Zihai Li

Mammalian heat shock protein gp96 is an obligate chaperone for multiple integrins and TLRs, the mechanism of which is largely unknown. We have identified gp93 in Drosophila having high sequence homology to gp96. However, no functions were previously attributed to gp93. To determine whether gp93 and gp96 are functionally conserved, we have expressed gp93 in gp96-deficient mouse cells. Remarkably, the Drosophila gp93 is able to chaperone multiple murine gp96 clients including integrins α₄, α_L, and β₂ and TLR2 and TLR9. This observation has led us to examine the structural basis of the chaperone function of gp96 by a close comparison between gp96 and gp93. We report that whereas gp96 undergoes intermolecular disulfide bond formation via Cys¹³⁸, gp93 is unable to do so due to the absence of a cysteine near the same region. However, abrogation of disulfide bond formation by substituting C with A (C138A) in gp96 via site-directed mutagenesis did not compromise its chaperone function. Likewise, gp93 chaperone ability could not be improved by forcing intermolecular bond formation between gp93 N termini. We conclude that gp93 is the Drosophila ortholog of gp96 and that the chaperone function of the two molecules is conserved. Moreover, gp96 N-terminal disulfide bond formation is not critical for its function, underscoring the importance of N-terminal dimerization via non-disulfide bond-mediated interactions in client protein folding by gp96. Further study of gp96 from an evolutionary angle shall be informative to uncover the detailed mechanism of its chaperone function of client proteins in the secretory pathway.

Inflammation is a necessary albeit insufficient component of tumorigenesis in some cancers. Infectious agents directly implicated in tumorigenesis have been shown to induce inflammation. This process involves both the innate and adaptive components of the immune system which contribute to tumor angiogenesis, tumor tolerance and metastatic properties of neoplasms. Recently, heat-shock proteins have been identified as mediators of this inflammatory process and thus may provide a link between infection-mediated inflammation and subsequent cancer development. In this review, the role of heat-shock proteins in infection-induced inflammation and carcinogenesis will be discussed.

Endoplasmic reticulum (ER) unfolded protein response (UPR) plays pivotal roles in both early B-cell development and plasma cell differentiation. As a major ER chaperone to mediate the UPR and a master chaperone for Toll-like receptors (TLRs), HSP90b1 (grp94, gp96) has long been implicated to facilitate the assembly of immunoglobulin. We hereby critically and comprehensively examine the roles of HSP90b1 in B-cell biology in vivo using B-cell–specific HSP90b1-null mice. We found that knockout B cells developed normally. There were no apparent problems with plasma cell differentiation, Ig assembly, class-switching, and Ig production. Strikingly, although both mutant conventional and innatelike B cells failed to compartmentalize properly due to loss of select but not all integrins, HSP90b1 was required for neither germinal center formation nor memory antibody responses in vivo. The only significant defect associated with HSP90b1 ablation in B cells was an attenuated antibody production in the context of TLR stimulation. Thus, our study has resolved the long-standing question regarding HSP90b1 in B-cell biology: HSP90b1 optimizes the function of B cells by chaperoning TLRs and integrins but not immunoglobulin. This study also has important implications in resolving the controversial roles of TLR in B-cell biology.

We have previously reported that heat shock protein 70 (HSP70) vaccination induced natural killer (NK) cell activity in patients with chronic myelogenous leukemia (CML). In this study, HSP70 of both autologous and allogeneic origin was found to stimulate IFN-gamma production from peripheral blood mononuclear cells of CML patients, as well as of normal subjects. Further investigations demonstrated that the activity of HSP70 was dependent on both NK cells and dendritic cells (DCs). HSP70 did not induce significant IFN-gamma production from either NK cells or DCs alone. Mechanistically, we found that HSP70-mediated DC-NK cell crosstalk required cell-cell contact, which could be inhibited completely by neutralizing antibody against NK activating receptor NKG2D. The significance of NKG2D was further corroborated by the finding that HSP70 induced the expression of an NKG2D ligand, the MHC class I chain-related protein A (MICA), on DCs; HSP70-augmented IFN-gamma release was abrogated by antibody against MICA. Thus extracellular HSP70, released during either stress or inflammatory cell death, may serve as a critical link between NK and DCs in mounting immune responses against infections, cancers and self-antigens.

Proinflammatory cytokine IL‐23 but not IL‐12 is critical for the pathogenesis of organ‐specific autoimmune diseases including experimental autoimmune encephalitis and collagen‐induced arthritis. The contribution by IL‐23 in systemic autoimmune diseases such as lupus is undefined. We addressed this question in a murine lupus‐like disease model, initiated by enforced cell‐surface expression of an ER HSP gp96 in C57BL/6 background. We found a significant increase of p40 in the sera in these mice that preceded the onset of diseases. However, autoimmunity was abrogated in transgenic mice expressing membrane‐bound gp96 reconstituted with p35^–/– BM, but not with p19^–/– BM. Moreover, we found that dendritic cells (DC) but not macrophages were the main producers of p40. To dissect the roles of DC further, we depleted DC using a diphtheria toxin‐based inducible DC depletion system. We demonstrated that the integrity of DC was essential for autoimmunity. Our results thus revealed that IL‐12 and DC are critical for the pathogenesis of lupus‐like disease precipitated by cell surface gp96. This study further highlighted the significant biological differences between IL‐12 and IL‐23.

Purpose: Fenretinide (4-HPR) is a synthetic retinoid that has shown a preventive activity in prostate cancer animal models.