リンパ管新生における低分子量Gタンパク質Arf6の機能

筑波大学 (University of Tsukuba)201

Brag2 differentially regulates β1- and β3-integrin-dependent adhesion in endothelial cells and is involved in developmental and pathological angiogenesis

β1-Integrins are essential for angiogenesis. The mechanisms regulating integrin function in endothelial cells (EC) and their contribution to angiogenesis remain elusive. Brag2 is a guanine nucleotide exchange factor for the small Arf-GTPases Arf5 and Arf6. The role of Brag2 in EC and angiogenesis and the underlying molecular mechanisms remain unclear. siRNA-mediated Brag2-silencing reduced EC angiogenic sprouting and migration. Brag2-siRNA transfection differentially affected α5β1- and αVβ3-integrin function: specifically, Brag2-silencing increased focal/fibrillar adhesions and adhesion on β1-integrin ligands (fibronectin and collagen), while reducing the adhesion on the αVβ3-integrin ligand, vitronectin. Consistent with these results, Brag2-silencing enhanced surface expression of α5β1-integrin, while reducing surface expression of αVβ3-integrin. Mechanistically, Brag2-mediated αVβ3-integrin-recycling and β1-integrin endocytosis and specifically of the active/matrix-bound α5β1-integrin present in fibrillar/focal adhesions (FA), suggesting that Brag2 contributes to the disassembly of FA via β1-integrin endocytosis. Arf5 and Arf6 are promoting downstream of Brag2 angiogenic sprouting, β1-integrin endocytosis and the regulation of FA. In vivo silencing of the Brag2-orthologues in zebrafish embryos using morpholinos perturbed vascular development. Furthermore, in vivo intravitreal injection of plasmids containing Brag2-shRNA reduced pathological ischemia-induced retinal and choroidal neovascularization. These data reveal that Brag2 is essential for developmental and pathological angiogenesis by promoting EC sprouting through regulation of adhesion by mediating β1-integrin internalization and link for the first time the process of β1-integrin endocytosis with angiogenesis.Deutsche Forschungsgemeinschaft. Transregional Collaborative Research Centre. (SFB/TR23)Deutsche Forschungsgemeinschaft. Transregional Collaborative Research Centre. (Project A2)Deutsche Forschungsgemeinschaft. Transregional Collaborative Research Centre.(Project Z5)Else Kroner-Fresenius-Stiftung (2013_A2

Cytohesin 2/ARF6 regulates preadipocyte migration through the activation of ERK1/2

Preadipocyte migration is vital for the development of adipose tissue, which plays a crucial role in lipid metabolism. ADP-ribosylation factor 6 (ARF6) small GTPase, which regulates membrane trafficking, is activated by GTP-exchange factors (GEFs) such as cytohesin 2. Cytohesin 2 and ARF6 have previously been implicated in the regulation of 3T3-L1 preadipocyte migration. We investigated here the molecular mechanism underlying the cytohesin 2 and ARF6 mediated regulation of preadipocyte migration. Preadipocyte migration and the activation of ARF6 and ERK1/2 were studied by using a number of approaches, including pharmacological inhibitors, siRNA and the inhibitory peptides. The siRNA mediated down regulation of ARF6 and cytohesin 2 expression confirmed the requirement of both for migration of preadipocytes. Phosphatidylinositol 3-kinase (PI3K) and PI 4,5-bisphosphate (PIP2) have also found to be essential for the cytohesin 2/ARF6 induced preadipocyte migration. Pharmacological inhibition of the activation of ARF6, ERK1/2 or dynamin led to significant reduction in migration of 3T3-L1 preadipocytes. Furthermore, our study revealed the activation of ARF6 and ERK1/2 during migration of preadipocytes. In the migrating preadipocytes, ARF6 activation was inhibited with SecinH3 (cytohesin inhibitor) and LY294002 (PI3K inhibitor) whereas the ERK1/2 phosphorylation was inhibited with SecinH3, LY294002, PBP10 (a PIP2 sequester peptide) and PD98059 (MAPKK inhibitor). However, dynosore (dynamin inhibitor) had inhibited neither ARF6 activation nor ERK1/2 phosphorylation during preadipocyte migration. These results together suggest that cytohesin 2 activates ARF6 in a PI3K dependent manner and then the active ARF6 causes phosphorylation of ERK1/2 during preadipocyte migration

ARF1 and ARF4 regulate recycling endosomal morphology and retrograde transport from endosomes to the Golgi apparatus.

Small GTPases of the ADP-ribosylation factor (ARF) family, except for ARF6, mainly localize to the Golgi apparatus, where they trigger formation of coated carrier vesicles. We recently showed that class I ARFs (ARF1 and ARF3) localize to recycling endosomes, as well as to the Golgi, and are redundantly required for recycling of endocytosed transferrin. On the other hand, the roles of class II ARFs (ARF4 and ARF5) are not yet fully understood, and the complementary or overlapping functions of class I and class II ARFs have been poorly characterized. In this study, we find that simultaneous depletion of ARF1 and ARF4 induces extensive tubulation of recycling endosomes. Moreover, the depletion of ARF1 and ARF4 inhibits retrograde transport of TGN38 and mannose-6-phosphate receptor from early/recycling endosomes to the trans-Golgi network (TGN) but does not affect the endocytic/recycling pathway of transferrin receptor or inhibit retrograde transport of CD4-furin from late endosomes to the TGN. These observations indicate that the ARF1+ARF4 and ARF1+ARF3 pairs are both required for integrity of recycling endosomes but are involved in distinct transport pathways: the former pair regulates retrograde transport from endosomes to the TGN, whereas the latter is required for the transferrin recycling pathway from endosomes to the plasma membrane

GEP100/Arf6 Is Required for Epidermal Growth Factor-Induced ERK/Rac1 Signaling and Cell Migration in Human Hepatoma HepG2 Cells

BACKGROUND: Epidermal growth factor (EGF) signaling is implicated in the invasion and metastasis of hepatoma cells. However, the signaling pathways for EGF-induced motility of hepatoma cells remain undefined. METHODOLOGY/PRINCIPAL FINDINGS: We found that EGF dose-dependently stimulated the migration of human hepatoma cells HepG2, with the maximal effect at 10 ng/mL. Additionally, EGF increased Arf6 activity, and ectopic expression of Arf6 T27N, a dominant negative Arf6 mutant, largely abolish EGF-induced cell migration. Blocking GEP100 with GEP100 siRNA or GEP100-△PH, a pleckstrin homology (PH) domain deletion mutant of GEP100, blocked EGF-induced Arf6 activity and cell migration. EGF also increased ERK and Rac1 activity. Ectopic expression GEP100 siRNA, GEP100-△PH, or Arf6-T27N suppressed EGF-induced ERK and Rac1 activity. Furthermore, blocking ERK signaling with its inhibitor U0126 remarkably inhibited both EGF-induced Rac1 activation as well as cell migration, and ectopic expression of inactive mutant form of Rac1 (Rac1-T17N) also largely abolished EGF-induced cell migration. CONCLUSIONS/SIGNIFICANCE: Taken together, this study highlights the function of the PH domain of GEP100 and its regulated Arf6/ERK/Rac1 signaling cascade in EGF-induced hepatoma cell migration. These findings could provide a rationale for designing new therapy based on inhibition of hepatoma metastasis

Doctor of Philosophy

dissertationMelanoma is a malignant tumor that develops from melanocytes, which are pigmented cells primarily found in the epidermis, eye, and mucosal epithelia. Melanomas can occur in any tissue containing melanocytes. Although both cutaneous and uveal melanomas are derived from melanocytes, they have significant differences in terms of genetic alterations, metastatic pattern, tumorigenesis, and therapeutic response. In this dissertation, I present the results of studies that explore the role of the small GTPase ARF6 in cutaneous and uveal melanoma. These studies show that ARF6 is required both for invasion and metastasis in cutaneous melanoma and for orchestrating oncogenic G protein-mediated signaling pathways that promote uveal melanoma cell proliferation. In cutaneous melanoma cells, WNT5A-FZD4-LRP6 signaling activates ARF6 via the guanine nucleotide exchange factor (GEF) GEP100. ARF6 activation promotes the release of -catenin from cell-surface N-cadherin, thereby increasing the pool of cytoplasmic and nuclear -catenin with a subsequent induction of transcription, invasion, and metastasis. As WNT signaling is implicated in many cancers, these findings suggest that a WNT-ARFGEF-ARF signaling module may play an important role in the metastasis of multiple cancers. Uveal melanoma is the most common primary intraocular tumor. Activating mutations in GNAQ and GNA11, which encode members of the q class of the G-protein alpha subunit (Gq), are the primary drivers of uveal melanoma tumorigenesis. I show iv that oncogenic GNAQ forms a complex with GEP100 to activate ARF6, which in turn induces all known GNAQ-mediated signaling pathways as well as the relocalization of -catenin from the cell surface to the nucleus. These findings indicate that ARF6 is the primary immediate effector of an oncogenic GNAQ/GEP100 complex that regulates multiple signaling pathways shown to be important in the control of uveal melanoma tumorigenesis and growth. These results not only provide an improved understanding of the molecular mechanism underlying Gq-mediated tumorigenesis but also suggest a new target for therapeutic intervention in uveal melanoma

SynArfGEF is a guanine nucleotide exchange factor for Arf6 and localizes preferentially at post-synaptic specializations of inhibitory synapses

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 synArfGEF 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 β-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

Down-Regulation of GEP100 Causes Increase in E-Cadherin Levels and Inhibits Pancreatic Cancer Cell Invasion

AIMS: Invasion and metastasis are major reasons for pancreatic cancer death and identifying signaling molecules that are specifically used in tumor invasion is of great significance. The purpose of this study was to elucidate the role of GEP100 in pancreatic cancer cell invasion and metastasis and the corresponding molecular mechanism. METHODS: Stable cell lines with GEP100 knocked-down were established by transfecting GEP100 shRNA vector into PaTu8988 cells and selected by puromycin. qRT-PCR and Western blot were performed to detect gene expression. Matrigel-invasion assay was used to detect cancer cell invasion in vitro. Liver metastasis in vivo was determined by splenic injection of indicated cell lines followed by spleen resection. Immunofluorescence study was used to detect the intracellular localization of E-cadherin. RESULTS: We found that the expression level of GEP100 protein was closely related to the invasive ability of a panel of 6 different human pancreatic cancer cell lines. Down-regulation of GEP100 in PaTu8988 cells significantly decreased invasive activity by Matrigel invasion assay, without affecting migration, invasion and viability. The inhibited invasive activity was rescued by over-expression of GEP100 cDNA. In vivo study showed that liver metastasis was significantly decreased in the PaTu8988 cells with GEP100 stably knocked-down. In addition, an epithelial-like morphological change, mimicking a mesenchymal to epithelial transition (MET) was induced by GEP100 down-regulation. The expression of E-cadherin protein was increased 2-3 folds accompanied by its redistribution to the cell-cell contacts, while no obvious changes were observed for E-cadherin mRNA. Unexpectedly, the mRNA of Slug was increased by GEP100 knock-down. CONCLUSION: These findings provided important evidence that GEP100 plays a significant role in pancreatic cancer invasion through regulating the expression of E-cadherin and the process of MET, indicating the possibility of it becoming a potential therapeutic target against pancreatic cancer

ARF GTPases and their GEFs and GAPs: concepts and challenges

Detailed structural, biochemical, cell biological, and genetic studies of any gene/protein are required to develop models of its actions in cells. Studying a protein family in the aggregate yields additional information, as one can include analyses of their coevolution, acquisition or loss of functionalities, structural pliability, and the emergence of shared or variations in molecular mechanisms. An even richer understanding of cell biology can be achieved through evaluating functionally linked protein families. In this review, we summarize current knowledge of three protein families: the ARF GTPases, the guanine nucleotide exchange factors (ARF GEFs) that activate them, and the GTPase-activating proteins (ARF GAPs) that have the ability to both propagate and terminate signaling. However, despite decades of scrutiny, our understanding of how these essential proteins function in cells remains fragmentary. We believe that the inherent complexity of ARF signaling and its regulation by GEFs and GAPs will require the concerted effort of many laboratories working together, ideally within a consortium to optimally pool information and resources. The collaborative study of these three functionally connected families ( \u3e /=70 mammalian genes) will yield transformative insights into regulation of cell signaling

The EGFR-GEP100-Arf6-AMAP1 Signaling Pathway Specific to Breast Cancer Invasion and Metastasis†

Tumors are tissue-specific diseases, and their mechanisms of invasion and metastasis are highly diverse. In breast cancer, biomarkers that specifically correlate with the invasive phenotypes have not been clearly identified. A small GTPase Arf6 primarily regulates recycling of plasma membrane components. We have shown that Arf6 and its effector AMAP1 (DDEF1, DEF1, ASAP1 and centaurin β4) are abnormally overexpressed in some breast cancers and used for their invasion and metastasis. Overexpression of these proteins is independent of the transcriptional upregulation of their genes, and occurs only in highly malignant breast cancer cells. We recently identified GEP100 (BRAG2) to be responsible for the Arf6 activation to induce invasion and metastasis, by directly binding to ligand-activated epidermal growth factor receptor (EGFR). A series of our studies revealed that for activation of the invasion pathway of EGFR, it is prerequisite that Arf6 and AMAP1 both are highly overexpressed, and that EGFR is activated by ligands. Pathological analyses indicate that a significant large population of human ductal cancers may utilize the EGFR-GEP100-Arf6-AMAP1 pathway for their malignancy. Microenvironments have been highly implicated in the malignancy of mammary tumors. Our results reveal an aspect of the precise molecular mechanisms of some breast cancers, in which full invasiveness is not acquired just by intracellular alterations of cancer cells, but extracellular factors from microenvironments may also be necessary. Possible translation of our knowledge to cancer therapeutics will also be discussed