We have previously shown that generation of arachidonic acid and inflammatory cytokines in antigen-stimulated RBL-2H3 cells is primarily dependent on MAP kinases whereas secretion of granules is primarily regulated by protein kinase (PK) C. All responses require sustained increases in cytosolic calcium and the activation of phosphatidylinositol (PI) 3-kinase. With respect to the MAP kinases, p38 MAP kinase regulates expression of phospholipase A2 (the enzyme responsible for production of arachidonic acid) and other enzymes that catalyze formation of eicosanoids from arachidonic acid such as cyclooxygenase-2 (COX-2) and 5-lipoxygenase. ERK phosphorylates and thereby activates phospholipase A2. The ERK pathway is also critical for the activation of genes for several inflammatory cytokines. We have since found that p38 MAP kinase and PI 3-kinase are necessary for the survival of RBL-2H3 cells as inhibition of either enzyme leads to apoptosis. We have conducted parallel studies in human mast cells derived from peripheral blood stem cells (see previous report Z01 HL00099314) because these cells, unlike RBL-2H3 cells, can be stimulated by the mast cell growth factor, Stem Cell Factor (SCF). An intriguing paradox is that SCF stimulates the same array of kinases as antigen in these cells. However, SCF, fails to induce generation of inflammatory cytokines and eicosanoids and secretion. To further our understanding of the links between specific signalling events and functional responses we have attempted to identify critical differences between these two stimulants. Both stimulants cause rapid activation of MAP and phosphatidylinositol kinases and phosphorylation of downstream targets but with some notable differences. Activation of the MAP kinases and phosphorylation of downstream substrates in response to either SCF or antigen are not sustained but in the presence of SCF antigen causes sustained responses. Also, antigen causes rapid and sustained elevation of cytosolic calcium whereas SCF causes a slow and relatively small increase in cytosolic calcium. Another, possibly critical, difference is that antigen but not SCF induces rapid production of AP1 proteins, c-Jun and c-fos, in a PKC dependent manner and the phosphorylation of c-Jun by JNK. Other MAP kinase substrates are phosphorylated when cells are stimulated with either antigen or SCF with the possible exception of AFT-2 which shows minimal induction and phosphorylation in response to SCF compared to antigen. These results suggest that AP1 and ATF transcription factors may play important roles in antigen stimulated cells. The human mast cell may thus provide a useful model to elucidate linkages between antigen-induced signaling events and functional read-outs. Our previous studies (Z01 HL00099314) have also indicted that physiologic concentrations of glucocorticoids suppress activation of all MAP kinases, induction of COX-2, release of eicosanoids, and the generation of inflammatory cytokines. These studies demonstrated that dexamethasone, and presumably other glucocorticoids, act via the glucocorticoid receptor to cause disassociation of heat shock protein 70 and 90 from molecular complexes of the initiating kinases (i.e., Raf-1, MEKK-1, TAK) in the MAP kinase pathways. The kinases, bereft of heat shock proteins, fail to activate and translocate to the cell membrane. The reason for the loss of heat shock proteins from the complexes is still unclear but it may be associated with extensive dephosphorylation of the heat shock proteins that accompanies treatment of cells with dexamethasone. Whether the dephosphorylation is attributable to altered expression of a kinase or phosphatase is under investigation. Most recently we have found that PI 3-kinase-dependent phosphorylation of the PDK substrate, Akt(PKB) is suppressed in dexamethasone-treated cells. We are currently investigating whether or not these kinases are also associated with the heat shock proteins. These findings reveal novel mechanisms of action of glucocorticoids in addition to their known direct actions on transcription factors.