The Role of ARF6 in Biliary Atresia

Background & Aims

Altered extrahepatic bile ducts, gut, and cardiovascular anomalies constitute the variable phenotype of biliary atresia (BA).

Methods

To identify potential susceptibility loci, Caucasian children, normal (controls) and with BA (cases) at two US centers were compared at >550000 SNP loci. Systems biology analysis was carried out on the data. In order to validate a key gene identified in the analysis, biliary morphogenesis was evaluated in 2-5-day post-fertilization zebrafish embryos after morpholino-antisense oligonucleotide knockdown of the candidate gene ADP ribosylation factor-6 (ARF6, Mo-arf6).

Results

Among 39 and 24 cases at centers 1 and 2, respectively, and 1907 controls, which clustered together on principal component analysis, the SNPs rs3126184 and rs10140366 in a 3’ flanking enhancer region for ARF6 demonstrated higher minor allele frequencies (MAF) in each cohort, and 63 combined cases, compared with controls (0.286 vs. 0.131, P = 5.94x10^-7, OR 2.66; 0.286 vs. 0.13, P = 5.57x10^-7, OR 2.66). Significance was enhanced in 77 total cases, which included 14 additional BA genotyped at rs3126184 only (p = 1.58x10^-2, OR = 2.66). Pathway analysis of the 1000 top-ranked SNPs in CHP cases revealed enrichment of genes for EGF regulators (p<1 x10^-7), ERK/MAPK and CREB canonical pathways (p<1 x10^-34), and functional networks for cellular development and proliferation (p<1 x10^-45), further supporting the role of EGFR-ARF6 signaling in BA. In zebrafish embryos, Mo-arf6 injection resulted in a sparse intrahepatic biliary network, several biliary epithelial cell defects, and poor bile excretion to the gall bladder compared with uninjected embryos. Biliary defects were reproduced with the EGFR-blocker AG1478 alone or with Mo-arf6 at lower doses of each agent and rescued with arf6 mRNA.

Conclusions

The BA-associated SNPs identify a chromosome 14q21.3 susceptibility locus encompassing the ARF6 gene. arf6 knockdown in zebrafish implicates early biliary dysgenesis as a basis for BA, and also suggests a role for EGFR signaling in BA pathogenesis.     

Mycoplasma infection and hypoxia initiate succinate accumulation and release in the VM-M3 cancer cells

Succinate is known to act as an inflammatory signal in classically activated macrophages through stabilization of HIF-1α leading to IL-1β production. Relevant to this, hypoxia is known to drive succinate accumulation and release into the extracellular milieu. The metabolic alterations associated with succinate release during inflammation and under hypoxia are poorly understood. Data are presented showing that Mycoplasma arginini infection of VM-M3 cancer cells enhances the Warburg effect associated with succinate production in mitochondria and eventual release into the extracellular milieu. We investigated how succinate production and release was related to the changes of other soluble metabolites, including itaconate and 2-HG. Furthermore, we found that hypoxia alone could induce succinate release from the VM-M3 cells and that this could occur in the absence of glucose-driven lactate production. Our results elucidate metabolic pathways responsible for succinate accumulation and release in cancer cells, thus identifying potential targets involved in both inflammation and hypoxia. This article is part of a Special Issue entitled 20th European Bioenergetics Conference, edited by László Zimányi and László Tretter.     

AP-3 Mediates Tyrosinase but Not TRP-1 Trafficking in Human Melanocytes

Patients with Hermansky-Pudlak syndrome type 2 (HPS-2) have mutations in the beta 3A subunit of adaptor complex-3 (AP-3) and functional deficiency of this complex. AP-3 serves as a coat protein in the formation of new vesicles, including, apparently, the platelet's dense body and the melanocyte's melanosome. We used HPS-2 melanocytes in culture to determine the role of AP-3 in the trafficking of the melanogenic proteins tyrosinase and tyrosinase-related protein-1 (TRP-1). TRP-1 displayed a typical melanosomal pattern in both normal and HPS-2 melanocytes. In contrast, tyrosinase exhibited a melanosomal (i.e., perinuclear and dendritic) pattern in normal cells but only a perinuclear pattern in the HPS-2 melanocytes. In addition, tyrosinase exhibited a normal pattern of expression in HPS-2 melanocytes transfected with a cDNA encoding the beta 3A subunit of the AP-3 complex. This suggests a role for AP-3 in the normal trafficking of tyrosinase to premelanosomes, consistent with the presence of a dileucine recognition signal in the C-terminal portion of the tyrosinase molecule. In the AP-3-deficient cells, tyrosinase was also present in structures resembling late endosomes or multivesicular bodies; these vesicles contained exvaginations devoid of tyrosinase. This suggests that, under normal circumstances, AP-3 may act on multivesicular bodies to form tyrosinase-containing vesicles destined to fuse with premelanosomes. Finally, our studies demonstrate that tyrosinase and TRP-1 use different mechanisms to reach their premelanosomal destination.     

Tyrp1 and Oculocutaneous Albinism Type 3

Tyrosinase‐related protein 1 (Tyrp1) is a melanocyte‐specific gene product involved in eumelanin synthesis. Mutations in the mouse Tyrp1 gene are associated with brown pelage, and in the human TYRP1 gene with oculocutaneous albinism type 3 (OCA3). In the murine system, Tyrp1 expresses significant dihydroxyindole carboxylic acid oxidase (i.e. DHICA oxidase) activity. However, in humans, TYRP1 is enigmatic in that despite extensive efforts focused on the study of its function, its actual role in the human melanocyte is still unclear. There is mounting evidence demonstrating that in addition to its role in eumelanin synthesis, Tyrp1 is involved in maintaining stability of tyrosinase protein and modulating its catalytic activity. Tyrp1 is also involved in maintenance of melanosome ultrastructure and affects melanocyte proliferation and melanocyte cell death. The current review is an attempt to consolidate our understanding of the role of Tyrp1 in the melanocyte.     

Tyrp1 and oculocutaneous albinism type 3.

Tyrosinase-related protein 1 (Tyrp1) is a melanocyte-specific gene product involved in eumelanin synthesis. Mutations in the mouse Tyrp1 gene are associated with brown pelage, and in the human TYRP1 gene with oculocutaneous albinism type 3 (OCA3). In the murine system, Tyrp1 expresses significant dihydroxyindole carboxylic acid oxidase (i.e. DHICA oxidase) activity. However, in humans, TYRP1 is enigmatic in that despite extensive efforts focused on the study of its function, its actual role in the human melanocyte is still unclear. There is mounting evidence demonstrating that in addition to its role in eumelanin synthesis, Tyrp1 is involved in maintaining stability of tyrosinase protein and modulating its catalytic activity. Tyrp1 is also involved in maintenance of melanosome ultrastructure and affects melanocyte proliferation and melanocyte cell death. The current review is an attempt to consolidate our understanding of the role of Tyrp1 in the melanocyte.     

PIG3V, an immortalized human vitiligo melanocyte cell line, expresses dilated endoplasmic reticulum

Summary Vitiligo is an enigmatic pigmentary disorder of the skin. Factors potentially involved in the progressive loss of melanocytes from the basal layer of the epidermis include genetically determined aberrancies of the vitiligo melanocyte. It follows that analysis of melanocytes cultured from vitiligo donors can contribute to a further understanding of the etiopathomechanism. A setback for vitiligo research has been the limited availability of vitiligo-derived melanocytes. To overcome this limitation, we have generated a vitiligo melanocyte cell line according to a protocol established previously for the immortalization of normal human melanocytes. Vitiligo melanocytes Ma9308P4 were transfected with HPV16 E6 and E7 genes using the retroviral construct LXSN16E6E7. Successful transformants were selected using geneticin and subsequently cloned to ensure genetic homogeneity. The resulting cell line PIG3V has undergone more than 100 cell population doublings ince its establishment as a confluent primary culture, whereas untransfected melanocytes derived from adult skin senesce after a maximum of 50 population doublings. Cells immortalized by this transfection procedure retain lineage-specific characteristics and proliferate significantly faster than parental cells. In this study, the phenotype of PIG3V resembled melanocytes rather than melanoma cells in culture. Tyrosinase was processed properly and melanosomes remained pigmented. Importantly, ultrastructural characterization of PIG3V cells revealed dilated endoplasmic reticulum profiles characteristic of vitiligo melanocytes. An explanation for this dilation may be found in the retention of proteins with molecular weight of 37.5, 47.5, and 56.5 kDa, as determined by gel electrophoresis of microsomal proteins isolated from radiolabeled cells. Presented in part at the Annual Meeting of the Panamerican Society for Pigment Cell Research, Aspen, Colorado, 1998.