Retinoic Acid Causes Up-Regulation of the Gastrointestinal Glutathione Peroxidase (GI-GPx) Promoter and Concomitantly Down-Regulation of Hepatitis C Virus (HCV) Subgenomic RNA

Abstract

The mRNA expression patterns of three Hepatitis C Virus (HCV)-subgenomic RNA replicon cell lines were compared with those of mock transfected or untransfected HuH7 cells utilizing cDNA array filters. The gastrointestinal-glutathione peroxidase (GI- GPx) mRNA was drastically down-regulated (as low as 5 to 10% of controls) in all replicon cell lines, while the expression level of the classical cellular-glutathione peroxidase (cGPx) remained unaffected. These data were confirmed by Northern blot and Western blot analyses. GI-GPx is a selenoprotein belonging to a family of four members, responsible for the detoxification of peroxides. Measuring total cellular glutathione peroxidase activity, revealed that the replicon cells showed reduced glutathione peroxidase activity (approx. 50% of control cells). Accordingly, replicon cells demonstrated increased susceptibility towards paraquat, a compound producing oxidative stress, reflected by a reduced viability of the replicon cultures compared to mock-transfected cell lines. When replicon cells were incubated with interferon for four days to induce the innate immune response, the HCV-replicon became down-regulated. Concomitantly, expression of GI-GPx resumed to nearly normal levels. Interferon itself did not effect the expression of GI-GPx in mock transfected and naïve HuH7 cells. Furthermore, transient over-expression of the GI-GPx cDNA via adenoviral gene transfer induced a substantial and consistent down-regulation of the HCV RNA and the NS5a protein in replicon cells. In depth inspection of the 5rsquor; promoter region of the GI-GPx gene revealed the presence of two retinoic acid response elements (RARE). Treating replicon cultures with retinoic acid in the presence of selenite lead to increased expression of endogenous GI-GPx, followed by a dramatic down-regulation of the replicon. This decrease was even more pronounced, when cells were incubated with retinoic acid in the presence of selenite and Interferon alpha. Taken together, these data show, that (a) expression of GI-GPx and replication of HCV exclude each other and (b) retinoic acid might be a valuable tool for the treatment of HCV patients. Therefore, a clinical pilot trial at the University of Mainz with a population of interferon non-responders was initiated. The data of this clinical trial will be presented in parallel.