Differential effects of a novel IFN-zeta/limitin and IFN-alpha on signals for Daxx induction and Crk phosphorylation that couple with growth control of megakaryocytes

OBJECTIVE: Although a novel IFN-zeta/limitin uses IFN-alpha/beta receptor, it lacks some common activities of type I IFNs. We compared effects on megakaryocyte proliferation and differentiation as well as signals for their biological activities. MATERIALS AND METHODS: Recombinant IFN-zeta/limitin and IFN-alpha titrated with a cytopathic effect dye binding assay, were used in this study. Colony assays and serum-free suspension cultures for megakaryocytes were performed to compare their growth inhibitory effects. To analyze signals, megakaryocytes cultured in serum-free suspension cultures were stimulated and Western blotted with the indicated antibody. RESULTS: Both IFN-zeta/limitin and IFN-alpha suppressed the proliferation of megakaryocyte progenitors without influencing their differentiation. However, much higher concentrations of IFN-zeta/limitin were required for the growth inhibition than IFN-alpha. The growth inhibition by IFN-zeta/limitin and IFN-alpha was significantly reduced when either Tyk2 or STAT1 was disrupted. In addition, the antisense oligonucleotides against Crk and Daxx, downstream molecules of Tyk2, greatly rescued the IFN-zeta/limitin- and IFN-alpha-induced reduction of megakaryocyte colony numbers. In cultured megakaryocytes, IFN-zeta/limitin induced the expression of SOCS-1 as strongly as IFN-alpha. However, IFN-zeta/limitin induced weaker phosphorylation of Crk and lower induction of Daxx than IFN-alpha. CONCLUSIONS: Weaker signals for Crk and Daxx may participate in less megakaryocyte suppressive activity of IFN-zeta/limitin and may distinguish IFN-zeta/limitin from IFN-alpha in megakaryocytes. Our results extend the understanding about thrombocytopenia in patients with IFN-alpha treatment as well as the possibility for the clinical application of human homologue of IFN-zeta/limitin or an engineered cytokine with useful features of the IFN-zeta/limitin structure.