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LPS-binding protein (LBP) is a central mediator that transfers LPS to CD14 to initiate TLR4-mediated proinflammatory response. However, a possibility of another LPS transfer molecule has been suggested because LBP-deficient mice showed almost normal inflammatory response after LPS injection. In this study, we describe the novel finding that high mobility group box 1 protein (HMGB1) recently identified as a mediator of sepsis has a function of LPS transfer for a proinflammatory response. We used ELISA and surface plasmon resonance to show that HMGB1 binds LPS in a concentration-dependent manner and that the binding is stronger to lipid A moiety than to the polysaccharide moiety of LPS. This binding was inhibited by LBP and polymyxin B. Using native PAGE and fluorescence-based LPS transfer analyses, we show that HMGB1 can catalytically disaggregate and transfer LPS to both soluble CD14 protein and to human PBMCs in a dose-dependent manner. However, this effect was dramatically reduced to the baseline level when HMGB1 was heat inactivated. Furthermore, a mixture of HMGB1 and LPS treatment results in a higher increase in TNF-α production in human PBMCs and peripheral blood monocytes than LPS or HMGB1 treatment alone or their summation. Thus, we propose that HMGB1 plays an important role in Gram-negative sepsis by catalyzing movement of LPS monomers from LPS aggregates to CD14 to initiate a TLR4-mediated proinflammatory response.

Many transcription coactivators interact with nuclear receptors in a ligand-and C-terminal transactivation function (AF2)-dependent manner. We isolated a nuclear factor (designated ASC-2) with such properties by using the ligand-binding domain of retinoid X receptor as a bait in a yeast two-hybrid screening. ASC-2 also interacted with other nuclear receptors, including retinoic acid receptor, thyroid hormone receptor, estrogen receptor ␣, and glucocorticoid receptor, basal factors TFIIA and TBP, and transcription integrators CBP/p300 and SRC-1. In transient cotransfections, ASC-2, either alone or in conjunction with CBP/p300 and SRC-1, stimulated ligand-dependent transactivation by wild type nuclear receptors but not mutant receptors lacking the AF2 domain. Consistent with an idea that ASC-2 is essential for the nuclear receptor function in vivo, microinjection of anti-ASC-2 antibody abrogated the liganddependent transactivation of retinoic acid receptor, and this repression was fully relieved by coinjection of ASC-2-expression vector. Surprisingly, ASC-2 was identical to a gene previously identified during a search for genes amplified and overexpressed in breast and other human cancers. From these results, we concluded that ASC-2 is a bona fide transcription coactivator molecule of nuclear receptors, and its altered expression may contribute to the development of cancers.The nuclear receptor superfamily is a group of ligand-dependent transcriptional regulatory proteins that function by binding to specific DNA sequences named hormone response elements in the promoters of target genes (for a review, see Ref.1). The superfamily includes receptors for a variety of small hydrophobic ligands such as steroids, T3, 1 and retinoids as well as a large number of related proteins that do not have known ligands, referred to as orphan nuclear receptors (reviewed in Ref.2). Functional analysis of nuclear receptors has shown that there are two major activation domains. The activation function-2 (AF-2) at the extreme C-terminal region of the ligandbinding domain (LBD) exhibits ligand-dependent transactivation, whereas the N-terminal activation function-1 contains a ligand-independent transactivation domain. The AF-2 region is conserved among nuclear receptors, and deletion or point mutations in this region impair transcriptional activation without changing ligand and DNA binding affinities. X-ray crystallographic studies of the LBD of nuclear receptors revealed that the ligand binding induces a major conformational change in the AF-2 region (3-7), suggesting that this region may play a critical role in mediating transactivation by a ligand-dependent interaction with coactivators. As expected, many coactivators fail to interact with AF-2 mutants of nuclear receptors (8 -10). Transcriptional activation of most nuclear receptors involves at least two separate processes as follows: derepression and activation. Repression is mediated in part by interaction of unliganded receptors with corepressors such as N-CoR (11) and SMRT (12). H...