TRAF3 enhances type I interferon receptor signaling in T cells by modulating the phosphatase PTPN22.

Type I interferons (IFNs) are among the most powerful tools that host cells deploy against intracellular pathogens. Their effectiveness is due both to the rapid, directly antiviral effects of IFN-stimulated gene products and to the effects of type I IFN on responding immune cells. Type I IFN signaling through its receptor, IFNAR, is tightly regulated at multiple steps in the signaling cascade, including at the level of IFNAR downstream effectors, which include the kinase JAK1 and the transcriptional regulator STAT1. Here, we found that tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) enhanced the activation of JAK1 and STAT1 specifically in CD4+ T cells by preventing recruitment of the negative regulatory phosphatase PTPN22 to the IFNAR complex. The balance between signals through IFNAR and other cytokine receptors influences CD4+ T cell differentiation and function during infections. Our work reveals TRAF3 and PTPN22 as key regulators of CD4+ T cell activation by type I IFNs.

TRAF3 regulation of inhibitory signaling pathways in B and T lymphocytes by kinase and phosphatase localization.

This brief review presents current understanding of how the signaling adapter protein TRAF3 can both induce and block inhibitory signaling pathways in B and T lymphocytes, via association with kinases and phosphatases, and subsequent regulation of their localization within the cell. In B lymphocytes, signaling through the interleukin 6 receptor (IL-6R) induces association of TRAF3 with IL-6R-associated JAK1, to which TRAF3 recruits the phosphatase PTPN22 (protein tyrosine phosphatase number 22) to dephosphorylate JAK1 and STAT3, inhibiting IL-6R signaling. An important biological consequence of this inhibition is restraining the size of the plasma cell compartment, as their differentiation is IL-6 dependent. Similarly, in T lymphocytes, interleukin 2 receptor (IL-2R) signaling recruits TRAF3, which in turn recruits the phosphatase TCPTP (T cell protein tyrosine phosphatase) to dephosphorylate JAK3. The resulting inhibition of IL-2R signaling limits the IL-2-dependent size of the T regulatory cell (Treg) compartment. TRAF3 also inhibits type 1 IFN receptor (IFNαR) signaling to T cells by this mechanism, restraining expression of IFN-stimulated gene expression. In contrast, TRAF3 association with two inhibitors of TCR signaling, C-terminal Src kinase (Csk) and PTPN22, promotes their localization to the cytoplasm, away from the membrane TCR complex. TRAF3 thus enhances TCR signaling and downstream T cell activation. Implications are discussed for these regulatory roles of TRAF3 in lymphocytes, as well as potential future directions.

TRAF3 enhances TCR signaling by regulating the inhibitors Csk and PTPN22.

The adaptor protein TNF receptor associated factor (TRAF) 3 is required for effective TCR signaling and normal T cell effector functions, and associates with the CD3/CD28 complex upon activation. To determine how TRAF3 promotes proximal TCR signaling, we studied TRAF3-deficient mouse and human T cells, which showed a marked reduction in activating phosphorylation of the TCR-associated kinase Lck. The impact of TRAF3 on this very early signaling event led to the hypothesis that TRAF3 restrains one or both of two known inhibitors of Lck, C-terminal Src kinase (Csk) and protein tyrosine phosphatase N22 (PTPN22). TRAF3 associated with Csk, promoting the dissociation of Csk from the plasma membrane. TRAF3 also associated with and regulated the TCR/CD28 induced localization of PTPN22. Loss of TRAF3 resulted in increased amounts of both Csk and PTPN22 in T cell membrane fractions and decreased association of PTPN22 with Csk. These findings identify a new role for T cell TRAF3 in promoting T cell activation, by regulating localization and functions of early TCR signaling inhibitors.

TRAF3 deficiency promotes metabolic reprogramming in B cells.

The adaptor protein TNF receptor-associated factor 3 (TRAF3) is a critical regulator of B lymphocyte survival. B cell-specific TRAF3 deficiency results in enhanced viability and is associated with development of lymphoma and multiple myeloma. We show that TRAF3 deficiency led to induction of two proteins important for glucose metabolism, Glut1 and Hexokinase 2 (HXK2). This was associated with increased glucose uptake. In the absence of TRAF3, anaerobic glycolysis and oxidative phosphorylation were increased in B cells without changes in mitochondrial mass or reactive oxygen species. Chemical inhibition of glucose metabolism or glucose deprivation substantially attenuated the enhanced survival of TRAF3-deficient B cells, with a decrease in the pro-survival protein Mcl-1. Changes in Glut1 and Mcl-1 levels, glucose uptake and B cell number in the absence of TRAF3 were all dependent upon NF-κB inducing kinase (NIK). These results indicate that TRAF3 deficiency suffices to metabolically reprogram B cells, a finding that improves our understanding of the role of TRAF3 as a tumor suppressor, and suggests potential therapeutic strategies.

Roles of TRAF3 in T cells: many surprises.

Tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) is broadly involved in different receptor-mediated signaling pathways. Considerable progress was made recently in understanding the role of TRAF3 in T cell biology. Here we review these new findings about how TRAF3 participates in T cell development and function. The different roles of TRAF3 in distinct immune cells are also compared. That TRAF3 is required for T cell effector functions, and invariant Natural Killer T cell function and development, was unexpected. Another surprising finding is that TRAF3 normally restrains regulatory T cell development. It is now clear that TRAF3 regulates signaling to T cells not only through costimulatory members of the TNFR superfamily, but also through the T cell receptor complex, and cytokine receptors. The diverse roles it plays support the multifaceted nature of this molecule. How TRAF3 mediates integration of different signaling cascades is an important topic for future study.

VEGF/Flk1 signaling cascade transactivates Etv2 gene expression.

Previous reports regarding the genetic hierarchy between Ets related protein 71 (Er71/Etv2) and Flk1 is unclear. In the present study, we pursued a genetic approach to define the molecular cascade between Etv2 and Flk1. Using a transgenic Etv2-EYFP reporter mouse, we examined the expression pattern of Etv2 relative to Flk1 in the early conceptus. Etv2-EYFP was expressed in subset of Flk1 positive cells during primitive streak stages, suggesting that Flk1 is upstream of Etv2 during gastrulation. Analysis of reporter gene expression in Flk1 and Etv2 mutant mice further supports the hypothesis that Flk1 is necessary for Etv2 expression. The frequency of cells expressing Flk1 in Etv2 mutants is only modestly altered (21% decrease), whereas expression of the Etv2-EYFP transgenic reporter was severely reduced in the Flk1 null background. We further demonstrate using transcriptional assays that, in the presence of Flk1, the Etv2 promoter is activated by VEGF, the Flk1 ligand. Pharmacological inhibition studies demonstrate that VEGF mediated activation is dependent on p38 MAPK, which activates Creb. We identify the VEGF response element in the Etv2 promoter and demonstrate that Creb binds to this motif by EMSA and ChIP assays. In summary, we provide new evidence that VEGF activates Etv2 by signaling through Flk1, which activates Creb through the p38 MAPK signaling cascade.

Foxk1 promotes cell proliferation and represses myogenic differentiation by regulating Foxo4 and Mef2.

In response to severe injury, adult skeletal muscle exhibits a remarkable regenerative capacity due to a resident muscle stem/progenitor cell population. While a number of factors are expressed in the muscle progenitor cell (MPC) population, the molecular networks that govern this cell population remain an area of active investigation. In this study, utilizing knockdown techniques and overexpression of Foxk1 in the myogenic lineage, we observed dysregulation of Foxo and Mef2 downstream targets. Utilizing an array of technologies, we establish that Foxk1 represses the transcriptional activity of Foxo4 and Mef2 and physically interacts with Foxo4 and Mef2, thus promoting MPC proliferation and antagonizing the myogenic lineage differentiation program, respectively. Correspondingly, knockdown of Foxk1 in C2C12 myoblasts results in cell cycle arrest, and Foxk1 overexpression in C2C12CAR myoblasts retards muscle differentiation. Collectively, we have established that Foxk1 promotes MPC proliferation by repressing Foxo4 transcriptional activity and inhibits myogenic differentiation by repressing Mef2 activity. These studies enhance our understanding of the transcriptional networks that regulate the MPC population and muscle regeneration.