Dysregulation of gene expression in ABCC6 knockdown HepG2 cells.

ABCC6 protein is an ATP-dependent transporter that is mainly found in the basolateral plasma membrane of hepatocytes. ABCC6 deficiency is the primary cause of several forms of ectopic mineralization syndrome. Mutations in the human ABCC6 gene cause pseudoxanthoma elasticum (PXE), an autosomal recessive disease characterized by ectopic calcification of the elastic fibers in dermal, ocular and vascular tissues. Mutations in the mouse ABCC6 gene were also associated with dystrophic cardiac calcification. Reduced levels of ABCC6 protein were found in a ß-thalassemic mouse model. Moreover, some cases of generalized arterial calcification in infancy are due to ABCC6 mutations. In order to study the role of ABCC6 in the pathogenesis of ectopic mineralization, the expressions of genes involved in this process were evaluated in HepG2 cells upon stable knockdown of ABCC6 by small hairpin RNA (shRNA) technology. ABCC6 knockdown in HepG2 cells causes a significant upregulation of the genes promoting mineralization, such as TNAP, and a parallel downregulation of genes with anti-mineralization activity, such as NT5E, Fetuin A and Osteopontin. Although the absence of ABCC6 has been already associated with ectopic mineralization syndromes, this study is the first to show a direct relationship between reduced ABCC6 levels and the expression of pro-mineralization genes in hepatocytes.

Expression, purification and structural characterization of up-regulated gene 7 encoded protein.

Up-Regulated Gene 7 (URG7) is a host gene up-regulated in HBV infected hepatocytes that has been suggested to have an anti-apoptotic activity mediated by caspases 3 and 8 and an endoplasmic reticulum localization. Here we report the structural characterization of the encoded protein URG7 by circular dichroism and fluorescence spectroscopy in different solvent media: phosphate buffer and two membrane-mimetic solvents, i.e. 2,2,2-trifluoroethanol (TFE) and SDS micelles. In all solvents URG7 contains substantial amounts of secondary structures. To obtain information about the structural organization and stability of URG7, its thermal denaturation in a membrane environment was studied and intermediate states of thermal unfolding were observed. Furthermore, fluorescence results in SDS micelles could be compatible with different environments for the four tryptophan residues in URG7. Preliminary NMR data indicate that URG7 in TFE solution is quite flexible and not well folded. These data are the first structural information on URG7 and might provide an insight into its structure-function relationships.