Rac1 Signaling Research Articles

MiR-365 regulates liver cancer stem cells via RAC1 pathway.

Liver cancer stem cells (CSCs) were involved in tumorigenesis, progression, recurrence, and drug resistance of hepatocellular carcinoma (HCC). miR‐365 was downregulated in hepatocellular carcinoma and inhibited HCC cell proliferation and invasion. However, the role of miR‐365 in liver cancer stem cells was unknown. Herein, we observed a remarkable decrease of miR‐365 expression in CD133 or EpCAM‐positive liver CSCs as well as in CSC‐enriched hepatoma spheres. Up‐regulated miR‐365 suppressed liver CSC expansion by inhibiting the dedifferentiation of hepatoma cells and decreasing the self‐renewal ability of liver CSCs. Mechanistically, bioinformatic and luciferase reporter analysis identified Ras‐related C3 botulinum toxin substrate 1 (RAC1) as a direct target of miR‐365. Overexpression of miR‐365 in hepatoma cells downregulated the RAC1 mRNA and protein expression. RAC1 also could promote the expansion of liver CSCs. The special RAC1 inhibitor EHop‐106 or RAC1 overexpression abolished the discrepancy in liver CSC proportion and the self‐renewal capacity between miR‐365 overexpression hepatoma cells and control cells, which further confirmed that RAC1 was required in miR‐365‐suppressed liver CSCs expansion. miR‐365 was downregulated in liver CSCs and could inhibit HCC cells dedifferentiation and liver CSCs expansion by targeting RAC1 signaling.

Effects of TLR4-PI3K-Rac1 pathway on cytoskeleton rearrangement and phagocytosis of macrophage

Objective: To investigate the effects of Toll-like receptor 4 (TLR4)-phosphatidylinositol 3-kinase (PI3K) -Ras-related C3 botulinum toxin 1 (Rac1) signaling pathway on macrophage cytoskeleton rearrangement and phagocytosis. Methods: Mouse macrophage cell line RAW264.7 was divided into blank group, negative control group and TLR4-RNA interference (RNAi) group. The lentivirus carrying TLR4 short hairpin RNA (shRNA) and nonsense control sequence were respectively transfected into TLR4-RNAi group and negative control group. The cells in blank group were not transfected. The silencing efficiency of TLR4 was detected by Western blot. Real-time fluorescence quantitative PCR was used to detect the expression of PI3K and Rac1 mRNA in each group. The expressions of PI3K, p-Rac1 and Rac1 protein were detected by Western blot. Cytoskeleton was observed by laser scanning confocal microscopy. Mean fluorescence intensity (MFI) and the percentage of cells phagocytosing flurescein inothiocyanate-labeled Eseherichina coli (FITC-E.coli) (Phagocytosed cell %) were detected by flow cytometry. Results: The RAW264.7 cells can be successfully transfected by TLR4-shRNA lentivirus, and the transfection efficiency ranged from 80% to 90%. The silencing efficiency of TLR4 gene was (63±4)%. After silencing the TLR4 gene, the relative expression of TLR4 mRNA and protein (0.20±0.03, 0.37±0.04), PI3K mRNA and protein (0.64±0.06, 0.75±0.06), Rac1 mRNA, protein and p-Rac1 protein (0.75±0.04, 0.76±0.01, 0.74±0.05) in TLR4-RNAi group were significantly lower than those in negative control group and blank group (all P<0.01). The change of cytoskeleton: after silencing the TLR4 gene, the celluar pseudopods were short and stiff, with the impaired capacity of phagocytosing FITC-E.coli. Cells in blank group and negative control group extended good pseudopodia, and the capacity of phagocytosing FITC-E.coli was normal. The MFI and Phagocytosed cells % of TLR4-RNAi group[(7 453±564), (70.20±2.27)%]were significantly lower than those in the blank group and the negative control group (all P<0.01). Positive correlations were existed between mRNA, protein expression of TLR4, PI3K, Rac1 and MFI, Phagocytosed cells% (all P<0.05) in all groups. Conclusion: TLR4-PI3K-Rac1 pathway involves in the cytoskeleton rearrangement and impairs the phagocytosis of macrophages.

ITRAQ-based proteomics suggests LRP6, NPY and NPY2R perturbation in the hippocampus involved in CSDS may induce resilience and susceptibility

AimsAlthough decades of research have revealed numerous molecular abnormalities in the hippocampus associated with depression, the different mechanisms involved in the susceptibility and resilience of mice to chronic social defeat stress (CSDS)-induced depression remain poorly understand. Through the social defeat model, we can study the differences in molecular changes between the susceptible and resilient mice. Main methodsWe used a proteomic-based platform to compare hippocampal proteins in CSDS mice with those in control mice. Differentially expressed proteins were identified through isobaric tags for relative and absolute quantitation (iTRAQ) combined with LC-MS/MS. We then analyzed the results by ingenuity pathway analysis (IPA) and verified five proteins by western blotting. Key findingsMice were exposed to 10 days of CSDS, which successfully induced stress-susceptible and -resilient phenotypes. 161 and 134 proteins were significantly differentially expressed in the susceptible and resilient groups, respectively, compared with the levels in the control group. The Rac signaling and the GABA receptors signaling pathways were the common top-ranking pathways. We found that low-density lipoprotein receptor–related protein 6 (LRP6) was upregulated in resilient mice and neuropeptide Y (NPY) was downregulated in susceptible mice compared with the levels in control mice. Moreover, neuropeptide Y receptor type 2 (NPY2R) protein expression in susceptible mice was downregulated compared with that in the resilient group. SignificanceOur findings in the three groups potentially reveal the differences in molecular mechanisms underlying depression between susceptible and resilient mice. The results provide insight into molecular abnormalities of the hippocampus in CSDS mice and some potential drug targets for treating depression.

Rac1 Nanoscale Organization on the Plasma Membrane Is Driven by Lipid Binding Specificity Encoded in the Membrane Anchor.

Rac1 is a small guanine nucleotide binding protein that cycles between an inactive GDP-bound and active GTP-bound state to regulate cell motility and migration. Rac1 signaling is initiated from the plasma membrane (PM). Here, we used high-resolution spatial mapping and manipulation of PM lipid composition to define Rac1 nanoscale organization. We found that Rac1 proteins in the GTP- and GDP-bound states assemble into nonoverlapping nanoclusters; thus, Rac1 proteins undergo nucleotide-dependent segregation. Rac1 also selectively interacts with phosphatidic acid (PA) and phosphoinositol (3,4,5)-trisphosphate (PIP3), resulting in nanoclusters enriched in these lipids. These lipids are structurally important because depleting the PM of PA or PIP3 impairs both Rac1 PM binding and Rac1 nanoclustering. Lipid binding specificity of Rac1 is encoded in the amino acid sequence of the polybasic domain (PBD) of the C-terminal membrane anchor. Point mutations within the PBD, including arginine-to-lysine substitutions, profoundly alter Rac1 lipid binding specificity without changing the electrostatics of the protein and result in impaired macropinocytosis and decreased cell spreading. We propose that Rac1 nanoclusters act as lipid-based signaling platforms emulating the spatiotemporal organization of Ras proteins and show that the Rac1 PBD-prenyl anchor has a biological function that extends beyond simple electrostatic engagement with the PM.

Tendinosis Results From Oxygen Tension-Dependent Rac1 Signaling in Aging Tenocytes

Grant support received from: NIH R03 AG048118, OREF, AFSH, Hand Rehabiliation Foundation Royalty: Elsevier, Zimmer Biomet, Skeletal Dynamics (Osterman) Consulting Fee: Arthrex, NewClip, Acumed (Osterman) Contracted Research: Acumed (Osterman) Age-related tendon degeneration (tendinosis) is characterized by a phenotypic change in which tenocytes display a cellular and biochemical phenotype typical of fibrochondrocytes. As tendon degeneration has been noted in vivo in areas of varying vascularity, we hypothesized that altered vascularity causes tendinosis via decreased oxygen tension, and that this process can be recapitulated in vitro. Human tendinosis and normal tendon tissue were harvested according to IRB-approved protocols and examined for differential collagen I, collagen II, and phosphate expression via FTIR and Western blotting techniques. Aged and young human tenocytes were cultured in hypoxia (1) and normoxia (21% 02) and analyzed for collagen I and collagen II expression over time. Rac1/RhoA GTPase signaling was analyzed via biochemical assay and controlled via pharmacologic and adenoviral expression. Tendon explant culture was performed using normal tendon tissue cultured in hypoxia or normoxia, in the presence or absence of pharmacologic Rac1 inhibitor. Human tendinosis tissue demonstrated a fibrochondrocyte phenotype via histopathology, FTIR, and Western blot (Figure 59-1). Hypoxic (1% 02) culture of aged and young human tenocytes resulted in the immediate expression of a fibrochondrocyte phenotype in aged cells, but a belated response in young cells. Investigation of the molecular mechanism responsible for this response revealed that fibrochondrocyte differentiation in aged hypoxic tenocytes was accompanied by decreased Rac1 activity. In young hypoxic tenocytes, however, fibrochondrocyte differentiation was accompanied by decreased Rac1 activity coupled with increased RhoA activity. Using pharmacologic and adenoviral manipulation, we confirmed that these hypoxic effects on the tenocyte phenotype were linked directly to the activity of RhoA/Rac1 GTPase, and result in tendinosis of human tendon explant cultures in vitro (Figure 59-2). •Hypoxia drives development of human tendinosis via decreased Rac1 and increased RhoA GTPase activity.•By varying Rac1/RhoA activity, the tendinosis phenotype is recreated in a human tendon explant system.•These results provide a molecular insight into the development of human tendinosis that occurs with aging, and detail the mechanism by which it occurs.Figure 59-2Human tendon explant in normoxic (G-H) or hypoxic (I-J) culture conditions without (G, I) or with (H, J) Rac1 inhibitor.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT) This research was supported by a Basic Science Grant from the American Foundation for Surgery of the Hand.

NKCC1 promotes EMT-like process in GBM via RhoA and Rac1 signaling pathways.

Glioblastoma is the most common and lethal primary intracranial tumor. As the key regulator of tumor cell volume, sodium‐potassium‐chloride cotransporter 1 (NKCC1) expression increases along with the malignancy of the glioma, and NKCC1 has been implicated in glioblastoma invasion. However, little is known about the role of NKCC1 in the epithelial‐mesenchymal transition‐like process in gliomas. We noticed that aberrantly elevated expression of NKCC1 leads to changes in the shape, polarity, and adhesion of cells in glioma. Here, we investigated whether NKCC1 promotes an epithelial–mesenchymal transition (EMT)‐like process in gliomas via the RhoA and Rac1 signaling pathways. Pharmacological inhibition and knockdown of NKCC1 both decrease the expressions of mesenchymal markers, such as N‐cadherin, vimentin, and snail, whereas these treatments increase the expression of the epithelial marker E‐cadherin. These findings indicate that NKCC1 promotes an EMT‐like process in gliomas. The underlying mechanism is the facilitation of the binding of Rac1 and RhoA to GTP by NKCC1, which results in a significant enhancement of the EMT‐like process. Specific inhibition or knockdown of NKCC1 both attenuate activated Rac1 and RhoA, and the pharmacological inhibitions of Rac1 and RhoA both impair the invasion and migration abilities of gliomas. Furthermore, we illustrated that NKCC1 knockdown abolished the dissemination and spread of glioma cells in a nude mouse intracranial model. These findings suggest that elevated NKCC1 activity acts in the regulation of an EMT‐like process in gliomas, and thus provides a novel therapeutic strategy for targeting the invasiveness of gliomas, which might help to inhibit the spread of malignant intracranial tumors.

Cannabinoid receptor 2 agonist attenuates blood‑brain barrier damage in a rat model of intracerebral hemorrhage by activating the Rac1 pathway

Blood‑brain barrier (BBB) disruption and consequent edema formation are the most common brain injuries following intracerebral hemorrhage (ICH). Endocannabinoid receptors can alter the permeability of various epithelial barriers and have potential neuroprotective effects. The present study aimed to explore whether the selective cannabinoid receptor2 (CNR2) agonist, JWH133, can ameliorate BBB integrity and behavioral outcome by activating Ras‑related C3 botulinum toxin substrate1 (Rac1) following ICH. Autologous arterial blood was injected into the basal ganglia of rats to induce ICH. Animals were randomly divided into the following groups: Sham‑operated, ICH+vehicle, ICH+JWH133, ICH+JWH13+vehicle, ICH+JWH133+AM630 (a selective CNR2 antagonist), ICH+AM630, ICH+JWH133 +NSC23766 (a Rac1 antagonist) and ICH+NSC23766. JWH133 and AM630 were independently intraperitoneally administrated at 1h prior to ICH. NSC23766 was intracerebroventricularly (ICV) administered 30min prior to ICH. A modified Garcia test, corner test, Evans blue extravasation and brain water content analysis were performed at 24 and 72h following ICH. Western blotting and pull‑down assays were performed at 24h following ICH. The results demonstrated that JWH133 treatment improved neurofunctional deficits, reduced perihematomal brain edema and alleviated BBB damage at 24 and 72h following ICH. In addition, JWH133 treatment increased the protein expression levels of guanosine‑5'‑triphosphate‑Rac1 and of the adherens junction proteins occludin, zonula occludens‑1 and claudin‑5. However, these effects were reversed by AM630 and NSC23766 treatment. In conclusion, the present findings revealed that JWH133 treatment attenuated brain injury in a rat model of ICH via activation of the Rac1 signaling pathway, thus preserving BBB integrity.

Huntingtin-interacting protein 1 (HIP1) regulates arthritis severity and synovial fibroblast invasiveness by altering PDGFR and Rac1 signalling

ObjectivesWhile new treatments for rheumatoid arthritis (RA) have markedly improved disease control by targeting immune/inflammatory pathways, current treatments rarely induce remission, underscoring the need for therapies that target other aspects...

The Human FSGS-Causing ANLN R431C Mutation Induces Dysregulated PI3K/AKT/mTOR/Rac1 Signaling in Podocytes.

We previously reported that mutations in the anillin (ANLN) gene cause familial forms of FSGS. ANLN is an F-actin binding protein that modulates podocyte cell motility and interacts with the phosphoinositide 3-kinase (PI3K) pathway through the slit diaphragm adaptor protein CD2-associated protein (CD2AP). However, it is unclear how the ANLN mutations cause the FSGS phenotype. We hypothesized that the R431C mutation exerts its pathogenic effects by uncoupling ANLN from CD2AP. We conducted in vivo complementation assays in zebrafish to determine the effect of the previously identified missense ANLN variants, ANLNR431C and ANLNG618C during development. We also performed in vitro functional assays using human podocyte cell lines stably expressing wild-type ANLN (ANLNWT ) or ANLNR431C . Experiments in anln-deficient zebrafish embryos showed a loss-of-function effect for each ANLN variant. In human podocyte lines, expression of ANLNR431C increased cell migration, proliferation, and apoptosis. Biochemical characterization of ANLNR431C -expressing podocytes revealed hyperactivation of the PI3K/AKT/mTOR/p70S6K/Rac1 signaling axis and activation of mTOR-driven endoplasmic reticulum stress in ANLNR431C -expressing podocytes. Inhibition of mTOR, GSK-3β, Rac1, or calcineurin ameliorated the effects of ANLNR431C . Additionally, inhibition of the calcineurin/NFAT pathway reduced the expression of endogenous ANLN and mTOR. The ANLNR431C mutation causes multiple derangements in podocyte function through hyperactivation of PI3K/AKT/mTOR/p70S6K/Rac1 signaling. Our findings suggest that the benefits of calcineurin inhibition in FSGS may be due, in part, to the suppression of ANLN and mTOR. Moreover, these studies illustrate that rational therapeutic targets for familial FSGS can be identified through biochemical characterization of dysregulated podocyte phenotypes.

An Intellectual Disability-Related Missense Mutation in Rac1 Prevents LTP Induction.

The small GTPase Rac1 promotes actin polymerization and plays a critical and increasingly appreciated role in the development and plasticity of glutamatergic synapses. Growing evidence suggests that disruption of the Rac1 signaling pathway at glutamatergic synapses contributes to Autism Spectrum Disorder/intellectual disability (ASD/ID)-related behaviors seen in animal models of ASD/ID. Rac1 has also been proposed as a strong candidate of convergence for many factors implicated in the development of ASD/ID. However, the effects of ASD/ID-related mutations in Rac1 itself have not been explored in neurons. Here, we investigate a recently reported de novo missense mutation in Rac1 found in an individual with severe ID. Our modeling predicts that this mutation will strongly inhibit Rac1 activation by occluding Rac1’s GTP binding pocket. Indeed, we find that this de novo mutation prevents Rac1 function and results in a selective reduction in synaptic AMPA receptor function. Furthermore, this mutation prevents the induction of long-term potentiation (LTP), the cellular mechanism underlying learning and memory formation. Together, our findings strongly suggest that this mutation contributes to the development of ID in this individual. This research demonstrates the importance of Rac1 in synaptic function and plasticity and contributes to a growing body of evidence pointing to dysregulation of actin polymerization at glutamatergic synapses as a contributing factor to ASD/ID.

Abstract 27: Regulating Rac1 signaling proteins influences migratory and protrusive behaviors of metastatic breast cancer cells in aligned, 3D collagen and collagen-like matrices

Abstract Breast cancer is the most commonly diagnosed cancer in women, with one out of every eight women being diagnosed in their lifetime. Our lab has previously shown that aligned collagen promotes tumor progression to metastatic disease, and predicts poor prognosis for breast cancer patients. Aligned collagen increases the persistence of cellular migration by limiting the number of protrusions from the cell body. The regulation of Rac1 and Rho proteins are important for cell motility through formation of lamelliepodia. Deregulation of these proteins and those upstream of Rac1, can result in cancer cell migration, invasion and metastases. Understanding how these proteins are regulated in different 3D collagen tumor microenvironments is important for determining phenotypes of invasive breast cancers. Inhibition of cell motility through the Rac1 pathway may limit cancer cell migration and prevent metastatic disease. While many studies have investigated cell migration in 2D, little is known regarding the cellular mechanisms that regulate both breast cancer cell migration and protrusive events in 3D aligned collagen. In vitro models will be used to recapitulate 3D aligned, and random collagen environments and assess the mechanisms of cell migration and protrusion dynamics by live-cell, time-lapse microscopy. Previous work from our group determined that MDA-MB-231 cells seeded into collagen environments had fewer protrusions, which localized in the directions of collagen alignment, when migrating along aligned fibers compared to MDA-MB-231 cells in the random collagen matrices. Preliminary data demonstrates that signaling proteins upstream of Rac1 such as, small GTPases, DOCK180 and FilGAP, dictate cellular protrusions and migratory persistence. MDA-MB-231 cell lines with doxycycline-induced knock-down Rac1 GTPase activating protein (GAP), FilGAP, and guanine nucleotide exchange factor (GEF), DOCK180, demonstrate that the regulation of Rac1 activity through these specific proteins plays a role in breast cancer cell migration and protrusion dynamics in an aligned collagen environment. Understanding the regulation of breast cancer cell migration in aligned and random 3D collagen environments will enhance the current understanding of progression to metastatic disease on a cellular level. Citation Format: Rachel Van Doorn, Joseph Szulczewski, Suzanne Ponik. Regulating Rac1 signaling proteins influences migratory and protrusive behaviors of metastatic breast cancer cells in aligned, 3D collagen and collagen-like matrices [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 27.

Abstract 2376: AXL receptor tyrosine kinase stimulates cell softening and cancer progression via Rac signaling pathway

Abstract Cancer cells exhibit different biophysical properties, such as cell stiffness and elasticity, compared with normal cells. It is now well accepted that cell stiffness measured by atomic force microscope (AFM) is a quantitative indicator of cancer progression and metastasis. To study the role of cell softening in malignant progression, Using Transwell assays and AFM, six human non-small cell lung cancer cell lines were classified into two groups: a high motility-low stiffness (HMLS) group and a low motility-high stiffness (LMHS) group. We found a significant difference in expression of the AXL receptor tyrosine kinase, which belongs to the TAM (Tyro3, AXL, Mer) family, in the stimulation of motility and cell softening. HMLS cells expressed higher AXL and phosphorylated AXL levels than LMHS cells. Exogenous AXL gene expression in H1703 LMHS cells exhibited increased motility and decreased stiffness, along with lower levels of actin stress fibre formation. Conversely, the AXL-specific inhibitor R428 and AXL-targeting siRNA reduced motility and increased stiffness in H1299 HMLS cells. Knockdown of AXL gene stimulated both actin stress fibre formation and focal adhesion kinase phosphorylation, which inhibited tumour formation in a mouse xenograft model. Treatment with Ras/Rac inhibitor SCH 51344, which blocks disruption of actin stress fibres, exerted similar effects to AXL inactivation in H1299 and H1703-AXL cells, but not in H1703 cells (no AXL expression). Furthermore, treatment with an ROCK inhibitor, Y27632, did not show any effect in two AXL expressing cells, while it stimulated motility in H1703 cells. Considering all these results, we propose that the Ras/Rac pathway operates downstream of AXL and suppresses the Rho/ROCK pathway. Cell softening induced by AXL activation is a key biophysical property of cancer cells, and AXL is thus a new target for inhibition of malignant progression in non-small cell lung cancer. Citation Format: Masami Suganuma, Keisuke Iida, Shota Yokoyama, Anchalee Rawangkan, Kozue Namiki, Pattama Wongsirisin. AXL receptor tyrosine kinase stimulates cell softening and cancer progression via Rac signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2376.

Hepatic Rac1 GTPase contributes to liver-mediated basal immune homeostasis and LPS-induced endotoxemia

BackgroundThe Ras-homologous GTPase Rac1 plays a key role in the regulation of gene expression, cytoskeleton-associated processes and cell death as well as carcinogenesis and inflammation. Here, we investigated the impact of Rac1 signaling on liver-mediated immune homeostasis. MethodsWe employed a constitutive Alb-Cre-driven rac1 knock-out and a poly I:C-inducible Mx1-Cre-based knock-out model and analyzed cytokine expression profiles in liver and other organs under basal situation and following LPS-induced endotoxemia by flow cytometry, qRT-PCR and immunocytochemistry. ResultsConstitutive Alb-Cre-driven rac1 knockout in hepatocytes altered the basal distribution and activation of immune cells in the liver and likewise in kidney and lung. Early systemic alterations in cytokine serum levels following LPS treatment remained unaffected by Rac1. Furthermore, lack of Rac1 in hepatocytes of untreated animals shifted the liver to a chronic inflammatory state, as depicted by an enhanced mRNA expression of marker genes related to activated macrophages. Upon acute LPS-induced endotoxemia, increased IL-10 mRNA expression in the liver of Alb-Cre Rac1-deficient mice provided an anti-inflammatory response. Employing a poly I:C-inducible Mx1-Cre-based rac1 knock-out, which allows a more widespread rac1 deletion in both hepatocytes and non-hepatocytes, we observed substantial differences regarding both basal and LPS-stimulated cytokine expression profiles as compared to the Alb-Cre system. ConclusionsRac1-dependent mechanisms in hepatocytes and non-hepatocytes contribute to the maintenance of liver immune homeostasis under basal situation and following LPS-induced endotoxemia. Disturbed Rac1-regulated hepatocyte functions may promote liver damage under pathophysiological situation involving inflammatory stress.

Transcriptome analysis supports viral infection and fluoride toxicity as contributors to chronic kidney disease of unknown etiology (CKDu) in Sri Lanka.

Chronic kidney disease of unknown etiology (CKDu), having epidemic characteristics, is being diagnosed increasingly in certain tropical regions of the world, mainly Latin America and Sri Lanka. They have been observed primarily in farming communities and current hypotheses point toward many environmental and occupational triggers. CKDu does not have common etiologies of chronic kidney disease (CKD) such as hypertension, diabetes, or autoimmune disease. We aimed to understand the molecular processes underlying CKDu in Sri Lanka using transcriptome analysis. RNA extracted from whole blood was reverse transcribed and used for microarray analysis using the Human HT-12 v.4 array (Illumina). Pathway analysis was carried out using ingenuity pathway analysis (IPA-Qiagen). Microarray results were validated using real-time PCR of five selected genes. Pathways related to innate immune response, including interferon signaling, inflammasome signaling and TREM1 signaling had the most significant positive activation z scores, where as EIF2 signaling and mTOR signaling had the most significant negative activation z scores. Pathways previously linked to fluoride toxicity; G-protein activation, Cdc42 signaling, Rac signaling and RhoA signaling were activated in CKDu patients. The most significantly activated biological functions were cell death, cell movement and antimicrobial response. Significant toxicological functions were mitochondrial dysfunction, oxidative stress and apoptosis. Based on the molecular pathway analysis in CKDu patients and review of literature, viral infections and fluoride toxicity appear to be contributing to the molecular mechanisms underlying CKDu.

Peptidoglycan Suppresses Phagocytic Activities and Apoptosis of Macrophages in Colonic Mucosa Tissues of Crohn's Disease Patients and In Vitro.

BackgroundRac1 signaling plays a crucial role in controlling macrophage functions in CD. Peptidoglycan triggers several intracellular signaling pathways, including activation of Rac1, to regulate the function of macrophage. Suppressed Rac1 signaling in non-inflamed colonic mucosa of Crohn’s disease patients has been shown to correlate with increased innate immunity.Material/MethodsWe examined the effect of peptidoglycan on Rac1 signaling in macrophages and mucosal tissue samples collected from 10 patients with active Crohn’s disease and further investigated the effects of peptidoglycan on apoptosis and phagocytic activities of macrophages in vitro.ResultsMacrophage infiltration and Rac1 signaling was increased in inflamed mucosal tissues of Crohn’s disease patients. Immunoblotting assays revealed that peptidoglycan dose- and time-dependently increased the expression of Rac1-GTP, phosphorylated VAV1, and phosphorylated PAK1in RAW264.7 macrophages, which, however, was attenuated by 6-thioguanine. Peptidoglycan also dose-dependently inhibited phagocytic activities of human peripheral blood monocytic cells (PBMCs), which were partially abated by 6-thioguanine or NSC23766. Flow cytometry showed that peptidoglycan (3 μg/mL) decreased the proportion of apoptotic human PBMCs versus controls. The addition of 6-thioguanine or NSC3766 to peptidoglycan led to a sharper rise in the proportion of apoptotic human PBMCs than 6-thioguanine or NSC3766 alone.ConclusionsOur findings suggest that Rac1 signaling is a common molecular target shared by peptidoglycan and immunosuppressive treatment in intestinal macrophages. Inhibiting Rac1 activation may be crucial for optimizing macrophage immunity for treatment of Crohn’s disease.

Hyperactivity of Rac1-GTPase pathway impairs neuritogenesis of cortical neurons by altering actin dynamics

The small-GTPase Rac1 is a key molecular regulator linking extracellular signals to actin cytoskeleton dynamics. Loss-of-function mutations in RAC1 and other genes of the Rac signaling pathway have been implicated in the pathogenesis of Intellectual Disability (ID). The Rac1 activity is negatively controlled by GAP proteins, however the effect of Rac1 hyperactivity on neuronal networking in vivo has been poorly studied. ArhGAP15 is a Rac-specific negative regulator, expressed in the main subtypes of pyramidal cortical neurons. In the absence of ArhGAP15, cortical pyramidal neurons show defective neuritogenesis, delayed axonal elongation, reduced dendritic branching, both in vitro and in vivo. These phenotypes are associated with altered actin dynamics at the growth cone due to increased activity of the PAK-LIMK pathway and hyperphosphorylation of ADF/cofilin. These results can be explained by shootin1 hypo-phosphorylation and uncoupling with the adhesion system. Functionally, ArhGAP15−/− mice exhibit decreased synaptic density, altered electroencephalographic rhythms and cognitive deficits. These data suggest that both hypo- and hyperactivation of the Rac pathway due to mutations in Rac1 regulators can result in conditions of ID, and that a tight regulation of Rac1 activity is required to attain the full complexity of the cortical networks.

VEGF-neuropilin-2 signaling promotes stem-like traits in breast cancer cells by TAZ-mediated repression of the Rac GAP β2-chimaerin.

The role of vascular endothelial growth factor (VEGF) signaling in cancer is not only well known in the context of angiogenesis but also important in the functional regulation of tumor cells. Autocrine VEGF signaling mediated by its co-receptors called neuropilins (NRPs) appears to be essential for sustaining the proliferation and survival of cancer stem cells (CSCs), which are implicated in mediating tumor growth, progression, and drug resistance. Therefore, understanding the mechanisms involved in VEGF-mediated support of CSCs is critical to successfully treating cancer patients. The expression of the Hippo effector TAZ is associated with breast CSCs and confers stem cell-like properties. We found that VEGF-NRP2 signaling contributed to the activation of TAZ in various breast cancer cells, which mediated a positive feedback loop that promoted mammosphere formation. VEGF-NRP2 signaling activated the GTPase Rac1, which inhibited the Hippo kinase LATS, thus leading to TAZ activity. In a complex with the transcription factor TEAD, TAZ then bound and repressed the promoter of the gene encoding the Rac GTPase-activating protein (Rac GAP) β2-chimaerin. By activating GTP hydrolysis, Rac GAPs effectively turn off Rac signaling; hence, the TAZ-mediated repression of β2-chimaerin resulted in sustained Rac1 activity in CSCs. Depletion of β2-chimaerin in non-CSCs increased Rac1 activity, TAZ abundance, and mammosphere formation. Analysis of a breast cancer patient database revealed an inverse correlation between β2-chimaerin and TAZ expression in tumors. Our findings highlight an unexpected role for β2-chimaerin in a feed-forward loop of TAZ activation and the acquisition of CSC properties.

Neutrophils Provide a Favorable IL-1-Mediated Immunometabolic Niche that Primes GLUT4 Translocation and Performance in Skeletal Muscles

Metabolic immunomodulation involving IL-1 has been investigated for unfavorable metabolic effects, including obesity, but a potentially favorable role for IL-1 remains unclear. Here, we find mechanistic interactions between working skeletal muscles and locally recruited neutrophils expressing IL-1β, which supports muscle performance through priming exercise-dependent GLUT4 translocation. Thus, during exercise, both IL-1α/β-deficient and neutrophil-depleted mice similarly exhibit increased fatigability associated with impaired muscle glucose homeostasis due to GLUT4 dysregulation. Deficiency of IL-1-producing neutrophils results in intrinsic abnormalities represented by aberrant Rac1 signaling and irregular GLUT4-storage vesicles, suggesting that these properties are maintained by local IL-1 produced by recruited neutrophils upon exercise, possibly on a daily basis. We propose that neutrophils are highly engaged in skeletal muscle performance via IL-1 regulation, which coordinates favorable inflammatory microenvironments supporting muscle glucose metabolism.

N‐Cadherin Juxtacrine Signaling Maintains Blood Brain Barrier and Cognitive Function

The blood brain barrier (BBB) consists of a specialized microvasculature with highly restricted permeability to both low and high molecular weight proteins. The BBB plays a critical role in maintaining tissue‐fluid homeostasis and preventing blood‐to‐brain paracellular diffusion of plasma components that can cause neuronal dysfunction. The BBB is composed of brain endothelial cells (BEC), which physically interact with pericytes at the basement membrane. BECs and pericytes express N‐cadherin, which forms homophilic adhesions to establish cross‐communications between these cell types. The role of N‐cadherin in the BBB is not well understood. We have demonstrated that an endothelial specific deletion of N‐cadherin gene (Cdh2) in mice resulted in increased permeability of the BBB in a size dependent manner, suggesting that N‐cadherin juxtacrine signaling in endothelial cells restricts BBB permeability. These changes in the BBB permeability were not associated with pericyte loss since we observed an increase in capillary coverage by pericytes in the cerebral cortex in these mice. To gain a holistic picture of N‐cadherin juxtacrine signaling in maintenance of the BBB, we utilized N‐cadherin biomimetic surfaces to induce signaling in a cell culture model. We found that Trio, a Rho guanine nucleotide exchange factor consisting of two GEFs, is recruited to N‐cadherin adhesions to induce Rac1 signaling through activation of GEF1. Genetic studies in humans provide a causal link between a deficiency of Trio GEF1 activity and neurodevelopmental diseases such as intellectual disability, suggesting that increased vascular leakage and associated neurotoxicity impairs cognitive function. Therefore, we tested whether N‐cadherin deficient mice exhibited cognitive deficit. Using the Morris water navigation task, we demonstrated that loss of N‐cadherin in endothelial cells results in a deficit in spatial learning and memory. This difference to control mice became indistinguishable with age, suggesting that deficiency of N‐cadherin juxtacrine signaling may be an underlying cause for cognitive dysfunction in the elderly population. Hence, mice lacking Cdh2 gene in endothelial cells may serve as an animal model to study vascular leakage and cognitive function in aging.Support or Funding InformationNIH T32 HL027829, NIH R01 HL103922This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of Rac1 signaling pathway of azathioprine and peptidoglycan in the regulation of monocyte-macrophage apoptosis in Crohn's disease

Objective: To evaluate the changes of macrophages and expression of Rac1 in the inflammatory site of Crohn's disease, and to investigate the effects of 6-thioguanine (6-TG) and peptidoglycan on apoptosis of human peripheral blood monocyte-macrophage by regulating Rac1 signaling pathway. Methods: Ten patients with Crohn's disease and eight healthy controls diagnosed were enrolled at Department of Gastroenterology and Hepatology, Tianjin Medical University General Hospital from January 2013 to January 2014. The number of macrophages, apoptosis and expression of Rac1 in the inflammation sites and non-inflammation sites of intestinal mucosa were detected in both patients and controls. Peripheral blood mononuclear cells (PBMCs) were sorted by CD14 immunomagnetic beads. The apoptosis of monocytes, expression of Rac1 and related apoptosis signaling molecules were detected in patients treated with peptidoglycan, 6-TG and Rac1 inhibitor NSC23766 and another 15 healthy donors. Results: The number of macrophages and apoptotic cells significantly increased in the inflammatory group of Crohn's disease patients compared with the non-inflammatory group. The expression of PAK1, downstream molecular of Rac1 signaling pathway of macrophages was also significantly higher in the inflammatory group of Crohn's disease patients than that in healthy controls and non-inflammatory group. Compared with control group, anti-apoptotic signals (NF-κB, Bcl-xL and STAT-3) in PBMCs increased in the peptidoglycan group, while slightly decreased in 6-TG group. 6-TG and NSC23766 significantly promoted peptidoglycan-related anti-apoptosis [peptidoglycan group (8.6±3.7)%, peptidoglycan+ 6-TG group (42.0±2.7)%, peptidoglycan+ NSC23766 group (58.5±6.9)%, P<0.05]. Conclusions: Peptidoglycan plays a role in the pathogenesis of Crohn's disease by recruiting macrophages. However, 6-TG inhibits peptidoglycan-induced activation of Rac1 signaling pathway leading to macrophage apoptosis.