BAFF Activates Akt and Erk Through BAFF-R in an IKK1-Dependent Manner in Primary Mouse B Cells

BAFF activates Akt and Erk through BAFF-R in an IKK1-dependent manner in primary mouse B cells

Kevin L. Otipoby*†, Yoshiteru Sasaki*†‡, Marc Schmidt-Supprian†§, Alina Patke¶, Ralph Gareusʈ**, Manolis Pasparakisʈ**, Alexander Tarakhovsky¶, and Klaus Rajewsky†,††

†Immune Disease Institute, Harvard Medical School, Boston, MA 02115; ¶Laboratory of Lymphocyte Signaling, The Rockefeller University, New York, NY 10021; ʈInstitute for Genetics, University of Cologne, Zulpicher Strasse 47, 50674 Cologne, Germany; and **Mouse Biology Unit, European Molecular Biology Laboratory, I-00016 Monterotondo, Italy

Contributed by Klaus Rajewsky, June 5, 2008 (sent for review April 10, 2008) B cell activating factor (BAFF) signals through BAFF-R to promote Results mature B cell survival. Recent analyses of BAFF-induced signaling BAFF-Mediated B Cell Size Increase Is Mediated by BAFF-R and revealed direct association between augmented B cell metabolic Requires IKK1. The TNF-family cytokine BAFF recognizes TACI fitness and activation of Akt, one of the key regulators of cell and, to a lesser extent, BCMA (3), but it is its interaction with survival. The strongest and most reproducible induction of Akt BAFF-R that is required for B cell survival (5, 6, 19, 20). It was occurs with significant delay (24 h) after BAFF treatment, where it recently reported that treatment with BAFF promotes metabolic precedes activation of anabolism. It was also recently shown that fitness of B cells as assayed by, among other things, increase in BAFF induces sustained Erk activation and increased turnover of cell size (14, 16, 21). To address the requirement of BAFF-R in the proapoptotic molecule Bim. Here we show that these BAFF- BAFF-mediated cell size increase, we used B cells that lack induced signaling pathways are mediated by BAFF-R and represent BAFF-R expression but survive because of overexpression of the previously unknown arms of I kappa B kinase (IKK)1-dependent antiapoptotic molecule Bcl-2. BAFF induced a slight but repro- signaling. In combination with the known role of IKK1 in regulating ducible increase in cell size of BAFF-R-sufficient (control, transcription of prosurvival genes, our data underscore the central bcl-2tg) but not BAFF-R-deficient (baff-rϪ/Ϫ, bcl-2tg) B cells, role of IKK1 in coordinating multiple BAFF-R-mediated signaling showing that exposure to BAFF increases B cell size in a pathways controlling mature B cell homeostasis. BAFF-R-dependent fashion (Fig. 1A). To address the potential role of alternative NF-␬B activation urvival of mature B cells depends critically on the TNF- in BAFF-mediated cell size regulation, we used IKK1-deficient Sfamily cytokine B cell activating factor (BAFF, also known B cells. Because constitutive ablation of IKK1 leads to perinatal as BlyS) (1, 2). Prosurvival signaling events induced by BAFF death, we crossed mice harboring a conditional ikk1 allele (ikk1f) allow peripheral B cells to complete their maturation and to mice that express Cre early in B cell development and accumulate in the peripheral lymphoid organs at physiological recombine loxP-flanked alleles with high efficiency (mb1-cre)to levels. BAFF is a ligand for three TNF receptor-family members, generate mice with IKK1-deficient B cells. Consistent with BAFF-R (also known as BR3), TACI, and BCMA [reviewed in previous results (22, 23), B cell development in ikk1f/f, mb1-cre (3)]. Similar to BAFF-deficient mice, mice lacking BAFF-R (B cell⌬IKK1) mice is apparently normal until the T1 stage, while expression display a strong reduction in late transitional and further maturation is strongly blocked (M.S.-S. and K.R., un- mature B-2 cell numbers (1, 2, 4–6). In contrast, mice with published results). Similar to BAFF-R deficiency (5, 6), trans- individual or combined deficiencies of TACI and BCMA contain genic expression of Bcl-2 partially rescues the survival defect of normal or even increased populations of mature B cells (6–8). IKK1-deficient B cells [(22) and M.S.-S. and K.R., unpublished This genetic evidence suggests that BAFF-R is the receptor that results], allowing us to obtain mature IKK1-deficient B cells. mediates BAFF-dependent B cell survival. Western blot analysis confirmed the ablation of IKK1 protein in The finding that BAFF activates alternative NF-␬B signaling, B cells from B cell⌬IKK1, bcl-2tg mice compared to control ikk1f/f, which requires IKK1 expression and promotes p100 processing bcl-2tg mice (Fig. 1B). Furthermore, IKK1-deficient B cells to p52, suggested a link between BAFF stimulation and antiapo- express normal levels of BAFF-R (Fig. 1C). The BAFF-induced ptotic gene activation as a mechanism for BAFF-mediated B cell increase in cell size of control, bcl-2tg B cells was not seen in survival (9–11). This was further supported by the impaired BAFF-treated IKK1-deficient B cells (see Fig. 1A). In contrast, survival of B cells with altered NF-␬B function (12, 13). The B cell antigen receptor (BCR) stimulation induced blast forma- solitary central role of NF-␬B in BAFF-mediated signaling has tion in the absence of BAFF-R or IKK1 expression [see sup- IMMUNOLOGY recently been challenged by the observation that B cells deficient porting information (SI) Fig. S1], showing that these cells have in PKC␤ expression show impaired responses to BAFF but normal p100 processing to p52 (14). Furthermore, BAFF treatment was shown to induce multiple signaling pathways, including Author contributions: K.L.O., Y.S., M.S.-S., A.P., A.T., and K.R. designed research; K.L.O., Akt and Erk1/2 (14–16). BAFF-induced Akt activation promotes Y.S., M.S.-S., and A.P. performed research; R.G. and M.P. contributed new reagents/analytic tools; K.L.O., Y.S., M.S.-S., A.P., A.T., and K.R. analyzed data; and K.L.O., A.T., and K.R. wrote an increase in the metabolic activity and protein synthesis, while the paper. sustained Erk1/2 activation leads to phosphorylation of the The authors declare no conflict of interest. proapoptotic Bcl-2 family member Bim and prevents its accu- *K.L.O. and Y.S. contributed equally to this work. mulation (14–16). ‡Present address: Laboratory for Stem Cell Biology, RIKEN Center for Developmental By using B cells that lack either BAFF-R or IKK1 expression, Biology, Kobe 650-0047, Japan. we show that BAFF-induced B cell growth is mediated by §Present address: Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 BAFF-R and requires IKK1 expression. We also show that late Martinsried, Germany. Akt and Erk1/2 phosphorylation are, like cell growth, dependent ††To whom correspondence should be addressed at: Immune Disease Institute, 200 Long- on BAFF-R and IKK1. These data identify IKK1, a molecule wood Avenue, Boston, MA 02115. E-mail: [email protected]. critical for alternative NF-␬B activation but also possessing other This article contains supporting information online at www.pnas.org/cgi/content/full/ signaling functions (17, 18), as a coordinator of multiple BAFF- 0805460105/DCSupplemental. R-mediated signaling pathways. © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0805460105 PNAS ͉ August 26, 2008 ͉ vol. 105 ͉ no. 34 ͉ 12435–12438 Downloaded by guest on September 26, 2021 A control baff-r-/- B cell ∇ IKK1 A baff-r+/- baff-r-/- WT bcl-2tg bcl-2tg bcl-2tg bcl-2tg bcl-2tg PBS BAFF PBS BAFF -IgM

100 α

80 10 min 24 hr 24 hr 0 10 min 10 min 24 hr 0 10 min 5 min 24 hr 60 Day 1 pAkt 40 (S473)

20 Akt

0 100 B control baff-r-/- 80 bcl-2tg bcl-2tg

60 Day 2 40 24 hr 24 hr 10 min 10 min

20 -IgM -IgM PBS BAFF PBS BAFF α α 0 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 pErk1/2 FSC PBS Erk1/2 BAFF

Fig. 2. BAFF induced Erk1/2 and late Akt phoshorylation are BAFF-R depen- ϩ Ϫ B C 100 baff-r-/- dent. (A) Splenic B cells from BAFF-R sufﬁcient (baff-r / , bcl-2tg) or deﬁcient Ϫ/Ϫ tg 80 (baff-r , bcl-2tg) mice were preincubated in 1% FCS containing medium for

2 bcl-2tg - bcl-2tg 60 1 h before addition of BAFF or anti-IgM in PBS or PBS alone. Puriﬁed B cells

bcl 40 +/- were also lysed immediately after the 1 h preincubation (time 0). Equal

IKK1 baff-r 20 bcl-2tg amounts of each lysate were used for SDS/PAGE and Western blot analysis for 0 phospho-Akt (S473). The membrane was then stripped and probed for Akt Ϫ/Ϫ 100 levels. (B) Lysates of splenic B cells from control, bcl-2tg or baff-r , bcl-2tg B cell control control ikk1f/f 80 mice stimulated with (BAFF) or without (PBS) BAFF for 24 h were analyzed for bcl-2tg 60 phospho-Erk1/2 and Erk1/2 levels. These data are representative of at least IKK1 three independent experiments. 40 B cell 20 bcl-2tg 0

β-actin 100 101 102 103 104 ∇ phosphorylation between 3- and 27-fold, which is entirely BAFF- BAFF-R R-dependent (Fig. 2A), thus making this signaling event more likely to be related to B cell survival. Fig. 1. BAFF-induced cell size increase is mediated by BAFF-R and down- Craxton and colleagues showed that BAFF stimulation acti- stream of IKK1. (A) Splenic B cells were cultured for 1 or 2 days in 1% FCS containing medium with either 200-ng/ml BAFF diluted in PBS (red histo- vates the Mek/Erk pathway in a B cell line (15). We observed grams) or PBS alone (ﬁlled histograms). ToPro-3 negative, B220 positive B cells similar results in primary B cells, namely that BAFF stimulation were gated and analyzed for forward scatter properties (FSC). (B) Puriﬁed for 24 h induces Erk1/2 phosphorylation, albeit less strongly than splenic B cells from ikk1f/f, bcl-2tg (control, bcl-2tg)orikk1f/f, mb1-cre, bcl-2tg following BCR stimulation (Fig. S3). We also found that BAFF- (B cell⌬IKK1, bcl-2tg) mice were analyzed for IKK1 expression. The membrane induced Erk1/2 phosphorylation depends on BAFF-R expres- was then stripped and blotted for ␤-actin as a loading control. (C) BAFF-R levels sion (Fig. 2B). These data demonstrate that BAFF stimulates Ϫ ϩ on gated AA4.1 B220 mature splenic B cells of the indicated genotypes late Akt and Erk1/2 phosphorylation through BAFF-R and not analyzed directly ex vivo. These data are representative of three independent TACI or BCMA. experiments. BAFF-R-Induced Phosphorylation of Akt and Erk1/2 Is IKK1-Dependent. the ability to grow. These data show that BAFF/BAFF-R- The timing of BAFF-R-dependent Akt activation overlaps with mediated cell size increase requires IKK1 expression. an array of transcription and signaling events controlled by IKK1. To test a possible link between IKK1 and BAFF-R- BAFF Induces Akt and Erk1/2 Phosphorylation Through BAFF-R. BAFF mediated Akt and Erk1/2 activation, we used bcl-2tg IKK1- treatment of B cells has been reported to induce Akt activation deficient B cells. In contrast to controls, IKK1-deficient B cells that initially occurs early (10 min to 1 h) and then again relatively were unable to induce Akt phosphorylation in response to BAFF late (24 h) (14, 16). Of note, the kinetics (14, 16) and magnitude treatment (Fig. 3A). Similar results were observed for BAFF- of early BAFF-mediated Akt activation vary significantly from induced Erk1/2 phosphorylation (see Fig. 3A). These effects no detectable activation after 10 min of BAFF stimulation (range were not because of a generalized defect in the PI-3K/Akt or of 0.6- to 1.3-fold relative to controls, n ϭ 5, K.L.O. and Y.S.) Mek/Erk pathways in IKK1-deficient B cells, because BCR to an average of 10-fold (range of 2.6- to 25.8-fold relative to stimulation induced normal phosphorylation of Akt and Erk1/2 controls, n ϭ 11, A.P. and A.T.). Paradoxically, when early Akt (see Fig. 3A). These data suggest that BAFF/BAFF-R interac- activation in response to BAFF is observed, it occurs in the tion induces Akt and Erk1/2 phosphorylation through IKK1- absence of BAFF-R yet is abrogated by the presence of soluble dependent signaling processes in B cells. decoy BAFF-R in the incubation medium (Fig. S2), which might Our findings place IKK1 as a central upstream mediator of Akt indicate a role for TACI or BCMA. Given the central role of and Erk1/2 activation in BAFF-R-mediated signaling events but BAFF-R in regulation of B cell survival, it seems that the do not exclude the possibility that BAFF/BAFF-R regulates B variable pattern of early Akt induction in response to BAFF in cell responses also through IKK1-independent signaling path- vitro may not mediate crucial functions in B cell physiology. In ways. Indeed, we found that BAFF treatment of WT or bcl-2tg contrast, BAFF treatment for 24 h consistently induced Akt primary B cells induces MHC class II up-regulation (Fig. 3B and

12436 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0805460105 Otipoby et al. Downloaded by guest on September 26, 2021 ␤ A ikk1f/f B cell ∆IKK1 response to BAFF is highlighted by the findings that PKC - bcl-2tg bcl-2tg deficient B cells show impaired Akt phosphorylation and no increase in cell size in response to BAFF (14). In addition, the -IgM PBS BAFF PBS BAFF -IgM mTOR inhibitor rapamycin partially blocks BAFF-mediated α α rescue of WT B cells from atrophy (16). We found that BAFF-

hr induced Akt phosphorylation is, indeed, mediated by BAFF-R. 0 10 min 0 10 min 10 min 24 hr 10 min 24 24 hr 10 min 24 hr 10 min Furthermore, we found that IKK1 is an essential signaling pAkt molecule upstream of the PI-3K/Akt pathway in response to (S473) BAFF and that BAFF treated IKK1-deficient B cells are unable to grow. BAFF-R regulates both BAFF-induced survival and growth of Akt B cells, but the downstream signaling requirements for these effects are not the same. Although, as noted, blocking the activation of PI-3K/Akt/mTOR is sufficient to inhibit growth, BAFF-induced B cell survival under the same conditions is pErk1/2 relatively unaffected (14, 16) unless the B cells are also deficient for Pim2 expression, in which case BAFF is unable to promote B cell survival (16). Pim2 is a target of alternative NF-␬B Erk signaling and, as such, is induced in response to BAFF in an IKK1-dependent manner (11). Thus, the requirement of IKK1 expression for both BAFF-induced Pim2 expression and activation of the PI-3K/Akt pathway might explain the defective control baff-r-/- B cell ∆ IKK1 B bcl-2tg bcl-2tg bcl-2tg survival of IKK1-deficient B cells in response to BAFF (11). In 100 addition, we found that IKK1 expression is required for Erk1/2

80 phosphorylation in response to BAFF-R signaling. Both the Akt

60 Day 1 and Erk pathways have been shown to negatively regulate the 40 levels of the proapoptotic molecule Bim in B cells; Akt activation 20 represses Bim expression (25), while Erk activation promotes 0 ␬ 100 Bim protein turnover (15). BAFF mediated NF- B activation

80 also activates transcription of antiapoptotic Bcl-2 family mem-

60 bers (9, 16, 24, 26–28). BAFF-R signaling may thus promote B Day 2 40 cell survival in an IKK1-dependent manner by setting the levels 20 of pro- and antiapoptotic molecules in favor of survival by 0 regulating multiple downstream signaling pathways. 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 I-Ab Although the data presented here place IKK1 upstream of the PBS Akt and Erk pathways in response to BAFF-R-mediated signal- BAFF ing, we do not know how IKK1 functions to activate the Akt and Erk pathways. One possibility is that IKK1 induces (or represses) Fig. 3. IKK1 is necessary for Akt and Erk1/2 phosphorylation but not for MHC ␬ class II up-regulation in response to BAFF-R signaling. (A) Splenic B cells from downstream NF- B-dependent targets responsible for a second IKK1 sufficient (ikk1f/f, bcl-2tg) or deficient (ikk1f/f, bcl-2tg, mb1-cre) mice wave of signal transduction via the Akt and Erk pathways. were stimulated and analyzed as in Fig. 2A. Blots were probed with the Alternatively, IKK1 may signal more directly in a manner indicated antibodies. (B) B cells from mice with the indicated genotypes were independent of its role in NF-␬B activation or function inde- cultured and stimulated with PBS (filled histograms) or BAFF (red histograms) pendent of its kinase activity. Interestingly, we found that at least Ϫ ϩ as in Fig. 1A. Histograms show MHC class II (I-Ab) levels on ToPro-3 , B220 one BAFF-induced response, namely up-regulation of MHC cells. These data are representative of at least two independent experiments. class II expression, depends on BAFF-R but largely independent of IKK1 expression. In all other tested cases, however, IKK1 data not shown), while levels of other activation markers, such serves as a major coordinator of signal transduction downstream as CD69, CD80 or CD86 remain unchanged (see Fig. S1 and data of BAFF-R that regulates BAFF-induced B cell survival and not shown). The MHC class II up-regulation is completely growth. dependent on BAFF-R but only partially dependent on IKK1 Methods IMMUNOLOGY expression (see Fig. 3B). In contrast, BCR stimulation leads to Mouse Strains. C57BL/6, baff-rϪ/Ϫ (5), ikk1f/f (29), mb1-cre (30) and E␮-Bcl2 (31) a similar increase of MHC class II and CD69 expression in mice were bred and maintained in specific pathogen free conditions. All BAFF-R- or IKK1-deficient and control bcl-2tg B cells (see Fig. mouse protocols were approved by the Harvard University and the Immune S1). The IKK1-independent signaling pathways from BAFF-R Disease Institute Institutional Animal Care and Use Committees. that mediate increased MHC class II expression are not known but may include the canonical NF-␬B signaling pathway (24). Cell Purification and Stimulation. B cells were purified from spleen suspensions after red blood cell lysis by depleting CD43 positive cells with magnetic beads Discussion (Miltenyi). Purity was assayed by FACS and found to be Ͼ90% CD19 positive. BAFF protects B cells from atrophy and provides survival signals B cells were cultured at 37°C for 1 to3hin1%FCSBcell culture media [DMEM to B cells in vivo and in vitro (1, 2, 14, 16), and genetic evidence supplemented with 1% FCS (Biowhittacker), 10-mM hepes, 2-mM L- ϫ ϫ strongly supports the notion that BAFF promotes B cell survival glutamine, 1-mM sodium pyruvate, 1 penicillin/streptomycin, 1 nonessen- ␮ ␤ through only one of its three receptors, BAFF-R (1, 2, 4–8). We tial amino acids (Cellgro), and 50- M -mercaptoethanol] before stimulation with 200-ng/ml recombinant human BAFF (kindly provided by Biogen), 25- now show that the same is true for BAFF-dependent regulation ng/ml recombinant murine BAFF (R&D Systems) or 50-␮g/ml goat F(abЈ)2 of B cell growth. The PI-3K/Akt pathway plays an important role antimouse IgM (Jackson Immunoresearch) in PBS for the indicated amount of in regulating cellular metabolism and growth by activating the time. Equal volumes of PBS were added to controls. Cells were harvested on ice mammalian target of rapamycin (mTOR). The importance of and either stained or washed with ice-cold PBS before lysis. For some exper- the PI-3K/Akt/mTOR pathway in regulating B cell size in iments (see Fig. S2), mature B cells were purified from spleen and lymph nodes

Otipoby et al. PNAS ͉ August 26, 2008 ͉ vol. 105 ͉ no. 34 ͉ 12437 Downloaded by guest on September 26, 2021 by CD43 depletion followed by CD62L enrichment (Miltenyi). Puriﬁed B cells Immunoblot Analysis. Whole-cell lysates were prepared in lysis buffer [40-mM were preincubated overnight in B cell medium containing 10% FCS to reduce Tris (pH 7.6), 150-mM NaCl, 2-mM EDTA, 1% Nonidet P-40, 0.25% sodium the background BAFF signaling caused by endogenous BAFF in vivo (9). After deoxycholate, 1ϫ EDTA-free protease inhibitor mixture (Roche), 1-mM PMSF, preincubation, live cells were separated over a Ficoll gradient (Cedar Lanes). 5-mM NaF, 0.5 ␮M, 1-mM Na3VO4]. The protein concentration was determined Cells were resuspended in culture medium and stimulated with BAFF (25- by detergent-compatible protein assay kit (Bio-Rad). Equal amounts of pro- ng/ml recombinant murine BAFF or 200-ng/ml recombinant human BAFF) tein were separated by SDS/PAGE and transferred to PVDF membranes (Mil- diluted in culture medium or an equal volume of culture medium without lipore). The membranes were immunoblotted with indicated antibodies. BAFF for control samples. We found that recombinant murine BAFF (R&D Antibodies used here are phospho-Akt (S473), Akt, phospho-Erk1/2, Erk1/2 ␤ Systems) consistently caused stronger BAFF-mediated cell survival and growth (Cell Signaling), IKK1 (Imgenex), and -actin (Sigma–Aldrich). compared to recombinant human BAFF (Biogen) (data not shown). ACKNOWLEDGMENTS. We thank Drs. Yen-Ming Hsu and Martin Scott for the gift of recombinant human BAFF. We are grateful to Junrong Xia, Dvora FACS Analysis. Single cell suspensions of ex vivo splenocytes or cultured B cells Ghitza, and Anthony Monti for technical support and Emmanuel Derudder were stained with antibodies to BAFF-R (5), B220 (RA3–6B2, BD PharMingen), and Jane Seagal for critical reading of the manuscript. This work was sup- AA4.1 (CD93), MHC class II (I-Ab), or CD69 (ebioscience). Data were collected on ported by National Institute of Health Grants AI057947 and AI054636. Y.S. FACScalibur (Becton Dickenson) and analyzed with FloJo software (Treestar). received a fellowship from the Uehara Memorial Foundation.

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