Analysis of the IFN-gamma-signaling pathway in macrophages at different stages of maturation.
We previously demonstrated that the macrophage cell lines RAW 264.7 and WEHI-3 exhibit distinct patterns of gene expression in response to IFN-gamma. 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 gamma-activated sequence DNA regulatory element in response to stimulation with IFN-gamma, while other aspects of IFN-gamma gene induction remain intact. In the work described here, we examined the components of the IFN-gamma 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-gamma 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-gamma stimulation. These results demonstrate that the components of the IFN-gamma signal transduction pathway are intact in WEHI-3 cells, but stimulation of these cells by IFN-gamma does not result in STAT1 activation.
Lucas
D M
DM
Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison 53706, USA.
Lokuta
M A
MA
McDowell
M A
MA
Doan
J E
JE
Paulnock
D M
DM
D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000502:physiology; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D053613:Janus Kinase 1; D053614:Janus Kinase 2; D008264:Macrophages / Q000166:cytology / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000502:physiology; D011518:Proto-Oncogene Proteins; D050794:STAT1 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D015534:Trans-Activators / Q000502:physiology; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D015534:Trans-Activators; D007371:Interferon-gamma; D011505:Protein-Tyrosine Kinases; C507904:JAK1 protein, human; C507924:JAK2 protein, human; D053613:Janus Kinase 1; D053614:Janus Kinase 2