Allergic diseases affect about 20% of the population. Mast cells are the primary effectors of immediate type allergic reactions and play a role in sustaining allergic inflammation. Targeting of mast cells or mast cell-derived mediators is an important aspect of therapy. Binding of IgE to high affinity receptors on the mast cell surface initiates signal transduction cascades that control degranulation and cytokine gene transcription. These cascades utilize sequential protein kinase reactions. In the mast cell, the activation pathways regulating degranulation and cytokine transcription are independent and largely parallel. The focus of this proposal is to define the sequential protein kinases which control the transcriptional regulation of cytokine production following aggregation of FceRI. We have cloned, characterized and identified a family of (four) serine/threonine protein kinases, the MEK kinases, in the regulation of cytokine production, particularly through the regulation of the activation of c-Jun kinase. The broad aims of this proposal are to define the sequential protein kinase pathways involving MEKK proteins and c-Jun kinase in cytokine gene transcription and production and the integration of FceRI and stem cell factor (SCF) signaling in the regulation of cytokine production. Utilizing both a mast cell line and differentiated cultures of embryonic stem cell derived mast cells (ESMC), we will define and distinguish the role of the different MEKKs in response to FceRI aggregation and ligation of the SCFR. Using cyclosporin A, we will delineate the novel role of calcineurin in the regulation of the JNK pathway and the interplay between the JNK and NFAT pathways, in the cyclosporin-inhibitable regulation of cytokine expression. Using ESMC having targeted deletion of specific protein kinases, we will define their function in differentiation, cytokine production and degranulation. Characterization of these pathways has implications for the understanding of the pathogenesis and control of allergic diseases.
