Rac1 Activation Research Articles

Dipotassium glycyrrhizate and hinokitiol enhance macrophage efferocytosis by regulating recognition, uptake, and metabolism of apoptotic cells invitro.

Efferocytosis is a process whereby macrophages remove apoptotic cells, such as neutrophils, that have accumulated in tissues, which is required for resolution of inflammation. Efferocytosis is impaired in individuals with increasing age and in those with various systemic diseases. Recently, efferocytosis has been reported to be related to the pathogenesis and progression of periodontitis, and enhancement of efferocytosis, especially in the subjects with impaired efferocytosis, was suggested to lead to periodontitis prevention and care. Various anti-inflammatory ingredients are used in oral care products, but their effect on efferocytosis is unclear. Here, we aimed to identify ingredients contained in oral care products that are effective for efferocytosis regulation. The ability of dead cells to induce inflammation in human gingival fibroblast (HGF) cells were evaluated by measuring IL-6 secretion. Six ingredients in oral care products used as anti-inflammatory agents were evaluated for their effect on efferocytosis using flow cytometry. The expression of various efferocytosis-related molecules, such as MERTK and LRP1 involved in recognition, and LXRα and ABCA1 that function in metabolism, were measured in RAW264.7 cells with or without ingredient treatment. Rac1 activity, which is related to the uptake of dead cells, was measured using the G-LISA kit. Dead cells elicited IL-6 secretion in HGF cells. Among the six ingredients, GK2 and hinokitiol enhanced efferocytosis activity. GK2 and hinokitiol significantly increased the expression of MERTK and LRP1, and also enhanced LXRα and ABCA1 expression after efferocytosis. Furthermore, they increased Rac1 activity in the presence of dead cells. Among the six ingredients tested, GK2 and hinokitiol promoted efferocytosis by regulating apoptotic cell recognition, uptake, and metabolism-related molecules. Efferocytosis upregulation may be one of the mechanisms of GK2 and hinokitiol in the treatment of inflammatory diseases, such as periodontitis.

ANKS1B encoded AIDA-1 regulates social behaviors by controlling oligodendrocyte function

Heterozygous deletions in the ANKS1B gene cause ANKS1B neurodevelopmental syndrome (ANDS), a rare genetic disease characterized by autism spectrum disorder (ASD), attention deficit/hyperactivity disorder, and speech and motor deficits. The ANKS1B gene encodes for AIDA-1, a protein that is enriched at neuronal synapses and regulates synaptic plasticity. Here we report an unexpected role for oligodendroglial deficits in ANDS pathophysiology. We show that Anks1b-deficient mouse models display deficits in oligodendrocyte maturation, myelination, and Rac1 function, and recapitulate white matter abnormalities observed in ANDS patients. Selective loss of Anks1b from the oligodendrocyte lineage, but not from neuronal populations, leads to deficits in social preference and sensory reactivity previously observed in a brain-wide Anks1b haploinsufficiency model. Furthermore, we find that clemastine, an antihistamine shown to increase oligodendrocyte precursor cell maturation and central nervous system myelination, rescues deficits in social preference in 7-month-old Anks1b-deficient mice. Our work shows that deficits in social behaviors present in ANDS may originate from abnormal Rac1 activity within oligodendrocytes.

Rho GTPase activity crosstalk mediated by Arhgef11 and Arhgef12 coordinates cell protrusion-retraction cycles

Rho GTPases play a key role in the spatio-temporal coordination of cytoskeletal dynamics during cell migration. Here, we directly investigate crosstalk between the major Rho GTPases Rho, Rac and Cdc42 by combining rapid activity perturbation with activity measurements in mammalian cells. These studies reveal that Rac stimulates Rho activity. Direct measurement of spatio-temporal activity patterns show that Rac activity is tightly and precisely coupled to local cell protrusions, followed by Rho activation during retraction. Furthermore, we find that the Rho-activating Lbc-type GEFs Arhgef11 and Arhgef12 are enriched at transient cell protrusions and retractions and recruited to the plasma membrane by active Rac. In addition, their depletion reduces activity crosstalk, cell protrusion-retraction dynamics and migration distance and increases migration directionality. Thus, our study shows that Arhgef11 and Arhgef12 facilitate exploratory cell migration by coordinating cell protrusion and retraction by coupling the activity of the associated regulators Rac and Rho.

TIMP2 ameliorates blood-brain barrier disruption in traumatic brain injury by inhibiting Src-dependent VE-cadherin internalization.

Blood-brain barrier (BBB) disruption is a serious pathological consequence of traumatic brain injury (TBI), for which there are limited therapeutic strategies. Tissue inhibitor of metalloproteinase-2 (TIMP2), a molecule with dual functions of inhibiting MMP activity and displaying cytokine-like activity through receptor binding, has been reported to inhibit VEGF-induced vascular hyperpermeability. Here, we investigate the ability of TIMP2 to ameliorate BBB disruption in TBI and the underlying molecular mechanisms. Both TIMP2 and AlaTIMP2, a TIMP2 mutant without MMP-inhibiting activity, attenuated neurological deficits and BBB leakage in TBI mice; they also inhibited junctional protein degradation and translocation to reduce paracellular permeability in human brain microvascular endothelial cells (ECs) exposed to hypoxic plus inflammatory insult. Mechanistic studies revealed that TIMP2 interacted with α3β1 integrin on ECs, inhibiting Src activation-dependent VE-cadherin phosphorylation, VE-cadherin/catenin complex destabilization, and subsequent VE-cadherin internalization. Notably, localization of VE-cadherin on the membrane was critical for TIMP2-mediated EC barrier integrity. Furthermore, TIMP2-mediated increased membrane localization of VE-cadherin enhanced the level of active Rac1, thereby inhibiting stress fiber formation. All together, our studies have identified an MMP-independent mechanism by which TIMP2 regulates EC barrier integrity after TBI. TIMP2 may be a therapeutic agent for TBI and other neurological disorders involving BBB breakdown.

Centrosome clustering control in osteoclasts through CCR5-mediated signaling

Osteoclasts uniquely resorb calcified bone matrices. To exert their function, mature osteoclasts maintain the cellular polarity and directional vesicle trafficking to and from the resorbing bone surface. However, the regulatory mechanisms and pathophysiological relevance of these processes remain largely unexplored. Bone histomorphometric analyses in Ccr5-deficient mice showed abnormalities in the morphology and functional phenotype of their osteoclasts, compared to wild type mice. We observed disorganized clustering of nuclei, as well as centrosomes that organize the microtubule network, which was concomitant with impaired cathepsin K secretion in cultured Ccr5-deficient osteoclasts. Intriguingly, forced expression of constitutively active Rho or Rac restored these cytoskeletal phenotypes with recovery of cathepsin K secretion. Furthermore, a gene-disease enrichment analysis identified that PLEKHM1, a responsible gene for osteopetrosis, which regulates lysosomal trafficking in osteoclasts, was regulated by CCR5. These experimental results highlighted that CCR5-mediated signaling served as an intracellular organizer for centrosome clustering in osteoclasts, which was involved in the pathophysiology of bone metabolism.

Unveiling Key Biomarkers and Therapeutic Drugs in Polycystic Ovary Syndrome (PCOS) Through Pathway Enrichment Analysis and Hub Gene-miRNA Networks.

Polycystic ovary syndrome (PCOS) affects women of reproductive age globally with an incidence rate of 5% - 26%. Growing evidence reports important roles for microRNAs (miRNAs) in the pathophysiology of granulosa cells (GCs) in PCOS. The objectives of this study were to identify the top differentially expressed miRNAs (DE-miRNAs) and their corresponding targets in hub gene-miRNA networks, as well as identify novel DE-miRNAs by analyzing three distinct microarray datasets. Additionally, functional enrichment analysis was performed using bioinformatics approaches. Finally, interactions between the 5 top-ranked hub genes and drugs were investigated. Using bioinformatics approaches, three GC profiles from the gene expression omnibus (GEO), namely gene expression omnibus series (GSE)-34526, GSE114419, and GSE137684, were analyzed. Targets of the top DE-miRNAs were predicted using the multiMiR R package, and only miRNAs with validated results were retrieved. Genes that were common between the "DE-miRNA prediction results" and the "existing tissue DE-mRNAs" were designated as differentially expressed genes (DEGs). Gene ontology (GO) and pathway enrichment analyses were implemented for DEGs. In order to identify hub genes and hub DE-miRNAs, the protein-protein interaction (PPI) network and miRNA-mRNA interaction network were constructed using Cytoscape software. The drug-gene interaction database (DGIdb) database was utilized to identify interactions between the top-ranked hub genes and drugs. Out of the top 20 DE-miRNAs that were retrieved from the GSE114419 and GSE34526 microarray datasets, only 13 of them had "validated results" through the multiMiR prediction method. Among the 13 DE-miRNAs investigated, only 5, namely hsa-miR-8085, hsa-miR-548w, hsa-miR-612, hsa-miR-1470, and hsa-miR-644a, demonstrated interactions with the 10 hub genes in the hub gene-miRNA networks in our study. Except for hsa-miR-612, the other 4 DE-miRNAs, including hsa-miR-8085, hsa-miR-548w, hsa-miR-1470, and hsa-miR-644a, are novel and had not been reported in PCOS pathogenesis before. Also, GO and pathway enrichment analyses identified "pathogenic E. coli infection" in the Kyoto encyclopedia of genes and genomes (KEGG) and "regulation of Rac1 activity" in FunRich as the top pathways. The drug-hub gene interaction network identified ACTB, JUN, PTEN, KRAS, and MAPK1 as potential targets to treat PCOS with therapeutic drugs. The findings from this study might assist researchers in uncovering new biomarkers and potential therapeutic drug targets in PCOS treatment.

Inhibition of spinal Rac1 attenuates chronic inflammatory pain by regulating the activation of astrocytes

BackgroundSpinal astrocyte-mediated neuroinflammation is an important mechanism for the maintenance of chronic inflammatory pain. Previous studies have investigated that Ras-related C3 botulinum toxin substrate 1 (Rac1) is closely related to astrocyte activation after central nervous system injury. However, the role of Rac1 in astrocyte activation in chronic inflammatory pain has not been reported. MethodsComplete Freund's adjuvant (CFA)-induced chronic inflammatory pain model and LPS-stimulated astrocytes were used to investigate the role of Rac1 in astrocyte activation and the underlying mechanism. Rac1-interfering adeno-associated virus (AAV) targeting astrocytes was delivered to spinal astrocytes by intrathecal administration and a Rac1 specific inhibitor, NSC23766, was used to block cultured astrocytes. The glial fibrillary acidic protein (GFAP), proinflammatory cytokines, p-NF-κB, and nod-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome were detected by RT-qPCR, Western blotting, and immunofluorescence to investigate the activation of astrocytes. ResultsCFA induced spinal astrocyte activation and increased the expression of active Rac1 in spinal astrocytes. Knockdown of astrocyte Rac1 alleviated chronic inflammatory pain and inhibited astrocyte activation. Inhibition of Rac1 activation in cultured astrocytes decreased the expression of GFAP and proinflammatory cytokines. Knockdown of Rac1 inhibited the increase of expression of NLRP3 inflammasome and phosphorylation of NF-κB in the spinal lumbar enlargement after CFA injection. Similarly, the inhibition of Rac1 suppressed the increase of NLRP3 inflammasome and p-NF-κB protein level after LPS stimulation. ConclusionKnockdown of astrocyte Rac1 attenuated CFA-induced hyperalgesia and astrocyte activation possibly by blocking the expression of NLRP3 inflammasome and phosphorylation of NF-κB.

Conditional Astrocyte Rac1KO Attenuates Hyperreflexia after Spinal Cord Injury.

Spasticity is a hyperexcitability disorder that adversely impacts functional recovery and rehabilitative efforts after spinal cord injury (SCI). The loss of evoked rate-dependent depression (RDD) of the monosynaptic H-reflex is indicative of hyperreflexia, a physiological sign of spasticity. Given the intimate relationship between astrocytes and neurons, that is, the tripartite synapse, we hypothesized that astrocytes might have a significant role in post-injury hyperreflexia and plasticity of neighboring neuronal synaptic dendritic spines. Here, we investigated the effect of selective Rac1KO in astrocytes (i.e., adult male and female mice, transgenic cre-flox system) on SCI-induced spasticity. Three weeks after a mild contusion SCI, control Rac1wt animals displayed a loss of H-reflex RDD, that is, hyperreflexia. In contrast, transgenic animals with astrocytic Rac1KO demonstrated near-normal H-reflex RDD similar to pre-injury levels. Reduced hyperreflexia in astrocytic Rac1KO animals was accompanied by a loss of thin-shaped dendritic spine density on α-motor neurons in the ventral horn. In SCI-Rac1wt animals, as expected, we observed the development of dendritic spine dysgenesis on α-motor neurons associated with spasticity. As compared with WT animals, SCI animals with astrocytic Rac1KO expressed increased levels of the glial-specific glutamate transporter, glutamate transporter-1 in the ventral spinal cord, potentially enhancing glutamate clearance from the synaptic cleft and reducing hyperreflexia in astrocytic Rac1KO animals. Taken together, our findings show for the first time that Rac1 activity in astrocytes can contribute to hyperreflexia underlying spasticity following SCI. These results reveal an opportunity to target cell-specific molecular regulators of H-reflex excitability to manage spasticity after SCI.Significance Statement Spinal cord injury leads to stretch reflex hyperexcitability, which underlies the clinical symptom of spasticity. This study shows for the first time that astrocytic Rac1 contributes to the development of hyperreflexia after SCI. Specifically, astrocytic Rac1KO reduced SCI-related H-reflex hyperexcitability, decreased dendritic spine dysgenesis on α-motor neurons, and elevated the expression of the astrocytic glutamate transporter-1 (GLT-1). Overall, this study supports a distinct role for astrocytic Rac1 signaling within the spinal reflex circuit and the development of SCI-related spasticity.

2-hydroxy-4-methoxy benzaldehyde from Hemidesmus indicus root extract suppresses toll-like receptor2-mediated migration and invasive mechanisms in rheumatoid arthritis

IntroductionElevated expression of Toll-like receptor2 (TLR2) in synovial fibroblasts, endothelial and other infiltrating cells in rheumatoid arthritis promotes their migration and invasion. Our aim was to isolate a TLR2 inhibitor from Hemidesmus indicus root extract and test its effectiveness in regulating migration and invasion of fibroblast-like synoviocytes, endothelial cells and ex vivo synovial explant outgrowth. MethodFractionation of TLR2 inhibitor by a bioassay, followed by testing the inhibitor in the cultures of isolated fibroblast-like synoviocytes, endothelial cells and ex vivo synovial explant outgrowth in the presence of a TLR2 agonist, Pam3CSK4 was undertaken. ResultsTLR2 inhibition using 2-hydroxy-4-methoxy benzaldehyde significantly lowered Pam3CSK4-induced cell migration, invasion, MMP-1, MMP-3, Pro-MMP-2 and Pro-MMP-9 expression in fibroblast-like synovial cells isolated from the discarded biopsy specimens of rheumatoid arthritis patients as well as in ex vivo synovial explant culture. TLR2 inhibition lowered active Rac1 protein and β1 integrin mRNA levels in synovial fibroblasts and suppressed filopodia and lamellipodia formation in response to Pam3CSK4. In endothelial cells, 2-hydroxy-4-methoxy benzaldehyde inhibited Pam3CSK4-stimulated vascular endothelial growth factor (VEGF) and ICAM-1 expression and thereby lowered tube formation and endothelial migration. TLR2 inhibition using 2-hydroxy-4-methoxy benzaldehyde also attenuated the Pam3CSK4-evoked outgrowth formation in ex vivo synovial explants. 2-hydroxy-4-methoxy benzaldehyde targeted TLR2-MyD88 interactions and prevented dimerization. Conclusions2-hydroxy-4-methoxy benzaldehyde is the active constituent in the roots of H indicus that appears to act through TLR2 inhibition, followed by modulation of the agonist-induced cell migration, invasion and angiogenesis.

RBIO-08. GTP SIGNALING LINKS ALTERED METABOLISM, DNA REPAIR AND TREATMENT RESISTANCE IN GLIOBLASTOMA

Abstract OBJECTIVE We sought to define the mechanisms by which purines regulate DNA repair and therapy response. METHODS Phosphoproteomics was used to identify GTP-dependent (de)phosphorylation events after radiation (RT) and antibodies generated against novel sites. Animal models of glioblastoma (GBM) and normal tissues were used to assess DNA repair and treatment responses in vivo. RESULTS Pharmacogenomic inhibition of GTP (but not ATP) synthesis sensitized GBM cells to RT by inhibiting the activity of non-homologous end joining, but not homologous recombination. We found a GTP-dependent RT-induced dephosphorylation event on Abl interactor 1 (Abi-1) serine 323 (S323) using phosphoproteomics. We generated a new antibody for p-Abi-1 (S323), validated its specificity, and confirmed that RT causes a GTP-dependent dephosphorylation of Abi-1 (S323). Knockout of Abi-1 slowed RT-induced double-strand break (DSB) repair, and this was rescued by re-expression of dephosphomimetic Abi-1 (S323A) but not phosphomimetic Abi-1 (S323D). Abi-1 canonically binds to G protein Rac1. Expression of constitutively active Rac1 promoted but dominant negative Rac1 blocked the dephosphorylation of Abi-1 (S323) and DSB repair. Knock-down or inhibition of protein phosphatase 5 reversed the GTP- and Rac1-mediated dephosphorylation of Abi-1 and DSB repair. In GBM PDX samples, p-Abi-1 (S323) levels negatively correlated with Rac1 activity and predicted favorable efficacy of genotoxic treatments. In orthotopic GBM mouse models, inhibiting Rac1 enhanced RT responses and suppressed Abi-1 (S323) dephosphorylation. Abi-1 knockout enhanced efficacy of genotoxic treatments and could be rescued by Abi-1 S323A (but not Abi-1 S323D) re-expression. This regulation is generalizable beyond brain cancer, as GTP supplementation promoted DNA repair and p-Abi1 (S323) dephosphorylation in non-malignant cells and protected mice from RT-mediated gastrointestinal injury and bleomycin-induced pulmonary fibrosis. CONCLUSION The GTP-Rac1-PP5-Abi-1 signaling axis links metabolism and DNA repair. Disrupting this pathway can overcome GBM resistance to genotoxic therapy while augmenting it can mitigate genotoxic injury of normal tissues.

Human epicardial adipose tissue-secreted miR-92a-3p regulates myocardial redox state via paracrine signalling: implications for cardiovascular clinical outcomes

Abstract Background Visceral obesity is directly linked to increased cardiovascular risk, including heart failure. Purpose We explored the ability of human epicardial adipose tissue (EAT)-derived microRNAs (miRNAs) to regulate the myocardial redox state and clinical outcomes. Methods The 5 study arms included 466 patients undergoing cardiac surgery to perform: 1) the discovery phase in which we screened for 351 miRNAs expressed and released from human EAT; 2) correlation analyses between EAT microRNAs and myocardial expression of their targets or myocardial superoxide production in paired EAT/atrial biopsies; 3) genome-wide association screening for miR-92a-3p expression in EAT to test causality/directionality; 4) ex vivo experiments to investigate the underlying mechanisms that were also studied in vitro and in vivo; 5) an 8-year follow-up study to test the prognostic value of the discovered miRNA. Superoxide (O2.-) generation was measured by lucigenin chemiluminescence with NADPH 100μM stimulation as indicator of NADPH-oxidases activity. Vas2870 400 μM (a specific pan-NADPH oxidase inhibitor) was used to obtain the Vas2870-inhibitable O2.- signal which constitutes a more specific index of NADPH oxidase activity. Activation of Rac1, a key NADPH-oxidases subunit, was evaluated by a commercially available kit. Differentiated H9c2 cells were used as an in vitro model of cardiomyocytes. Doxycycline-inducible Wnt5a-overexpressing mice were used for in vivo experiments. Results The EAT secretome profiling on study 1 patients led us to identify 3 microRNAs both expressed and released by EAT whose levels in EAT correlated with oxidative stress in human myocardium. Among these miRNAs only miR-92a-3p reduced NADPH-oxidase-derived superoxide (O2.-) in cardiomyocytes (A). A genetic screening identified 7 SNPs that were associated with high miR-92a-3p levels in EAT (EAT-miR-92a-3p SNPs) and were related to lower myocardial superoxide production (B). miR-92a-3p decreased both activation of Rac1 (not shown) and Wnt5a protein levels in vitro (C). Patients with high EAT miR-92a-3p levels had lower WNT5A levels in the myocardium (not shown). Ex vivo, in vivo and in vitro experiments showed opposite effects of Wnt5a on Rac1-mediated NADPH-oxidases activity (D and not shown). Finally, we found an association of miR-92a-3p levels in EAT with lower relative risk of adverse cardiovascular events (E). Conclusions EAT-derived miRNAs exert paracrine effects on the human heart. Indeed miR-92a-3p suppresses the Wnt5a/Rac1/NADPH oxidase axis and improves myocardial redox state. EAT-derived miR-92a-3p is related with improved clinical outcomes and is a rational therapeutic target for the prevention and treatment of obesity-related heart disease.

Si-Ni-San alleviates early life stress-induced depression-like behaviors in adolescence via modulating Rac1 activity and associated spine plasticity in the nucleus accumbens.

Background: Early life stress (ELS) is a major risk factor for depression in adolescents. The nucleus accumbens (NAc) is a key center of the reward system, and spine remodeling in the NAc contributes to the development of depression. The Si-Ni-San formula (SNS) is a fundamental prescription for treating depression in traditional Chinese medicine. However, little is known about the effects of SNS on behavioral abnormalities and spine plasticity in the NAc induced by ELS. Purpose: This study aimed to investigate the therapeutic effect and the modulatory mechanism of SNS on abnormal behaviors and spine plasticity in the NAc caused by ELS. Methods: We utilized a model of ELS that involved maternal separation with early weaning to explore the protective effects of SNS on adolescent depression. Depressive-like behaviors were evaluated by the sucrose preference test, the tail suspension test, and the forced swimming test; anxiety-like behaviors were monitored by the open field test and the elevated plus maze. A laser scanning confocal microscope was used to analyze dendritic spine remodeling in the NAc. The activity of Rac1 was detected by pull-down and Western blot tests. Viral-mediated gene transfer of Rac1 was used to investigate its role in ELS-induced depression-like behaviors in adolescence. Results: ELS induced depression-like behaviors but not anxiety-like behaviors in adolescent mice, accompanied by an increase in stubby spine density, a decrease in mushroom spine density, and decreased Rac1 activity in the NAc. Overexpression of constitutively active Rac1 in the NAc reversed depression-related behaviors, leading to a decrease in stubby spine density and an increase in mushroom spine density. Moreover, SNS attenuated depression-like behavior in adolescent mice and counteracted the spine abnormalities in the NAc induced by ELS. Additionally, SNS increased NAc Rac1 activity, and the inhibition of Rac1 activity weakened the antidepressant effect of SNS. Conclusion: These results suggest that SNS may exert its antidepressant effects by modulating Rac1 activity and associated spine plasticity in the NAc.

SrGAP3 Deficiency Mediates Podocyte Injury by Promoting Cdc42 and Rac1 Activity in Diabetic Nephropathy

SrGAP3 Deficiency Mediates Podocyte Injury by Promoting Cdc42 and Rac1 Activity in Diabetic Nephropathy

The PMA phorbol ester tumor promoter increases canonical Wnt signaling via macropinocytosis

Activation of the Wnt pathway lies at the core of many human cancers. Wnt and macropinocytosis are often active in the same processes, and understanding how Wnt signaling and membrane trafficking cooperate should improve our understanding of embryonic development and cancer. Here, we show that a macropinocytosis activator, the tumor promoter phorbol 12-myristate 13-acetate (PMA), enhances Wnt signaling. Experiments using the Xenopus embryo as an in vivo model showed marked cooperation between the PMA phorbol ester and Wnt signaling, which was blocked by inhibitors of macropinocytosis, Rac1 activity, and lysosome acidification. Human colorectal cancer tissue arrays and xenografts in mice showed a correlation of cancer progression with increased macropinocytosis/multivesicular body/lysosome markers and decreased GSK3 levels. The crosstalk between canonical Wnt, focal adhesions, lysosomes, and macropinocytosis suggests possible therapeutic targets for cancer progression in Wnt-driven cancers.

The PMA phorbol ester tumor promoter increases canonical Wnt signaling via macropinocytosis.

Activation of the Wnt pathway lies at the core of many human cancers. Wnt and macropinocytosis are often active in the same processes, and understanding how Wnt signaling and membrane trafficking cooperate should improve our understanding of embryonic development and cancer. Here, we show that a macropinocytosis activator, the tumor promoter phorbol 12-myristate 13-acetate (PMA), enhances Wnt signaling. Experiments using the Xenopus embryo as an in vivo model showed marked cooperation between the PMA phorbol ester and Wnt signaling, which was blocked by inhibitors of macropinocytosis, Rac1 activity, and lysosome acidification. Human colorectal cancer tissue arrays and xenografts in mice showed a correlation of cancer progression with increased macropinocytosis/multivesicular body/lysosome markers and decreased GSK3 levels. The crosstalk between canonical Wnt, focal adhesions, lysosomes, and macropinocytosis suggests possible therapeutic targets for cancer progression in Wnt-driven cancers.

Diverse gut pathogens exploit the host engulfment pathway via a conserved mechanism

Macrophages clear infections by engulfing and digesting pathogens within phagolysosomes. Pathogens escape this fate by engaging in a molecular arms race; they use WxxxE motif-containing "effector" proteins to subvert the host cells they invade and seek refuge within protective vacuoles. Here, we define the host component of the molecular arms race as an evolutionarily conserved polar "hot spot" on the PH domain of ELMO1 (Engulfment and Cell Motility protein 1), which is targeted by diverse WxxxE effectors. Using homology modeling and site-directed mutagenesis, we show that a lysine triad within the "patch" directly binds all WxxxE effectors tested: SifA (Salmonella), IpgB1 and IpgB2 (Shigella), and Map (enteropathogenic Escherichia coli). Using an integrated SifA-host protein-protein interaction network, in silico network perturbation, and functional studies, we show that the major consequences of preventing SifA-ELMO1 interaction are reduced Rac1 activity and microbial invasion. That multiple effectors of diverse structure, function, and sequence bind the same hot spot on ELMO1 suggests that the WxxxE effector(s)-ELMO1 interface is a convergence point of intrusion detection and/or host vulnerability. We conclude that the interface may represent the fault line in coevolved molecular adaptations between pathogens and the host, and its disruption may serve as a therapeutic strategy.

Cell aggregation activates small GTPase Rac1 and induces CD44 cleavage by maintaining lipid raft integrity

Lipid rafts are highly ordered membrane domains that are enriched in cholesterol and glycosphingolipids and serve as major platforms for signal transduction. Cell detachment from the extracellular matrix (ECM) triggers lipid raft disruption and anoikis, which is a barrier for cancer cells to metastasize. Compared to single circulating tumor cells (CTCs), our recent studies have demonstrated that CD44-mediatd cell aggregation enhances the stemness, survival and metastatic ability of aggregated cells. Here, we investigated whether and how lipid rafts are involved in CD44-mediated cell aggregation. We found that cell detachment, which mimics the condition when tumor cells detach from the ECM to metastasize, induced lipid raft disruption in single cells, but lipid raft integrity was maintained in aggregated cells. We further found that lipid raft integrity in aggregated cells was required for Rac1 activation to prevent anoikis. In addition, CD44 and γ-secretase coexisted at lipid rafts in aggregated cells, which promoted CD44 cleavage and generated CD44 intracellular domain (CD44 ICD) to enhance stemness of aggregated cells. Consequently, lipid raft disruption inhibited Rac1 activation, CD44 ICD generation, and metastasis. Our findings reveal two new pathways regulated by CD44-mediated cell aggregation via maintaining lipid raft integrity. These findings also suggest that targeting cell aggregation-mediated pathways could be a novel therapeutic strategy to prevent CTC cluster-initiated metastasis.

The HACE1 E3 ligase mediates RAC1-dependent control of mTOR signaling complexes.

HACE1 is a HECT family E3 ubiquitin-protein ligase with broad but incompletely understood tumor suppressor activity. Here, we report a previously unrecognized link between HACE1 and signaling complexes containing mammalian target of rapamycin (mTOR). HACE1 blocks mTORC1 and mTORC2 activities by reducing mTOR stability in an E3 ligase-dependent manner. Mechanistically, HACE1 binds to and ubiquitylates Ras-related C3 botulinum toxin substrate 1 (RAC1) when RAC1 is associated with mTOR complexes, including at focal adhesions, leading to proteasomal degradation of RAC1. This in turn decreases the stability of mTOR to reduce mTORC1 and mTORC2 activity. HACE1 deficient cells show enhanced mTORC1/2 activity, which is reversed by chemical or genetic RAC1 inactivation but not in cells expressing the HACE1-insensitive mutant, RAC1K147R . In vivo, Rac1 deletion reverses enhanced mTOR expression in KRasG12D -driven lung tumors of Hace1-/- mice. HACE1 co-localizes with mTOR and RAC1, resulting in RAC1-dependent loss of mTOR protein stability. Together, our data demonstrate that HACE1 destabilizes mTOR by targeting RAC1 within mTOR-associated complexes, revealing a unique ubiquitin-dependent process to control the activity of mTOR signaling complexes.

Vav2 promotes ductus arteriosus anatomic closure via the remodeling of smooth muscle cells by Rac1 activation.

The ductus arteriosus (DA), bridging the aorta and pulmonary artery, immediately starts closing after birth. Remodeling of DA leads to anatomic obstruction to prevent repatency. Several histological changes, especially extracellular matrices (ECMs) deposition and smooth muscle cells (SMCs) migration bring to anatomic closure. The genetic etiology and mechanism of DA closure remain elusive. We have previously reported a novel copy number variant containing Vav2 in patent ductus arteriosus (PDA) patients, but its specific role in DA closure remains unknown. The present study revealed that the expression of Vav2 was reduced in human patent DA, and it was less enrichment in the adjacent aorta. Matrigel experiments demonstrated that Vav2 could promote SMC migration from PDA patient explants. Smooth muscle cells with Vav2 overexpression also presented an increased capacity in migration and downregulated contractile-related proteins. Meanwhile, SMCs with Vav2 overexpression exhibited higher expression of collagen III and lessened protein abundance of lysyl oxidase, and both changes are beneficial to DA remodeling. Overexpression of Vav2 resulted in increased activity of Rac1, Cdc42, and RhoA in SMCs. Further investigation noteworthily found that the above alterations caused by Vav2 overexpression were particularly reversed by Rac1 inhibitor. A heterozygous, rare Vav2 variant was identified in PDA patients. Compared with the wild type, this variant attenuated Vav2 protein expression and weakened the activation of downstream Rac1, further impairing its functions in SMCs. In conclusion, Vav2 functions as an activator for Rac1 in SMCs to promote SMCs migration, dedifferentiation, and ECMs production. Deleterious variant potentially induces Vav2 loss of function, further providing possible molecular mechanisms about Vav2 in PDA pathogenesis. These findings enriched the current genetic etiology of PDA, which may provide a novel target for prenatal diagnosis and treatment. KEY MESSAGES: Although we have proposed the potential association between Vav2 and PDA incidence through whole exome sequencing, the molecular mechanisms underlying Vav2 in PDA have never been reported.This work, for the first time, demonstrated that Vav2 was exclusively expressed in closed DAs. Moreover, we found that Vav2 participated in the process of anatomic closure by mediating SMCs migration, dedifferentiation, and ECMs deposition through Rac1 activation. Our findings first identified a deleterious Vav2 c.701C>T variant that affected its function in SMCs by impairing Rac1 activation, which may lead to PDA defect. Vav2 may become an early diagnosis and an effective intervention target for PDA clinical therapy.

Key role for Rac in the early transcriptional response to ECM stiffness and the stiffness-dependent repression of ATF3.

The Rho family GTPases, Rac and Rho, play critical roles in transmitting mechanical information contained within the extracellular matrix (ECM) to the cell. Rac and Rho have well described roles in regulating stiffness-dependent actin remodeling, proliferation and motility. However, much less is known about the relative roles of these GTPases in stiffness-dependent transcription, particularly at the genome-wide level. Here, we selectively inhibited Rac and Rho in mouse embryonic fibroblasts cultured on deformable substrata and used RNA sequencing to elucidate and compare the contribution of these GTPases to the early transcriptional response to ECM stiffness. Surprisingly, we found that the stiffness-dependent activation of Rac is dominant over Rho in the initial transcriptional response to ECM stiffness. We also identified Activating Transcription Factor 3 (ATF3) as a major target of stiffness/Rac-mediated signaling and show that ATF3 repression by ECM stiffness helps to explain how the stiffness-dependent activation of Rac results in the induction of cyclin D1.