Analysis of the IFN-γ-Signaling Pathway in Macrophages at Different Stages of Maturation

Abstract

We previously demonstrated that the macrophage cell lines RAW 264.7 and WEHI-3 exhibit distinct patterns of gene expression in response to IFN-γ. This difference is controlled at the transcriptional level and results from a specific inability of the less mature WEHI-3 cells to utilize either the IFN-stimulated response element or the γ-activated sequence DNA regulatory element in response to stimulation with IFN-γ, while other aspects of IFN-γ gene induction remain intact. In the work described here, we examined the components of the IFN-γ signal transduction pathway in RAW 264.7 and WEHI-3 cells to determine whether differences in pathway components or activity exist in WEHI-3 cells that could give rise to this difference in transcriptional response. Reverse transcriptase-PCR (RT-PCR) and flow cytometric analyses indicated that the levels of IFN-γ receptor mRNA accumulation and protein expression are comparable for RAW 264.7 and WEHI-3 cells. RT-PCR and immunoblot analyses revealed that the principal components of this signaling pathway, including JAK1, JAK2, and STAT1, are present in both RAW 264.7 and WEHI-3 cells. However, analysis of STAT1 DNA-binding activity by electrophoretic mobility shift assay and of STAT1 phosphorylation by immunoblot revealed that this DNA-binding factor is active in RAW 264.7, but not in WEHI-3, cells after IFN-γ stimulation. These results demonstrate that the components of the IFN-γ signal transduction pathway are intact in WEHI-3 cells, but stimulation of these cells by IFN-γ does not result in STAT1 activation.

Regulation of c-myc expression by IFN-γ through Stat1-dependent and -independent pathways

“…Targeted disruption in mice has confirmed that Stat1 is obligatory for signaling in response to all IFNs and has revealed that Stat1 is also involved in immune surveillance and tumor suppression (Durbin et al ., 1996; Meraz et al ., 1996; Kaplan et al ., 1998). Some tumor cells and tumor‐derived cell lines express little or no Stat1 mRNA or protein (L.H.Wong et al ., 1997; Abril et al ., 1998; Sun et al ., 1998) or fail to activate Stat1 following treatment with IFNs (Lucas et al ., 1998). IFNs inhibit the growth of many cell types (Balkwill and Taylor‐Papadimitriou, 1978; Lin et al ., 1986; Kimchi, 1992), and Stat1 that is fully active transcriptionally is required for this effect (Bromberg et al ., 1996).…”

“…Some tumor cells and tumorderived cell lines express little or no Stat1 mRNA or protein (L.H. Abril et al, 1998;Sun et al, 1998) or fail to activate Stat1 following treatment with IFNs (Lucas et al, 1998). IFNs inhibit the growth of many cell types (Balkwill and Taylor-Papadimitriou, 1978;Lin et al, 1986;Kimchi, 1992), and Stat1 that is fully active transcriptionally is required for this effect (Bromberg et al, 1996).…”