Regulation of gene expression is an emerging and potentially important function of leukocyte chemoattractants. Accumulating evidence indicates that chemoattractants, including classic chemotactic factors and chemokines, can effectively induce the production of proinflammatory cytokines (e.g. IL-1beta) and chemokines (e.g. IL-8). Recent studies suggest that certain chemoattractants also regulate the expression of immunomodulatory cytokines such as IL-12 and therefore influence the emergence of adaptive immunity. In some cases, chemoattractants have been found to suppress LPS-induced IL-12 production by macrophages. Our studies conducted during the initial funding period have established an important role of NF-kappaB in chemoattractant-induced leukocyte gene expression. These agents stimulate NF-kappaB activation through coupling to heterotrimeric G proteins. This unique mechanism distinguishes chemoattractants from cytokines such as TNFalpha and IL-1beta in their abilities to induce transcription factor activation. However, despite our expanding knowledge of cytokine-induced NF-kappaB activation, little is known about how chemoattractants regulate transcription through G proteins. Studies proposed in this application will focus on the mechanisms by which chemoattractant receptors uses Galphai proteins to regulate leukocyte gene expression. All heptahelical chemoattractant receptors activate Galphai, which releases Gbeta gamma for activation of major functions of leukocytes.
In Aim 1, we will determine how Gbeta gamma mediates NF-kappaB activation. The involvement of a Gbeta gamma effector, PI-3 kinase-gamma, will be investigated using cells from normal and genetically altered mice.
Aim 2 is devoted to studies of Galpha16 for its direct coupling with chemoattractant receptors and its permissive activation of PLCbeta, a unique mechanism of cooperative activation that may explain why chemoattractants are effective transcriptional regulators only in hematopoietic cells.
In Aim 3, we will examine the potential role of Galphai in the inhibition of IL- 12 gene expression. Using in vitro reconstitution assays, we will compare chemoattractant receptors that mediate suppression of IL-12 production with those that lack this ability in order to identify the differences in their G protein-coupling properties. Collectively, these studies are designed to test the central hypothesis that chemoattractant receptors regulate gene expression through differential coupling to G proteins.
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