| Budget Amount *help |
¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2010: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2009: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2008: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
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| Research Abstract |
The transcription factor NF-κB regulates the expression of a wide range of genes, including various proinflammatory cytokines, cell adhesion proteins, and several anti-apoptotic molecules that together play pivotal roles in almost all aspects of immune and inflammatory responses. In resting cells, NF-κB associates with members of the inhibitory family of IκB proteins, resulting in retention of these complexes in the cytoplasm. Following appropriate stimulation, IκB proteins are phosphorylated on two specific NH_2-terminal serine residues by one of the catalytic subunits of the IκB kinase (IKK). Phosphorylated IκBs are subsequently ubiquitinated and degraded by the proteasome, leaving NF-κB free to translocate to the nucleus, where it binds to cognate enhancer/promoter elements in its cohort of target genes. However, we and others have shown that, besides the regulated degradation of IκBs, phosphorylation of nuclear NF-κB p65 is also an obligatory step for efficient transcription of NF-
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κB-dependent genes. Several different protein kinases and putative sites of phosphorylation on p65 have been identified, and, in general, it is believed that these phosphorylation events occur concomitantly with IKK-mediated phosphorylation of IκB proteins. We suggest that this additional step in the NF-κB activation pathway helps ensure that only NF-κB that enters the nucleus from the cytoplasm in response to appropriate inducing signals is able to trigger gene expression.
To address the biological importance of the putative sites of phosphorylation on p65, Ser 276 and Ser 534, we generated knock-in mice expressing a serine-to-alanine mutation. Instead of the expected embryonic lethality from hepatocyte apoptosis seen in the absence of NF-κB activity, the S276A knock-in embryos die at different embryonic days due to variegated developmental abnormalities. We demonstrate that this variegated phenotype is due to epigenetic repression resulting from the recruitment of histone deacetylases by the nonphosphorylatable form of NF-κB into the vicinity of genes positioned fortuitously near NF-κB-binding sites. Therefore, unphosphorylated nuclear NF-κB can affect expression of genes not normally regulated by NF-κB through epigenetic mechanisms. On the other hand, S534A knock-in mice demonstrated normal development with slight increasing body weight. Consistent with these results, we observed increased subcutaneous fat in S534A mice compared with control littermate.
To further determine whether changing the Ser 276 to a phospyhomimetic amino acid could mimic phosphorylation, and to examine the biological consequences of such a modification, we knocked in a mutant form of p65, where Ser 276 was changed to aspartic acid (S276D), into the genome. Mice bearing the p65 S276D mutation are born at normal Mendelian ratios, but soon begin to display a progressive, systemic hyperinflammatory condition that results in severe runting and, typically, death 8-20 d after birth. We demonstrated that a significant number of NF-κB target genes are up-regulated in these mice, thereby explaining the hyperinflammatory phenotype. Remarkably, crossing the knock-in strain with mice lacking TNF receptor 1 (TNFR1) leads to a complete rescue of the systemic inflammatory phenotype, but not in certain local inflammatory conditions-including one that resembles human keratoconjunctivitis sicca (KCS) or "dry eye". Therefore, the p65 S276D knock-in mice provide a unique model system, demonstrating the distinct roles of NF-κB in systemic inflammation and certain localized inflammatory diseases. Less
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