Figure 7
Figure 7. miR-142 controls B-cell homeostasis by targeting BAFF-R. (A) Upregulation of BAFF-R expression on the cell surface of miR-142−/− B cells. FACS analysis of CD19+B220+ B cells from WT (open histogram) and KO (shaded histogram) animals with anti-CD268 (BAFF-R) antibodies. (B) Mean fluorescence intensity (MFI) of BAFF-R expression in B cells from WT (n = 8) and KO (n = 6) animals. (C) Activation of noncanonical NF-κB and Akt pathways in miR-142−/− cells. Western blot analysis of p100/NFKB2 processing and Akt (S473) phosphorylation in purified CD19+ B cells isolated from WT and KO animals. Cells were stimulated with human BAFF ligand (500 ng/mL) for 15 hours. β-Actin and pan-Akt signals were used as loading controls. (D) Analysis of B-cell proliferation by 3H-thymidine incorporation assay. Purified WT (n = 2) and KO (n = 2) B cells were stimulated with human BAFF ligand (500 ng/mL) for 24 and 48 hours. (E-K) Lowering of the BAFF-R gene dose rescues the B-cell expansion defect in miR-142−/− mice. (E) Analysis of BAFF-R expression in WT (n = 6), miR-142−/− (n = 5), and miR-142−/−BAFF-R+/BCMD1 (n = 9) splenocytes by quantitative reverse-transcription polymerase chain reaction. Expression level of BAFF-R allele in WT mouse was arbitrarily set to 1. Mouse β-actin levels were used for normalization. (F-H) Rescue of the splenomegaly phenotype in miR-142−/−BAFF-R+/BCMD1 mice. (F) Representative image of spleens from WT, miR-142−/− and miR-142−/−BAFF-R+/BCMD1 mice. Spleen weights (G) and total splenocyte counts (H) in WT (n = 9), miR142−/− (n = 17), miR-142−/−BAFF-R+/BCMD1 (n = 12), and BAFF-R+/BCMD1 (n = 4) animals. (I) Total CD19+B220+ B-cell counts in WT (n = 9), miR-142−/− (n = 17), miR-142−/−BAFF-R+/BCMD1 (n = 12), and BAFF-R+/BCMD1 (n = 4) spleens. (J) Normal number of MZ B cells in miR-142−/−BAFF-R+/BCMD1 mice. Total number of MZ B cells in WT (n = 9), miR-142−/− (n = 17), miR-142−/−BAFF-R+/BCMD1 (n = 12), and BAFF-R+/BCMD1 (n = 4) spleens. (K) Lack of rescue of the B1 B-cell defect in miR-142−/−BAFF-R+/BCMD1 mice. Total number of B1 B cells in the peritoneal cavities of WT (n = 6), miR-142 KO (n = 16), and miR-142−/−BAFF-R+/BCMD1 (n = 11) mice. Results are plotted as means ± SD. P values were calculated using Student t test or 2-way analysis of. *P ≤ .05; **P ≤ .01; ***P ≤ .001; ****P ≤ .0001. ns, not significant.

miR-142 controls B-cell homeostasis by targeting BAFF-R. (A) Upregulation of BAFF-R expression on the cell surface of miR-142−/− B cells. FACS analysis of CD19+B220+ B cells from WT (open histogram) and KO (shaded histogram) animals with anti-CD268 (BAFF-R) antibodies. (B) Mean fluorescence intensity (MFI) of BAFF-R expression in B cells from WT (n = 8) and KO (n = 6) animals. (C) Activation of noncanonical NF-κB and Akt pathways in miR-142−/− cells. Western blot analysis of p100/NFKB2 processing and Akt (S473) phosphorylation in purified CD19+ B cells isolated from WT and KO animals. Cells were stimulated with human BAFF ligand (500 ng/mL) for 15 hours. β-Actin and pan-Akt signals were used as loading controls. (D) Analysis of B-cell proliferation by 3H-thymidine incorporation assay. Purified WT (n = 2) and KO (n = 2) B cells were stimulated with human BAFF ligand (500 ng/mL) for 24 and 48 hours. (E-K) Lowering of the BAFF-R gene dose rescues the B-cell expansion defect in miR-142−/− mice. (E) Analysis of BAFF-R expression in WT (n = 6), miR-142−/− (n = 5), and miR-142−/−BAFF-R+/BCMD1 (n = 9) splenocytes by quantitative reverse-transcription polymerase chain reaction. Expression level of BAFF-R allele in WT mouse was arbitrarily set to 1. Mouse β-actin levels were used for normalization. (F-H) Rescue of the splenomegaly phenotype in miR-142−/−BAFF-R+/BCMD1 mice. (F) Representative image of spleens from WT, miR-142−/− and miR-142−/−BAFF-R+/BCMD1 mice. Spleen weights (G) and total splenocyte counts (H) in WT (n = 9), miR142−/− (n = 17), miR-142−/−BAFF-R+/BCMD1 (n = 12), and BAFF-R+/BCMD1 (n = 4) animals. (I) Total CD19+B220+ B-cell counts in WT (n = 9), miR-142−/− (n = 17), miR-142−/−BAFF-R+/BCMD1 (n = 12), and BAFF-R+/BCMD1 (n = 4) spleens. (J) Normal number of MZ B cells in miR-142−/−BAFF-R+/BCMD1 mice. Total number of MZ B cells in WT (n = 9), miR-142−/− (n = 17), miR-142−/−BAFF-R+/BCMD1 (n = 12), and BAFF-R+/BCMD1 (n = 4) spleens. (K) Lack of rescue of the B1 B-cell defect in miR-142−/−BAFF-R+/BCMD1 mice. Total number of B1 B cells in the peritoneal cavities of WT (n = 6), miR-142 KO (n = 16), and miR-142−/−BAFF-R+/BCMD1 (n = 11) mice. Results are plotted as means ± SD. P values were calculated using Student t test or 2-way analysis of. *P ≤ .05; **P ≤ .01; ***P ≤ .001; ****P ≤ .0001. ns, not significant.

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