Related Experiment Videos
Phospholipase A2 activation and autoinduction of tumor necrosis factor gene expression by tumor necrosis factor
D R Spriggs1, M L Sherman, K Imamura
1Laboratory of Clinical Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115.
Abstract:
Tumor necrosis factor (TNF) acts via a cell surface receptor to induce a variety of cellular events including cytolysis, differentiation, and mitogenesis. The mechanisms underlying the cell specific actions of TNF are not known. In the present study, postreceptor events associated with the autoinduction of TNF expression were examined in HL-60 cells. There was no detectable alteration in phospholipase C activity as measured by inositol phosphate generation or release of choline metabolites following TNF stimulation. However, TNF increased the release of arachidonic acid metabolites from HL-60 cells. This increase in arachidonic acid metabolism was associated with a 40% increase in phospholipase A2 activity. Furthermore, the release of arachidonic acid metabolites was blocked by inhibitors of phospholipase A2. Taken together, these findings indicated that TNF stimulates phospholipase A2 and arachidonic acid metabolism in HL-60 cells. The results also demonstrate that TNF expression is induced 15-30 min after stimulation with TNF and that this effect is associated with an increase in the rate of TNF transcription. This autoinduction of TNF mRNA was blocked by inhibitors of phospholipase A2. While the cyclooxygenase inhibitor indomethacin had no detectable effect, ketoconazole and nordihydroguaiaretic acid, inhibitors of lipoxygenase, also blocked the induction of TNF expression by TNF. These findings suggest that phospholipase A2 and lipoxygenase activity are required for the transcriptional activation of TNF gene expression associated with TNF stimulation of HL-60 cells.
Insights
Tumor necrosis factor (TNF) stimulates phospholipase A2 and arachidonic acid metabolism in HL-60 cells. This pathway is crucial for the autoinduction of TNF gene expression, as indicated by blocked TNF mRNA levels when phospholipase A2 and lipoxygenase are inhibited.
Area of Science:
- Cellular and Molecular Biology
- Immunology
- Biochemistry
Background:
- Tumor necrosis factor (TNF) mediates diverse cellular responses like cytolysis and differentiation through cell surface receptors.
- The precise mechanisms governing TNF's cell-specific actions and its autoinduction remain incompletely understood.
Purpose of the Study:
- To investigate post-receptor signaling events involved in TNF-induced autoinduction of TNF expression in HL-60 cells.
- To elucidate the role of phospholipase enzymes and arachidonic acid metabolism in TNF signaling pathways.
Main Methods:
- Stimulation of HL-60 cells with TNF and measurement of phospholipase C and A2 activities.
- Analysis of arachidonic acid metabolite release and TNF mRNA transcription.
- Assessment of the effects of phospholipase A2 inhibitors and lipoxygenase inhibitors (ketoconazole, nordihydroguaiaretic acid) on TNF expression.
Main Results:
- TNF stimulation significantly increased phospholipase A2 activity and arachidonic acid metabolite release in HL-60 cells.
- TNF autoinduction of TNF expression was observed within 15-30 minutes, linked to increased TNF transcription.
- Inhibition of phospholipase A2 or lipoxygenase blocked TNF-induced TNF expression, while cyclooxygenase inhibition had no effect.
Conclusions:
- TNF activates phospholipase A2 and subsequent arachidonic acid metabolism in HL-60 cells.
- Phospholipase A2 and lipoxygenase pathways are essential for the transcriptional activation of TNF gene expression following TNF stimulation.
- These findings reveal a novel mechanism for TNF autoinduction involving lipid mediator signaling.