IB-is an inducible nuclear protein that interacts with nuclear factor-B (NF-B) via its carboxyl-terminal ankyrin-repeats. Previous studies using an NF-B reporter have shown that IB-inhibits the activity of NF-B. In the present study, we dissected the aminoterminal region of IB-, which shows no homology to any other proteins. Indirect immunofluorescence studies demonstrated the presence of a bipartite nuclear localization signal spanning amino acids 163-178. Using GAL4 fusion proteins, we found that internal fragments containing amino acids 329 -402 possessed intrinsic transcriptional activation activity. Interestingly, the activity was not detected in GAL4 fusion proteins of the full-length IB-. On the other hand, the GAL4-dependent transcriptional activity was generated by co-expression of the GAL4-NF-B p50 subunit fusion protein and the full-length IB-, neither of which exhibited the activity on their own. A new splicing variant, IB-(D), with a deletion of amino acids 236 -429, was found to lack transactivation activity. Forced expression of IB-, but not IB-(D), augmented interleukin-6 production, indicating the functional significance of the transactivation activity. In contrast, tumor necrosis factor-␣ production was inhibited by expression of IB-, highlighting the dual functions of this molecule. These results indicate that IB-harbors latent transcriptional activation activity, and that the activity is expressed upon interaction with the NF-B p50 subunit. In addition to the inhibitory activity on NF-B-mediated transcription, the transcriptional activation activity of IBshould be crucial for the regulation of inflammation.

Toll-like receptor 4 (TLR4) and MD-2 are pivotal components that elicit inflammatory responses to lipopolysaccharide (LPS). They have been shown to form a physical complex on the cell surface that responds directly to LPS. However, the functional region of TLR4 required for association with MD-2 and LPS responsiveness is poorly understood. To identify the region of TLR4, we created a series of mutants with deletions in the extracellular domain and examined their activities in human embryonic kidney 293 cells. A mutant with a 317-amino acid deletion from the membrane proximal region of TLR4 was capable of associating with MD-2, while only a 9-amino acid truncation of the N terminus severely impaired the interaction. The association between the two molecules was well correlated with TLR4 maturation into an endoglycosidase H-resistant form and the cell surface expression. Mouse MD-2 bound to human TLR4, but its activity to facilitate the cell surface expression of TLR4 and confer LPS responsiveness was much weaker than that of human MD-2, indicating species specificity. A chimeric receptor composed of the N-terminal region of human TLR4 and the adjacent region of mouse TLR4 showed preference for human MD-2 in its transport to the cell surface and responsiveness to LPS. Taken together, the N-terminal region of TLR4 is essential for association with MD-2, which is required for the cell surface expression and hence the responsiveness to LPS.

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