Tumor necrosis factor (TNF) is a multifunctional cytokine secreted primarily by mitogen-activated macrophages. The biological properties of TNF include modulation of differentiation, promotion of inflammation, chemoattraction of neutrophils and macrophages, cytotoxicity towards certain cells and induction of expression of genes involved in immune function and inflammation (such as IL-1 and Il-6). Expression of TNF is associated with endotoxic shock, with inflammatory manifestations associated with rheumatoid arthritis, with cachexia associated with cancer and AIDS and with other diseases. The ability of TNF to induce specific gene expression is controlled (at least in part) by its ability to induced the DNA-binding activity of pre-existing transcription factor known as NF-kB. NF-kB binds to regulatory elements in genes such as Il-1, IL-6, MHC classI, IL-2 and TNFalpha itself and stimulates transcription. Classic NF-kB is composed of a heterodimer of a 50kD and 65kD subunit, both of which are related to the product of the c-rel proto-oncogene. Interestingly, the 50kD subunit is derived by proteolytic processing from a precursor protein known as IkB. In vitro studies have shown the phosphorylation of IB likely plays a role in release of IkB from NF-kB. We recently identified a cDNA encoding one form of IkB (IkBalpha/MAD-3) and demonstrated that it contains multiple copies of the so-called ankyrin/SWI6 repeats. Our data demonstrate that TNFalpha induces the rapid in vivo phosphorylation of IkBalpha. Furthermore, we have shown that IkBa is rapidly lost format he TNF-treated cells and two forms of NF-kB associated with IkB concomitantly appear in the nucleus. Within 1-2 hrs, the normal levels of IkBalpha have returned to the TNF-treated cells. Intriguingly, okadaic acid (an inhibitor of type 1 and type 2A phosphatases) induced a similar response to that induced by TNF (the apparent phosphorylation and loss of IkBalpha). In this NIH application we propose to: (1) determine the molecular basis of the phosphorylation of IkBalpha induced b TNF (2) the rapid resynthesis of IkBalpha following release of NF-kB (experiments focus n the promoter of the IkBalpha gene (4) characterize the signal transduction pathways involved in the phosphorylation of IkB, determining whether inactivation of a phosphatase is involved in the process (5) determine whether such a phosphatase is a target of oxygen radicals which are known to induce NF-kB, (6) assay the precursor forms of NF-kB and another inhibitor Bcl-3 to determine if they are also targets of TNF, and (7) characterize two TNF responsive elements, one in the c-jun promoter and one in the IL-8 promoter. These experiments will lead to the enhancement of our knowledge of the mechanisms of action of TNF and of the transduction pathways involved in its signaling.
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