INTRODUCTION: Antigen-activated mast cells release preformed inflammatory factors from secreted granules and produce inflammatory lipids from arachidonic acid and numerous cytokines through gene transcription. As noted in HL000993-22 LMI, our aim is to define the signaling pathways that lead to these responses and how they are modulated by physiologic and therapeutic agents. These responses are synergistically enhanced when mast cells are co-stimulated with antigen (via FceRI)and agonists of receptors for adenosine, stem cell factor (Kit), and Toll-like receptors (TLRs)(see previous reports in this series). Some co-stimulants enhance degranulation and production of cytokines (e.g. adenosine and SCF)whereas others selectively enhance the generation of cytokines thus altering the profile of response to antigen (e.g. TLR ligands) (Qiao et al., Blood 107:610, 2006). An underlying theme is that in allergic disease, FceRI-mediated signals operate in the context of physiologic (e.g., stem cell factor) and pathologic factors (e.g., adenosine and TLR ligands). Glucocorticoids remain the most effective drugs for treatment of allergic diseases. For this reason, we have investigated the effects of glucocorticoids on signaling pathways to gain a better understanding of their therapeutic actions. ? ? CURRENT OBJECTIVES: This year we have extended our study of potential co-stimulants to include PGE2 because, like adenosine, its receptors are expressed on mast cells, it accumulates in inflamed tissues, and by itself minimally stimulates mast cells. We have followed up on previous findings that glucocorticoids induce synthesis of several inhibitory regulators of signaling events. We now find that these regulators inhibit both FceRI and TLR mediated pathways. As an alternative therapeutic approach to allergic disease, we have screened agents that simultaneously inhibit Kit and FceRI mediated signals because of the marked synergy between these two receptor systems and found one suitable candidate, the KIT inhibitor hypothemycin. Finally, a multi-laboratory collaborative study was initiated to gain insight on the homeostatic regulation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) by the phosphatase PTEN with the expectation that such insight might reveal new therapeutic targets and a role for PTEN in allergic disease (see Refs. 1 and 2). ? ? PGE2 MARKEDLY POTENTIATE RESPONSES TO ANTIGEN: PGE2, acting through its G protein-coupled EP3 receptor, markedly potentiated FceRI-mediated activation of PLC, calcium signal, calcium-dependent PKC isoforms, degranulation, and production of cytokines. Of note, activation of both EP3-linked PLCbeta and FceRI-linked PLCgamma were amplified by co-stimulation to reveal unexpected cross talk between these two PLCs (manuscript submitted). These and previous studies with adenosine (Ali et al., J. Biol. Chem. 268:16887, 1993) and TLR ligands, illustrate the remarkable ability of co-stimulants to sensitize mast cells to antigen. ? ? IDENTIFICATION OF A NOVEL MECHANISM OF ACTION OF GLUCORTICOIDS: As noted in previous reports, glucocorticoids disrupt critical FceRI-mediated signaling events. These include the activation of Syk and downstream events(Andrade et al., J. Immunol. 172:7254, 2004 and Ref.3) and the Raf1/Erk/PLA2/arachidonic acid pathway(Cissel and Beaven, J. Biol. Chem. 275:7066, 2000). These disruptions can now be attributed to upregulation of inhibitory regulators such as Dok-1 which inhibits Ras activation, MAP kinase phosphatase-1 which dephosphorylates Erk (Hiragun et al., Mol. Pharmacol. 67:598, 2005), and """"""""Src-like adaptor protein"""""""", SLAP, which inhibits Syk activity (Ref. 3). The upregulation of these inhibitors is of slow onset and apparent with clinically relevant concentratiions (1 nM to 10 nM) of dexamethasone--features that were consistent with the inhibition of the Syk and Ras/Raf dependent pathways by dexamethasone. The role of thesse dexamethasone-inducible inhibitors in suppressing activation of Ras, ERK, and Syk, as well as downstream events has been established by various genetic approaches(see Ref. 3). Additional candidate inhibitory regulators are now under study (see next section).? ? Our findings add a new dimension to our understanding of the anti-inflammatory actions of glucocorticoids. In the past these have been attributed to suppression of cytokine gene transcription by a process refered to as """"""""transrepression"""""""". We believe that the anti-inflammatory actions also include induction of inhibitory signaling regulators through interaction of the glucocorticoid receptor with glucocorticoid response elements (GREs), a process refered to as """"""""transactivation"""""""". We have found that expression of a mutated GR which lacks the ability to interact with GREs fails to mediate inhibition of degranulation and release of arachidonic acid but retains the ability to mediate suppression of cytokine production. The GRE for SLAP has now been identified, cloned, and shown to negatively regulate gene transcription in the presence of dexamethasone. These findings are relevant to the development by pharmaceutical companies of """"""""disociated"""""""" steroids that lack the """"""""transactivation"""""""" potential of current glucococorticoids, a potential that was previously thought to account for the undesirable side-effects of glucocortoids. ? ? SYNERGY BETWEEN ANTIGEN AND TLR LIGANDS IS SUPPRESSED BY LOW CONCENTRATIONS OF GLUCOCORTICOIDS: In addition to mediating allergic reactions via FceRI, mast cells protect against microbial infections by production of inflammatory cytokines through the activation of TLRs. Co-stimulation of mast cells via FceRI and TLRs markedly enhances production of these inflammatory cytokines .The enhanced production was associated with synergistic activation of MAP kinases and downstream transcription factors and the engagement of a more effective repertoire of transcription factors for cytokine gene transcription (see last years report).? ? Glucocorticoids potently inhibit production of cytokines in response TLR ligands alone or in combination with antigen (see previous reports). As with antigen stimulation, the inhibitory effects were apparent after a delay of several hours on exposure to low concentrations of dexamethasone and were mediated via the glucocorticoid receptor. Again in common with antigen stimulation, activation of MAP kinase and NFkB pathways were particularly sensitive to the inhibitory actions of dexamethasone. We now find that activation of upstream TAK1 by TRAF6 is a key target for dexamethasone in TLR activated mast cells. Upstream events, the engagement of MyD88 and IRAK, were unaffected. Use of gene chip analysis and RT-PCR revealed two additional dexamethasone-inducible inhibitory regulators, the suppressor of cytokine signaling (SOCS1) and its cognate CIS. Of these two, SOCS1 was found to associate with TRAF6 and enhance its degradation. Use of SOCS1-deficient mast cells and other genetic approaches are being used to confirm that induction of SOCS1 by dexamethasone is responsible for the inhibition of TLR signaling at the level of TRAF6/TAK1. These results suggest that glucocorticoids might be especially effective when allergic disease is exacerbated by viral or bacterial infection.? ? SEARCH FOR ALTERNATIVE THERAPEUTIC APPROACHES: Examination of various inhibitors of Kit revealed that hypothemycin blocked not only Kit kinase activity, as expected, but also FceRI-mediated degranulation and cytokine production and the potentiation of these responses by Kit ligand. The inhibition of FceRI-dependent signaling events occurred at the level of Btk at concentrations that blocked degranulation. Hypothemycin potently inhibited passive cutaneous anaphylaxis in mice to indicate that simultaneous targeting of Kit and FceRI may have therapeutic utility (manuscript submitted).
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