Interferon-a and regulation of TPO synthesis in hepatocytes

Interferon-a (IFN) therapy in patients with chronic hepatitis C results in an average drop in platelet counts of 30% and can lead to severe thrombocytopenia and withdrawal of antiviral therapy. The drop in platelet count does not lead to the expected counterregulatory increase in the most potent thrombopoetic growth factor thrombopoetin (TPO), which is synthesized in the hepatocytes. We could show that IFN-therapy results in a change of TPO- mRNA splice-variants within the 5-untranslated region of the TPO-mRNA. This leads to a markedly reduced production of TPO protein in the liver cells during IFN-therapy and could explain the lack of a sizable increase in TPO-levels in patients with IFN-induced thrombocytopenia. This represents not only a novelty with regard to TPO-regulation but describes for the first time a new gene regulatory pathway for IFN. The development of substances that can interfere in this way with TPO-metabolism could lead to new therapeutic avenues for increasing the endogenous TPO-expression in cases of thrombocytopenia. This mode of IFN-induced gene regulation could on the other hand bring interesting new insights more generally into the mode of action of IFN in antiviral and oncologic therapies. Aims of the study: Are the changes in TPO-secretion brought about by IFN-therapy caused through alternative splicing of TPO- mRNA or through splice-independent mechanisms like changes in the subcellular distribution of TPO. From the recent literature it was known that in the hepatocyte several different TPO-mRNA species can be found, which differ in the 5`-untranslated region only through transcription from alternative promoters and differential mRNA-splicing but show very different translation efficiencies. The mRNA species with good translation efficiency amounts to only 2% of the total hepatocellular TPO-mRNA. We hypothesized that only a minute shift between the expression of the different TPO-mRNA species through IFN-therapy could lead to a marked change in the expression of the TPO-protein. Results: IFN results in a dose-dependent reduction in TPO protein production in several different cell lines. This can only partly be explained though a reduction in TPO mRNA production, since this reduced mRNA production can only be detected with very sensitive assays. Indeed, Taqman quantitative real time PCR was able to detect a shift in the TPO-splice variants under IFN-therapy towards the less efficiently translated mRNA species, at least in one of the cell lines tested. These results still have to be confirmed in other cell lines but hint to a completely new way of regulation of protein expression by IFN. Proof of an effective pharmacologic intervention being able to influence TPO protein expression could revive the interest in the search for substances to effectively augment the expression of the more efficiently translated TPO mRNA species.