Inhibition of Interferon Regulatory Factor-1 Expression Results in Predominance of Cell Growth Stimulatory Effects of Interferon-γ Due to Phosphorylation of Stat1 and Stat3

Abstract

AbstractInterferon-γ (IFN-γ) is a potent inhibitor of hematopoiesis in vitro and has been implicated in the pathophysiology of human bone marrow failure syndromes. IFN-γ both inhibits cell cycling and induces expression of the Fas-receptor, resulting in subsequent apoptosis of hematopoietic progenitor cells. IFN regulatory factor-1 (IRF-1) mediates some of these suppressive effects by activation of downstream inducible genes, such as double-stranded RNA-activatable protein kinase and inducible nitric oxide synthase. However, under certain experimental conditions, IFN-γ appears to stimulate proliferation of hematopoietic cells. Based on the hypothesis that IFN-γ–receptor triggering may activate diverse signaling cascades, we designed experiments to determine which intracellular mechanisms (in addition to the IRF-1 transduction pathway) influence the biologic effects of IFN-γ. Using antisense technique, we inhibited the IRF-1–mediated pathway in KG1a cells stimulated with IFN-γ. In contrast to the suppressive effects of IFN-γ observed in control cells, untreated and IFN-γ–treated KG-1a cells that were transduced with retroviral vectors expressing IRF-1 antisense mRNA showed enhanced proliferation. The increased growth rate was associated with decreased levels of IRF-1 mRNA and protein but unchanged levels of IRF-2. We inferred that IFN-γ could also activate a stimulatory transduction pathway that, under specific conditions, may control the cellular response to this cytokine. The family of Stat proteins is involved in signal transduction of hematopoietic growth factors. We showed that, in KG-1a cells, IFN-γ also induced phosphorylation of Stat1 and Stat3, whereas p42 MAP kinase was phosphorylated regardless of the presence of IFN-γ. Using electrophoresis mobility shift assays, IFN-γ enhanced Stat1-Stat1 homodimer and Stat1-Stat3 heterodimer formation, suggesting that, in addition to inhibitory signals mediated by IRF-1, IFN-γ may activate proliferative signals by phosphorylation of Stat1 and Stat3 proteins. The observations made in experiments with KG-1a cells were confirmed in primary hematopoietic cells. After inhibition of the IRF-1 pathway by transduction of an antisense IRF-1 retrovirus into human CD34+ cells, IFN-γ produced an aberrant stimulatory effect on hematopoietic colony formation. Conversely, in control vector-transduced CD34+ cells, the typical inhibitory response to IFN-γ was seen. Our results indicate that inhibitory cytokines such as IFN-γ may exhibit diverse biologic effects depending on the intracellular balance of transcriptional regulators, in turn influenced by the activation and differentiation status of the target cells.