Endothelial-Specific Reduction in Arf6 Impairs Insulin-Stimulated Vasodilation and Skeletal Muscle Blood Flow Resulting in Systemic Insulin Resistance in Mice.

BACKGROUND: Much of what we know about insulin resistance is based on studies from metabolically active tissues such as the liver, adipose tissue, and skeletal muscle. Emerging evidence suggests that the vascular endothelium plays a crucial role in systemic insulin resistance; however, the underlying mechanisms remain incompletely understood. Arf6 (ADP ribosylation factor 6) is a small GTPase that plays a critical role in endothelial cell function. Here, we tested the hypothesis that the deletion of endothelial Arf6 will result in systemic insulin resistance. METHODS: We used mouse models of constitutive endothelial cell-specific Arf6 deletion (Arf6f/- Tie2Cre+) and tamoxifen-inducible Arf6 knockout (Arf6f/f Cdh5CreER+). Endothelium-dependent vasodilation was assessed using pressure myography. Metabolic function was assessed using a battery of metabolic assessments including glucose and insulin tolerance tests and hyperinsulinemic-euglycemic clamps. We used a fluorescence microsphere-based technique to measure tissue blood flow. Skeletal muscle capillary density was assessed using intravital microscopy. RESULTS: Endothelial Arf6 deletion impaired insulin-stimulated vasodilation in white adipose tissue and skeletal muscle feed arteries. The impairment in vasodilation was primarily due to attenuated insulin-stimulated nitric oxide bioavailability but independent of altered acetylcholine-mediated or sodium nitroprusside-mediated vasodilation. Endothelial cell-specific deletion of Arf6 also resulted in systematic insulin resistance in normal chow-fed mice and glucose intolerance in high-fat diet-fed obese mice. The underlying mechanisms of glucose intolerance were reductions in insulin-stimulated blood flow and glucose uptake in the skeletal muscle and were independent of changes in capillary density or vascular permeability. CONCLUSIONS: Results from this study support the conclusion that endothelial Arf6 signaling is essential for maintaining insulin sensitivity. Reduced expression of endothelial Arf6 impairs insulin-mediated vasodilation and results in systemic insulin resistance. These results have therapeutic implications for diseases that are associated with endothelial cell dysfunction and insulin resistance such as diabetes.

Enterovirus 71 enters human brain microvascular endothelial cells through an ARF6-mediated endocytic pathway.

Infection of the central nervous system caused by enterovirus 71 (EV71) remains the main cause of death in hand-foot-and-mouth disease. However, the mechanism responsible for how EV71 breaks through the blood-brain barrier to infect brain cells has yet to be elucidated. By performing a high-throughput small interfering RNA (siRNA) screening and validation, we found that the infection of human brain microvascular endothelial cells (HBMECs) by EV71 was independent of the endocytosis pathways mediated by caveolin, clathrin, and macropinocytosis but dependent on ADP-ribosylation factor 6 (ARF6), a small guanosinetriphosphate (GTP)-binding protein of the Ras superfamily. The specific siRNA targeting ARF6 markedly inhibited HBMECs susceptibility to EV71. EV71 infectivity was inhibited by NAV-2729, a specific inhibitor of ARF6, in a dose-dependent manner. The subcellular analysis demonstrated the co-localization of the endocytosed EV71 and ARF6, while knockdown of ARF6 with siRNA remarkably influenced EV71 endocytosis. By immunoprecipitation assays, we found a direct interaction of ARF6 with EV71 viral protein. Furthermore, ARF1, another small GTP-binding protein, was also found to participate in ARF6-mediated EV71 endocytosis. Murine experiments demonstrated that NAV-2729 significantly alleviated mortality caused by EV71 infection. Our study revealed a new pathway by which EV71 enters the HBMECs and provides new targets for drug development.

[Arf6 regulates endometriotic epithelial-mesenchymal transition and mitochondrial distribution].

Objective: To investigate the role of adenosine diphosphate ribosylation factor 6 (Arf6) in the pathogenesis of endometriosis. Methods: Endometrial tissues were sampled from women who were hospitalized in the Affiliated Hospital of Medical School of Ningbo University and Ningbo Women and Children's Hospital from November 2020 to May 2021 with endometriosis (n=44, endometriosis group) and without endometriosis (n=17, control group). The expression of Arf6 protein in the endometrial tissues was detected by western blot. Endometrial epithelial cells from both groups were primary cultured and the distribution of intracellular mitochondria was detected by immunofluorescence. The expression of Arf6 protein was down-regulated by small interference RNA (siRNA), the distribution of mitochondria in cells with decreased Arf6 protein expression was observed, and the expression of mitochondria-related proteins development and differentiation enhancing factor 1 (DDEF1, also called AMAP1), reactive oxygen species 1 (ROS1) and epithelial-mesenchymal transition (EMT)-related proteins E-cadherin, vimentin were detected. Transwell assay was used to detect the changes in the migration ability of the cells. Results: Compared with the control group, ectopic endometrial tissue of endometriosis group showed high expression of Arf6 protein (0.174±0.019 vs 0.423±0.033; t=29.630, P<0.01); and in ectopic endometrial epithelial cells, mitochondria were distributed near the edge of the cell membrane. While Arf6 expression was down-regulated by siRNA, the distribution of mitochondria in ectopic cells returned to natural, close to the control level. In addition, the expression levels of AMAP1 and ROS1 in ectopic cells after Arf6 protein knockdown were significantly decreased. Transwell assay results indicated that knockdown of Arf6 could reduce the migration ability of ectopic epithelial cells [migration cell count: (34.3±7.5) cells]; and immunofluorescence verified low expression of E-cadherin but high expression of vimentin in ectopic epithelial cells, whereas knockdown of Arf6 protein E-cadherin expression increased but vimentin expression decreased. Conclusions: High expression of Arf6 protein in ectopic endometrial epithelial cells leads to the distribution of mitochondria tending to membrane marginalization, while inducing EMT, which are involved in the mechanism of endoheterosis pathogenesis.

Activation of the GTPase ARF6 regulates invasion of human vascular smooth muscle cells by stimulating MMP14 activity.

Hormones and growth factors stimulate vascular smooth muscle cells (VSMC) invasive capacities during the progression of atherosclerosis. The GTPase ARF6 is an important regulator of migration and proliferation of various cell types, but whether this small G protein can be activated by a variety of stimuli to promote invasion of VSMC remains unknown. Here, we aimed to define whether Platelet-derived growth factor (PDGF), a mitogenic stimulant of vascular tissues, and Angiotensin II (Ang II), a potent vasoactive peptide, can result in the activation of ARF6 in a human model of aortic SMC (HASMC). We demonstrate that these two stimuli can promote loading of GTP on this ARF isoform. Knockdown of ARF6 reduced the ability of both PDGF and Ang II to promote invasion suggesting that this GTPase regulates key molecular mechanisms mediating degradation of the extracellular matrix and migration. We report that PDGF-BB-mediated stimulation of ARF6 results in the activation of the MAPK/ERK1/2, PI3K/AKT and PAK pathways essential for invasion of HASMC. However, Ang II-mediated stimulation of ARF6 only promotes signaling through the MAPK/ERK1/2 and PAK pathways. These ARF6-mediated events lead to activation of MMP14, a membrane-bound collagenase upregulated in atherosclerosis. Moreover, ARF6 depletion decreases the release of MMP2 in the extracellular milieu. Altogether, our findings demonstrate that the GTPase ARF6 acts as a molecular switch to regulate specific signaling pathways that coordinate invasiveness of HASMC.

ADP-ribosylation factor 6 expression increase in oesophageal adenocarcinoma suggests a potential biomarker role for it.

ADP-ribosylation factor 6 small GTPase plays an important role in cell migration, invasion and angiogenesis, which are the hallmarks of cancer. Although alterations in ARF6 expression and activity have been linked to metastatic cancer in one or two tissues, the expression of ARF6 in cancers over a wide range of tissues has not been studied so far. In this report, we analysed the expression of ARF6 mRNA in cancers and corresponding healthy controls from 17 different tissues by real-time qualitative polymerase chain reaction (RT-qPCR). We further evaluated ARF6 protein expression in oesophageal adenocarcinoma (EAC) tissue microarray cores by immunohistochemistry. The ARF6 gene expression levels are highly variable between healthy and cancer tissues. Our findings suggest that the ARF6 gene expression is up-regulated highest in oesophageal cancer. In EAC TMAs, ARF6 protein expression increase correlated with EAC progression. This is the first study to investigate ARF6 gene expression in a wide array of cancer tissues and demonstrate that ARF6 expression, at both mRNA and protein levels, is significantly upregulated in higher grades of EAC, which may be useful in targeting ARF6 for cancer diagnostic and therapeutic purposes.

Arf6 anchors Cdr2 nodes at the cell cortex to control cell size at division.

Fission yeast cells prevent mitotic entry until a threshold cell surface area is reached. The protein kinase Cdr2 contributes to this size control system by forming multiprotein nodes that inhibit Wee1 at the medial cell cortex. Cdr2 node anchoring at the cell cortex is not fully understood. Through a genomic screen, we identified the conserved GTPase Arf6 as a component of Cdr2 signaling. Cells lacking Arf6 failed to divide at a threshold surface area and instead shifted to volume-based divisions at increased overall size. Arf6 stably localized to Cdr2 nodes in its GTP-bound but not GDP-bound state, and its guanine nucleotide exchange factor (GEF), Syt22, was required for both Arf6 node localization and proper size at division. In arf6Δ mutants, Cdr2 nodes detached from the membrane and exhibited increased dynamics. These defects were enhanced when arf6Δ was combined with other node mutants. Our work identifies a regulated anchor for Cdr2 nodes that is required for cells to sense surface area.

MiR-1299 functions as a tumor suppressor to inhibit the proliferation and metastasis of gastric cancer by targeting ARF6.

BACKGROUND: MiRNAs belong to non-coding RNAs that are involved in cancer development. Acting as a mediator, they could regulate the expression level of numerous gens. However, the expression and function of miR-1299 in gastric cancer (GC) are not clear. OBJECTIVE: To explore the role of miR-1299 in the process of GC. METHODS: We detected the levels of miR-1299 in clinical samples of GC and investigated the role of miR-1299 in the regulation of the GC cells proliferation, apoptosis and metastasis. Luciferase reporter assay was employed to verify the target of miR-1299. Additionally, the proliferation, apoptosis and metastasis of AGS and SGC7901 cells were analyzed after the overexpression of miR-1299. RESULTS: Our study showed the expression of miR-1299 was decreased in GC tissues and cell lines. It indicated that the cell proliferation, migration and invasion was inhibited, while the cell apoptosis was promoted by the over-expressed miR-1299. Also, we found that miR-1299 could directly target the 3'-untranslated region (3'UTR) of ARF6 genes. In addition, rescue assay demonstrated that miR-1299 overexpression promoted the cell apoptosis and inhibited cell growth, which could be attenuated by the overexpression of ARF6. CONCLUSIONS: These findings indicate that miR-1299 regulates cell progression in GC by targeting ARF6 genes, and suggest that miR-1299 may be a tumor suppressor in the GC progression.

Control of cell signaling by Arf GTPases and their regulators: Focus on links to cancer and other GTPase families.

The ADP-ribosylation factors (Arfs) comprise a family of regulatory GTP binding proteins. The Arfs regulate membrane trafficking and cytoskeleton remodeling, processes critical for eukaryotes and which have been the focus of most studies on Arfs. A more limited literature describes a role in signaling and in integrating several signaling pathways to bring about specific cell behaviors. Here, we will highlight work describing function of Arf1, Arf6 and several effectors and regulators of Arfs in signaling.

A Brucella effector modulates the Arf6-Rab8a GTPase cascade to promote intravacuolar replication.

Remodeling of host cellular membrane transport pathways is a common pathogenic trait of many intracellular microbes that is essential to their intravacuolar life cycle and proliferation. The bacterium Brucella abortus generates a host endoplasmic reticulum-derived vacuole (rBCV) that supports its intracellular growth, via VirB Type IV secretion system-mediated delivery of effector proteins, whose functions and mode of action are mostly unknown. Here, we show that the effector BspF specifically promotes Brucella replication within rBCVs by interfering with vesicular transport between the trans-Golgi network (TGN) and recycling endocytic compartment. BspF targeted the recycling endosome, inhibited retrograde traffic to the TGN, and interacted with the Arf6 GTPase-activating Protein (GAP) ACAP1 to dysregulate Arf6-/Rab8a-dependent transport within the recycling endosome, which resulted in accretion of TGN-associated vesicles by rBCVs and enhanced bacterial growth. Altogether, these findings provide mechanistic insight into bacterial modulation of membrane transport used to promote their own proliferation within intracellular vacuoles.

Arf6 exacerbates allergic asthma through cell-to-cell transmission of ASC inflammasomes.

Asthma is a chronic inflammatory disease of the airways associated with excess production of Th2 cytokines and lung eosinophil accumulation. This inflammatory response persists in spite of steroid administration that blocks autocrine/paracrine loops of inflammatory cytokines, and the detailed mechanisms underlying asthma exacerbation remain unclear. Here, we show that asthma exacerbation is triggered by airway macrophages through a prion-like cell-to-cell transmission of extracellular particulates, including ASC protein, that assemble inflammasomes and mediate IL-1ß production. OVA-induced allergic asthma and associated IL-1ß production were alleviated in mice with small GTPase Arf6-deficient macrophages. The extracellular ASC specks were slightly engulfed by Arf6-/- macrophages, and the IL-1ß production was reduced in Arf6-/- macrophages compared with that in WT macrophages. Furthermore, pharmacological inhibition of the Arf6 guanine nucleotide exchange factor suppressed asthma-like allergic inflammation in OVA-challenged WT mice. Collectively, the Arf6-dependent intercellular transmission of extracellular ASC specks contributes to the amplification of allergic inflammation and subsequent asthma exacerbation.

Cytohesin-2 mediates group I metabotropic glutamate receptor-dependent mechanical allodynia through the activation of ADP ribosylation factor 6 in the spinal cord.

Group I metabotropic glutamate receptors (mGluRs), mGluR1 and mGluR5, in the spinal cord are implicated in nociceptive transmission and plasticity through G protein-mediated second messenger cascades leading to the activation of various protein kinases such as extracellular signal-regulated kinase (ERK). In this study, we demonstrated that cytohesin-2, a guanine nucleotide exchange factor for ADP ribosylation factors (Arfs), is abundantly expressed in subsets of excitatory interneurons and projection neurons in the superficial dorsal horn. Cytohesin-2 is enriched in the perisynapse on the postsynaptic membrane of dorsal horn neurons and forms a protein complex with mGluR5 in the spinal cord. Central nervous system-specific cytohesin-2 conditional knockout mice exhibited reduced mechanical allodynia in inflammatory and neuropathic pain models. Pharmacological blockade of cytohesin catalytic activity with SecinH3 similarly reduced mechanical allodynia and inhibited the spinal activation of Arf6, but not Arf1, in both pain models. Furthermore, cytohesin-2 conditional knockout mice exhibited reduced mechanical allodynia and ERK1/2 activation following the pharmacological activation of spinal mGluR1/5 with 3,5-dihydroxylphenylglycine (DHPG). The present study suggests that cytothesin-2 is functionally associated with mGluR5 during the development of mechanical allodynia through the activation of Arf6 in spinal dorsal horn neurons.

Pals1 prevents Rac1-dependent colorectal cancer cell metastasis by inhibiting Arf6.

Loss of apical-basal polarity and downregulation of cell-cell contacts is a critical step during the pathogenesis of cancer. Both processes are regulated by the scaffolding protein Pals1, however, it is unclear whether the expression of Pals1 is affected in cancer cells and whether Pals1 is implicated in the pathogenesis of the disease.Using mRNA expression data and immunostainings of cancer specimen, we show that Pals1 is frequently downregulated in colorectal cancer, correlating with poorer survival of patients. We further found that Pals1 prevents cancer cell metastasis by controlling Rac1-dependent cell migration through inhibition of Arf6, which is independent of the canonical binding partners of Pals1. Loss of Pals1 in colorectal cancer cells results in increased Arf6 and Rac1 activity, enhanced cell migration and invasion in vitro and increased metastasis of transplanted tumor cells in mice. Thus, our data reveal a new function of Pals1 as a key inhibitor of cell migration and metastasis of colorectal cancer cells. Notably, this new function is independent of the known role of Pals1 in tight junction formation and apical-basal polarity.

Inhibition of mutant KRAS-driven overexpression of ARF6 and MYC by an eIF4A inhibitor drug improves the effects of anti-PD-1 immunotherapy for pancreatic cancer.

Many clinical trials are being conducted to clarify effective combinations of various drugs for immune checkpoint blockade (ICB) therapy. However, although extensive studies from multiple aspects have been conducted regarding treatments for pancreatic ductal adenocarcinoma (PDAC), there are still no effective ICB-based therapies or biomarkers for this cancer type. A series of our studies have identified that the small GTPase ARF6 and its downstream effector AMAP1 (also called ASAP1/DDEF1) are often overexpressed in different cancers, including PDAC, and closely correlate with poor patient survival. Mechanistically, the ARF6-AMAP1 pathway drives cancer cell invasion and immune evasion, via upregulating ß1-integrins and PD-L1, and downregulating E-cadherin, upon ARF6 activation by external ligands. Moreover, the ARF6-AMAP1 pathway enhances the fibrosis caused by PDAC, which is another barrier for ICB therapies. KRAS mutations are prevalent in PDACs. We have shown previously that oncogenic KRAS mutations are the major cause of the aberrant overexpression of ARF6 and AMAP1, in which KRAS signaling enhances eukaryotic initiation factor 4A (eIF4A)-dependent ARF6 mRNA translation and eIF4E-dependent AMAP1 mRNA translation. MYC overexpression is also a key pathway in driving cancer malignancy. MYC mRNA is also known to be under the control of eIF4A, and the eIF4A inhibitor silvestrol suppresses MYC and ARF6 expression. Using a KPC mouse model of human PDAC (LSL-Kras(G12D/+); LSL-Trp53(R172H/+)); Pdx-1-Cre), we here demonstrate that inhibition of the ARF6-AMAP1 pathway by shRNAs in cancer cells results in therapeutic synergy with an anti-PD-1 antibody in vivo; and furthermore, that silvestrol improves the efficacy of anti-PD-1 therapy, whereas silvestrol on its own promotes tumor growth in vivo. ARF6 and MYC are both essential for normal cell functions. We demonstrate that silvestrol substantially mitigates the overexpression of ARF6 and MYC in KRAS-mutated cells, whereas the suppression is moderate in KRAS-intact cells. We propose that targeting eIF4A, as well as mutant KRAS, provides novel methods to improve the efficacy of anti-PD-1 and associated ICB therapies against PDACs, in which ARF6 and AMAP1 overexpression, as well as KRAS mutations of cancer cells are biomarkers to identify patients with drug-susceptible disease. The same may be applicable to other cancers with KRAS mutations. Video abstract.

Rab22a regulates the establishment of epithelial polarity.

Membrane trafficking establishes and maintains epithelial polarity. Rab22a has a polarized distribution in activated T-cells, but its role in epithelial polarity has not been investigated. We showed previously that Rab14 acts upstream of Arf6 to establish the apical membrane initiation site (AMIS), but its interaction with Rab22a is unknown. Here we show that Rab14 and Rab22a colocalize in endosomes of both unpolarized and polarized MDCK cells and Rab22a localizes to the cell:cell interface of polarizing cell pairs. Knockdown of Rab22a results in a multi-lumen phenotype in three-dimensional culture. Further, overexpression of Rab22a in Rab14 knockdown cells rescues the multi-lumen phenotype observed with Rab14 knockdown, suggesting that Rab22a is downstream of Rab14. Because of the relationship between Rab14 and Arf6, we investigated the effect of Rab22a knockdown on Arf6. We find that Rab22a knockdown results in decreased active Arf6 and that Rab22a co-immunoprecipitates with the Arf6 GEF EFA6. In addition, EFA6 is retained in intracellular puncta in Rab22a KD cells. These results suggest that Rab22a acts downstream of Rab14 to traffic EFA6 to the AMIS to regulate Arf6 in the establishment of polarity.

Cytohesin-2/ARNO: A Novel Bridge Between Cell Migration and Immunoregulation in Synovial Fibroblasts.

The guanine nucleotide exchange factor cytohesin-2 (ARNO) is a major activator of the small GTPase ARF6 that has been shown to play an important role(s) in cell adhesion, migration and cytoskeleton reorganization in various cell types and models of disease. Interestingly, dysregulated cell migration, in tandem with hyper-inflammatory responses, is one of the hallmarks associated with activated synovial fibroblasts (SFs) during chronic inflammatory joint diseases, like rheumatoid arthritis. The role of ARNO in this process has previously been unexplored but we hypothesized that the pro-inflammatory milieu of inflamed joints locally induces activation of ARNO-mediated pathways in SFs, promoting an invasive cell phenotype that ultimately leads to bone and cartilage damage. Thus, we used small interference RNA to investigate the impact of ARNO on the pathological migration and inflammatory responses of murine SFs, revealing a fully functional ARNO-ARF6 pathway which can be rapidly activated by IL-1ß. Such signalling promotes cell migration and formation of focal adhesions. Unexpectedly, ARNO was also shown to modulate SF-inflammatory responses, dictating their precise cytokine and chemokine expression profile. Our results uncover a novel role for ARNO in SF-dependent inflammation, that potentially links pathogenic migration with initiation of local joint inflammation, offering new approaches for targeting the fibroblast compartment in chronic arthritis and joint disease.