Ced-4 and Apaf-1 belong to a major class of apoptosis regulators that contain caspase-recruitment (CARD) and nucleotide-binding oligomerization domains. Nod1, a protein with an NH 2 -terminal CARD-linked to a nucleotide-binding domain and a COOH-terminal segment with multiple leucine-rich repeats, was identified. Nod-1 was found to bind to multiple caspases with long prodomains, but specifically activated caspase-9 and promoted caspase-9-induced apoptosis. As reported for Apaf-1, Nod1 required both the CARD and P-loop for function. Unlike Apaf-1, Nod1 induced activation of nuclear factor-kappa-B (NF-B) and bound RICK, a CARDcontaining kinase that also induces NF-B activation. Nod1 mutants inhibited NF-B activity induced by RICK, but not that resulting from tumor necrosis factor-␣ stimulation. Thus, Nod1 is a leucine-rich repeatcontaining Apaf-1-like molecule that can regulate both apoptosis and NF-B activation pathways.

IL-21 costimulates B cell proliferation and cooperatively with IL-4 promotes T cell-dependent Ab responses. Somewhat paradoxically, IL-21 also induces apoptosis of B cells. The present study was undertaken to more precisely define the expression of the IL-21R, using a novel mAb, and the circumstances by which IL-21 promotes B cell growth vs death. The IL-21R was first detected during T and B cell development, such that this receptor is expressed by all mature lymphocytes. The IL-21R was further up-regulated after B and T activation, with the highest expression by activated B cells. Functional studies demonstrated that IL-21 substantially inhibited proliferation and induced Bim-dependent apoptosis for LPS or CpG DNA-activated B cells. In contrast, IL-21 induced both costimulation and apoptosis for anti-CD40-stimulated B cells, whereas IL-21 primarily costimulated B cells activated by anti-IgM or anti-IgM plus anti-CD40. Upon blocking apoptosis using C57BL/6 Bim-deficient or Bcl-2 transgenic B cells, IL-21 readily costimulated responses to anti-CD40 while proliferation to LPS was still inhibited. Engagement of CD40 or the BCR plus CD40 prevented the inhibitory effect by IL-21 for LPS-activated B cells. Collectively, these data indicate that there are three separable outcomes for IL-21-stimulated B cells: apoptosis, growth arrest, or costimulation. We favor a model in which IL-21 promotes B cell maturation during a productive T cell-dependent B cell response, while favoring growth arrest and apoptosis for nonspecifically or inappropriately activated B cells.

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We have identified and characterized Diva, which is a novel regulator of apoptosis. Sequence analysis revealed that Diva is a member of the Bcl-2 family of proteins containing Bcl-2 homology domain 1, 2, 3, and 4 (BH1, BH2, BH3, and BH4) regions and a carboxyl-terminal hydrophobic domain. The expression of Diva mRNA was detected in multiple embryonic tissues but was restricted to the ovary and testis in adult mice. The expression of Diva promoted the death of 293T, Ramsey, and T47D cells as well as that of primary sensory neurons, indicating that Diva is a proapoptotic protein. Significantly, Diva lacks critical residues in the conserved BH3 region that mediate the interaction between BH3-containing proapoptotic Bcl-2 homologues and their prosurvival binding partners. Consistent with this, Diva did not bind to cellular Bcl-2 family members including Bcl-2, Bcl-X L , Bcl-w, Mcl-1, and A1/Bfl-1. Furthermore, mutants of Diva lacking the BH3 region fully retained their proapoptotic activity, confirming that Diva promotes apoptosis in a BH3-independent manner. Significantly, Diva interacted with a viral Bcl-2 homologue (vBcl-2) encoded by the Kaposi's sarcoma-associated herpesvirus. Consistent with these associations, apoptosis induced by Diva was inhibited by vBcl-2 but not by Bcl-X L . Importantly, Diva interacted with Apaf-1, an adapter molecule that activates caspase-9, a central death protease of the apoptotic pathway. The expression of Diva inhibited the binding of Bcl-X L to Apaf-1, as determined by immunoprecipitation assays. Thus, Diva represents a novel type of proapoptotic Bcl-2 homologue that promotes apoptosis independently of the BH3 region through direct binding to Apaf-1, thus preventing Bcl-X L from binding to the caspase-9 regulator Apaf-1.Apoptosis, a morphologically distinguished form of programmed cell death, is critical during development and tissue homeostasis and plays a role in the pathogenesis of a variety of diseases (1). Several regulatory components of the apoptotic pathway have been identified in various living organisms including man (1, 2). A family of proteins that includes Bcl-2, Bcl-X L , Bcl-w, Mcl-1, and A1/Bfl-1 shares conserved regions termed Bcl-2 homology domains 1, 2, 3, and 4 (BH1, BH2, BH3, and BH4) 1 and function by repressing apoptosis (3). The biochemical process by which Bcl-2 family members regulate cell death is poorly understood. Analyses of the nematode cell death regulators CED-3, CED-4, and CED-9 have provided important insight into the biochemical mechanism that regulates apoptosis. CED-9, the nematode homologue of Bcl-2 and Bcl-X L , binds to CED-4 and represses cell death by interacting and inhibiting the killing activity of CED-3 through CED-4 (4 -7). Recently, it was shown that Bcl-X L associates with Apaf-1, a mammalian homologue of CED-4, and inhibits the activation of procaspase-9 (8 -11).Whereas proteins like Bcl-2 and Bcl-X L inhibit cell death, structurally related proteins including Bax, Bak, Bad, Bik/ Nbk, Bid, Hrk, Bim, Bok/Mtd, and BNIP3 promote apo...

Disseminated metastasis accounts for over 90% of breast cancer deaths. Recently, elevated serum levels of a glycoprotein known as chitinase-3 like-protein-1 (CHI3L1) has been correlated with poor prognosis and shorter survival of patients with metastatic breast cancer. In this study, we show that there are increased levels of CHI3L1 in plasma of tumor-bearing mice and that both tumor cells and immune cells express and secrete CHI3L1. However, the biological and physiological functions of CHI3L1 are still unclear. We demonstrate that while CHI3L1 has an inhibitory role in the expression of interferon-gamma (IFN-γ), CHI3L1 up-regulates pro-inflammatory mediators, C-chemokine ligand 2 (CCL2), Chemokine CX motif ligand 2 (CXCL2) and matrix metalloproteinase-9 (MMP-9) all of which contribute to tumor growth and metastasis. We found that in vitro inhibition of CHI3L1 by siRNA suppressed the production of CCL2, CXCL2 and MMP-9 by macrophages. In vivo treatment of mammary tumor-bearing mice with chitin (β-(1–4)-poly-N-acetyl D-glucosamine), a TH1 adjuvant and a ligand for CHI3L1, promoted immune effector functions with increased production of IFN-γ and decreased CCL2, CXCL2 and MMP-9 expression. In vivo administration of chitin to mammary tumor-bearing mice significantly decreased lung metastasis. These studies show that CHI3L1 plays a role in tumor progression and that chitin can inhibit the pleiotropic effects of CHI3L1 giving support to the idea that CHI3L1 is a useful therapeutic target for treatment of breast cancer.