Roles of the kinase TAK1 in TRAF6-dependent signaling by CD40 and its oncogenic viral mimic, LMP1.

The Epstein-Barr virus (EBV)-encoded protein latent membrane protein 1 (LMP1) is essential for EBV-mediated B cell transformation and plays a critical role in the development of post-transplant B cell lymphomas. LMP1 also contributes to the exacerbation of autoimmune diseases such as systemic lupus erythematosus (SLE). LMP1 is a functional mimic of the tumor necrosis factor receptor (TNFR) superfamily member CD40, and relies on TNFR-associated factor (TRAF) adaptor proteins to mediate signaling. However, LMP1 activation signals to the B cell are amplified and sustained compared to CD40 signals. We previously demonstrated that LMP1 and CD40 use TRAF molecules differently. Although associating with CD40 and LMP1 via separate mechanisms, TRAF6 plays a significant role in signal transduction by both. It is unknown whether TRAF6 mediates CD40 versus LMP1 functions via distinct or shared pathways. In this study, we tested the hypothesis that TRAF6 uses the kinase TAK1 to trigger important signaling pathways following both CD40 and LMP1 stimulation. We determined that TAK1 was required for JNK activation and interleukin-6 (IL-6) production mediated by CD40 and LMP1, in both mouse and human B cells. Additionally, TRAF3 negatively regulated TRAF6-dependent, CD40-mediated TAK1 activation by limiting TRAF6 recruitment. This mode of regulation was not observed for LMP1 and may contribute to the dysregulation of LMP1 compared to CD40 signals

Role of TAK1 in IL-6 production by CD40 and LMP1.

<p>A) WT mouse splenic B cells were treated with DMSO or TAK1i prior to stimulation with control insect cells (control i) or those expressing mCD154 (mCD154 i), as described in Methods. Cells were cultured for 24 h and supernatants were collected for IL-6 ELISAs. B) Similar to panel <i>A</i>, except that B cells were from a mCD40LMP1 Tg mouse. C) Similar to panel <i>A</i>, except that human B cells were stimulated. D) Similar to panel <i>A</i>, except that CH12.hCD40LMP1 mouse B cells were treated with medium alone (control) or IPTG to induce DN-TAK1 prior to stimulation. Data in all panels are mean values ± SEM of triplicate samples, and are representative of at least two independent experiments.</p

Role of TAK1 in CD40 and LMP1 early signaling events.

<p>A) Mouse CH12.LX B cells were treated with DMSO or TAK1i for 30 min prior to stimulation with anti-mCD40 Ab for 0, 2, 5, 10, or 30 min. Western blots show pTAK1, pJNK, pIκBα, and total IκBα, with total JNK and actin as loading controls. B) Similar to panel <i>A</i>, except that CH12.hCD40LMP1 mouse B cells were stimulated with anti-hCD40 Ab for 0, 10, 30, or 60 min. Data shown are representative of three independent experiments.</p

Requirement of TAK1 in CD40- and LMP1-mediated Ab production.

<p>CH12.hCD40 or CH12.hCD40LMP1 cells were treated with medium alone or IPTG for 18 h to induce DN-TAK1 prior to stimulation with anti-CD40 Abs, where anti-mCD40 Ab induced signaling by endogenous mCD40, and anti-hCD40 Ab induced signaling via either hCD40 (left) or hCD40LMP1 (right). After 72 h, samples were assayed for IgM production by hemolytic plaque assay. Data are presented as plaque-forming cells (Pfc, IgM-producing cells) per 10⁶ viable recovered cells, mean values ± SEM of replicate samples, and are representative of 3 independent experiments.</p

Requirement for TAK1 in CD40- and LMP1-mediated activation of the JNK pathway.

<p>A) WT mouse splenic B cells were treated with DMSO or TAK1i for 30 min prior to stimulation with anti-mCD40 Ab for 0, 5, 10, or 30 min. Western blots show protein levels for pJNK, with actin as a loading control. B) Similar to panel <i>A</i>, except that splenic B cells from a mCD40LMP1 Tg mouse were stimulated for 0, 10, 30, or 60 min. C) Mouse CH12.LX B cells were treated with DMSO or TAK1i for 30 min prior to stimulation with anti-mCD40 Ab for 0, 2, 4, or 6 h. Western blots show p-c-jun, with actin as a loading control. D) Similar to panel <i>C</i>, except that CH12.hCD40LMP1 mouse B cells were stimulated with anti-hCD40 Ab. Data shown are representative of at least two independent experiments. E) CH12.hCD40LMP1 cells were treated with DMSO or JNKi prior to stimulation with control insect cells (control i) or cells expressing either mCD154 (mCD154 i) or hCD154 (hCD154 i). Cells were cultured for 24 h and supernatants were collected for IL-6 ELISA assay. Data are mean values ± SEM of triplicate samples from two independent experiments.</p

Requirement of TRAFs in CD40-mediated TAK1 activation.

<p>A) A20.TRAF3^+/+ or A20.TRAF3^−/− mouse B cells were stimulated with anti-mCD40 Ab for 0, 5, 10, or 30 min. Western blots show pTAK1 and TRAF3, with actin as a loading control. B) Similar to panel <i>A</i>, except that A20.TRAF6^+/+ or A20.TRAF6^−/− mouse B cells were used. Western blots show pTAK1 and TRAF6, with actin as a loading control. These images were part of the same membrane, but the middle lanes were omitted here for clarity. Data shown are representative of at least four independent experiments.</p

Association of TRAF6 with CD40 in the presence or absence of TRAF3.

<p>A) CH12.TRAF3^+/+ or CH12.TRAF3^−/− mouse B cells expressing hCD40 were stimulated with control (−) or anti-hCD40 (+) Ab-coated beads for 10 min. hCD40 was immunoprecipitated using the same beads. B) Quantitation by luminescence imaging of the results of 3 independent experiments, mean values ± SEM. *p<0.05.</p

Impact of Mll3 loss-of-function on hematopoietic progenitor populations.

<p>Shown is <b>A</b>, the total number of BM cells, and the percentages of <b>B</b>, B cells and <b>C</b>, Gr1⁺ Mac1⁺ myeloid cells, where each point on the graph represents one mouse. Values in panels <i>B</i> and <i>C</i> are expressed as percentages of total cells. <b>D</b>, Representative H&E stains from one individual <i>Mll3</i>^+/+ and <i>Mll3</i>^Δ/Δ mouse of sternum sections taken at 400X magnification. Images depict the entire field of view (top panels) and a zoomed-in view (bottom panels). Scale bars are 50μm. <b>E-I</b>, Similar to panels <i>B</i> and <i>C</i>, except that the percentages of LSK cells, CLP, CMP, MEP, and GMP are shown, respectively. <b>B, C, I</b>, *p<0.05, **p<0.01, ***p<0.005 as determined by the Student’s <i>t</i>-test. <b>G</b>, *p<0.05 as determined by the Mann-Whitney test.</p