Abstract
Treatment of quiescent primary human fibroblasts with tumor necrosis factor (TNF) alpha, TNF-beta, interleukin-1, interferon (IFN) alpha, IFN beta, or IFN gamma induced Egr-1 mRNA. In primary human fibroblasts TNF-alpha and TNF-beta were mildly mitogenic and IFN alpha and IFN gamma were growth inhibitory. However, in HeLa cells TNF but not IFN induced the expression of Egr-1 mRNA, while both cytokines inhibited HeLa cell division. Kinetic measurements of Egr-1 gene expression showed that TNF-alpha, TNF-beta, and IFN gamma increased the cellular concentration of Egr-1 mRNA within 30 min. A maximum induction of Egr-1 mRNA was detected at approximately 60 min which dropped to basal level by 180 min. Induction was inhibited by H7 and staurosporine but not by HA1004, indicating the involvement of a functional protein kinase C. The Egr-1 message was translated and the cellular Egr-1 protein detected within 60 min of cytokine treatment. Despite similar Egr-1 mRNA induction, the amount of Egr-1 protein translated in IFN alpha- and IFN gamma-treated cells was lower than in those treated with TNF-alpha and TNF-beta, and highest in the EGF-treated primary human fibroblasts. Indeed, the level of Egr-1 protein translated in these cells correlated proportionally with both the phosphorylation of cap-binding protein (eukaryotic initiation factor) and the amount of cellular DNA synthesis in the variously treated fibroblasts. These results suggest that both growth stimulatory and inhibitory cytokines can regulate Egr-1 gene expression at the transcriptional and translational level. However, the combination of these regulatory controls may determine the cellular concentration of the Egr-1 gene product and hence, its effect on cell proliferation.
Highlights
From the Signal TransductionLaboratory, Institute of Molecular and Cell Biology, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511 and the $Howard Hughes Medical Institute, Departments of Medicine and Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637
The latterwere included because they are eithergrowth inhibitory or stimulatory depending on the cells used [23].IL-1 was included because the IL-1 signal transduction pathway and biological activity oncells is almost identicalwith tumor necrosis factor (TNF) [24]
The tory (TNF and EGF) but not growth inhibitory (IFN) cytolevel of Egr-1 protein translated in these cells corre- kines increased the phosphorylation of cap-binding protein. lated proportionally with both the phosphorylationof Egr-1 gene expression was induced in HeLa cells by TNF cap-binding protein and but notby IFN even though bothclasses of cytokine inhibited the amount of cellular DNA synthesis in the variously HeLa cell proliferation. treated fibroblasts.These results suggest that both growth stimulatory and inhibitory cytokines can regulate Egr-1gene expressionat the transcriptional and translational level
Summary
Lated proportionally with both the phosphorylationof Egr-1 gene expression was induced in HeLa cells by TNF cap-binding protein (eukaryotic initiation factor) and but notby IFN even though bothclasses of cytokine inhibited the amount of cellular DNA synthesis in the variously HeLa cell proliferation. Effects of TNF, IFN, and EGFon DNA Synthesis in Primary Human Fibroblasts-Primary human fibroblasts grown to confluency in plastic tissue culture wells were maintained in a quiescent state in regular growth medium containing 0.25% fetal calf serum for 48 h.
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