation causes α4 integrin phosphorylation. These results reveal a novel mechanism in which the expression of HPSE, acting by causing Sdc1 shedding, potentially regulates angiogenesis and the extravasation and invasion of cancer cells and identifies SSTNVEGFR2 as a promising cancer therapeutic. Citation Format: Oisun Jung, Alan Rapraeger. Heparanase-induced shedding of syndecan-1 promotes cancer cell invasion : prevention by inhibitory synstatin peptide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 894. doi:10.1158/1538-7445.AM2017-894

Cell adhesion controlled by adhesion G protein–coupled receptor GPR124/ADGRA2 is mediated by a protein complex comprising intersectins and Elmo–Dock

Developmental angiogenesis and the maintenance of the blood-brain barrier involve endothelial cell adhesion, which is linked to cytoskeletal dynamics. GPR124 (also known as TEM5/ADGRA2) is an adhesion G protein-coupled receptor family member that plays a pivotal role in brain angiogenesis and in ensuring a tight blood-brain barrier. However, the signaling properties of GPR124 remain poorly defined. Here, we show that ectopic expression of GPR124 promotes cell adhesion, additive to extracellular matrix-dependent effect, coupled with filopodia and lamellipodia formation and an enrichment of a pool of the G protein-coupled receptor at actin-rich cellular protrusions containing VASP, a filopodial marker. Accordingly, GPR124-expressing cells also displayed increased activation of both Rac and Cdc42 GTPases. Mechanistically, we uncover novel direct interactions between endogenous GPR124 and the Rho guanine nucleotide exchange factors Elmo/Dock and intersectin (ITSN). Small fragments of either Elmo or ITSN1 that bind GPR124 blocked GPR124-induced cell adhesion. In addition, Gβγ interacts with the C-terminal tail of GPR124 and promotes the formation of a GPR124-Elmo complex. Furthermore, GPR124 also promotes the activation of the Elmo-Dock complex, as measured by Elmo phosphorylation on a conserved C-terminal tyrosine residue. Interestingly, Elmo and ITSN1 also interact with each other independently of their GPR124-recognition regions. Moreover, endogenous phospho-Elmo and ITSN1 co-localize with GPR124 at lamellipodia of adhering endothelial cells, where GPR124 expression contributes to polarity acquisition during wound healing. Collectively, our results indicate that GPR124 promotes cell adhesion via Elmo-Dock and ITSN. This constitutes a previously unrecognized complex formed of atypical and conventional Rho guanine nucleotide exchange factors for Rac and Cdc42 that is putatively involved in GPR124-dependent angiogenic responses.

Abstract 1468: MiR-200b targets ARHGAP18 and suppresses triple negative breast cancer metastasis

Abstract Triple negative breast cancer (TNBC) refers to a group of highly heterogeneous malignant tumors that lack estrogen receptor and progesterone receptor expression, and human epidermal growth factor receptor 2 amplification, accounting for 10-20% of newly diagnosed breast cancer cases. Compared to other subtypes of breast cancer, TNBCs usually show worse clinical features such as rapid tumor growth, earlier recurrence, and more aggressive metastasis. Unfortunately, the mechanism of TNBC metastatic behavior has not been well understood. Moreover, no efficient targeted therapies for TNBCs are currently available, representing a real unmet need for effective new therapies. MiR-200 family members are among the first miRNAs reported to function as potent inhibitors of cancer metastasis. However, the mechanism of miR-200 family on cancer metastasis has not been well understood. In this study, we investigated the effect of miR-200b, one member of the miR-200 family, on TNBC metastasis using cell culture and mouse orthotopic mammary xenograft tumor models. We found that the expression level of miR-200b is significantly lower in TNBC cells and tissues than that in other types of breast cancer. Stably expressing miR-200b significantly reduced TNBC cell migration and invasion and suppressed TNBC metastasis in a mouse orthotopic mammary xenograft tumor model. Mechanistic studies revealed that miR-200b overexpression in TNBC cells caused drastic changes in cellular actin cytoskeleton organization patterns as evidenced by reduced lamellipodia formation but increased stress fiber formation. In consistent with these findings, Rho GTPase pulldown assays demonstrated that stably expressing miR-200b significantly increased the Rho GTPase Rho A activation, but reduced the Rho GTPase Rac1 activation. Moreover, inhibition of Rho A signaling impaired the inhibitory effect of miR-200b on TNBC cell migration. Bioinformatics analysis indicated that ARHGAP18, a specific Rho A GTPase activating protein (GAP), is a predicate target of miR-200b. Further Q-PCR, Western blot and 3’UTR reporter analysis confirmed that ARHGAP18 is a target of miR-200b. Knocking down ARHGAP18 in TNBC cells using siRNAs significantly increased Rho A activation but reduced Rac1 activation. To further determine the role of ARHGAP18 in TNBC, ARHGAP knockout TNBC cells were generated using the CRISPR technology. It was found that knockout ARHGAP18 phenocopied the effect of miR-200b overexpression. Moreover, overexpressing ARHGAP18 in miR-200b stable expression cells overcome the inhibitory effect of miR-200b on TNBC metastasis. Together, these findings suggest that miR-200b suppresses triple negative breast cancer metastasis by targeting ARHGAP18 and enhancing Rho A activation. Note: This abstract was not presented at the meeting. Citation Format: Chengfeng Yang, Brock Humphries, Yunfei Li, Zhishan Wang. MiR-200b targets ARHGAP18 and suppresses triple negative breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1468. doi:10.1158/1538-7445.AM2017-1468

The Amyloid Precursor Protein Is a Conserved Receptor for Slit to Mediate Axon Guidance.

The amyloid precursor protein (APP) is a receptor-like membrane protein. Although APP processing and β-amyloid production play a central role in Alzheimer’s disease (AD) pathogenesis, the physiological function of APP remains elusive. Here, we identify APP as a novel receptor for Slit that mediates axon guidance and neural circuit formation. APP deficiency abolishes the Slit repulsive effect in a 3D olfactory explant culture, consistent with its callosal projection deficit in vivo and reminiscent of Slit loss. Inactivation of APP ortholog APL-1 in Caenorhabditis elegans results in pioneer axon mistargeting and genetic analysis places APL-1 in the SLT-1 (Slit)/SAX-3 (Robo) repulsive pathway. Slit binds to APP through the E1 domain, which triggers APP ectodomain shedding and recruitment of the intracellular FE65 and Pak1 complex and associated Rac1 GTPase activation. Our study establishes APP as a novel receptor for Slit ligand mediating axon guidance and neural circuit formation.

Βeta-eudesmol reduces stem cell factor-induced mast cell migration

Previously, we showed the inhibitory effects of β-eudesmol on mast cell-mediated allergic inflammatory responses. Stem cell factor (SCF) participates in allergic reactions through the differentiation and migration of mast cells. However, the effects of β-eudesmol on SCF-mediated allergic reactions are poorly understood. Herein, we showed that a treatment of rat peritoneal mast cells (RPMCs) with β-eudesmol markedly suppressed SCF-induced mast cell migration and morphological alterations in a concentration-dependent manner. β-eudesmol also reduced F-actin formation. The activation of Fyn kinase, Rac1 GTPase, and p38 mitogen-activated protein kinases as increased by SCF was reduced by β-eudesmol. Moreover, β-eudesmol significantly abolished SCF-induced tumor necrosis factor-α and intercellular adhesion molecule-1 production without cytotoxicity. These results demonstrate that β-eudesmol has an anti-allergic inflammatory effect through the inhibition of mast cell migration and inflammatory cytokine production.

The shift in GH3 cell shape and cell motility is dependent on MLCK and ROCK

Cytoskeletal organization, actin-myosin contractility and the cell membrane together regulate cell morphology in response to the cell environment, wherein the extracellular matrix (ECM) is an indispensable component. Plasticity in cell shape enables cells to adapt their migration mode to their surroundings. GH3 endocrine cells respond to different ECM proteins, acquiring different morphologies: a rounded on collagen I-III (C I-III) and an elongated on collagen IV (C IV). However, the identities of the molecules that participate in these responses remain unknown. Considering that actin-myosin contractility is crucial to maintaining cell shape, we analyzed the participation of MLCK and ROCK in the acquisition of cell shape, the generation of cellular tension and the cell motility mode. We found that a rounded shape with high cortical tension depends on MLCK and ROCK, whereas in cells with an elongated shape, MLCK is the primary protein responsible for cell spreading. Further, in cells with a slow and directionally persistent motility, MLCK predominates, while rapid and erratic movement is ROCK-dependent. This behavior also correlates with GTPase activation. Cells on C I-III exhibited higher Rho-GTPase activity than cells on C IV and vice versa with Rac-GTPase activity, showing a plastic response of GH3 cells to their environment, leading to the generation of different cytoskeleton and membrane organizations and resulting in two movement strategies, rounded and fibroblastoid-like.

109 - The anticontractile inhibitor, secinH3 inhibits ARF6, but not Rac or RhoA GTPase activities in the human prostate: A new role for ARF6 in smooth muscle contraction?

109 - The anticontractile inhibitor, secinH3 inhibits ARF6, but not Rac or RhoA GTPase activities in the human prostate: A new role for ARF6 in smooth muscle contraction?

Plectin deficiency in liver cancer cells promotes cell migration and sensitivity to sorafenib treatment

ABSTRACTPlectin involved in activation of kinases in cell signaling pathway and plays important role in cell morphology and migration. Plectin knockdown promotes cell migration by activating focal adhesion kinase and Rac1-GTPase activity in liver cells. Sorafenib is a multi-targeting tyrosine kinase inhibitor that improves patient survival on hepatocellular carcinoma. The aim of this study is to investigate the correlation between the expression of plectin and cell migration as well as the sensitivity of hepatoma cell lines exposing to sorafenib. Hepatoma cell lines PLC/PRF/5 and HepG2 were used to examine the level of plectin expression and cell migration in comparison with Chang liver cell line. In addition, sensitivity of the 3 cell lines to sorafenib treatment was also measured. Expression of plectin was lower in PLC/PRF/5 and HepG2 hepatoma cells than that of Chang liver cells whereas HepG2 and PLC/PRF/5 cells exhibit higher rate of cell migration in trans-well migration assay. Immunohistofluorecent staining on E-cadherin revealed the highest rate of collective cell migration in HepG2 cells and the lowest was found in Chang liver cells. Likewise, HepG2 cell line was most sensitive to sorafenib treatment and Chang liver cells exhibited the least sensitivity. The drug sensitivity to sorafenib treatment showed inverse correlation with the expression of plectin. We suggest that plectin deficiency and increased E-cadherin in hepatoma cells were associated with higher rates of cell motility, collective cell migration as well as higher drug sensitivity to sorafenib treatment.

Low-intensity pulsed ultrasound promotes cell motility through vinculin-controlled Rac1 GTPase activity.

Low-intensity pulsed ultrasound (LIPUS) is a therapy used clinically to promote healing. Using live-cell imaging we show that LIPUS stimulation, acting through integrin-mediated cell-matrix adhesions, rapidly induces Rac1 activation associated with dramatic actin cytoskeleton rearrangements. Our study demonstrates that the mechanosensitive focal adhesion (FA) protein vinculin, and both focal adhesion kinase (FAK, also known as PTK2) and Rab5 (both the Rab5a and Rab5b isoforms) have key roles in regulating these effects. Inhibiting the link of vinculin to the actin-cytoskeleton abolished LIPUS sensing. We show that this vinculin-mediated link was not only critical for Rac1 induction and actin rearrangements, but was also important for the induction of a Rab5-dependent increase in the number of early endosomes. Expression of dominant-negative Rab5, or inhibition of endocytosis with dynasore, also blocked LIPUS-induced Rac1 signalling events. Taken together, our data show that LIPUS is sensed by cell matrix adhesions through vinculin, which in turn modulates a Rab5-Rac1 pathway to control ultrasound-mediated endocytosis and cell motility. Finally, we demonstrate that a similar FAK-Rab5-Rac1 pathway acts to control cell spreading upon fibronectin.

Popdc1/Bves Functions in the Preservation of Cardiomyocyte Viability While Affecting Rac1 Activity and Bnip3 Expression.

The Popeye domain containing1, also called Bves (Popdc1/Bves), is a transmembrane protein that functions in muscle regeneration, heart rate regulation, hypoxia tolerance, and ischemia preconditioning. The expression of Popdc1/Bves is elevated in cardiomyocytes maintained in serum free defined medium. We hypothesized that Popdc1/Bves is important for cardiomyocyte survival under the stress of serum deprivation and investigated the mechanisms involved. A deficit in Popdc1/Bves, achieved by siRNA-mediated gene silencing, results in cardiomyocyte injury and death, upregulation of the pro-apoptotic protein Bcl-2/adenovirus E1B 19-kDa interacting protein3 (Bnip3), as well as reduction in Rac1-GTPase activity and in Akt phosphorylation. Combined Popdc1/Bves and Bnip3 silencing attenuated cell injury and prevented Bnip3 upregulation induced by the silencing of Popdc1/Bves alone. Chromatin immunoprecipitation indicated an increased binding of the transcription factor FoxO3 to the Bnip3 promoter although augmentation of FoxO3 in the nuclei was not detected. By contrast, the transcription factor NFκB was excluded from the nuclei of Popdc1/Bves deficient cardiomyocytes and exhibited decreased binding to the Bnip3 promoter. The data indicates that Popdc1/Bves plays a role in the preservation of cardiomyocyte viability under serum deficiency through the alteration of Rac1 activity and the regulation of Bnip3 expression by FoxO3 and NFκB transcription factors pointing to Popdc1/Bves as a potential target to enhance heart protection. J. Cell. Biochem. 118: 1505-1517, 2017. © 2016 Wiley Periodicals, Inc.

The Clinical and Biological Effects of the Expression of Dedicator of Cytokinesis 1 (DOCK1) in Acute Myeloid Leukemia

Abstract Background: DOCK1, also known as DOCK180, is the prototype member of the evolutionarily conserved DOCK superfamily. DOCK1 acts as a guanine nucleotide exchange factor (GEF) which regulates the activation of GTPase Rac, thereby influencing several cellular pathways including cell motility, polarity, invasion, phagocytosis and clearance of apoptotic cells. Overexpression of DOCK1 has been associated with cancer cell migration and invasion, however, its significance in acute myeloid leukemia (AML) remains unexplored. Materials and Methods: Expression patterns of DOCK1 were extracted from 347 AML patients recruited in the National Taiwan University Hospital (NTUH) (Illumina HumanHT-12 v4 Expression BeadChip, Illumina, San Diego, CA). We employed several public datasets, including microarray data of various lineages of normal hematopoiesis and AML marrow (GSE12662, GSE24006, and GSE24759) and The Cancer Genome Atlas (TCGA) RNA sequencing data of AML. In our cohort we analyzed the impacts of DOCK1 expression on various clinical parameters and genetic abnormalities. Survival was analyzed in 227 patients who received standard chemotherapy. The global gene expression profiles associated with DOCK1 expression was analyzed by bioinformatics tools. Two independent cohorts, TCGA (N=183) and GSE12417 (N=61), were used for validating the prognostic significance of DOCK1. Results: DOCK1 is a moderately expressed gene in AML (RPKM, 4.2; ranking, 50.5%). Higher DOCK1 expression level was associated with higher percentages of peripheral blasts (p=0.005), but inversely correlated with favorable cytogenetics such as t(8;21) and t(15;17). Moreover, mutations in CEBPA, c-KIT and IDH2 were less often seen in the patients with higher DOCK1 expression, while FLT3-ITD, and mutations in PTPN11, MLL, NPM1, RUNX1, ASXL1 and DNMT3A happened more often in those with higher DOCK1 expression. The Gene Set Enrichment Analysis (GSEA) of published stem cell signatures suggested DOCK1 as a hematopoietic and leukemic stem cell marker (GSEA P < 0.001 and = 0.004, respectively). Furthermore, it was generally co-expressed with homeobox genes, with average correlations ranging from 0.18 to 0.33 in normal samples, and 0.10 to 0.71 in AML datasets. With a median follow up of 57 months, we observed strong correlation between higher DOCK1 expression levels and inferior overall survival (OS) (median 18.0 months vs 116.8 months, P < 0.001) and disease free survival (DFS) (median 6.4 months vs 96.4 months, P < 0.001) (Figure 1A and 1B). These survival impacts could be validated in the two independent cohorts. In addition, multivariate analysis revealed higher DOCK1 expression as a strong unfavorable prognostic factor for OS (p=0.017) independent of age, karyotype, FLT3-ITD, and mutations of PTPN11, RUNX1, MLL and TP53. Conclusion: To our knowledge, we are the first to present the unique clinical, prognostic, and genetic impacts of DOCK1 in AML. Studies on large prospective cohorts are necessary to confirm our observation. The mechanistic functions of DOCK1 in AML await for further studies. Figure 1A Overall survival of AML patients with higher expression versus lower expression of DOCK1 Figure 1A. Overall survival of AML patients with higher expression versus lower expression of DOCK1 Figure 1B Disease free survival of AML patients with higher expression versus lower expression of DOCK1 Figure 1B. Disease free survival of AML patients with higher expression versus lower expression of DOCK1 Disclosures No relevant conflicts of interest to declare.

Regulation of Rac1 GTPase activity by quinine through G-protein and bitter taste receptor T2R4.

Rac1 belongs to the Rho family of small GTPases and regulates actin cytoskeleton reorganization. T2R4 is a bitter taste receptor belonging to the G protein-coupled receptor family of proteins. In addition to mediating bitter taste perception from the tongue, T2R4s are found in extra-oral tissues, e.g., nasal epithelium, airways, brain, testis suggesting a much broader physiological function for these receptors. Anti-malarial drug and a bitter tasting compound, quinine, is a known agonist for T2R4, whereas BCML (Nα,Nα-Bis(carboxymethyl)-L-lysine) acts as an inverse agonist. Using western blot and Ca++ mobilization assays, the effects of quinine on Rac1 activity in HEK293T cells stably expressing T2R4/Gα16/44, T2R4, or Gα16/44 and transiently transfected with HA-Rac1 were investigated. Quinine treatment caused a significant reduction in the amount of active Rac1, whereas in the presence of BCML, quinine failed to cause any significant change in active Rac1. No significant change in Rac1 activity was observed in BAPTA-AM plus quinine-treated Gα16/44 cells, suggesting possibility of a pathway in addition to the canonical Ca++-dependent pathway. A noticeable role for Gα16/44 independent of T2R4 is observed in quinine-mediated Rac1 inactivation. Further, a significant difference in quinine-induced Ca++ response in T2R4/Gα16/44 or T2R4 cells was observed validating the partial role of calcium and importance of Gα16/44. This study is the first to show an inhibitory downstream action of a T2R4 agonist on Rac1 function. Further investigation will help in better understanding the downstream signal transduction network of T2R4 and its extra-oral physiological roles.

Antibody Binding to CD4 Induces Rac GTPase Activation and Alters T Cell Migration.

The use of nondepleting Abs specific for CD4 and CD8 is an effective strategy to tolerize CD4+ and CD8+ T cells in a tissue-specific manner. We reported that coreceptor therapy reverses diabetes in new onset NOD mice. A striking feature of coreceptor-induced remission is the purging of T cells from the pancreatic lymph nodes (PLN) and islets of NOD mice. Evidence indicates that Abs binding to the coreceptors promotes T cell egress from these tissues. The present study examined how coreceptor therapy affects the migration of CD4+ T cells residing in the PLN of NOD mice. Anti-CD4 Ab treatment resulted in an increased frequency of PLN but not splenic CD4+ T cells that exhibited a polarized morphology consistent with a migratory phenotype. Furthermore, PLN CD4+ T cells isolated from anti-CD4 versus control Ab-treated animals displayed increased in vitro chemotaxis to chemoattractants such as sphingosine-1-phosphate and CXCL12. Notably, the latter was dependent on activation of the small Rho GTPases Rac1 and Rac2. Rac1 and Rac2 activation was increased in Ab-bound CD4+ T cells from the PLN but not the spleen, and knockdown of Rac expression blocked the heightened reactivity of Ab-bound PLN CD4+ T cells to CXCL12. Interestingly, Rac1 and Rac2 activation was independent of Rac guanine nucleotide exchange factors known to regulate T cell activity. Therefore, Ab binding to CD4 initiates a novel pathway that involves inflammation-dependent activation of Rac and establishment of altered T cell migratory properties.