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.


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.
Ishida
Naoko
N
Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Osaka, Japan.
Oritani
Kenji
K
Shiraga
Masamichi
M
Yoshida
Hitoshi
H
Kawamoto
Sin-Ichiro
S
Ujiie
Hidetoshi
H
Masaie
Hiroaki
H
Ichii
Michiko
M
Tomiyama
Yoshiaki
Y
Kanakura
Yuzuru
Y
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D056970:Co-Repressor Proteins; D016207:Cytokines / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D016898:Interferon-alpha / Q000494:pharmacology*; D007372:Interferons / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics*; D008533:Megakaryocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018832:Molecular Chaperones; D009687:Nuclear Proteins / Q000235:genetics*; D010766:Phosphorylation; D050737:Proto-Oncogene Proteins c-crk; D015398:Signal Transduction / Q000187:drug effects
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D056970:Co-Repressor Proteins; C476798:Crk protein, mouse; D016207:Cytokines; C491473:Daxx protein, mouse; D016898:Interferon-alpha; D047908:Intracellular Signaling Peptides and Proteins; D018832:Molecular Chaperones; D009687:Nuclear Proteins; D050737:Proto-Oncogene Proteins c-crk; C410906:limitin; D007372:Interferons