Role of ADP-ribosylation factor 6 (ARF6) in gastric acid secretion

ADP-ribosylation factor (ARF) proteins are monomeric GTPases that are essential for membrane transport and exocytosis in a number of secretory cells. We investigated ARF6, the activation of which is insensitive to brefeldin A, to determine whether it regulates membrane traffic in gastric parietal cells. ARF6 translocated from cytosol to tubulovesicle in the presence of GTPgammaS, a potential inhibitor of acid secretion in permeabilized cells, whereas under the Mg2+-chelated condition where activity of ARF-GTPase activating protein is inhibited, ARF6 translocated to the apical secretory membrane. Immunohistochemical examination revealed that ARF6 mainly located in parietal cell within the gastric glands, and it translocated from the cytosol to the intracellular canaliculi when the glands were stimulated. These results indicated that the distribution of ARF6 between cytosol and the two different membranes was regulated by its GTPase activity. In cultured gastric glands infected with adenovirus expressing ARF6 Q67L, a mutant lacking GTP hydrolysis activity, gastric acid secretion was inhibited. These results suggest that ARF6 regulates gastric acid secretion in parietal cell and that the GTP hydrolysis cycle of ARF6 is essential for the activation pathway.     

ADP-ribosylation factor 6 (ARF6) defines two insulin-regulated secretory pathways in adipocytes.

ADP-ribosylation factor 6 (ARF6) appears to play an essential role in the endocytic/recycling pathway in several cell types. To determine whether ARF6 is involved in insulin-regulated exocytosis, 3T3-L1 adipocytes were infected with recombinant adenovirus expressing wild-type ARF6 or an ARF6 dominant negative mutant (D125N) that encodes a protein with nucleotide specificity modified from guanine to xanthine. Overexpression of these ARF6 proteins affected neither basal nor insulin-regulated glucose uptake in 3T3-L1 adipocytes, nor did it affect the subcellular distribution of Glut1 or Glut4. In contrast, the secretion of adipsin, a serine protease specifically expressed in adipocytes, was increased by the expression of wild-type ARF6 and was inhibited by the expression of D125N. These results indicate a requirement for ARF6 in basal and insulin-regulated adipsin secretion but not in glucose transport. Our results suggest the existence of at least two distinct pathways that undergo insulin-stimulated exocytosis in 3T3-L1 adipocytes, one for adipsin release and one for glucose transporter translocation.     

Expression of vasorin (Vasn) during embryonic development of the mouse

The murine vasorin (Vasn) gene, initially known as Slit-like 2, encodes a transmembrane protein that shares structural similarities with the eponymous Slit proteins. However, whether it also shares functional similarities with these large secreted proteins remains to be elucidated. Here, we report expression of Vasn during embryonic and fetal development of the mouse using whole-mount in situ hybridization (WISH) and histochemical detection of β-galactosidase expressed from a targeted Vasn(lacZ) knock-in allele. Comparison of whole-mount staining patterns of both approaches showed identical expression domains, confirming that Vasn promoter-driven β-galactosidase expression faithfully reflects endogenous Vasn expression. Vasn is highly expressed in vascular smooth muscle cells (hence the name), a finding consistent with a previous report on its human homolog VASN, whose extracellular domain was shown to function as a TGF-β trap (Ikeda et al., 2004). Most striking, however, is Vasn's prominent expression in the developing skeletal system, starting as early as the first mesenchymal condensations appear. Moreover, distinct expression domains outside the bones, e.g., in the developing kidneys and lungs, suggest further roles for this gene in the mouse. Recently, it was shown that mitochondria-localized Vasn protects cells from TNFα- and hypoxia-induced apoptosis, and partial deletion of the Vasn coding sequence leads to increased sensitivity of hepatocytes to TNFα-induced apoptosis (Choksi et al., 2011). By providing a first comprehensive analysis of the Vasn expression pattern during mouse embryonic development, our study will help to further elucidate its biological functions.     

A regulatory role for ADP-ribosylation factor 6 (ARF6) in activation of the phagocyte NADPH oxidase

In activated neutrophils NADPH oxidase is regulated through various signaling intermediates, including heterotrimeric G proteins, kinases, GTPases, and phospholipases. ADP-ribosylation factor (ARF) describes a family of GTPases associated with phospholipase D (PLD) activation. PLD is implicated in NADPH oxidase activation, although it is unclear whether activation of PLD by ARF is linked to receptor-mediated oxidase activation. We explored whether ARF participates in NADPH oxidase activation by formyl-methionine-leucine-phenylalanine (fMLP) and whether this involves PLD. Using multicolor forward angle light scattering analyses to measure superoxide production in differentiated neutrophil-like PLB-985 cells, we tested enhanced green fluorescent fusion proteins of wild-type ARF1 or ARF6, or their mutant counterparts. The ARF6(Q67L) mutant defective in GTP hydrolysis caused increased superoxide production, whereas the ARF6(T27N) mutant defective in GTP binding caused diminished responses to fMLP. The ARF1 mutants had no effect on fMLP responses, and none of the ARF proteins affected phorbol 12-myristate 13-acetate-elicited oxidase activity. PLD inhibitors 1-butanol and 2, 3-diphosphoglycerate, or the ARF6(N48R) mutant assumed to be defective in PLD activation, blocked fMLP-elicited oxidase activity in transfected cells. The data suggest that ARF6 but not ARF1 modulates receptor-mediated NADPH oxidase activation in a PLD-dependent mechanism. Because PMA-elicited NADPH oxidase activation also appears to be PLD-dependent, but ARF-independent, ARF6 and protein kinase C may act through distinct pathways, both involving PLD.     

ADP-ribosylation factor (ARF) interaction is not sufficient for yeast GGA protein function or localization

Golgi-localized gamma-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) facilitate distinct steps of post-Golgi traffic. Human and yeast GGA proteins are only ~25% identical, but all GGA proteins have four similar domains based on function and sequence homology. GGA proteins are most conserved in the region that interacts with ARF proteins. To analyze the role of ARF in GGA protein localization and function, we performed mutational analyses of both human and yeast GGAs. To our surprise, yeast and human GGAs differ in their requirement for ARF interaction. We describe a point mutation in both yeast and mammalian GGA proteins that eliminates binding to ARFs. In mammalian cells, this mutation disrupts the localization of human GGA proteins. Yeast Gga function was studied using an assay for carboxypeptidase Y missorting and synthetic temperature-sensitive lethality between GGAs and VPS27. Based on these assays, we conclude that non-Arf-binding yeast Gga mutants can function normally in membrane trafficking. Using green fluorescent protein-tagged Gga1p, we show that Arf interaction is not required for Gga localization to the Golgi. Truncation analysis of Gga1p and Gga2p suggests that the N-terminal VHS domain and C-terminal hinge and ear domains play significant roles in yeast Gga protein localization and function. Together, our data suggest that yeast Gga proteins function to assemble a protein complex at the late Golgi to initiate proper sorting and transport of specific cargo. Whereas mammalian GGAs must interact with ARF to localize to and function at the Golgi, interaction between yeast Ggas and Arf plays a minor role in Gga localization and function.     

ADP-ribosylation factor (ARF)-like 4, 6, and 7 represent a subgroup of the ARF family characterization by rapid nucleotide exchange and a nuclear localization signal.

The novel ARF-like GTPase ARL7 is a close relative of ARL4 and ARL6 (71% and 59%) identical amino acids). A striking characteristic of these GTPases is their basic C-terminus which, when fused to the C-terminus of green fluorescent protein (GFP), targets the constructs to the nucleus of transfected COS-7 cells. Full length ARL4 was detected in both nuclear and extranuclear compartments, whereas a construct of ARL4 lacking its C-terminus was excluded from the nucleus. Nucleotide exchange rates of recombinant ARL4, ARL6 and ARL7 were similar and appeared considerably higher than those of other members of the ARF family (ARF1, ARP). It is concluded that ARL4, ARL6 and ARL7 form a subgroup within the ARF family with similar, possibly nuclear, function.     

Expression of the transcription factor Zfp191 during embryonic development in the mouse

The human zinc finger protein 191 (ZNF191) is a Krüppel-like protein and can specifically interact with the widespread TCAT motif which constitutes the HUMTH01 microsatellite in the tyrosine hydroxylase (TH) gene (encoding the rate-limiting enzyme in the synthesis of catecholamines). Allelic variations of HUMTH01 are known to have a quantitative silencing effect on TH gene expression and to correlate with quantitative and qualitative changes in the binding by ZNF191. This factor has been isolated from bone marrow and promyelocytic leukemia cell lines indicating that ZNF191 also plays a role in hematopoiesis. Thus, ZNF191 could participate in the regulation of several genes implicated in different functions. Moreover, mice that are deficient in Zfp191, the murine homologue of ZNF191, have been shown to be severely retarded in development and to die approximately at embryonic day 7.5. In order to gain further insight into its biological functions, we have analysed the localisation of Zfp191 throughout mouse development. Expression was detected early during embryogenesis in ectodermal, endodermal, mesodermal and extra-embryonic tissues. In particular, Zfp191 was observed in the developing central nervous system. Interestingly, its expression levels were prominent in areas of proliferation such as the subventricular zone. Zfp191 expression pattern during development can account for the phenotypic features of Zfp191(-/-) embryos.     

ADP-ribosylation factor (ARF)-like 7 (ARL7) is induced by cholesterol loading and participates in apolipoprotein AI-dependent cholesterol export

Here, we identify ADP-ribosylation factor (ARF)-like 7 (ARL7) as the only ARF- and ARL-family member whose mRNA-expression is induced by liver X-receptor/retinoid X-receptor agonists or cholesterol loading in human macrophages. Moreover, subcellular distribution of mutant and wild type ARL7-enhanced green fluorescent protein (EGFP) supports that ARL7 may be involved in a vesicular transport step between a perinuclear compartment and the plasma membrane. Therefore, we investigated the effect of ARL7 over-expression on the cholesterol secretory pathway. We found that expression of wild type and dominant active ARL7-EGFP stimulated the rate of apolipoprotein AI-specific cholesterol efflux 1.7- and 2.8-fold. In contrast, expression of the dominant negative form of ARL7-EGFP led to approximately 50% inhibition of cholesterol efflux. This data is consistent with a model in which ARL7 is involved in transport between a perinuclear compartment and the plasma membrane apparently linked to the ABCA1-mediated cholesterol secretion pathway.     

Nuclear ADP-ribosylation in the chick lens during embryonic development

Nuclear ADP-ribosyltransferase is present in cells from the chick lens throughout embryonic development. The activity does not decrease when the cells become post-mitotic and commence terminal differentiation but declines slowly in both epithelia and fibre cells. At all stages studied the enzyme retains its ability to be activated by DNA strand breaks induced either by X-irradiation or by the action of an endogenous endonuclease. There is no correlation between the enzyme activity or the levels of its substrate NAD+ and the changes in DNA repair capacity which have been observed during the development of the lens.     

Calcium-regulated exocytosis of dense-core vesicles requires the activation of ADP-ribosylation factor (ARF)6 by ARF nucleotide binding site opener at the plasma membrane

Vacuole fusion requires a coordinated cascade of priming, docking, and fusion. SNARE proteins have been implicated in the fusion itself, although their precise role in the cascade remains unclear. We now report that the vacuolar SNAP-23 homologue Vam7p is a mobile element of the SNARE complex, which moves from an initial association with the cis-SNARE complex via a soluble intermediate to the docking site. Soluble Vam7p is specifically recruited to vacuoles and can rescue a fusion reaction poisoned with antibodies to Vam7p. Both the recombinant Vam7p PX domain and a FYVE domain construct of human Hrs block the recruitment of Vam7p and vacuole fusion, demonstrating that phosphatidylinositol 3-phosphate is a primary receptor of Vam7p on vacuoles. We propose that the Vam7p cycle is linked to the availability of a lipid domain on yeast vacuoles, which is essential for coordinating the fusion reaction prior to and beyond docking.     

Appearance of a kidney-specific ribosomal protein during mouse embryonic development

Ribosomes were prepared from adult mouse liver and kidney and the protein components examined by SDS polyacrylamide gel electrophoresis. A kidney specific protein was identified and was found to be associated with the large ribosomal subunit. $\text{In}$$\text{vitro}$ labelling of 11- and 14-day embryonic kidneys and subsequent analysis of the ribosomal proteins indicated a change in the ribosomal population during development. The kidney specific protein was synthesized during the first four days of kidney organogenesis.     

Expression of carbonic anhydrases IX and XII during mouse embryonic development

Background  

Of the thirteen active carbonic anhydrase (CA) isozymes, CA IX and XII have been linked to carcinogenesis. It has been suggested that these membrane-bound CAs participate in cancer cell invasion, which is facilitated by an acidic tumor cell environment. Since active cell migration is a characteristic feature of embryonic development, we set out to explore whether these isozymes are expressed in mouse embryos of different ages. The studies were focused on organogenesis stage.     

Hepatocyte growth factor (HGF) receptor expression and role of HGF during embryonic mouse testis development

The hepatocyte growth factor (HGF) receptor, c-met, transduces the HGF multiple biological activities. During embryonic development the system HGF/c-met regulates the morphogenesis of different organs and tissues. In this study we examined c-met gene expression during mouse testis development and, by means of Northern blot and in situ hybridization, we report the receptor expression pattern. C-met expression is not detectable in male genital ridges isolated from embryos at 11.5 days postcoitum (dpc). In testes isolated from 12.5 and 13.5 dpc, c-met expression is detectable and essentially localized in the developing cords. Male genital ducts do not express c-met at the reported ages, whereas female ducts appear c-met positive. Moreover, we report that HGF is able to induce testicular morphogenesis in vitro. Male genital ridges isolated from embryos at 11.5 dpc are morphologically nonorganized. Culturing 11.5 dpc urogenital ridges in the presence of HGF we obtained testis organization and testicular cord formation. Our data demonstrate that c-met is expressed during the beginning period of testis differentiation and that HGF is able to support testicular differentiation in vitro. All these data indicate that this growth factor, besides its role as mitogenic factor, plays a fundamental role during testicular cord formation probably inducing cell migration and/or cell differentiation.