NIK overexpression amplifies, whereas ablation of its TRAF3-binding domain replaces BAFF:BAFF-R-mediated survival signals in B cells - PubMed (original) (raw)

NIK overexpression amplifies, whereas ablation of its TRAF3-binding domain replaces BAFF:BAFF-R-mediated survival signals in B cells

Yoshiteru Sasaki et al. Proc Natl Acad Sci U S A. 2008.

Abstract

BAFF-R-dependent activation of the alternative NF-kappaB pathway plays an essential role in mature B cell survival. Mutations leading to overexpression of NIK and deletion of the TRAF3 gene are implicated in human multiple myeloma. We show that overexpression of NIK in mouse B lymphocytes amplifies alternative NF-kappaB activation and peripheral B cell numbers in a BAFF-R-dependent manner, whereas uncoupling NIK from TRAF3-mediated control causes maximal p100 processing and dramatic hyperplasia of BAFF-R-independent B cells. NIK controls alternative NF-kappaB signaling by increasing the protein levels of its negative regulator TRAF3 in a dose-dependent fashion. This mechanism keeps NIK protein levels below detection even when they cause B cell hyperplasia, so that contributions of NIK to B cell pathologies can easily be overlooked.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

NIKΔT3 expression strongly induces the alternative NF-κB pathway. (A) NIK or NIKΔT3 are expressed under control of the ROSA26 promoter after Cre-mediated deletion of the loxP-flanked STOP cassette. Flag-tagged wild-type NIK and NIKΔT3, which lacks the T3BD (amino acids 78–84) were used for this approach. KD, kinase domain. (B) B cell-specific expression of the IRES-eGFP-containing construct can be verified by FACS analysis. (C–H) Western blot analysis of extracts from control, NIKtg, and NIKΔT3tg B cells, as indicated above the individual blots. The levels of β-actin, tubulin, and lamin B1 are shown as loading controls for whole-cell, cytoplasmic (C), and nuclear (N) extracts, respectively. Numbers in red indicate average quantifications (normalized to the respective loading controls) relative to controls of four (D), five (E), three to six (F), four (G), or three to four (H) experiments.

Fig. 2.

Fig. 2.

B cell hyperplasia induced by NIK or NIKΔT3 expression. (A) Macroscopic appearance of spleen and lymph nodes. Bold red numbers below the spleens or lymph nodes indicate the average number of B cells (× 106) in three age-matched mice, respectively. (B) FACS analysis shows the proportions of transitional B220+AA4.1+ and mature B220+AA4.1− B cells in the spleens of control, B cellNIK, and B cellNIKΔT3 mice. Numbers next to individual gates refer to the percentages of transitional and mature splenic B cells of total lymphocytes. (C) Immunohistochemical analyses of spleen sections with anti-B220 (blue) and anti-MOMA1 (red) shows the ring of metallophilic macrophages (red) encircling the B cell (blue) follicles (FO). Blue staining outside the follicles represents MZ B cells. (Magnification: Upper, ×40; Lower, ×200.) (D) Hematoxylin/eosin staining of Peyer's patch and nasal-associated lymphoid tissue (NALT) sections from a B cellNIKΔT3 mouse. (E) FACS analysis of cell-surface marker expression on NIKΔT3tg and CD19-cre control B cells. Results are representative of at least three independent experiments. (F) Susceptibility of ex vivo isolated cells of the indicated genotypes to Fas-induced apoptosis. The percentages of life Fas-treated cells of life control antibody-treated cells are shown as mean and standard deviation of three independent experiments. (G) DNA-content analysis of ex vivo isolated control, NIKtg, and NIKΔT3tg B cells. The percentages of cells in different phases of the cell cycle (G0/G1, G2/M, and S) are indicated in red; one representative plot of three independent experiments is shown.

Fig. 3.

Fig. 3.

Uncoupling of NIK from TRAF3-mediated negative regulation renders B cells independent of BAFF-R signaling. (A) FACS analysis of splenic B cell populations. Bold red numbers indicate the average number of mature B220+AA4.1− splenic B cells (× 106) (n = 2–4). The numbers next to individual gates refer to the percentages of transitional B220+AA4.1+ and mature splenic B cells (Upper) or of mature follicular IgMlowIgDhigh B cells (Lower) of total lymphocytes. (B) Immunohistochemical analysis of spleen sections. Anti-B220 (blue) stains follicular B cells inside the ring of MOMA1+ (red) metallophilic macrophages. (Magnification: ×200.) (C) Cytoplasmic (C) and nuclear (N) levels of PKCδ, p100/p52, RelB, IκBα, p105/p50, and RelA analyzed by Western blotting in extracts prepared from B cells cultured in medium with or without 500 ng of BAFF/ml for 24 h. Tubulin and lamin B1 levels demonstrate purity and equal loading of cytosolic and nuclear fractions, respectively. This experiment was repeated once with identical results.

Fig. 4.

Fig. 4.

NIK protein level and activity are controlled by a negative feedback loop involving TRAF2/3. (A) Western blot analysis of TRAF2 and TRAF3 protein levels in purified splenic B cells of the indicated genotypes. (B) Western blot analysis of cytoplasmic (C) and nuclear (N) protein amounts of NIK, p100/p52, RelB, TRAF2, and TRAF3 in splenic B cells. (C) Northern blot analysis of Traf2, Traf3, and _IκB_α mRNA levels in CD43-depleted splenic B cells. Numbers in red indicate average quantifications (normalized to the respective loading controls) relative to controls of three (A), one (B), or two to three (C) experiments.

Fig. 5.

Fig. 5.

Scheme of BAFF:BAFF-R-induced alternative NF-κB activation via TRAF2, TRAF3, and NIK. During normal B cell physiology (A), BAFF:BAFF-R interactions induce the degradation of TRAF3, which binds to NIK at the T3BD and together with TRAF2 negatively regulates NIK protein levels. NIK activity stimulates B cell survival via the activation of predominantly alternative NF-κB and increases the TRAF2 and, more prominently, TRAF3 protein levels in a negative feedback interaction. (B) In the absence of BAFF:BAFF-R interactions, TRAF3 accumulates and together with TRAF2 induces the complete degradation of NIK, leading to shutdown of alternative NF-κB activity and cellular apoptosis. (C) In the presence of BAFF, increased expression of NIK leads to a steady state of enhanced NF-κB signaling and B cell survival and enhanced levels of TRAF2 and TRAF3, which keep NIK protein level below detection. Absence of BAFF (B) still results in TRAF2/3-mediated degradation of NIK and apoptosis. The activity of increased de novo generated NIK mostly likely slightly increases BAFF-independent survival. (D) NIKΔT3 induces BAFF-independent maximal p100 processing and B cell survival and very high TRAF2 and TRAF3 protein levels, which, however, cannot impact on NIKΔT3 protein levels.

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