We have previously shown that degranulation of antigen-stimulated cultured RBL-2H3 mast cells is regulated primarily by changes in cytosolic Ca2+ (the calcium signal), protein kinase (PK) C, and phospholipase (PL) D (see accompanying report HL000993-17 LMI) whereas the generation of inflammatory lipids and cytokines is dependent on the mitogen activated protein (MAP) kinases in addition to a calcium signal and PKC. With respect to the inflammatory lipids, p38 MAP kinase regulates expression of PLA2 (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. The activation of PLA2 is dependent on phosphorylation by the MAP kinase, ERK1/2(see previous reports in this series). This year we have investigated in more detail the regulation of cytokine production and in particular the question of why a background stimulation by stem cell factor (SCF), a mast cell growth factor, is required for optimal production of cytokines in response to antigen stimulation via the receptor for IgE (Fc-epsilon-RI). Studies were conducted with cultured bone marrow-derived mast cells and human mast cells that were derived from peripheral blood pluripotent CD34+ cells because the receptor for SCF, c-KIT, is constitutively active in RBL-2H3 cells. Also, we have extended studies of our earlier observations that glucocorticoids potently inhibit intermediate signalling events that are essential for mast cell activation. The new findings are as follows. Fc-epsilon-RI and c-KIT (CD117) are reported to mediate many signaling pathways in common although the c-KIT primarily promotes mast cell maturation, survival, and optimal physiologic responses to antigen whereas antigen induces release of inflammatory mediators. However, we find that there are critical differences in signaling processes initiated by the two receptors. In cells temporarily deprived of SCF, SCF failed to activate PKC and degranulation. Nevertheless, SCF stimulated PLCgamma, IP3 production, a calcium signal, and acted in synergy with antigen to augment the calcium signal and degranulation. SCF, like antigen, stimulated activating phosphorylations of all MAP kinases, phosphatidylinositol 3-kinase, and Akt. SCF and antigen together augmented these phosphorylations. The failure to of SCF to activate PKC, resulted in lack of induction and phosphorylation of c-Jun a critical component of the AP1 transcription factor complex. However, SCF did augment the induction of c-Jun and phosphorylation of other AP1 components, c-Fos and ATF-2, by antigen and activated JAK3/STAT5 pathway which antigen did not. Both stimulants activated the IkappaB and NF-kappaB pathway to an equal extent. By itself, antigen was a relatively weak stimulant of cytokine mRNA production (as determined by Rnase protection assay kits and film arrays) as compared to SCF which stimulated an array of cytokine mRNAs. In combination with SCF, antigen appeared to enhance production of these mRNAs by SCF and stimulate production of additional cytokine mRNAs including TNFalpha, IL-3, and IL-5. When human mast cells are continuously exposed to SCF, the normal physiologic condition, none of the acute responses to SCF noted above were apparent save for the phosphatidylinositol 3-kinase/Akt pathway which remained continuously activated (the status of JAK/STAT pathway remains to be determined). The addition of antigen reactivated the pathways described above and stimulated production of the same array of cytokine mRNAs as did the combination of antigen and SCF to SCF-deprived cells. These studies thus reveal differences in the requirements for AP1 and the JAK/STAT pathways in the production of individual cytokines. Also for certain cytokines, the combination SCF and antigen is required to provide all the necessary signals for transcription in particular the activation of PKC and AP1 components by antigen and the activation of the JAK/STAT5 pathway by SCF. This topic will be studied in more detail in future work. Suppression of cytokine production by glucocorticoids is attributed to repression of activity of transcriptional factors that promote cytokine gene transcription but no mechanism has been described for the suppression of degranulation in mast cells. As described in previous reports, physiologic concentrations of glucocorticoids suppress activation of all MAP kinases and, as a consequence, induction of COX-2, release of eicosanoids, and the generation of TNFalpha. We now find that dexamethasone and other glucocorticoids inhibit certain early critical signalling events linked to the activation of phosphatidylinositol (PI) 3-kinase. The primary perturbation appears to be the failure of the regulatory p85 subunit of PI 3-kinase to engage with the adaptor protein Gab2. PLCgamma2 phosphorylation, production of inositol 1,4,5-trisphosphate, the calcium signal, and degranulation are markedly inhibited, and to the same degree, by dexamethasone and PI 3-kinase inhibitors. Transcription of antigen-inducible cytokine genes and phosphorylation of certain transcription factors are also markedly suppressed by dexamethasone and PI 3-kinase inhibitors. Molecules activated through another docking molecule LAT, primarily Btk and PLC1gamma, are not affected by dexamethasone or inhibitors of PI 3-kinase. Of note, the inhibitory effects of dexamethasone are receptor specific in so far as the activation of PI 3-kinase is not inhibited but rather enhanced when cells are stimulated through c-KIT by SCF. Dexamethasone treated RBL-2H3 cells also exhibit enhanced responses to stimulation through the G-protein-coupled adenosine A3 receptor. Therefore, the actions of dexamethasone are not generically inhibitory but must target systems unique to Fc-epsilon-RI signaling processes. Gab2 would be one candidate as c-KIT interacts directly with p85 to activate PI 3-kinase and as far as we are aware G-protein coupled receptors do not recruit Gab proteins for signaling processes. On the basis of these finding we conclude that dexamethasone selectively inhibits the GAB2 cluster of signalling molecules. Also, PLCgamma2 is associated with GAB2 and not, as commonly assumed, LAT and that PLC-gamma-2, not PLC-gamma-1, is the predominant regulator of calcium mobilization and degranulation in mast cells. Our findings reveal novel actions of glucocorticoids on specific biochemical signalling pathways that regulate cytokine production and degranulation in mast cells. As these actions are apparent at 1 to 10 nM dexamethasone, or concentrations that fall within the range of therapeutic plasma levels in humans (5 to 20 nM), they likely contribute to the anti-inflammatory effects of glucocorticoids in addition to the known direct actions of glucocorticoids on transcriptional regulation.
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