Previous studies have elucidated the signalling pathways for release of secretory granules, the generation of arachidonic acid, and the production of inflammatory cytokines such as TNF-alpha in antigen stimulated RBL-2H3 mast cells (see Z01 HL 00937-14 LMI cells). Production of inflammatory cytokines (and arachidonic acid) is regulated by the p42 MAP kinase, ERK2, whereas secretion of the newly formed cytokines via Golgi-derived vesicles is regulated by protein kinase C (see the accompanying report Z01 HL 00993-12 LMI for description of regulation of secretion of preformed granules). We have continued studies with inhibitors of the MAP kinase pathways and found that antigen-induced production of TNF-alpha and arachidonic acid is not impaired by inhibitors of the JNK or p28 MAP kinase pathways to indicate that ERK2 is the primary kinase regulating these responses. Previous studies had also shown that production of arachidonic acid and TNF-alpha was inhibited by prior treatment of cells with nanomolar concentrations of the glucocorticoid, dexamethasone probably by inhibiting the ERK2 activation cascade. We now find that this drug inhibits activation of Raf-1 and downstream events,the activation of MEK, ERK2 and cytosolic phospholipase A2. Upstream events, the phosphorylation of the IgE receptor and of proteins that associate with this receptor (Lyn, Syk, Shc, and SHIP) as well as other events that are thought to lead to the activation of Raf-1 via Ras are not inhibited. The inhibition of Raf-1 is associated with dissociation of a Raf-1/heat shock protein 90 complex. This effect appears to be specific in that other proteins known to associate with Raf-1 (14-3-3, cdc37, FKBP65) are unaffected. The role of heat shock protein in Raf-1 signalling is unclear but our results suggest that it critical for such signalling. Also, because the effects of dexamethasone on TNF-alpha production, Raf-1 activation, and the interaction of heat shock protein with Raf-1 exhibit the same dependency on dexamthasone concentration (IC50, approx. 6 nM), we believe that we have identified a therapeutically relevant site of action for this drug.
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