Conditional inactivation of Mef2c in B cells. (A) A conditional KO strategy, using Cre recombinase driven by the CD19 locus, was used to excise Mef2c flanked by LoxP sites (Mef2c^flox/flox). All mice were backcrossed into a C57J/BL6 background for nine generations. For all experiments, CD19^Cre/+; Mef2c^flox/+ littermates were used to control for the effect of CD19 heterozygosity. (B) Deletion of the second coding exon of Mef2c in sorted Fo B cells was confirmed by Southern blot analysis. Conditional KO (ko) mice had the null allele (Tm1) but not the wild type (wt) allele, which was present in control littermates (ctrl). Note that the conversion of the “floxed” allele (flox) to the recombined form occurred in both control and KO Fo B cells with high efficiency because of the presence of the CD19^Cre allele. (C) Mef2c B cell KO animals (CD19^Cre/+; Mef2c^flox/−) were born at the expected 1:4 ratio.

MEF2C regulates B-cell proliferation in response to BCR stimulation. [³H]-thymidine incorporation assay shows a nearly 90% reduction in proliferation of sorted Fo B cells from KO (ko) mice compared with controls (ctrl) on stimulation of the BCR with α-IgM (lanes 3, 4) but not LPS (lanes 5, 6). Immature B cells (imm.) from either population failed to proliferate on BCR stimulation (lanes 1, 2). Data represent the mean plus SEM for three independent assays.

BCR stimulation results in phosphorylation of MEF2C. 2D immunoblot demonstrates that stimulation of BCR by α-IgG resulted in a shift in the isoelectric point of MEF2C (B, C), whereas P-mut MEF2C did not (D). Encircled is MEF2C; the arrows point to shifts in the isoelectric point of MEF2C. Spots were identified by molecular weight (MEF2C-FLAG = ∼53 kDa, IgG = ∼150 kDa) compared with a standard protein ladder run in nondenaturing conditions.

Mef2c is required for efficient humoral immune response against T-dependent antigens. (A) Mef2c B-cell conditional KO (ko, blue circles) mice exhibit reduced NP-specific IgG1 titers compared with control (ctrl, red circles) mice at 14 and 21 days after immunization with NP-chicken gamma globulin. (B) Immunohistochemistry on sections from lymph nodes depicts a reduced GC response (PNA, green) at 14 days after immunization in Mef2c B-cell KO (bottom row) mice compared with control (top row) mice (white arrows indicate GCs). Lymphoid follicle structure (IgD, red) remained intact in both groups. (C) Quantification of the GC defect by flow cytometry shows a ≈80% reduction in total PNA⁺, FAS⁺, B220⁺ GC B cells in lymph nodes of Mef2c B-cell KO mice compared with control mice.

Activation of MEF2C by BCR stimulation requires p38 MAPK. (A) BCR stimulation (α-IgG) of 2PK3 B cells transfected with an MEF2-dependent reporter plasmid (wt) showed a 10-fold increase in reporter activity compared with PBS-treated cells (lanes 1, 2), whereas mutant reporter plasmid (mut) did not (lanes 5, 6). Addition of a p38-specific inhibitor (SB203580) blocked BCR-induced reporter activity (lanes 3, 4). (B) Cotransfection with MKK6_EE and stimulation with α-IgG showed a synergistic increase in MEF2-dependent reporter activity (lane 4) compared with transfection of MKK6_EE without stimulation (lane 3) or reporter alone stimulated with α-IgG (lane 2). Addition of p38 inhibitor blocked this activation (lane 5). (C) p38 phosphorylation sites on MEF2C are necessary for its activity. Addition of α-IgG to cells transfected with P-mut MEF2C failed to activate the MEF2-dependent reporter (lanes 2, 4). Data in all panels represent the mean plus SEM for five independent transfections and analyses.

Expression of a P-mut form of MEF2C severely reduces the proliferative capacity of primary Fo B cells. Wild type primary Fo B cells were infected with lentivirus expressing either MEF2C wild type (wt), MEF2C(P-mut), or GFP control (ctrl) and were stimulated to proliferate with either α-IgM (BCR stimulation) (lanes 1–4) or LPS (Toll-like receptor stimulation) (lanes 5–8). Expression of MEF2C(P-mut) severely inhibited proliferation compared with expression of MEF2C wt (lanes 3, 4; P < 0.001). No difference in proliferation was observed between cells infected with MEF2C wt versus MEF2C(P-mut) when stimulated with LPS (lanes 7, 8). Data represent the mean plus SEM for three independent assays of primary Fo B cells isolated from three different wild type C57J/BL6 mice.