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Logo of hhmipaAbout Author manuscriptsSubmit a manuscriptHHMI Howard Hughes Medical Institute; Author Manuscript; Accepted for publication in peer reviewed journal
Nat Immunol. Author manuscript; available in PMC Aug 20, 2008.
Published in final edited form as:
Published online Apr 27, 2008. doi:  10.1038/ni.1609
PMCID: PMC2518613
HHMIMSID: HHMIMS56618

Transcription factor Mef2c is required for B cell proliferation and survival after antigen receptor stimulation

Abstract

Calcineurin is required for B cell receptor (BCR)–induced proliferation of mature B cells. Paradoxically, loss of NFAT transcription factors, themselves calcineurin targets, induces hyperactivity, which suggests that calcineurin targets other than NFAT are required for BCR-induced proliferation. Here we demonstrate a function for the calcineurin-regulated transcription factor Mef2c in B cells. BCR-induced calcium mobilization was intact after Mef2c deletion, but loss of Mef2c caused defects in B cell proliferation and survival after BCR stimulation in vitro and lower T cell–dependent antibody responses and germinal center formation in vivo. Mef2c activity was specific to BCR stimulation, as Toll-like receptor and CD40 signaling induced normal responses in Mef2c-deficient B cells. Mef2c-dependent targets included the genes encoding cyclin D2 and the prosurvival factor Bcl-xL. Our results emphasize an unrecognized but critical function for Mef2c in BCR signaling.

B cell receptor (BCR) signaling coordinates the development, maintenance, activation and tolerance of B cells. BCR engagement induces the phosphorylation of tyrosine residues in immunoreceptor tyrosine-based activation motifs of immunoglobulin-α and immunoglobulin-β by Src family kinases1. Subsequently, other kinases and adaptor molecules orchestrate signaling cascades that eventually activate transcription factors required for the proliferation, survival and differentiation of B cells, including those of the NFAT, AP-1 and NF-κB families25. BCR engagement can induce opposite outcomes depending on the stage of B cell development6. In mature B cells, BCR signaling induces proliferation, affinity maturation and class-switch recombination during T cell–dependent responses and ultimately drives the differentiation of B cells into memory cells and antibody-secreting plasma cells7. Alternatively, in immature B cells in the bone marrow and recent bone marrow emigrants in the spleen, BCR signaling leads to cell death6. Less than 10% of immature B cells in the bone marrow ever exit to the periphery, and fewer reach maturity8, presumably because of the deletion of self-reactive B cells in response to BCR engagement by self antigens.

Calcium mobilization is a central component of BCR signaling9. BCR-dependent activation of phospholipase C-γ causes hydrolysis of phosphatidylinositol-4,5-bisphosphate to diacylglycerol and inositol-1,4,5-trisphosphate. Inositol-1,4,5-trisphosphate induces the release of calcium from endoplasmic reticulum stores and eventual influx of calcium across the plasma membrane9. Increasing intracellular calcium concentrations activate calmodulin and a variety of calcium-calmodulin–dependent proteins, including calcium-calmodulin–dependent kinases and the serine-threonine phosphatase calcineurin9. Calcineurin is critical in B cell activation and BCR-induced proliferation10. Conditional deletion of the gene encoding calcineurin b1, the regulatory subunit of the calcineurin complex, in B cells causes a cell-intrinsic defect in BCR-induced proliferation in vitro and results in lower antibody responses to T cell–dependent antigens10. NFAT family transcription factors are the most well characterized targets of calcineurin activity in B cells9. If NFAT proteins were the only relevant targets of calcineurin in B cells, deficiencies in calcineurin and NFAT might be expected to show general similarities. However, single or combined deficiencies in NFAT factors cause B cells to develop ‘hyperactive’ phenotypes rather than causing a defect in BCR-induced proliferation, as seen in B cells lacking calcineurin activity2,1012. Such data suggest that additional targets of calcineurin may be required for BCR-induced proliferation.

NFAT proteins interact with a variety of other transcription factors, including members of the AP-1 (ref. 13), C/EBP14, IRF4 (ref. 15) and myocyte-enhancer factor 2 (Mef2) families16,17. The four genes of the mammalian Mef2 family, Mef2a, Mef2b, Mef2c and Mef2d, have overlapping but distinct expression patterns in embryonic and adult tissues18. Mef2 family members are defined by an amino-terminal MADS domain and an adjacent Mef2 domain, which mediate DNA binding and protein-protein interactions, respectively19. Mef2 factors bind DNA and regulate gene expression through interactions with other factors, including NFAT16,17, basic helix-loop-helix transcription factors20,21, GATA factors22 and histone deacetylases23. Mef2c (A001503) can repress gene expression by interacting with histone deacetylases and responds to various signaling pathways to activate gene expression after calcium influx, activation of calcineurin and activation of the p38 mitogen-activated protein kinase16,2426. Mef2c is critical in muscle cell development, and deletion of Mef2c causes embryonic death around day 9.5 due to defects in cardiac and vascular development2729. Mef2c also regulates chondrocyte, bone and craniofacial development29,30.

In examining gene expression in many tissues and immune cells, we found unexpectedly high expression of Mef2c in mature B cells relative to that of other tissues, even skeletal muscle and heart, where Mef2c is known to function29. As Mef2c deficiency is lethal, we used CD19-Cre31 and a conditional gene-targeting approach to test the function of Mef2c in B cells. Mef2c-deficient B cells had a substantial defect in proliferation and survival in response to BCR stimulation, which could be restored after treatment with lipopolysaccharide (LPS), and mice with B cell–specific deletion of Mef2c had a much lower immunoglobulin G1 (IgG1) antibody response to a T cell–dependent antigen and less induction of splenic germinal center B cells after immunization. Our results emphasize an unrecognized but critical function for Mef2c in mature B cell survival and proliferation after BCR stimulation and may provide an explanation for the differing outcomes of calcineurin and NFAT deficiency in B cells.

RESULTS

High expression of Mef2c in mature B cells

We examined a variety of mouse tissues and immune cell types by microarray to identify transcription factors with restricted expression patterns in cells or tissues. This analysis showed that among all the tissues and cells examined, B220+ splenic B cells had the highest expression of Mef2c (Supplementary Fig. 1 online). This finding was somewhat unexpected, as Mef2c is known to regulate developmental programs in neuronal, skeletal and cardiac tissues27,29,32 but its function in the immune system has not been explored. We first examined Mef2c expression in various lymphocyte subsets. Consistent with our gene expression array data and a published report identifying Mef2c as a transcription factor expressed by B cells33, Mef2c expression was high in splenic B cells but not in CD4+ and CD8+ splenic T cells (Fig. 1a). In subsets of developing B cells, Mef2c expression was low in pro–B cells and pre–B cells, intermediate in immature B cells in the bone marrow, and highest in mature recirculating B cells (Fig. 1b). Mef2c expression in B220+ AA4.1+ splenic transitional cells was approximately half that in mature B220+ AA4.1 splenic B cells (Fig. 1b). Thus, Mef2c is selectively expressed in B cells but not in T cells, and its expression increases as B cells mature.

Figure 1
Mef2c has high expression in mature B cells and is efficiently deleted in splenic B cells of Mef2c-cKO mice. (a) Mef2c expression in CD4+, CD8+ and CD19+ splenic lymphocyte subsets, normalized to Hprt expression and presented relative to Mef2c expression ...

B cell–specific deletion of Mef2c

We generated mice lacking Mef2c activity specifically in the B cell compartment by crossing mice with two loxP-flanked Mef2c alleles (Mef2cfl/fl)34 onto a ‘CD19-Cre–deleter’ strain31. We assessed the efficiency of Cre recombinase–mediated deletion in DNA obtained from B cells versus that in DNA obtained from tail tissue in the resultant ‘Mef2cfl/flCd19+/cre’ mice (called ‘Mef2c-cKO mice’ here; Fig. 1c). As expected, there was no deletion of the Mef2cfl allele in DNA obtained from tail tissue of Mef2c-cKO mice. In contrast, there was approximately 90% deletion of the Mef2cfl allele in splenic B220+ cells, which indicated efficient conditional deletion of Mef2c in peripheral B cells. The deletion of the Mef2cfl allele in B cell subsets in the bone marrow and spleen increased progressively from approximately 70% deletion in pro–B cells and pre–B cells in the bone marrow to 90% in mature recirculating B cells in the bone marrow and mature B cells in the spleen (Supplementary Fig. 2 online). We confirmed deletion of the Mef2cfl allele in peripheral splenic B cells with antibodies specific for Mef2c protein. Two full-length splice variants of Mef2c protein were readily detected in splenic B cells of control mice but were nearly undetectable in splenic B cells from Mef2c-cKO mice (Fig. 1d).

We next analyzed B cell development in the bone marrow of Mef2c-cKO and control mice (Fig. 2a), as defined by expression of B220, CD43, BP-1, CD24, IgM and IgD35. Control mice included Mef2cfl/flCd19+/+ and Mef2c+/flCd19+/+ mice, which produced results similar to those of Mef2c+/+Cd19+/cre mice. Among B220+CD43hi cells, there were similar percentages of CD24BP-1, CD24+ BP-1 and CD24+ BP-1+ populations (Hardy fractions A, B and C35) in Mef2c-cKO and control mice, which indicated there were no differences in the frequency of pro–B cells or pre–B cells. Among B220+ CD43 cells, there were similar frequencies of IgMIgD, IgMhiIgDint and IgMintIgDhi populations (Hardy fractions D, E and F35) in Mef2c-cKO and control mice, which indicated there were no differences in late pre–B cells, immature B cells and mature recirculating B cells. Additionally, total numbers of B220+ cells in the bone marrow were not altered in Mef2c-cKO mice (Supplementary Fig. 3 online). Although B cell development in the bone marrow seemed unaffected in Mef2c-cKO mice, deletion of Mef2c with CD19-driven Cre recombinase was incomplete during early stages of B cell development (Supplementary Fig. 2) and we cannot exclude the possibility that Mef2c is involved in B cell development in the bone marrow.

Figure 2
B cell development in Mef2c-cKO mice. (a) Flow cytometry of bone marrow cells from control and Mef2c-cKO mice to identify B cell developmental subsets. (b) Flow cytometry of splenocytes and cells from inguinal lymph nodes and the peritoneal cavity for ...

The maturation state of B cells, as defined by expression of IgM and IgD, seemed normal in the spleen and lymph nodes of Mef2c-cKO mice (Fig. 2b). Likewise, there were normal numbers of B220+ cells in the inguinal lymph nodes of Mef2c-cKO mice and normal numbers and frequencies of B-1 cells (B220+CD11b+) and B-2 cells (B220+CD11b) in the peritoneum of Mef2c-cKO mice, with normal allocation to the B-1a and B-1b cell subsets (Fig. 2b and Supplementary Fig. 3). The splenic numbers and distribution of mature B cells (B220+AA4.1) and immature transitional B cells (B220+AA4.1+) were similar in Mef2c-cKO and control mice (Fig. 2c,d). Additionally, the frequency of splenic follicular cells (B220+AA4.1IgMintCD21/35int) and marginal zone cells (B220+AA4.1IgMhiCD21/35hi) seemed normal in Mef2c-cKO mice relative to that in control mice (Fig. 2d). However, there were differences in the surface phenotypes of transitional B cell stages, as defined by expression of CD23 and IgM36. For B220+ AA4.1+ transitional B cells, Mef2c-cKO mice showed atypical distribution to the T1 (IgMhi CD23), T2 (IgMhi CD23+) and T3 (IgMint CD23+) transitional stage gates because of lower CD23 expression (Fig. 2d). Similarly, the spleens of Mef2c-cKO mice lacked a population of B220+AA4.1IgMintCD23+ cells, a surface phenotype used to identify mature follicular B cells, which again seemed to result from lower CD23 expression. Despite the lower CD23 expression on splenic B cells, the spleen showed normal localization of B cells to lymphoid follicles and the marginal zone (Fig. 2e). In summary, B cell populations, as defined by a variety of markers and localization, all seemed to develop normally in the absence of Mef2c, except for lower CD23 expression on mature B cells.

BCR-induced proliferation and survival require Mef2c

We next measured B cell proliferative responses to mitogenic stimuli. Mef2c-cKO splenocytes proliferated poorly relative to control cells in response to a range of doses of antibody to IgM (anti-IgM), as assessed by incorporation of tritiated thymidine (Fig. 3a, top). In contrast, LPS-induced proliferation seemed normal in Mef2c-cKO splenocytes (Fig. 3a, bottom). We considered that the failure to incorporate tritated thymidine in response to BCR stimulation could have been due to failure of the cells to survive or their failure to enter cell cycle. To distinguish those possibilities, we labeled purified B cells with the cytosolic dye CFSE and measured CFSE dilution and cell recovery in response to anti-IgM. As expected, control B cells stimulated with anti-IgM increased in size and granularity, as is typical of ‘blasting’ cells (Fig. 3b). In contrast, there was a lower frequency of anti-IgM-stimulated Mef2c-cKO B cells in the live cell gate, and these cells remained small rather than becoming blasts (Fig. 3b). Most anti-IgM-stimulated control B cells underwent at least one round of cellular division, whereas Mef2c-cKO B cell cells did not proliferate in response to BCR stimulation, as shown by an absence of CFSE dilution (Fig. 3c). This proliferative defect was specific for BCR stimulation, as Mef2c-cKO B cells stimulated with LPS or anti-CD40 responded with similar changes in forward-scatter and side-scatter characteristics and proliferated to an extent equal to that of control B cells (Fig. 3b,c). In accordance with the observed proliferative defect of Mef2c-cKO B cells in response to BCR stimulation, we recovered significantly fewer viable Mef2c-cKO B cells than control B cells from cultures after anti-IgM stimulation (Fig. 3d). Whereas stimulation of control B cells with anti-IgM resulted in the recovery of more total viable cells relative to that of unstimulated control B cells, stimulation of Mef2c-cKO B cells with anti-IgM resulted in much lower recovery of viable cells relative to that of unstimulated Mef2c-cKO B cells, which indicated that BCR stimulation of Mef2c-cKO B cells induces B cell death (Fig. 3d). In contrast, we recovered similar numbers of control and Mef2c-cKO B cells after stimulation with LPS and anti-CD40 (Fig. 3d).

Figure 3
Mef2c is required for B cell proliferation and survival in response to BCR stimulation. (a) Proliferation of splenocytes stimulated with anti-IgM or LPS (5 µg/ml), assessed as [3H]thymidine incorporation. (b,c) Viability (b) and cell division ...

Consistent with the observation that Mef2c-cKO B cells were dying in response to BCR stimulation (Fig. 3d), a greater percentage of viable Mef2c-cKO B cells bound annexin V after 48 h of anti-IgM stimulation, which demonstrated that more Mef2c-cKO B cells were undergoing early apoptotic events (Fig. 3e). Additionally, the proliferative defect was cell autonomous, as mixing control B cells with Mef2c-cKO B cells did not inhibit the proliferative response of control B cells or restore the ability of Mef2c-cKO B cells to proliferate (Fig. 3f). These data indicate a cell-autonomous requirement for Mef2c in B cell survival and proliferation in response to BCR stimulation.

Costimulation ‘rescues’ defects in Mef2c-cKO B cells

We further characterized the responses of Mef2c-cKO B cells to a variety of B cell stimuli, including anti-IgM, LPS, anti-CD40, interleukin 4 (IL-4), the survival factor BAFF, and antibody to the Toll-like receptor homolog RP105. We found that without additional costimulation, Mef2c-cKO B cells failed to survive or proliferate in response to BCR stimulation over a range of anti-IgM concentrations (Fig. 4 and Supplementary Fig. 4 online). In contrast, in response to various concentrations of LPS, forward- and side-scatter characteristics, proliferation and the total number of viable cells recovered were similar for control and Mef2c-cKO B cells (Supplementary Fig. 4). Both control and Mef2c-cKO B cells stimulated with anti-CD40, IL-4 or BAFF alone responded with similar changes in forward- and side-scatter characteristics and in the total number of viable cells recovered, but did not undergo substantial proliferation (Supplementary Fig. 4). B cell proliferation and survival induced by stimulation with anti-RP105, which requires the Src-family protein kinase Lyn, protein kinase C-β, Erk2, the adaptor molecule Vav and CD19 (refs. 37,38), seemed normal in Mef2c-cKO B cells (Supplementary Fig. 4). Thus, Mef2c-cKO B cells show a survival and proliferation defect after BCR stimulation but respond normally to stimulation with LPS, anti-CD40, IL-4, BAFF and anti-RP105.

Figure 4
Costimulation can restore the survival and proliferation of BCR-stimulated Mef2c-cKO B cells. (a,b) Viability (a) and cell division (b) of control and Mef2c-cKO B cells labeled with CFSE and cultured for 72 h without stimulation or stimulated with anti-IgM ...

We next sought to determine whether the failure of Mef2c-cKO B cells to survive and proliferate was due to a dominant inhibitory effect of BCR signaling or whether costimulation with additional agents could ‘rescue’ these defects. Costimulation of Mef2c-cKO B cells with anti-IgM and LPS together induced proliferation and survival profiles similar to those of control B cells, as shown by equivalent changes in forward- and side-scatter characteristics (Fig. 4a) and CFSE dilution (Fig. 4b) and a slightly lower recovery of viable cells from the culture that was not significantly different from that of control B cells (Fig. 4c). Likewise, costimulation of Mef2c-cKO B cells with anti-IgM plus anti-CD40 or BAFF restored their survival, as indicated by the percentage of cells in the live gate and the total number of viable cells recovered from the culture relative to that of unstimulated Mef2c-cKO B cells and Mef2c-cKO B cells stimulated with anti-IgM alone (Fig. 4a,c). In contrast, costimulation of Mef2c-cKO B cells with anti-IgM plus anti-CD40 or BAFF did not restore the proliferative response (Fig. 4b). Costimulation with anti-IgM and IL-4 together restored Mef2c-cKO B cell survival but incompletely restored the proliferative response (Fig. 4). These data show that there is a specific requirement for Mef2c in the BCR-induced pathway of B cell proliferation and survival. Signaling pathways activated by LPS, anti-CD40, IL-4 or BAFF do not require Mef2c and can compensate for Mef2c deficiency after BCR stimulation.

Altered immunoglobulin responses of Mef2c-cKO mice

We characterized the in vivo antibody responses of Mef2c-cKO mice. Serum titers of IgG1 and IgG3 in unimmunized mice were altered slightly, being lower in Mef2c-cKO mice (Fig. 5a). Their baseline titers of IgM, IgG2a, and IgG2b were normal, whereas serum IgA was slightly higher (Fig. 5a). We also evaluated the antibody responses of Mef2c-cKO mice to immunization with trinitrophenyl-Ficoll (TNP-Ficoll), a type 2 T cell–independent antigen. Mef2c-cKO mice showed significantly higher antigen-specific IgM and IgG3 responses than those of control mice at day 7 after immunization with this T cell–independent antigen (Fig. 5b). Thus, Mef2c is required for normal basal immunoglobulin titers and regulates the magnitude of T cell–independent antibody responses.

Figure 5
Mef2c-cKO mice have altered basal immunoglobulin titers and mount enhanced antibody responses to a T cell–independent antigen. (a) ELISA of basal serum immunoglobulin titers control and Mef2c-cKO mice. (b) IgM and IgG3 TNP-specific antibody responses ...

To evaluate T cell–dependent antibody responses, we immunized control and Mef2c-cKO mice with a hapten-conjugated protein and measured hapten-specific antibody responses at various times. T cell–dependent hapten-specific IgM responses in Mef2c-cKO were lower than but not significantly different from those of control mice (Fig. 6a, left). However, the T cell–dependent IgG1 antibody response was significantly lower at both 14 d and 21 d after immunization (Fig. 6a, right). We further characterized antibody responses of Mef2c-cKO mice to sheep red blood cells as a polyclonal antigen. At days 10 and 15 after immunization with sheep red blood cells, control mice showed the expected increase in the total number and frequency of splenic germinal center B cells (defined as B220+IgDloFas+GL7+; Fig. 6b). In contrast, the total number and frequency of germinal center B cells in the spleens of Mef2c-cKO mice increased only slightly after immunization with sheep red blood cells and were significantly less than those in spleens of immunized control mice (Fig. 6b). Consistent with the data reported above, histological examination showed fewer germinal center reactions in spleens of Mef2c-cKO mice immunized with sheep red blood cells than in control mice, although some germinal centers could still be identified (Fig. 6c). In summary, mice with Mef2c-deficient B cells have modest alterations in basal serum immunoglobulin titers, develop higher antibody responses to a T cell–independent antigen and have significantly lower IgG1 antibody responses and germinal center formation after immunization with a T cell–dependent antigen.

Figure 6
Mef2c-cKO mice have deficient responses to T cell–dependent antigens. (a) IgM and IgG1 NP-specific antibody responses of control and Mef2c-cKO mice immunized with NP-conjugated chicken γ-globulin in alum, analyzed in blood obtained before ...

BCR signaling in Mef2c-deficient B cells

We sought to determine whether the lower survival and proliferation of B cells was the result of a defect in early BCR signaling events caused by Mef2c deficiency. As many BCR-initiated signaling events depend on calcium influx, we first measured intracellular calcium mobilization in response to BCR stimulation. Mef2c-cKO and control B cells had similar changes in intracellular calcium in response to BCR stimulation (Fig. 7a), which indicated that there was no apparent defect in proximal calcium mobilization in Mef2c-cKO B cells. The slight difference in calcium mobilization seen after the addition of ionomycin varied slightly among experiments but was not reproducibly higher or lower in Mef2c-cKO B cells. We next evaluated phosphorylation of Jnk and of Erk1 and Erk2 in response to anti-IgM stimulation. Phosphorylation of Jnk and of Erk1 and Erk2 induced by BCR stimulation was intact in Mef2c-cKO B cells (Fig. 7b,c). Mef2c-cKO B cells had slightly more Jnk phosphorylation than did control B cells (Fig. 7b). Similarly, there was slightly more phosphorylation of Erk1 and Erk2 in Mef2c-cKO B cells (Fig. 7c). We also measured the pattern of total tyrosine phosphorylation in control and Mef2c-cKO B cells after BCR stimulation. Unstimulated control B cells and Mef2c-cKO B cells had very little tyrosine-phosphorylated protein before stimulation with anti-IgM (Supplementary Fig. 5 online). We found that after treatment with anti-IgM, Mef2c-cKO B cells had normal patterns of global tyrosine phosphorylation compared with those of control B cells and had slightly less tyrosine-phosphorylated proteins at 10 min (Supplementary Fig. 5). In summary, although Mef2c-cKO B cells showed slight alterations in patterns of tyrosine phosphorylation, there was no evidence of substantial defects in proximal BCR signaling.

Figure 7
Early BCR-mediated signaling events in Mef2c-cKO B cells. (a) Intracellular calcium flux in control and Mef2c-cKO splenocytes loaded with Fluo-4–acetoxymethyl ester after the addition (arrows) of anti-IgM and ionomycin (Iono). Data are representative ...

Bcl-XL and cyclin D2 are Mef2c-dependent targets

To identify transcriptional targets, direct or indirect, of Mef2c activity, we analyzed global gene expression of control and Mef2c-cKO B cells by microarray. We first evaluated differences between unstimulated control and Mef2c-cKO B cells. In this condition, we identified very few differences in gene expression. However, Mef2c-cKO B cells had lower expression of the genes encoding the immunoglobulin joining chain and the serine-threonine kinase Srpk3, each reported targets of Mef2c39,40, than did control B cells in the absence of BCR stimulation (Supplementary Fig. 6 online). We considered the possibility that Mef2c-dependent expression of the gene encoding Srpk3 could have accounted for the defects in B cell survival and proliferation if Srpk3 were an unrecognized component of BCR-induced signal cascades. However, in response to stimulation with anti-IgM, LPS or anti-CD40, Srpk3-deficient B cells showed changes in forward- and side-scatter characteristics similar to those of control cells and proliferated as well as control cells, which demonstrated that the defect in Mef2c-cKO B cells was not solely due to low Srpk3 expression (Supplementary Fig. 7 online).

We next characterized differences in gene expression in control versus Mef2c-cKO B cells after BCR stimulation. We identified the genes encoding Bcl-xL (Bcl2l1), cyclin D2, cyclin E2, cyclin A2, cyclin B1 and cyclin F as Mef2c-dependent gene targets of BCR stimulation (Fig. 8a). Bcl-xL is a member of the Bcl-2 family of prosurvival molecules, and cyclin proteins are critical regulators of cell cycle entry and progression. As Bcl2l1 encodes at least three isoforms41, we specifically evaluated by real-time PCR expression of Bcl-xL, which is the long isoform. Bcl-xL was not induced in Mef2c-cKO B cells, whereas it was upregulated approximately threefold after 12 h of BCR stimulation and more than fivefold after 48 h of BCR stimulation in control cells (Fig. 8b). Additionally, we confirmed that the expression of cyclin D2, which regulates progression through gap-phase 1 of the cell cycle42, increased progressively with anti-IgM stimulation in control B cells but was not induced in Mef2c-cKO cells (Fig. 8c). These data suggest that Mef2c is required for BCR-induced expression of Bcl-xL and cyclin D2, which may promote survival and cell proliferation, respectively.

Figure 8
BCR-stimulated Mef2c-cKO B cells fail to induce Bcl-xL and cyclin D2. (a) Microarray analysis of the expression of genes involved in cell cycle regulation and survival in control and Mef2c-cKO B cells stimulated for 0–48 h (above plot) with anti-IgM ...

DISCUSSION

Here we have identified an unexpected requirement for Mef2c in B cell activation and differentiation. Our results indicate that Mef2c is absolutely required for B cell proliferation and survival in response to BCR stimulation in vitro. In vivo, Mef2c is required for efficient IgG1 antibody responses to T cell–dependent antigens and for normal induction of germinal center B cells. Consistent with those effects, we have identified Bcl-xL and cyclin D2 as two Mef2c-dependent targets of BCR signaling that might explain the inability of Mef2c-cKO B cells to survive and proliferate.

Mef2c-cKO splenic B cells had lower CD23 expression, a marker commonly used to identify splenic follicular B cells. Our data have shown that the lower CD23 expression on Mef2c-cKO B cells reflected only a failure to express a marker used to distinguish splenic B cell subsets and did not reflect a developmental defect resulting in the absence of B cell subsets expressing CD23, as mature follicular B cells could be identified at normal numbers and frequency in the spleen, lymph nodes and bone marrow of Mef2c-cKO mice through the use of alternative phenotypic markers. CD23, the low-affinity IgE receptor, is not required for the development or proliferation of B cells after BCR stimulation but acts as a negative feedback component of IgE regulation43,44, which suggests that the lower CD23 expression on Mef2c-cKO B cells may lead to aberrant IgE responses. However, the functional consequences of lower CD23 expression are probably complicated by the profound survival and proliferative defects of Mef2c-cKO B cells.

Immunization of Mef2c-cKO mice with T cell–dependent antigens showed a selective defect in IgG1 antibody responses that correlated with the induction of fewer germinal center B cells. Although B cell survival and proliferation in vitro after BCR stimulation was absolutely dependent on Mef2c, our data suggest that Mef2c regulates the induction, magnitude or duration of germinal center responses in vivo. During the germinal center reaction, B cells undergo clonal expansion, hypermutation of immunoglobulin genes and affinity selection to promote the generation and export of high-affinity plasma cells and memory cells7. Additionally, B cells can undergo class-switch recombination during germinal center reactions. Isotype-switched B cells appear only after B cells undergo many cell divisions45, which suggests the diminished IgG1 responses in Mef2c-cKO mice may have resulted from the proliferative defect of BCR-stimulated Mef2c-deficient B cells. B cells receive cognate T cell help in germinal centers in the form of cytokine stimulation, CD40–CD40 ligand engagement and additional signals that are still being elucidated46. Our in vitro results have shown that costimulation with LPS, cytokines and anti-CD40 selectively ‘rescued’ defects in B cell survival and proliferation after antigen receptor stimulation. Additionally, whereas Mef2c was absolutely required for B cell proliferation in vitro in response to anti-IgM stimulation, Mef2c seemed to negatively regulate antibody responses to a T cell–independent antigen, which demonstrated that in vitro stimulation with anti-IgM does not directly correspond to T cell–independent responses in vivo. Marginal zone and B-1 B cells are chief contributors to T cell–independent responses, whereas conventional B-2 B cells are central to T cell–dependent responses, which suggests that Mef2c may have distinct functions in the antibody responses emanating from different B cell subsets. Consequently, the precise function of Mef2c in vivo may depend on antigen composition, the nature of T cell help, the microenvironment in secondary lymphoid structures, and Toll-like receptor stimulation during immunization or infection.

The suppression and promotion of B cell apoptosis is regulated by Bcl-2 family members47. Bcl-2 and Bcl-xL prevent apoptosis in part by preserving mitochondrial membrane integrity and preventing the release of cytochrome c into the cytoplasm, whereas other Bcl-2 family members, including Bax, Bak and Bad, promote apoptosis by antagonizing the function of Bcl-2 and Bcl-xL (ref. 47). Here we have identified Bcl-xL as a Mef2c-dependent target ‘downstream’ of BCR stimulation, which suggests that the failure of Mef2c-cKO B cells to survive after anti-IgM stimulation may have resulted from a lack of the prosurvival actions of Bcl-xL. Notably, costimulation with LPS, IL-4, BAFF and anti-CD40 restored the survival of Mef2c-cKO B cells treated with anti-IgM; each of these factors is known to rapidly induce Bcl-xL in peripheral B cells4851. Bcl-xL has also been linked to the clonal selection and apoptosis of germinal center B cells after immunization52 and the maintenance of the germinal center reaction53. Together with our observation that Mef2c-deficient mice had lower IgG1 responses and generated fewer germinal center B cells after immunization, these data suggest that Mef2c-dependent Bcl-xL expression may be important in regulating the germinal center reaction in vivo.

B cell entry into and progression through the cell cycle after BCR stimulation is regulated by the coordinated activities of cyclin-dependent kinases and their regulatory cyclins54. Progression through gap-phase 1 of the cell cycle is regulated by the induced expression of D-type cyclins and their subsequent association with and activation of cyclin-dependent kinases 4 and 6, which inactivate the retinoblastoma tumor suppressor and commit cells to progression through the cell cycle55. We have identified cyclin D2 as a Mef2c-dependent target of BCR stimulation. Notably, similar to the proliferative defect of Mef2c-cKO B cells, cyclin D2–deficient B cells show a selective defect in BCR-induced but not LPS-induced proliferation42, which suggests that the failure of Mef2c-cKO B cells to increase expression of cyclin D2 may be the chief cause of the proliferative defect.

Although we found no evidence of substantial defects in proximal BCR signaling in Mef2c-cKO B cells, there were modest differences in the strength and kinetics of phosphorylation of Jnk and of Erk1 and Erk2 after stimulation with anti-IgM. It is possible that naive Mef2c-cKO B cells are poised to respond with alterations in the quality of BCR-transduced signals that ultimately lead to failed proliferation and the induction of B cell death. However, the transactivating activity of Mef2c is known to be influenced by signaling pathways activated ‘downstream’ of the BCR16,17,56,57. Thus, we suggest that the lack of BCR-induced Mef2c activity in Mef2c-cKO B cells results in the failed transcriptional induction of factors such as Bcl-xL and cyclin D2, which are required for B cell survival and proliferation.

BCR signaling in mature B cells induces B cell proliferation and survival, whereas BCR signaling in immature B cells results in cell death6. Similar to the activity of immature B cells in the bone marrow, mature Mef2c-cKO B cells failed to proliferate in response to BCR stimulation and instead died. In contrast, both immature B cells and Mef2c-deficient mature B cells proliferated and survived in response to LPS stimulation. The progressive increase in Mef2c expression during B cell development in the bone marrow and spleen also suggested potential involvement of Mef2c in B cell tolerance mechanisms, and Mef2c has been identified as a gene that is differentially expressed in a model of B cell tolerance58. Self-reactive B cells can be deleted in response to autoantigenic stimulation during the immature B cell stage in the bone marrow, when Mef2c expression is normally low relative to that of mature B cells59. After the introduction of a transgene encoding Bcl-xL, self-reactive B cells are not deleted and instead survive but do not proliferate after BCR stimulation60. Our data have shown that Bcl-xL and cyclin D2 are BCR-induced Mef2c-dependent targets in mature B cells. Thus, it is possible that BCR stimulation of self-reactive immature B cells, which express less Mef2c than do mature B cells, results in failed expression of Bcl-xL and the subsequent deletion of autoreactive B cells in a way analogous to the induction of apoptosis in BCR-stimulated Mef2c-deficient mature B cells.

Here we have identified Mef2c as a transcriptional effector of BCR signaling required for B cell activation and normal antibody responses. Notably, many signaling pathways are known to modulate Mef2 transcriptional activity. The transactivation domains of Mef2 proteins are phosphorylated by p38 and Erk5, which results in increased transcriptional activity56,61. Additionally, many calcium-dependent pathways have also been shown to converge on Mef2 activity. Calcium-calmodulin–dependent kinases are known to serine-phosphorylate Mef2-associated class II histone deacetylases in other cell types62. Once phosphorylated, histone deacetylases are shuttled from the nucleus by 14-3-3 proteins to activate Mef2 proteins6264. Furthermore, the calcium-dependent activation of calcineurin phosphatase activity can promote Mef2 transcriptional activity in at least two ways. First, calcineurin activity results in dephosphorylation of Mef2 and stimulation of Mef2 transcriptional activity57. Second, calcineurin directly dephosphorylates NFAT, causing its translocation to the nucleus, where it can act together with Mef2 proteins in a variety of cell types16,17,57. Notably, B cells lacking calcineurin activity and Mef2c-deficient B cells both show a proliferative defect in response to BCR stimulation that correlates with failed induction of cyclin D2 (ref. 10), which suggests that Mef2c may be a previously unknown calcium-calcineurin–responsive transcription factor involved in the execution of genetic programs after B cell activation.

In summary, our evaluation of mice lacking Mef2c activity specifically in the B cell compartment has identified a requirement for Mef2c in B cell survival and proliferation after BCR stimulation. Mef2c is responsive to calcium mobilization and calcineurin phosphatase activity16,17,57,6264, and it is likely that calcium flux induced by BCR engagement positively influences Mef2c transcriptional activity in B cells, either alone or together with activated transcription factors of the NFAT family. Thus, Mef2c may represent a previously unrecognized effector of calcium mobilization in B cells that is required for B cell survival and proliferation, although demonstration of a direct connection between BCR-induced signaling and Mef2c activation will require further experimentation.

METHODS

Mice

Mice with loxP sites flanking the second coding exon of Mef2c34 (Mef2cfl/fl) were crossed with CD19-Cre (Cd19+/cre) mice31 to generate mice with conditional loss of Mef2c activity in the B cell compartment (Mef2cfl/flCd19+/cre; called ‘Mef2c-cKO’ here). Control mice included Mef2cfl/flCd19+/+ and Mef2c+/flCd19+/+ mice, which produced results similar to those of Mef2c+/+Cd19+/cre mice (Supplementary Fig. 8 online). Mice were bred and housed in the Washington University animal facility. Mice lacking the muscle-specific kinase Srpk3 (Srpk3−/−) were housed in the animal facility of The University of Texas Southwestern Medical Center39. Live animal experiments were approved by the Animal Studies Committee at Washington University.

Flow cytometry

Phycoerythrin-indotricarbocyanine–conjugated anti-B220 (RA3-6B2), anti-CD11b (M1/70) and anti-FAS (Jo2), allophycocyanin conjugated anti-B220 (RA3-6B2), biotin-conjugated anti-CD43 (S7), phycoerythrin-conjugated anti-BP1 (BP-1) and anti-CD23 (B3B4), fluorescein isothiocyanate–conjugated anti-CD24 (M1/69), anti-IgM (II/41) and anti-GL7 (GL7), fluorescein isothiocyanate–annexin V, 7-amino-actinomycin D staining solution and streptavidin-allophycocyanin were from BD Biosciences; allophycocyanin-conjugated anti-AA4.1 (AA4.1) and phycoerythrin-conjugated anti-IgD (11-26c) were from eBioscience. All flow cytometry data were collected on a FACSCalibur (BD Biosciences) and were analyzed with FlowJo software (Tree Star).

B cell isolation and proliferation

Splenic B cells were enriched by negative selection of CD43-expressing cells with CD43 microbeads (Miltenyi Biotech) according to the manufacturer’s instructions. B cell samples were routinely enriched to over 96% B220+ cells, as assessed by flow cytometry. For analysis of Mef2c deletion efficiency, splenocyte samples depleted of CD43-expressing cells were purified to over 99% B220+ cells by cell sorting with anti-B220, followed by isolation of genomic DNA from purified B cells for analysis. Cells were labeled with CFSE (carboxyfluorescein diacetate succinimidyl diester; Sigma-Aldrich) by being incubated for 8 min at 25 °C with 1 µM CFSE at a density of 20 × 106 cells per ml in PBS. Cells were incubated for 1 min with an equal volume of FCS and were washed twice with media containing 10% (vol/vol) FCS before use. Enriched B cells (2 × 105 cells at a density of 1 × 106 cells/ml) were cultured in 96-well plates and were treated with F(ab′)2 goat anti–mouse IgM (115-006-020; Jackson ImmunoResearch), LPS from Escherichia coli O111:B4 (Sigma-Aldrich), anti-CD40 (3/23; BD Biosciences) and recombinant mouse BAFF and IL-4 (R&D Systems). CFSE dilution was assessed by flow cytometry after 72 h of stimulation. For analysis of [3H]thymidine incorporation, purified B cells were plated in triplicate at a density of 2 × 105 cells per 200 µl in 96-well plates with various stimuli and were cultured for a total of 72 h with 0.4 µCi of [3H]thymidine added for the final 14 h of culture.

Intracellular staining

Phosphorylated Jnk, Erk1 and Erk2 were analyzed by intracellular staining as follows. Splenocyte suspensions were equilibrated to 37 °C in a humidified incubator and were stimulated for various times with F(ab′)2 goat anti–mouse IgM (10 µg/ml; Jackson ImmunoResearch). Stimulation was stopped by the addition of an equal volume of 4% (vol/vol) paraformaldehyde and incubation for 15 min at 37 °C. Cells were made permeable and stained in PBS containing 0.5% (vol/vol) Triton-X-100 (Sigma-Aldrich) and 10% (vol/vol) FCS. Cells were stained with antibody to phosphorylated Erk1 and Erk2 (9101) or antibody to phosphorylated Jnk (9251; both from Cell Signaling) and allophycocyanin-conjugated anti-B220 for the identification of B cells. Cells were washed and were stained with phycoerythrin-labeled goat anti–rabbit IgG (heavy and light chain; 111–116–144; Jackson ImmunoResearch) as a secondary detection antibody. Data were collected on a FACSCalibur and analyzed with FlowJo software.

Calcium flux

Splenocyte suspensions at a density of 20 × 106 cells per ml in complete media with 10% (vol/vol) FCS were ‘loaded’ for 30 min at 37 °C with the fluorescent Ca2+ indicator Fluo-4–acetoxymethyl ester (2.5 µg/ml; Invitrogen) with occasional mixing. Cells were labeled with allophycocyanin-conjugated anti-B220 for identification of B cells. A 20-second baseline reading was obtained for each sample before the addition of F(ab′)2 goat anti–mouse IgM. Calcium flux was monitored for 6 min with a FACSCalibur before the addition of 1 µM ionomycin (Sigma-Aldrich) for an additional 1 min. Flow cytometry data were analyzed with FlowJo software.

Quantitative real-time PCR

Genomic DNA isolated from tail tissue and from purified splenic B cells was analyzed by quantitative real-time PCR with the following primers, which amplify the span of the Mef2c genomic sequence flanked by loxP sites34 excised by Cre recombinase: M2c-genomic-forward, 5′-CATACGCCACATACGAGTAC-3′, and M2c-genomic-reverse, 5′-ATGAT CAGTGCAATCTCACAG-3′. The following primers were used to amplify a genomic segment of Gapdh (encoding glyceraldehyde phosphate dehydrogenase) as a control for genomic DNA input: Gapdh-genomic-forward, 5′-CAGTATTCCACTCTGAAGAAC-3′, and Gapdh-genomic-reverse, 5′-ATACGGCCAAATCTGAAAGAC-3′.

For gene expression analysis, total RNA and cDNA were prepared from various cell types with the RNeasy Mini Kit (Qiagen) and Superscript III reverse transcriptase (Invitrogen). Primers used to evaluate relative expression were as follows: Mef2c (M2c-forward, 5′-AGATCTGACATCCGGTGCAG-3′, and M2c-reverse, 5′-TCTTGTTCAGGTTACCAGGTG-3′), Ccnd2 (cyclind2-forward, 5′-GAGTGGGAACTGGTAGTGTTG-3′, and cyclind2-reverse, 5′-CGCACAGAGCGATGAAGGT-3′), Bcl2l1 (bclxl-forward, 5′-GACAAGGAGATGCAGGTATTGG-3′, and bclxl-reverse, 5′-TCCCGTAGAGATCCACAAAAGT-3′), and Hprt1 (encoding hypoxanthine guanine phosphoribosyl transferase (normalization control); hprt-forward, 5′-AGCCTAAGATGAGCGCCAAGT-3′, and hprt-reverse, 5′-TTACTAGGCAGATGGCCACA-3′).

For real-time PCR, the relative standard curve method, SYBR Green PCR master mix and an ABI7000 machine (Applied Biosystems) were used according to the manufacturer’s instructions. PCR conditions were 2 min at 50 °C and 10 min at 95 °C, followed by 40 two-step cycles consisting of 15 s at 95 °C and 1 min at 60 °C.

Immunization and enzyme-linked immunosorbent assay (ELISA)

Sex- and age-matched mice 8–11 weeks of age were immunized and their antibodies were measured. Basal serum immunoglobulin titers were quantified by ELISA with the horseradish peroxidase–conjugated SBA Clonotyping System (SouthernBiotech). For evaluation of T cell–dependent or T cell–independent antibody responses, mice were immunized intraperitoneally with 50 µg nitrophenol (NP)–conjugated chicken γ-globulin or 25 µg TNP-Ficoll, respectively (Biosearch Technologies). Anti-NP and anti-TNP titers were measured by ELISA with plate-bound NP- or TNP-conjugated BSA (Biosearch Technologies) and isotype-specific horseradish peroxidase–conjugated secondary antibodies (SouthernBiotech). Titers are presented as the greatest serum dilution that provided an average optical density exceeding 1.5-fold over the average background optical density at 405 nm. Germinal center formation was evaluated by flow cytometry after intraperitoneal immunization of mice with 400 µl of 5% (vol/vol) sheep red blood cells in PBS (Sigma-Aldrich) with allophycocyanin-conjugated anti-B220, phycoerythrin-conjugated anti-IgD, fluorescein isothiocyanate–conjugated anti-GL7 and phycoerythrin-indotricarbocyanine–conjugated anti-Fas (BD Biosciences).

Immunohistochemistry

Tissue sections 6 µm in thickness were prepared from spleens frozen in Tissue-Tek (Sakura Finetek). Sections were fixed for 10 min at 4 °C in acetone, were air-dried, were rehydrated in PBS and were treated for 15 min with a blocking solution consisting of 10% (vol/vol) FCS in PBS. Sections were washed in PBS and were stained for 18 h with the appropriate primary antibodies. Sections were washed again and incubated for 2 h with secondary antibody. Antibodies included rat anti-IgD (11-26c.2a; BD Pharmingen), biotin-conjugated peanut agglutinin (Vector Laboratories), biotinylated MOMA-1 (marker specific for metallophilic macrophages; MP Biomedicals), Alexa Fluor 488–conjugated anti-B220 (RA3-6B2; BD Pharmingen), Alexa Fluor 555–conjugated streptavidin (Invitrogen) and Alexa Fluor 488–conjugated anti–rat IgG (heavy and light chains; Invitrogen). Finally, slides were washed and mounted in GVAMount (Zymed Laboratories).

Gene expression profiling

B cells were stimulated for various times with F(ab′)2 goat anti–mouse IgM (5 µg/ml) and total RNA was collected with the RNeasy Mini Kit (Qiagen). Biotinylated antisense cRNA was prepared with two cycles of in vitro amplification, and biotinylated cRNA was fragmented and hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 arrays. Data were normalized and model-based expression values were generated with DNA-Chip analyzer software (http://biosun1.harvard.edu/complab/dchip/).

Statistical analysis

A Student’s unpaired two-tailed t-test was used for statistical analyses of cell recovery data, basal serum immunoglobulin concentrations, and the number and frequency of germinal center B cells. The nonparametric Mann-Whitney or Wilcoxon signed-rank test was used for analysis of antigen-specific antibody titers. Differences with P values of 0.05 or less are considered significant.

Supplementary Material

2

Note: Supplementary information is available on the Nature Immunology website.

ACKNOWLEDGMENTS

Supported by the Howard Hughes Medical Institute (K.M.M.) and the National Institutes of Health (5P01AI031238 and 5T32HL007317).

Footnotes

Accession codes. UCSD-Nature Signaling Gateway (http://www.signaling-gateway.org): A001503; ArrayExpress: microarray data, E-MEXP-1518 and E-MEXP-1511.

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