- Paper Report
- Open Access
The MAPK p38 pathway controls TNF synthesis
- Andy Clark1
© Current Science Ltd 1999
- Published: 3 September 1999
The biosynthesis of tumor necrosis factor (TNF) is blocked at the translational level by pyridinyl imidazole compounds such as SB203580, which inhibit the mitogen activated protein kinase (MAPK) p38. MAPK p38 is activated by various pro-inflammatory and "cell-stress" stimuli, and phosphorylates substrates including transcription factors (such as MEF2C) and kinases (such as MAPKAPK2, the subject of this paper). In spite of intense interest in the p38 pathway as a potential target for anti-inflammatory therapeutic strategies, little is known about how it regulates TNF synthesis. To generate a mouse knock-out line in order to examine the role of the p38-regulated kinase MAPKAPK2 in the lipopoysaccharide (LPS)-induced synthesis of TNF and other cytokines.
Homozygous knockout mice also had impaired hsp25 phosphorylation in LPS-treated heart tissue. Compared to wild type, -/- mice showed a significantly improved survival of LPS-induced endotoxic shock (50% compared to 10% survival at 24 hours), but no difference in survival after intravenous TNF injection. Spleen cells from MAPKAPK2-deficient mice showed a marked (>70%) decrease in LPS-induced production of TNF, interleukin-6 (IL-6) and interferon (IFN) -?. The production of IL-1? and IL-10 was less profoundly affected (30-40%). Decreases in cytokine production were accompanied by decreases in mRNA in the cases of IL-1? and IL-6 only. There was no evidence of inhibition of TNF secretion, or of altered TNF mRNA stability in MAPKAPK2-deficient spleen cells. Nor did there appear to be any impairment in germinal centre formation following immunisation, although some evidence for decreased apoptosis in the germinal centres was found by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling) assay. FACS analysis of spleen or blood cells showed no defect in CD14+, CD3+, CD4+ or CD8+ populations in the MAPKAPK2-deficient mouse (not shown).
The knockout line was generated by standard methods, involving homologous recombination in embryonic stem cells. Confirmation of the knockout was provided by northern and Southern blotting, and by an in-gel kinase assay for MAPKAPK2. The in vivo phosphorylation of the MAPKAPK2 substrate hsp25 was also investigated by two-dimensional electrophoresis and western blotting. Survival of wild-type(+/+), homozygous knockout (-/-) and heterozygous (+/-) mice was measured following endotoxic shock or TNF injection. Cytokine production by spleen cells was measured by ELISA, and immune cell populations were analysed by fluorescence-activated cell sorter (FACS) (although the latter data are not shown). Germinal centre architecture was examined by immuno-histochemistry. Northern blots were used to examine the expression of various cytokine-encoding mRNAs, and to examine mRNA stability in actinomycin D chase experiments.