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Murine leukemia viruses (MuLVs) are simple retroviruses that cause several diseases in mice. Retroviruses encode three basic genes: gag, pol, and env. Gag is translated as a polyprotein and moves to assembly sites where viral particles are shaped by cleavage of poly-Gag. Viral release depends on the intracellular trafficking of viral proteins, which is determined by both viral and cellular factors. ADP-ribosylation factor 6 (Arf6) is a small GTPase that regulates vesicular trafficking and recycling of different types of cargo in cells. Arf6 also activates phospholipase D (PLD) and phosphatidylinositol-4-phosphate 5-kinase (PIP5K) and produces phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). We investigated how Arf6 affected MuLV release with a constitutively active form of Arf6, Arf6Q67L. Expression of Arf6Q67L impaired Gag release by accumulating Gag at PI(4,5)P2-enriched compartments in the cytoplasm. Treatment of the inhibitors for PLD and PIP5K impaired or recovered MuLV Gag release in the cells expressing GFP (control) and Arf6Q67L, implying that regulation of PI(4,5)P2 through PLD and PIP5K affected MuLV release. Interference with the phosphoinositide 3-kinases, mammalian target of rapamycin (mTOR) pathway, and vacuolar-type ATPase activities showed further impairment of Gag release from the cells expressing Arf6Q67L. In contrast, mTOR inhibition increased Gag release in the control cells. The proteasome inhibitors reduced viral release in the cells regardless of Arf6Q67L expression. These data outline the differences in MuLV release under the controlled and overactivated Arf6 conditions and provide new insight into pathways for MuLV release. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Murine leukemia viruses (MuLVs) are simple retroviruses that cause several diseases in mice. Retroviruses encode three basic genes: gag, pol, and env. Gag is translated as a polyprotein and moves to assembly sites where viral particles are shaped by cleavage of poly-Gag. Viral release depends on the intracellular trafficking of viral proteins, which is determined by both viral and cellular factors. ADP-ribosylation factor 6 (Arf6) is a small GTPase that regulates vesicular trafficking and recycling of different types of cargo in cells. Arf6 also activates phospholipase D (PLD) and phosphatidylinositol-4-phosphate 5-kinase (PIP5K) and produces phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). We investigated how Arf6 affected MuLV release with a constitutively active form of Arf6, Arf6Q67L. Expression of Arf6Q67L impaired Gag release by accumulating Gag at PI(4,5)P2-enriched compartments in the cytoplasm. Treatment of the inhibitors for PLD and PIP5K impaired or recovered MuLV Gag release in the cells expressing GFP (control) and Arf6Q67L, implying that regulation of PI(4,5)P2 through PLD and PIP5K affected MuLV release. Interference with the phosphoinositide 3-kinases, mammalian target of rapamycin (mTOR) pathway, and vacuolar-type ATPase activities showed further impairment of Gag release from the cells expressing Arf6Q67L. In contrast, mTOR inhibition increased Gag release in the control cells. The proteasome inhibitors reduced viral release in the cells regardless of Arf6Q67L expression. These data outline the differences in MuLV release under the controlled and overactivated Arf6 conditions and provide new insight into pathways for MuLV release.

Shuo Wang,1 Tianjiao Wang,2 Pengcheng Gu1 1Department of Orthopedics Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310006, People’s Republic of China; 2Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, Zhejiang, 310051, People’s Republic of ChinaCorrespondence: Pengcheng GuDepartment of Orthopedics Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, 79 Qingchun Road, Hangzhou, Zhejiang, 310006, People’s Republic of ChinaTel +86-571-87236848Email 1192014@zju.edu.cnPurpose: Hepatocellular carcinoma (HCC) has the fourth highest rate of mortality among the different types of cancer worldwide. This study aimed to investigate the functions of microRNA-145-5p and AFR6 on migration, invasion and metastasis in HCC.Methods: A total of 150 pairs of tumor and their matched adjacent nontumor liver tissues were collected from HCC patients. Expressions of microRNA-145-5p and AFR6 were measured by real-time PCR in HCC tissues and in HCC cell lines. The correlations between microRNA-145-5p and HCC prognosis were investigated. The proliferation, migration, invasion, cell cycle progression, and apoptosis of HCCLM3 cells were evaluated with CCK8, wound healing, transwell, and flow cytometric experiments.Results: The expression of miR-145-5p was confirmed to be downregulated not only in HCC tissues but also in several HCC cell lines compared with normal controls. A low expression level of miR-145-5p was notably associated with poor prognosis in patients with HCC and certain characteristics of metastatic tumors. In vitro, miR-145-5p negatively regulated cell proliferation, migration, and invasion and induced apoptosis in HCCLM3 cells. Subsequent experiments further verified that ARF6 is a novel target of miR-145-5p and is significantly overexpressed in HCC tissues. Overexpression of ARF6 circumvented the effects of miR-145-5p in HCCLM3 cells.Conclusion: miR-145-5p may play a pivotal role in HCC metastasis via regulating ARF6, and these findings may both provide further insights into the key factors of HCC metastasis and prove to be useful in the development of novel treatment options for HCC.Keywords: microRNA-145-5p, ADP-ribosylation factor 6, hepatocellular carcinoma, proliferation, invasion, metastasis

Diabetic nephropathy (DN), one of the most common complications of Diabetes Mellitus, is the leading cause of end-stage renal diseases worldwide. Our previous study proved that hepatocyte growth factor (HGF) alleviated renal damages in mice with type 1 Diabetes Mellitus by suppressing overproduction of reactive oxygen species (ROS) in podocytes, while the further mechanism of how HGF lessens ROS production had not been clarified yet. ADP-ribosylation factor 6 (ARF6), the member of the small GTPases superfamilies, is widely spread among epithelial cells and can be activated by the HGF/c-Met signaling. Thus, this study was aimed to explore whether HGF could function on mitochondrial homeostasis, the main resource of ROS, in podocytes exposed to diabetic conditions via ARF6 activation. Our in vivo data showed that HGF markedly ameliorated the pathological damages in kidneys of db/db mice, especially the sharp decline of podocyte number, which was mostly blocked by the ARF6 inhibitor SecinH3. Correspondingly, our in vitro data revealed that HGF protected against high glucose-induced podocyte injuries by increasing ARF6 activity. Besides, this ARF6-dependent beneficial effect of HGF on podocytes was accompanied by improved mitochondrial dynamics and declined DRP1 translocation from cytosol to mitochondria. Collectively, our findings confirm the ability of HGF maintaining mitochondrial homeostasis in diabetic podocytes via decreasing ARF6-dependent DRP1 translocation and shed light on the novel mechanism of HGF treatment for DN. • HGF protects mice kidney especially the podocytes from high glucose-induced injury via activating ARF6. • The mitochondrial homeostasis of high glucose-cultured podocytes was improved by the effect of HGF on mitochondrial dynamics. • ARF6 is required for HGF-enhanced DRP1 mitochondrial translocation and its phosphorylation in high glucose-treated podocytes. • ARF6-dependent regulation of mitochondrial dynamics contributes to the beneficial effects of HGF on diabetic nephropathy. [ABSTRACT FROM AUTHOR]

Cocaine is an addictive drug that acts in brain reward areas. Recent evidence suggests that cocaine stimulates synthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) in midbrain, increasing dopamine neuron activity via disinhibition. Although a mechanism for cocaine-stimulated 2-AG synthesis is known, our understanding of 2-AG release is limited. In NG108 cells and mouse midbrain tissue, we find that 2-AG is localized in non-synaptic extracellular vesicles (EVs) that are secreted in the presence of cocaine via interaction with the chaperone protein sigma-1 receptor (Sig-1R). The release of EVs occurs when cocaine causes dissociation of the Sig-1R from ADP-ribosylation factor (ARF6), a G-protein regulating EV trafficking, leading to activation of myosin light chain kinase (MLCK). Blockade of Sig-1R function, or inhibition of ARF6 or MLCK also prevented cocaine-induced EV release and cocaine-stimulated 2-AG-modulation of inhibitory synapses in DA neurons. Our results implicate the Sig-1R-ARF6 complex in control of EV release and demonstrate that cocaine-mediated 2-AG release can occur via EVs.

Background: Experimental evidence demonstrated that endurance training (ET) could be beneficial for the diabetic peripheral neuropathy (DPN), but its mechanism is not clear. Due to the critical role of the ADP-ribosylation factor (Arf6) in the nervous system, the current study aimed at evaluating the effects of ET on gene expression of Arf6 in cerebellum of male Wistar rats with DPN. Methods: Animals were randomly divided into three groups: Control (C), diabetic control (DC), and diabetic trained (DT). Diabetes induction was performed by a single intraperitoneal injection of streptozotocin (STZ). Behavioral nociception assessment was conducted by Von Frey filaments and tail flick tests. Treadmill protocol consisted of six weeks of moderate intensity endurance training. The Arf6 cerebellar gene expression was assessed by the real-time PCR. Results: In the current study, the Arf6 gene expression levels in the DC group were significantly higher than those of the C group (P = 0.003). Also, ET could damp this elevation in diabetic rats (P = 0.004). Conclusions: It seems that Arf6 is an important mediator of stress-induced plasticity in the nervous system in response to ET and neurodegeneration disease, but the exact physiological functions of Arf6 are not clear. Hence, further studies are required to identify these issues. [ABSTRACT FROM AUTHOR]

ADP-ribosylation factor 6 (Arf6), a member of the ADP-ribosylation factor family, is overexpressed in different types of cancer cell and promotes invasion, metastasis and drug resistance. However, the potential functions of Arf6 in gastric cancer (GC), and the molecular mechanism underlying these functions, remain to be fully elucidated. In the present study, the results demonstrated that in vitro knockdown of Arf6 decreased proliferation, colony formation, migration and invasion in SGC-7901 cells. Arf6 knockdown also markedly decreased the activity of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. Furthermore, knockdown of Arf6 was associated with elevated chemosensitivity of SGC-7901 cells to 5-fluorouracil through inactivation of the ERK1/2 signaling pathway. Taken together, these results suggest that Arf6 is involved in regulating proliferation, migration, invasion and drug resistance in GC, and may be a potential therapeutic target for the treatment of GC. [ABSTRACT FROM AUTHOR]

Though significant progress has been made in the availability of diagnostic techniques and treatment strategies, pancreatic cancer remains a disease of high mortality rates. Therefore, there is an urgent need for a better understanding of the molecular mechanisms that governs the oncogenesis and metastasis process of pancreatic cancer. In our study, by using the Cancer Genome Atlas (TCGA) dataset analysis, we demonstrated that the small guanosine triphosphatase (GTPase) ADP-ribosylation factor 6 (ARF6) serves as a biomarker for predicting prognosis of pancreatic cancer. In vitro studies demonstrated that silencing ARF6 expression reduced cell proliferation and attenuated the Warburg effect. Moreover, we observed that ARF6 was a downstream target of Kras/ERK signaling pathway, and the strong correlation of expression between Kras and ARF6 in the TCGA dataset further confirmed this observation. Taken together, our novel findings suggest ARF6, a target of mutant Kras, may promote pancreatic cancer development by enhancing the Warburg effect. [ABSTRACT FROM AUTHOR]

ADP-ribosylation factor 6 ( ARF6) is an evolutionarily conserved molecule that has an essential function in intracellular trafficking and organelle structure. To better understand its role during presymbiosis between plant roots and compatible filamentous fungi, the full-length cDNA sequence of ARF6 from Pisolithus tinctorius was cloned and a variety of bioinformatics analyses performed. The full-length sequence was 849 bp long and contained a 549 bp open reading frame encoding a protein of 182 amino acids. A phylogenetic analysis showed that ptARF6 was the ortholog of the ADP ribosylation factor 6/GTPase SAR1 gene from the white-rot basidiomycete Trametes versicolor. A domain architecture analysis of the ARF6 protein revealed a repeat region, which is a common feature of ARF6 in other species. Recombinant ARF6 protein was expressed with an N-terminal 6×His tag and purified using Ni2+-NTA affinity chromatography. The molecular mass of the recombinant protein was estimated by SDS-PAGE to be 25 kDa. The recombinant ARF6 protein bound strongly to 18:1 and 18:2 phosphatidic acids. Thus, ARF6 may participate in the signaling pathways involved in membrane phospholipid composition. The intracellular distribution of ptADP6 in HEK239T cells also indicates that ptADP6 may function not only in plasma membrane events but also in endosomal membranes events. Real-time quantitative PCR revealed that the differential expression of ptARF6 was associated with the presymbiotic stage. ptARF6 may be induced by presymbiosis during the regulation of mycorrhizal formation. [ABSTRACT FROM AUTHOR]

SynArfGEF, also known as BRAG3 or IQSEC3, is a member of the brefeldin A-resistant Arf-GEF/IQSEC family and was originally identified by screening for mRNA species associated with the post-synaptic density fraction. In this study, we demonstrate that synArfGEF activates Arf6, using Arf pull down and transferrin incorporation assays. Immunohistochemical analysis reveals that synArfGEF is present in somata and dendrites as puncta in close association with inhibitory synapses, whereas immunoelectron microscopic analysis reveals that synArfGEF localizes preferentially at post-synaptic specializations of symmetric synapses. Using yeast two-hybrid and pull down assays, we show that syn ArfGEF is able to bind utrophin/dystrophin and S-SCAM/ MAGI-2 scaffolding proteins that localize at inhibitory synapses. Double immunostaining reveals that synArfGEF co-localizes with dystrophin and S-SCAM in cultured hippocampal neurons and cerebellar cortex, respectively. Both b-dystroglycan and S-SCAM were immunoprecipitated from brain lysates using anti-synArfGEF IgG. Taken together, these findings suggest that synArfGEF functions as a novel regulator of Arf6 at inhibitory synapses and associates with the dystrophin-associated glycoprotein complex and S-SCAM. [ABSTRACT FROM AUTHOR]

Abstract: IQ-ArfGEF/BRAG1, a guanine nucleotide exchange factor for Arf1 and Arf6, is localized at the postsynaptic density (PSD) and interacts with PSD-95. In this study, we identified a novel interaction of IQ-ArfGEF/BRAG1 with insulin receptor tyrosine kinase substrate of 53 kDa (IRSp53), also known as brain-specific angiogenesis inhibitor 1-associated protein 2. The interaction was mediated by the binding of the C-terminal proline-rich sequence of IQ-ArfGEF/BRAG1 to the SH3 domain of IRSp53. IQ-ArfGEF/BRAG1 and IRSp53 were colocalized at the PSD of excitatory synapses of certain neuronal populations. Our present findings suggest that IQ-ArfGEF/BRAG1 may play roles downstream of NMDA receptors through the interaction with multivalent PSD proteins such as IRSp53 and PSD-95. [Copyright &y& Elsevier]

Abstract: The EFA6 family is a member of guanine nucleotide exchange factors (GEFs) that can activate ARF6 specifically in vitro. In this study, we determined the complete primary sequence of mouse EFA6D encoding a protein of 1004 amino acids with a calculated molecular weight of 111,207 Da. In ARF pull-down assay, EFA6D showed a preferential GEF activity toward ARF6. RT-PCR analysis revealed the widespread tissue distribution of EFA6D and the high expression of EFA6A, C and D in the brain. In situ hybridization analysis demonstrated a distinct spatiotemporal expression pattern of EFA6D from those of EFA6A and C in mouse brain. Furthermore, immunoblot analysis revealed that EFA6D was highly concentrated in the postsynaptic density fraction. These findings suggest differential spatiotemporal regulation of ARF6 by three members of the EFA6 family in the brain. [Copyright &y& Elsevier]

Abstract: Actin cytoskeletal reorganization and membrane trafficking are important for spine morphogenesis. Here we investigated whether the small GTPase, ADP-ribosylation factor 6 (ARF6), which regulates actin dynamics and peripheral vesicular trafficking, is involved in the regulation of spine formation. The developmental expression pattern of ARF6 in mouse hippocampus was similar to that of the post-synaptic density protein-95, and these molecules colocalized in mouse hippocampal neurons. Overexpression of a constitutively active ARF6 mutant in cultured hippocampal neurons decreased the spine density, whereas a dominant-negative ARF6 mutant increased the density. These results demonstrate a novel function for ARF6 as a key regulator of spine formation. [Copyright &y& Elsevier]

Previous studies have reported that the expression levels of microRNA (miR)-28-3p are downregulated in prostate cancer (PCa) compared with those in adjacent normal tissues. However, to the best of our knowledge, the function and underlying mechanisms of miR-28-3p in PCa have not been reported. The present study aimed to explore the role of miR-28-3p and its mechanism in the development of PCa. In the present study, miR-28-3p and ADP-ribosylation factor 6 (ARF6) expression levels were analyzed using reverse transcription-quantitative PCR (RT-qPCR). Cell proliferation, colony formation, apoptosis, migration and invasion were determined using Cell Counting Kit-8, colony forming, flow cytometry and Transwell assays, respectively. The association between miR-28-3p and ARF6 was investigated using a dual luciferase reporter assay. ARF6, Rac1, Erk1/2 and phosphorylated (p)-Erk1/2 protein expression levels were analyzed using western blotting. The results of the present study revealed that miR-28-3p expression levels were downregulated, whereas ARF6 expression levels were upregulated in PCa cell lines (LNCaP, 22Rv-1, PC-3 and DU145) compared with those in the normal prostate line RWPE-1. The overexpression of miR-28-3p promoted cell apoptosis, and inhibited cell proliferation, colony formation, migration and invasion. However, the knockdown of miR-28-3p exerted the opposite results. The results of the dual luciferase reporter assays, RT-qPCR and western blotting indicated that ARF6 was a target gene of miR-28-3p. Finally, rescue experiments demonstrated that ARF6 overexpression attenuated the effects of the miR-28-3p mimic by upregulating Rac1 and p-Erk1/2 expression in PCa cells. In conclusion, these findings indicated that miR-28-3p may inhibit the biological behaviors of PCa cells by targeting ARF6, and therefore may represent a novel therapeutic candidate for PCa. [ABSTRACT FROM AUTHOR]

Nucleic acid cellular uptake into endosomes is critical in eliciting nucleotide-sensing toll-like receptors (TLRs) innate immune responses. ADP-ribosylation factor 6 (ARF6) is a member of the Ras superfamily, which is critical to a wide variety of cellular events including endocytosis. Our previous report indicated that ARF6 plays a critical role in CpG ODN/TLR9-mediated responses. Here, we further explored that the basal level of active ARF6 is nonspecifically responsible for initiation of ODNs uptake, which is relatively CpG motif independent. While the initiation of CpG ODN uptake but not GpC ODN uptake can promote TLR9 responses thereby enhancing ARF6 activation which may lead to further nonspecifically increase of cellular uptake of stimulatory CpG ODN as well as nonstimulatory GpC ODN. Because nucleotide-sensing TLR9 plays a role in contributing to immune diseases, selective activation or inhibition of ARF6 might be useful in certain immunological or therapeutic applications. [ABSTRACT FROM AUTHOR]