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-Signaling Pathway in Macrophages at Different Stages of Maturation1
Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison, WI 53706
We previously demonstrated that the macrophage cell lines RAW 264.7
and WEHI-3 exhibit distinct patterns of gene expression in response to
IFN-
. 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 -activated
sequence DNA regulatory element in response to stimulation with
IFN-, while other aspects of IFN- gene induction remain intact.
In the work described here, we examined the components of the IFN-
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- 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- stimulation. These results demonstrate that the
components of the IFN- signal transduction pathway are intact in
WEHI-3 cells, but stimulation of these cells by IFN- does not result
in STAT1 activation.
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