Small GTP-binding Protein Rac1 Research Articles

Expression Analysis of the Small GTP-Binding Protein Rac in Pterygium

To determine the roles of small GTP-binding proteins Rac1, Rac2, and Rac3 expression in pterygial tissue and to compare these expressions with normal conjunctival tissue. Seventy-eight patients with primary pterygium were enrolled. Healthy conjunctival graft specimens obtained during pterygium surgery were used as control tissue. The real-time polymerase chain reaction method on the BioMark HD dynamic array system was utilized in genomic mRNA for the gene expression analysis. Protein expressions were analyzed using western blot and immunohistochemical methods. RAC1, RAC2, and RAC3 gene expressions in pterygial tissues were not markedly elevated when compared to the control specimens (p>0.05). As a very low level of RAC1 gene expression was observed, further protein expression analysis was performed for the Rac2 and Rac3 proteins. Western blot and immunohistochemical analysis of Rac2 and Rac3 protein expression revealed no significant differences between pterygial and healthy tissues (p>0.05). This is the first study to identify the contribution of Rac proteins in pterygium. Our results indicate that the small GTP-binding protein Rac may not be involved in pterygium pathogenesis.

Enterovirus-A71 exploits peripherin and Rac1 to invade the central nervous system.

Enterovirus-A71 (EV-A71) has been associated with severe neurological forms of hand, foot, and mouth disease (HFMD). EV-A71 infects motor neurons at neuromuscular junctions (NMJs) to invade the central nervous system (CNS). Here, we investigate the role of peripherin (PRPH) during EV-A71 infection, a type III intermediate neurofilament involved in neurodegenerative conditions. In mice infected with EV-A71, PRPH co-localizes with viral particles in the muscles at NMJs and in the spinal cord. In motor neuron-like and neuroblastoma cell lines, surface-expressed PRPH facilitates viral entry, while intracellular PRPH influences viral genome replication through interactions with structural and non-structural viral components. Importantly, PRPH does not play a role during infection with coxsackievirus A16, another causative agent of HFMD rarely associated with neurological complications, suggesting that EV-A71 ability to exploit PRPH represents a unique attribute for successful CNS invasion. Finally, we show that EV-A71 also exploits some of the many PRPH-interacting partners. Of these, small GTP-binding protein Rac1 represents a potential druggable host target to limit neuroinvasion of EV-A71.

Molecular pathway network of EFNA1 in cancer and mesenchymal stem cells

Abundant molecules are dynamically activated in cancer and stem cells. To investigate the role of ephrin A1 (EFNA1) in cancer and stem cell signaling pathways, we analyzed the gene expression and molecular network of EFNA1 in mesenchymal stem cells (MSCs) and diffuse-type gastric cancer (GC). Diffuse-type GC has more mesenchymal-like feature and malignant characteristics compared to intestinal-type GC. The signaling and molecular network of EFNA1 in cancer and stem cells were analyzed using several databases, including cBioPortal for Cancer Genomics, Kyoto Encyclopedia of Genes and Genomes (KEGG). The gene expression of EFNA1 was up-regulated in diffuse-type GC compared to MSCs. The molecular pathway network of EFNA1 includes cadherin 1 (CDH1), catenin beta 1 (CTNNB1), ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1) (RAC1), EPH receptor A5 (EPHA5), and the KRAS proto-oncogene, GTPase (KRAS). We summarized molecular pathway network of EFNA1 in cancer and stem cells. The results revealed a network model for EFNA1 in cancer and stem cells.

Cytotoxic and antimigratory effects of Cratoxy formosum extract against HepG2 liver cancer cells.

The aim of the present study was to investigate the molecular mechanisms underlying Cratoxylum formosum (CF) Dyer-induced cancer cell death and antimigratory effects in HepG2 liver cancer cells. The cytotoxic, antiproliferative and antimigratory effects of CF leaf extract on human liver cancer HepG2 cell lines were evaluated using sulforhodamine B, colony formation, and wound healing assays. In addition, apoptosis induction mechanisms were investigated via reactive oxygen species (ROS) formation, caspase 3 activities, and mitochondrial membrane potential (ΔΨm) disruption. Gene expression and apoptosis-associated protein levels were measured by reverse transcription-quantitative polymerase chain reaction and western blotting. CF induced HepG2 cell death in a time- and dose-dependent manner with half maximal inhibitory concentration values of 219.03±9.96 and 124.90±6.86 µg/ml at 24 and 48 h, respectively. Treatment with CF caused a significant and dose-dependent decrease in colony forming ability and cell migration. Furthermore, the present study demonstrated that CF induced ROS formation, increased caspase 3 activities, decreased the ΔΨm, and caused HepG2 apoptosis. CF marginally decreased the expression level of the cell cycle regulatory protein, ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1) and the downstream protein, cyclin dependent kinase 6. Additionally, CF significantly enhanced p21 levels, reduced cyclin D1 protein levels and triggered cancer cell death. CF leaf extracts induced cell death, stimulated apoptosis and inhibited migration in HepG2 cells. Thus, CF may be useful for developing an anticancer drug candidate for the treatment of liver cancer.

The role of oxidative stress and NADPH oxidase in the pathogenesis of atherosclerosis.

Reactive oxygen species (ROS) play a key role in the pathogenesis of atherosclerosis. The main mechanisms which are involved are low-density lipoprotein oxidative modification, inactivation of nitric oxide and modulation of redox-sensitive signaling pathways. ROS contribute to several aspects of atherosclerosis including endothelial cell dysfunction, monocyte/macrophage recruitment and activation, stimulation of inflammation, and inducing smooth muscle cell migration and proliferation. NADPH oxidase is the main source of ROS in the vasculature. This enzyme consists of a membrane-bound heterodimer of gp91phox and p22phox, cytosolic regulatory subunits p47phox, p67phox and p40phox, and small GTP-binding proteins rac1 and rac 2. Seven distinct isoforms of this enzyme have been identified, of which four (NOX1, 2, 4 and 5) may have cardiovascular function. In this paper, we review the current state of knowledge concerning the role of oxidative stress and NOX enzymes in pathogenesis of atherosclerosis. Moreover, we analyze the experimental studies that explore the relationship between the NOX family and atherosclerosis.

The role of oxidative stress and NADPH oxidase in the pathogenesis of atherosclerosis

Reactive oxygen species (ROS) play a key role in the pathogenesis of atherosclerosis. The main mechanisms which are involved are low-density lipoprotein oxidative modification, inactivation of nitric oxide and modulation of redox-sensitive signaling pathways. ROS contribute to several aspects of atherosclerosis including endothelial cell dysfunction, monocyte/macrophage recruitment and activation, stimulation of inflammation, and inducing smooth muscle cell migration and proliferation. NADPH oxidase is the main source of ROS in the vasculature. This enzyme consists of a membrane-bound heterodimer of gp91phox and p22phox, cytosolic regulatory subunits p47phox, p67phox and p40phox, and small GTP-binding proteins rac1 and rac 2. Seven distinct isoforms of this enzyme have been identified, of which four (NOX1, 2, 4 and 5) may have cardiovascular function. In this paper, we review the current state of knowledge concerning the role of oxidative stress and NOX enzymes in pathogenesis of atherosclerosis. Moreover, we analyze the experimental studies that explore the relationship between the NOX family and atherosclerosis.

Abstract PD3-04: PAK-1 amplified breast cancer cell lines are preferentially sensitive to PAK inhibition with G-5555

Abstract The small GTP-binding proteins Rac1 and Cdc42 stimulate activity of the serine/threonine kinase p21-activated kinase-1 (PAK-1) to drive growth factor signaling networks and Ras-driven tumorigenesis. Genomic amplification and over-expression of PAK1 are prevalent in luminal breast cancer and correlate with poor clinical outcome. Here we use a novel and selective small molecule inhibitor, G-5555, of the group I PAKs (PAK1, 2, and 3) to evaluate the importance of PAK1 in promoting growth of PAK1 amplified breast cancer cells. Cell lines with amplification of PAK1 were found to be more sensitive to PAK1 inhibition than non-amplified cell lines. Additionally, reverse phase protein array (RPPA) was used to assess the effects of PAK1 inhibition on a wide range of signaling pathways in both amplified and non-amplified cell lines. Reduced levels of phosphorylation of MEK S298 was observed in all cell lines exposed to G-5555 irrespective of amplification status, consistent with PAK1 inhibition in these cell lines. However, modulation of this downstream PAK1 substrate did not correlate with inhibition of cell proliferation or induction of cell death. Cell lines that showed inhibition of proliferation in response to G-5555 also showed enhanced levels of cell death along with apoptosis. Moreover, G-5555 reduced tumor growth in the PAK1 amplified MDA-MB-175 xenograft tumor model. Finally, we compared the in vitro activity of G-5555 with palbociclib, a recently approved inhibitor of the cyclin-dependent kinases CDK4 and CDK6, in PAK1 amplified luminal breast cancer cell lines. Our data supports PAK1 as an attractive target in PAK1 amplified cells and tumors and suggests that inhibiting PAK1 rather than CDK4/6 in this context may be a more attractive therapeutic strategy. Citation Format: Ong CC, Blackwood E, Jakubiak D, Daemen A, Ramaswamy S, Heise C, Schmidt M, Sanders L, Wilson TR, Huw L, Ndubaku C, Rudolph J, Hoeflich KP, Friedman L, O'Brien T. PAK-1 amplified breast cancer cell lines are preferentially sensitive to PAK inhibition with G-5555. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD3-04.

Leucine-rich repeat kinase 2 interacts with p21-activated kinase 6 to control neurite complexity in mammalian brain.

Leucine‐rich repeat kinase 2 (LRRK2) is a causative gene for Parkinson's disease, but the physiological function and the mechanism(s) by which the cellular activity of LRRK2 is regulated are poorly understood. Here, we identified p21‐activated kinase 6 (PAK6) as a novel interactor of the GTPase/ROC domain of LRRK2. p21‐activated kinases are serine‐threonine kinases that serve as targets for the small GTP binding proteins Cdc42 and Rac1 and have been implicated in different morphogenetic processes through remodeling of the actin cytoskeleton such as synapse formation and neuritogenesis. Using an in vivo neuromorphology assay, we show that PAK6 is a positive regulator of neurite outgrowth and that LRRK2 is required for this function. Analyses of post‐mortem brain tissue from idiopathic and LRRK2 G2019S carriers reveal an increase in PAK6 activation state, whereas knock‐out LRRK2 mice display reduced PAK6 activation and phosphorylation of PAK6 substrates. Taken together, these results support a critical role of LRRK2 GTPase domain in cytoskeletal dynamics in vivo through the novel interactor PAK6, and provide a valuable platform to unravel the mechanism underlying LRRK2‐mediated pathophysiology. We propose p21‐activated kinase 6 (PAK6) as a novel interactor of leucine‐rich repeat kinase 2 (LRRK2), a kinase involved in Parkinson's disease (PD). In health, PAK6 regulates neurite complexity in the brain and LRRK2 is required for its function, (a) whereas PAK6 is aberrantly activated in LRRK2‐linked PD brain (b) suggesting that LRRK2 toxicity is mediated by PAK6.

Retraction Note to: Analyzing time-series microarray data reveals key genes in spinal cord injury.

Although many scholars have utilized high-throughput microarrays to delineate gene expression patterns after spinal cord injury (SCI), no study has evaluated gene changes in raphe magnus (RM) and somatomotor cortex (SMTC), two areas in brain primarily affected by SCI. In present study, we aimed to analyze the differentially expressed genes (DEGs) of RM and SMTC between SCI model and sham injured control at 4, 24 h, 7, 14, 28 days, and 3 months using microarray dataset GSE2270 downloaded from gene expression omnibus and unpaired significance analysis of microarray method. Protein–protein interaction (PPI) network was constructed for DEGs at crucial time points and significant biological functions were enriched using DAVID. The results indicated that more DEGs were identified at 14 days in RM and at 4 h/3 months in SMTC after SCI. In the PPI network for DEGs at 14 days in RM, interleukin 6, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), FBJ murine osteosarcoma viral oncogene homolog (FOS), tumor necrosis factor, and nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) were the top 5 hub genes; In the PPI network for DEGs at 3 months in SMTC, the top 5 hub genes were ubiquitin B, Ras‐related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1), FOS, Janus kinase 2 and vascular endothelial growth factor A. Hedgehog and Wnt signaling pathways were the top 2 significant pathways in RM. These hub DEGs and pathways may be underlying therapeutic targets for SCI.

Ras, Rac1, and phosphatidylinositol-3-kinase (PI3K) signaling in nitric oxide induced endothelial cell migration

The small GTP-binding proteins Ras and Rac1 are molecular switches exchanging GDP for GTP and converting external signals in response to a variety of stimuli. Ras and Rac1 play an important role in cell proliferation, cell differentiation, and cell migration. Rac1 is directly involved in the reorganization and changes in the cytoskeleton during cell motility. Nitric oxide (NO) stimulates the Ras – ERK1/2 MAP kinases signaling pathway and is involved in the interaction between Ras and the phosphatidyl-inositol-3 Kinase (PI3K) signaling pathway and cell migration. This study utilizes bradykinin (BK), which promotes endogenous production of NO, in an investigation of the role of NO in the activation of Rac1 in rabbit aortic endothelial cells (RAEC). NO-derived from BK stimulation of RAEC and incubation of the cells with the s-nitrosothiol S-nitrosoglutathione (GSNO) activated Rac1. NO-derived from BK stimulation promoted RAEC migration over a period of 12 h. The use of RAEC permanently transfected with the dominant negative mutant of Ras (RasN17) or with the non-nitrosatable mutant of Ras (RasC118S); and the use of specific inhibitors of: Ras, PI3K, and Rac1 resulted in inhibition of NO-mediated Rac1 activation. BK-stimulated s-nitrosylation of Ras in RAEC mediates Rac1 activation and cell migration. Inhibition of NO-mediated Rac1 activation resulted in inhibition of endothelial cell migration. In conclusion, the NO indirect activation of Rac1 involves the direct participation of Ras and PI3K in the migration of endothelial cells stimulated with BK.

Anti-inflammatory property of propolis.

Dear Editor-in-Chief, The recent article by Weina Gao and colleagues, on July issue of this Journal, showed that Brazilian green propolis have a positive effect on innate and adaptive immunity in aged mice.(1) This role should be mainly attributed to flavonoids contained in propolis.(2) The Authors reported that Brazilian propolis had 189.12 mg/g total polyphenols, 98.46 mg/g flavonoids, 1.95 mg/g cinnamic acid and 23 mg/g artepillin-C.(1) Yet, the participation of any single molecule to the reported evidence in aged animals might have involved different spectra of activity and different molecular targets in promoting the beneficial action observed by the Authors.(1) Artepillin C, namely (E)-3-[4-hydroxy-3,5-bis(3-methylbut-2-enyl)phenyl]prop-2-enoic acid, is one of the major component in Brazilian propolis and recent reports have stressed its ability to block the serine/theonine protein kinase known as PAK-1.(3) PAK proteins are critical effectors that link the Rho family of GTPases to the cytoskeleton reorganization and nuclear signaling; These proteins serve as targets for the small GTP binding proteins Cdc42 and Rac and have been implicated in a wide range of biological activities, for example PAK1 regulates cell motility and morphology, furthermore the role of Cdc42 GTPases activating protein in aging has been recently reported.(4) The involvement of PAK1 in inflammatory and neuro-degenerative disorders suggests that this molecules is targeted in aging mechanism. Few natural PAK1-blockers such as rosmarinic acid, curcumin and caffeic acid extend the lifespan of the nematode Caenorhabditis elegans or fruit flies.(5) PAK1 promotes reproduction, whereas it inactivates HSP16.2 gene and shortens lifespan, as do also PI-3 kinase (AGE-1), mTOR, and insulin-like signalling (ILS) (Daf-2) in this nematode.(5) It is arguable that caffeic acid and particularly artepillin-C play a role in slowing aging cellular mechanism and synergistically exert an anti-inflammatory action with propolis flavonoids.(4) This suggestive speculation may appear contradictory when data from serum in aged mice, showing a marked increase in inflammatory cytokines on increasing Brazilian propolis assumption, are considered.(1) The Authors concluded that this evidence assessed the improvement in inflammatory response caused by propolis in aged mice.(1) This circumstance addresses the positive role exhibited by nature-derived phytochemicals on stressed, dysregulated, aged or infected (inflamed) cells. Therefore, while a wide literature exists reporting the anti-inflammatory role of flavonoids,(6) many data from clinics should suggest a pro-inflammatory role exerted by polyphenols in restoring health, prevent cancer onset and degenerative disorders.(7) Fundamentally, as like as in propolis, the most proper idea about the activity of nature-derived polyphenols and phenolic substances, should deal with the concept of immune modulation, depending on the cell nature and homeostatic balance, not merely an improvement given by inhibition or promotion coming from the molecular nature of these molecules. Propolis is widely used as an anti-inflammatory raw substance, expecially during allergy and airway inflammatory disorders. The interesting paper by Weina Gao et al., would suggest that in elderly people Brazilian propolis may ensure a health promoting action by activating the immune response.(1) This is formally true but literature reports many data with potentially misleading evidence about. A combination therapy (anti-inflammatory drugs plus propolis) in aged adults showed encouraging results(8) and, moreover, due to yet contradictory results from clinics,(9) respect to in vitro and animal evidence,(1,10) a more cautious, critical and less enthusiastic debate about this substance has to be taken into account in the next future. Looking forward to a kind reply from yours.

MEMBRANE TYPE 1-MATRIX METALLOPROTEINASE (MT1-MMP) IDENTIFIED AS A MULTIFUNCTIONAL REGULATOR OF VASCULAR RESPONSES.

Membrane type 1-matrix metalloproteinase (MT1-MMP) functions as a signaling molecules in addition to a transmembrane metalloprotease, which degrades interstitial collagens and extracellular matrix components. This review focuses on the multifunctional roles of MT1-MMP as a signaling molecule in vascular responses to pro-atherosclerotic stimuli in the pathogenesis of cardiovascular diseases. First, the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1)-MT1-MMP signaling axis contributes to endothelial dysfunction, which is mediated via small GTP-binding protein RhoA and Rac1 activation. Second, MT1-MMP plays a crucial role in reactive oxygen species (ROS) generation through the activation of receptor for advanced glycation end products (AGEs) in smooth muscle cells, indicating that MT1-MMP may be a therapeutic target for diabetic vascular complications. Third, MT1-MMP is involved in RhoA/Rac1 activation and Ca(2+) signaling in the mechanism of thrombin-stimulated endothelial dysfunction and oxidant stress. Fourth, the inhibition of the MT1-MMP/Akt signaling pathway may be an attractive strategy for treating endothelial disordered hemostasis in the development of vascular diseases linked to TNF-α-induced inflammation. Fifth, MT1-MMP through RAGE induced RhoA/Rac1 activation and tissue factor protein upregulation through NF-κB phosphorylation in endothelial cells stimulated by high-mobility group box-1, which plays a key role in the systemic inflammation. These findings suggest that the MT1-MMP-mediated signaling axis may be a promising target for treating atherosclerosis and subsequent cardiovascular diseases.

Transient receptor potential cation channel subfamily M member 2 (TRPM2); Ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1) (RAC1)

Transient receptor potential cation channel subfamily M member 2 (TRPM2); Ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1) (RAC1)

RETRACTED ARTICLE: Analyzing time-series microarray data reveals key genes in spinal cord injury

Although many scholars have utilized high-throughput microarrays to delineate gene expression patterns after spinal cord injury (SCI), no study has evaluated gene changes in raphe magnus (RM) and somatomotor cortex (SMTC), two areas in brain primarily affected by SCI. In present study, we aimed to analyze the differentially expressed genes (DEGs) of RM and SMTC between SCI model and sham injured control at 4, 24 h, 7, 14, 28 days, and 3 months using microarray dataset GSE2270 downloaded from gene expression omnibus and unpaired significance analysis of microarray method. Protein-protein interaction (PPI) network was constructed for DEGs at crucial time points and significant biological functions were enriched using DAVID. The results indicated that more DEGs were identified at 14 days in RM and at 4 h/3 months in SMTC after SCI. In the PPI network for DEGs at 14 days in RM, interleukin 6, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), FBJ murine osteosarcoma viral oncogene homolog (FOS), tumor necrosis factor, and nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) were the top 5 hub genes; In the PPI network for DEGs at 3 months in SMTC, the top 5 hub genes were ubiquitin B, Ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1), FOS, Janus kinase 2 and vascular endothelial growth factor A. Hedgehog and Wnt signaling pathways were the top 2 significant pathways in RM. These hub DEGs and pathways may be underlying therapeutic targets for SCI.

FZD7 drives in vitro aggressiveness in Stem-A subtype of ovarian cancer via regulation of non-canonical Wnt/PCP pathway

Ovarian cancer (OC) can be classified into five biologically distinct molecular subgroups: epithelial-A (Epi-A), Epi-B, mesenchymal (Mes), Stem-A and Stem-B. Among them, Stem-A expresses genes relating to stemness and is correlated with poor clinical prognosis. In this study, we show that frizzled family receptor 7 (FZD7), a receptor for Wnt signalling, is overexpressed in the Stem-A subgroup. To elucidate the functional roles of FZD7, we used an RNA interference gene knockdown approach in three Stem-A cell lines: CH1, PA1 and OV-17R. Si-FZD7 OC cells showed reduced cell proliferation with an increase in the G0/G1 sub-population, with no effect on apoptosis. The cells also displayed a distinctive morphologic change by colony compaction to become more epithelial-like and polarised with smaller internuclear distances and increased z-axis height. Immunofluorescence (IF) staining patterns of pan-cadherin and β-catenin suggested an increase in cadherin-based cell–cell adhesion in si-FZD7 cells. We also observed a significant rearrangement in the actin cytoskeleton and an increase in tensile contractility in si-FZD7 OC cells, as evident by the loss of stress fibres and the redistribution of phospho-myosin light chain (pMLC) from the sites of cell–cell contacts to the periphery of cell colonies. Furthermore, there was reciprocal regulation of RhoA (Ras homolog family member A) and Rac1 (Ras-related C3 botulinum toxin substrate 1 (Rho family, small GTP-binding protein Rac1)) activities upon FZD7 knockdown, with a significant reduction in RhoA activity and a concomitant upregulation in Rac1 activity. These changes in pMLC and RhoA, as well as the increased TopFlash reporter activities in si-FZD7 cells, suggested involvement of the non-canonical Wnt/planar cell polarity (PCP) pathway. Selected PCP pathway genes (cadherin EGF LAG seven-pass G-type receptor 3 (CELSR3), prickle homolog 4 (Drosophila) (PRICKLE4), dishevelled-associated activator of morphogenesis 1 (DAAM1), profilin 2 (PFN2), protocadherin 9 (PCDH9), protocadherin α1 (PCDHA1), protocadherin β17 pseudogene (PCDHB17), protocadherin β3 (PCDHB3), sprouty homolog 1 (SPRY1) and protein tyrosine kinase 7 (PTK7)) were found to be more highly expressed in Stem-A than non Stem-A subgroup of OC. Taken together, our results suggest that FZD7 might drive aggressiveness in Stem-A OC by regulating cell proliferation, cell cycle progression, maintenance of the Mes phenotype and cell migration via casein kinase 1ɛ-mediated non-canonical Wnt/PCP pathway.

Phosphatidylinositol‐3,4,5‐triphosphate‐dependent Rac exchange factor 1 regulates epinephrine‐induced exocytosis of Weibel–Palade bodies

Weibel-Palade bodies (WPBs) function as storage vesicles for von Willebrand factor (VWF) and a number of other bioactive compounds, including angiopoietin-2 and insulin-like growth factor-binding protein 7. WPBs release their content following stimulation with agonists that increase the level of intracellular Ca²⁺, such as thrombin, or agonists that increase intracellular levels of cAMP, such as epinephrine. Previously, we have shown that the exchange protein activated by cAMP, exchange protein activated by cAMP, and the small GTPase Rap1 are involved in cAMP-mediated release of WPBs. In this study, we explored potential downstream effectors of Rap1 in cAMP-mediated WPB release. Studies were performed in primary human umbilical vein endothelial cells. Activation of the small GTP-binding protein Rac1 was monitored by its ability to bind to the CRIB domain of the serine/threonine kinase P21-activated kinase (PAK)1. Downstream effectors of Rap1 were identified with a proteomic screen using a glutathione-S-transferase fusion of the Ras-binding domain of RalGDS. Functional involvement of candidate proteins in WPB release was determined by RNA interference (RNAi)-mediated knockdown of gene expression. Depletion of Rac1 by RNAi prevented epinephrine-induced VWF secretion. Also, the Rac1 inhibitor EHT1864 reduced epinephrine-induced WPB release. We identified the phosphatidylinositol-3,4,5-triphosphate-dependent Rac exchange factor 1 (PREX1) and the regulatory β-subunit of phosphatidylinositol 3-kinase (PI3K) as downstream targets of Rap1. The PI3K inhibitor LY294002 reduced epinephrine-induced release of VWF. RNAi-mediated downregulation of PREX1 abolished epinephrine-induced but not thrombin-induced release of WPBs. Our findings show that PREX1 regulates epinephrine-induced release of WPBs.

The small GTPase Rac1 is required for smooth muscle contraction

The role of the small GTP-binding protein Rac1 in smooth muscle contraction was examined using small molecule inhibitors (EHT1864, NSC23766) and a novel smooth muscle-specific, conditional, Rac1 knockout mouse strain. EHT1864, which affects nucleotide binding and inhibits Rac1 activity, concentration-dependently inhibited the contractile responses induced by several different modes of activation (high-K+, phenylephrine, carbachol and protein kinase C activation by phorbol-12,13-dibutyrate) in several different visceral (urinary bladder, ileum) and vascular (mesenteric artery, saphenous artery, aorta) smooth muscle tissues. This contractile inhibition was associated with inhibition of the Ca2+ transient. Knockout of Rac1 (with a 50% loss of Rac1 protein) lowered active stress in the urinary bladder and the saphenous artery consistent with a role of Rac1 in facilitating smooth muscle contraction. NSC23766, which blocks interaction between Rac1 and some guanine nucleotide exchange factors, specifically inhibited the α1 receptor responses (phenylephrine) in vascular tissues and potentiated prostaglandin F2α and thromboxane (U46619) receptor responses. The latter potentiating effect occurred at lowered intracellular [Ca2+]. These results show that Rac1 activity is required for active contraction in smooth muscle, probably via enabling an adequate Ca2+ transient. At the same time, specific agonists recruit Rac1 signalling via upstream modulators, resulting in either a potentiation of contraction via Ca2+ mobilization (α1 receptor stimulation) or an attenuated contraction via inhibition of Ca2+ sensitization (prostaglandin and thromboxane receptors).

Inhibition of the TRPC5 ion channel protects the kidney filter

An intact kidney filter is vital to retention of essential proteins in the blood and removal of waste from the body. Damage to the filtration barrier results in albumin loss in the urine, a hallmark of cardiovascular disease and kidney failure. Here we found that the ion channel TRPC5 mediates filtration barrier injury. Using Trpc5-KO mice, a small-molecule inhibitor of TRPC5, Ca2+ imaging in isolated kidney glomeruli, and live imagining of podocyte actin dynamics, we determined that loss of TRPC5 or its inhibition abrogates podocyte cytoskeletal remodeling. Inhibition or loss of TRPC5 prevented activation of the small GTP-binding protein Rac1 and stabilized synaptopodin. Importantly, genetic deletion or pharmacologic inhibition of TRPC5 protected mice from albuminuria. These data reveal that the Ca2+-permeable channel TRPC5 is an important determinant of albuminuria and identify TRPC5 inhibition as a therapeutic strategy for the prevention or treatment of proteinuric kidney disease.

Emerging role of oxidative stress in metabolic syndrome and cardiovascular diseases: important role of Rac/NADPH oxidase

'Oxidative stress' is a term defining states of elevated reactive oxygen species (ROS) levels. Normally, ROS control several physiological processes, such as host defence, biosynthesis of hormones, fertilization and cellular signalling. However, oxidative stress has been involved in different pathologies, including metabolic syndrome and numerous cardiovascular diseases. A major source of ROS involved in both metabolic syndrome and cardiovascular pathophysiology is the NADPH oxidase (NOX) family of enzymes. NOX is a multi-component enzyme complex that consists of membrane-bound cytochrome b-558, which is a heterodimer of gp91phox and p22phox, cytosolic regulatory subunits p47phox and p67phox, and the small GTP-binding protein Rac1. Rac1 plays many important biological functions in cells, but perhaps the most unique function of Rac1 is its ability to bind and activate the NOX complex. Furthermore, Rac1 has been reported to be a key regulator of oxidative stress through its co-regulatory effects on both nitric oxide (NO) synthase and NOX. Therefore, the main goal of this review is to give a brief outline about the important role of the Rac1-NOX axis in the pathophysiology of both metabolic syndrome and cardiovascular disease.

The Small GTPase RALA Controls c-Jun N-terminal Kinase-mediated FOXO Activation by Regulation of a JIP1 Scaffold Complex

FOXO (forkhead box O) transcription factors are tumor suppressors and increase the life spans of model organisms. Cellular stress, in particular oxidative stress caused by an increase in levels of reactive oxygen species (ROS), activates FOXOs through JNK-mediated phosphorylation. Importantly, JNK regulation of FOXO is evolutionarily conserved. Here we identified the pathway that mediates ROS-induced JNK-dependent FOXO regulation. Following increased ROS, RALA is activated by the exchange factor RLF (RalGDS-like factor), which is in complex with JIP1 (C-Jun-amino-terminal-interacting protein 1) and JNK. Active RALA consequently regulates assembly and activation of MLK3, MKK4, and JNK onto the JIP1 scaffold. Furthermore, regulation of FOXO by RALA and JIP1 is conserved in C. elegans, where both ral-1 and jip-1 depletion impairs heat shock-induced nuclear translocation of the FOXO orthologue DAF16.