The overall objective of this application is to investigate the regulatory mechanisms involved in macrophage activation and apoptosis in response to bacterial component lipopolysaccharide (LPS). It is well known that LPS activates the stress response transcription factor NF-kappaB (nuclear factor kappa B) and induces macrophage secretion of inflammatory mediators including tumor necrosis factor (TNF) and interleukin-1 (IL-1) in oral inflammatory diseases. Additionally, the studies indicated that LPS prolonged macrophage survival, which may be responsible for the chronic and persistent inflammation. The results from the principal investigator's laboratory have demonstrated that NF-kappaB maintains the novel function--anti- apoptosis by regulating the expression of anti-apoptotic genes such as inhibitors of apoptosis (IAPs). Thus, control of macrophage activation and survival may be of primary importance for the host to limit the extent of the inflammatory response. The central hypothesis to be tested in this application is that LPS-induced macrophage activation an survival are tightly coupled-events which are transcriptionally controlled by NF- kappaB. In order to understand the regulatory mechanism of macrophage activation and apoptosis, we propose the following three specific aims.
Aim 1 is to explore the molecular mechanisms by which LPS prevents apoptosis of macrophages. The caspase assays and Western blot analysis will be utilized to determine whether LPS-stimulated NF-kappaB activation suppresses Fas-mediated caspase cascades. The Apoptosis Expression Array will be performed to identify the LPS-stimulated anti-apoptotic genes.
Aim 2 is to determine whether LPS-mediated protein kinase cascades modify the transactivation domain of NF-kappaB subunit p65/RelA and thereby potentiate NF-kappaB transcription activity. The biological significance of phosphorylation of the p65 transactivation domain will be explored.
Aim 3 is to apply the two-hybrid assay to identify novel regulators of IKKbeta activation, a central step in LPS mediated NF-kappaB activation. IKKbeta-interacting proteins will be characterized using both the kinase assay and NF-kappaB-responsive luciferase reporter assay. In summary, these studies will provide new insights into the mechanisms of macrophage activation and apoptosis, and have important implication in treatment of oral an systemic inflammatory diseases and septic shock.
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| Yang, Fan; Tang, Eric; Guan, Kunliang et al. (2003) IKK beta plays an essential role in the phosphorylation of RelA/p65 on serine 536 induced by lipopolysaccharide. J Immunol 170:5630-5 |
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