Toll-like receptors (TLRs) play a central role in innate and adaptive immunity. TLRs have evolved to detect infection by recognizing the conserved molecular patterns of microorganisms, and such recognition leads to the induction of innate and adaptive immunity through the activation and maturation of dendritic cells (DCs), the most potent professional antigen-presenting cells (APCs). The central question regarding the induction of both innate and adaptive immune responses is how the immune receptor-mediated signals are transduced intracellularly, thereby leading to the nuclear gene expression and immune effector functions. The mitogen-activated protein kinase (MAPK) and the IkappaBalpha kinase (IKK) are the main targets of the TLR pathways in DCs and other cells in immune system. MEKK3 is a serine/threonine protein kinase that belongs to the MAPK kinase kinase family. We disrupted the Mekk3 gene in mice and found that MEKK3 is a key signal transducer of the IL-1R and TLR4 signaling to the downstream MAPK and IKK pathways. Because the IL-1 receptor (IL-1R)/TLR gene family is central for the multiple aspects of DC biology, we hypothesize that MEKK3 plays an essential role in DC function. The proposed studies are designed to elucidate the molecular mechanisms of MEKK3 signaling and MEKK3 function in DCs through the IL-1R-TLR pathways.
Four Specific Aims are proposed: (1) to define the molecular mechanism of MEKK3 activation by IL-1R-TLR4. (2) to determine the structural basis of MEKK3 specificity in the IL-1R-TLR4 pathways and to isolate and clone MEKK3 regulators and targets in DCs. (3) to determine the role of MEKK3 in DC activation and maturation. (4) to investigate the role of MEKK3 in DC differentiation in vivo. We will determine MEKK3 intracellular localization, phosphorylation, enzymatic activity, and MEKK3 association with adaptor molecules in the IL-1R-TLR pathways. We will determine the MEKK3 functional domains and clone MEKK3 regulators in DCs. We will establish MEKK3-deficient DCs using Mekk3-/- embryos, Mekk3 siRNA, and Mekk3 floxed mice and determine the role of MEKK3 in MAPK and IKK-NF-kappaB activation, and in DC lineage differentiation, activation and maturation. In addition to revealing the regulation and function of MEKK3 in DCs, the outcome from this study will also provide conceptual and material resources for studying MEKK3 and its homologues in many other physiological and pathological processes. Most importantly, this study may discover novel molecular targets for therapeutic interventions for treating diseases such as arthritis, sepsis, atherosclerosis, autoimmunity, and cancer.
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