TRAF2 is believed to mediate the activation of NF-kappaB and JNK induced by the tumor necrosis factor receptor (TNFR) superfamily, which elicits pleiotropic responses in lymphocytes. We have investigated the physiological roles of TRAF2 in these processes by expressing a lymphocyte-specific dominant negative form of TRAF2, thereby blocking this protein's effector function. We find that the TNFR superfamily signals require TRAF2 for activation of JNK but not NF-kappaB. In addition, we show that TRAF2 induces NF-kappaB-independent antiapoptotic pathways during TNF-induced apoptosis. Inhibition of TRAF2 leads to splenomegaly, lymphadenopathy, and an increased number of B cells. These findings indicate that TRAF2 is involved in the regulation of lymphocyte function and growth in vivo.

Binding of microRNA (miRNA) to mRNA within the RNA-induced silencing complex (RISC) leads to either translational inhibition or to destruction of the target mRNA. Both of these functions are executed by Argo-naute 2 (Ago2). Using hematopoiesis in mice as a model system to study the physiological function of Ago2 in vivo, we found that Ago2 controls early development of lymphoid and erythroid cells. We show that the unique and defining feature of Ago2, the Slicer endonuclease activity, is dispensable for hematopoiesis. Instead, we identified Ago2 as a key regulator of miRNA homeosta-sis. Deficiency in Ago2 impairs miRNA biogenesis from precursor-miRNAs followed by a reduction in miRNA expression levels. Collectively, our data identify Ago2 as a highly specialized member of the Argonaute family with an essential nonredundant Slicer-independent function within the mammalian miRNA pathway. Supplemental material is available at http://www.genesdev.org.

4-1BB ligand (4-1BBL) is a member of the tumor necrosis factor (TNF) family expressed on activated antigen-presenting cells. Its receptor, 4-1BB, is a member of the TNF receptor family expressed on activated CD4 and CD8 T cells. We have produced a soluble form of 4-1BBL using the baculovirus expression system. When coimmobilized on plastic with anti-CD3, soluble 4-1BBL induces interleukin (IL)-2 production by resting CD28+ or CD28− T cells, indicating that 4-1BBL can function independently of other cell surface molecules, including CD28, in costimulation of resting T cell activation. At low concentrations of anti-CD3, 4-1BBL is inferior to anti-CD28 in T cell activation. However, when 4-1BB ligand is provided together with strong TCR signals, then 4-1BBL and anti-CD28 are equally potent in stimulation of IL-2 production by resting T cells. We find that TNF receptor–associated factor (TRAF)1 or TRAF2 associate with a glutathione S-transferase–4-1BB cytoplasmic domain fusion protein in vitro. In T cells, we find that association of TRAF1 and TRAF2 with 4-1BB requires 4-1BB cross-linking. In support of a functional role for TRAF2 in 4-1BB signaling, we find that resting T cells isolated from TRAF2-deficient mice or from mice expressing a dominant negative form of TRAF2 fail to augment IL-2 production in response to soluble 4-1BBL. Thus 4-1BB, via the TRAF2 molecule, can provide CD28-independent costimulatory signals to resting T cells.

The nature of signals that govern the development of immunoglobulin heavy chain-dependent B cells is largely unknown. Using mice deficient for the B cell-expressed Src-family protein tyrosine kinases (SFKs) Blk, Fyn and Lyn, we show an essential role of these kinases in pre-B cell receptor (pre-BCR)- mediated NF-kappaB activation and B cell development. This signaling defect is SFK specific, as a deficiency in Syk, which controls pre-B cell development, does not affect NF-kappaB induction. Impaired NF-kappaB induction was overcome by the activation of protein kinase C (PKC)-lambda, thus suggesting the involvement of PKC-lambda in pre-BCR-mediated SFK-dependent activation of NF-kappaB. Our data show the existence of a functionally distinct SFK signaling module responsible for pre-BCR-mediated NF-kappaB activation and B cell development.

Activation of the nuclear factor (NF)-κB transcription complex by signals derived from the surface expressed B cell antigen receptor controls B cell development, survival, and antigenic responses. Activation of NF-κB is critically dependent on serine phosphorylation of the IκB protein by the multi-component IκB kinase (IKK) containing two catalytic subunits (IKKα and IKKβ) and one regulatory subunit (IKKγ). Using mice deficient for protein kinase C β (PKCβ) we show an essential role of PKCβ in the phosphorylation of IKKα and the subsequent activation of NF-κB in B cells. Defective IKKα phosphorylation correlates with impaired B cell antigen receptor–mediated induction of the pro-survival protein Bcl-xL. Lack of IKKα phosphorylation and defective NF-κB induction in the absence of PKCβ explains the similarity in immunodeficiencies caused by PKCβ or IKKα ablation in B cells. Furthermore, the well established functional cooperation between the protein tyrosine kinase Bruton's tyrosine kinase (Btk), which regulates the activity of NF-κB and PKCβ, suggests PKCβ as a likely serine/threonine kinase component of the Btk-dependent NF-κB activating signal transduction chain downstream of the BCR.

Tumor necrosis factor receptor (TNFR)–associated factor 2 (TRAF2) and TRAF1 were found as components of the TNFR2 signaling complex, which exerts multiple biological effects on cells such as cell proliferation, cytokine production, and cell death. In the TNFR2-mediated signaling pathways, TRAF2 works as a mediator for activation signals such as NF-κB, but the role of TRAF1 has not been previously determined. Here we show in transgenic mice that TRAF1 overexpression inhibits antigen-induced apoptosis of CD8+ T lymphocytes. Our results demonstrate a biological role for TRAF1 as a regulator of apoptotic signals and also support the hypothesis that the combination of TRAF proteins in a given cell type determines distinct biological effects triggered by members of the TNF receptor superfamily.

No abstract

SummaryTumor necrosis factor (TNF) signaling leads to pleiotropic responses in a wide range of cell types, in part by activating antiapoptotic and proapoptotic signaling pathways. Thus, although TNF can cause apoptosis and may prove useful in the treatment of malignancies, most cells are resistant to TNF-induced cell death unless de novo protein synthesis is inhibited. Previous studies suggested that TNF activation of the nuclear factor (NF)-B transcription factor family antagonizes the proapoptotic signals initiated by TNF-␣ . TNF receptor-associated factor (TRAF)2 has also been shown to mediate crucial antiapoptotic signals during TNF stimulation, yet is not essential in activation of NF-B under physiologic conditions, thus raising questions about the relationship between these antiapoptotic pathways. We report here that inhibition of TRAF2 and NF-B function in primary cells, by coexpression of a constitutive repressor of multiple NF-B/Rel proteins (I B ␣ .DN) and a dominant negative form of TRAF2 (TRAF2.DN), synergistically enhanced TNF-induced apoptosis. The effects were stimulus dependent, such that neither inhibitory molecule affected Fas-and daunorubicin-induced apoptosis to the same degree as TNF-induced death. These findings indicate that the NF-B and TRAF2 pathways activate independent antiapoptotic mechanisms which act in concert to suppress the proapoptotic signals induced by TNF-␣ .