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. 2013 Jul 16;8(7):e68804.
doi: 10.1371/journal.pone.0068804. Print 2013.

The Serine/Threonine Phosphatase PP4 Is Required for pro-B Cell Development Through Its Promotion of Immunoglobulin VDJ Recombination

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Free PMC article

The Serine/Threonine Phosphatase PP4 Is Required for pro-B Cell Development Through Its Promotion of Immunoglobulin VDJ Recombination

Yu-Wen Su et al. PLoS One. .
Free PMC article

Abstract

PP4 phosphatase regulates a number of crucial processes but the role of PP4 in B cells has never been reported. We generated B cell-specific pp4 knockout mice and have identified an essential role for PP4 in B cell development. Deficiency of PP4 in B lineage cells leads to a strong reduction in pre-B cell numbers, an absence in immature B cells, and a complete loss of mature B cells. In PP4-deficient pro-B cells, immunoglobulin (Ig) DJ(H) recombination is impaired and Ig µ heavy chain expression is greatly decreased. In addition, PP4-deficient pro-B cells show an increase of DNA double-strand breaks at Ig loci. Consistent with their reduced numbers, residual PP4-deficient pre-B cells accumulate in the G1 phase, exhibit excessive DNA damage, and undergo increased apoptosis. Overexpression of transgenic Ig in PP4-deficient mice rescues the defect in B cell development such that the animals have normal numbers of IgM(+) B cells. Our study therefore reveals a novel function for PP4 in pro-B cell development through its promotion of V(H)DJ(H) recombination.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. PP4 deficiency induces a severe block in B cell development.
(A) Flow cytometric profiles of: total BM lymphocytes from WT and cKO mice analyzed for B220 vs CD43 expression (left panel); B220+CD43+ BM lymphocytes analyzed for CD24 vs BP-1 (middle panel); and B220+CD43 BM lymphocytes analyzed for IgM vs IgD (right panel). (B) Quantitative cell count analysis of Frs. A to F cells from WT and cKO mice. (C) Quantitative cell count analysis of: splenic B220+ B cells from WT and cKO mice. (D) Flow cytometric profiles of BM, spleen and MLN lymphocytes from WT and cKO mice (n = 4/group) analyzed for B220 vs IgM; B220+ splenocytes analyzed for CD21 vs CD23; and peritoneal lymphocytes analyzed for IgM vs CD5, as indicated. For A-D, results are representative of three independent experiments. For B and C, data are the mean ± SD (n = 4) (*p≤0.05; **p≤0.005).
Figure 2
Figure 2. Efficiency of pp4c deletion in B cells.
(A) Schematic illustration of the targeting strategy used to delete pp4c and the primers designed to detect deletion efficiency. For genomic PCR, forward primers “a” (5′-ACGTGATTTGCGAAAGCCTCTCA-3′) and “b” (5′-CTTGGTAGAAGAGAGCAACGTGCAG-3′), and reverse primer “c” (5′-TGCTCTGGTGGCAGGAGATGTGTG-3′), were used as indicated. The PCR products of the WT pp4c allele (427 bp; 1); the pp4c floxp allele before cre-mediated deletion (480 bp; 2); and the pp4c floxp allele after cre-mediated deletion (550 bp; 3) are shown. (B) PCR analysis of genomic DNA from Frs. A, B and C cells from WT and cKO mice (n = 1/group) showing products representing the WT pp4c allele (+/+), the pp4c floxp allele (F/F), and the deleted pp4c allele (deleted F/F). Percentages shown are deletion efficiencies. CD14, loading control. Results are representative of three independent experiments. (C) RT-PCR analysis of PP4 and HPRT mRNAs in Frs. A, B and C cells from WT and cKO mice (n = 2/group). Numbers are the relative mRNA levels quantified by Image J and normalized to the WT Fr. A value (set to 1). P, positive control for PP4. HPRT, loading control. Results are representative of three independent experiments. (D) Quantitation of the mRNA levels in the cells in (C) after normalization to HPRT values.
Figure 3
Figure 3. Reduced Ig µ HC expression and impaired DJH recombination in PP4-deficient pro-B cells.
(A) Flow cytometric profiles of Frs. A, B and C cells from WT and cKO mice (n = 3/group) analyzed for B220 vs intracellular µ HC (upper panel), or B220 vs isotype control (lower panel). Results are representative of two independent experiments. (B) Quantitation of percentages of the Fr. A, B and C cells in (A) expressing intracellular µ HC. Data are the mean ± SD (n = 2). (C) Confocal images of intracellular µ HC staining in Fr. B cells from WT and cKO mice (n = 2/group). DAPI, nuclei. Results are representative of three independent experiments. (D) Southern blot of genomic DNA from Fr. B cells of WT and cKO mice (n = 2/group) subjected to a DJH recombination assay. Genomic DNA was serially diluted from 1∶1 to 1∶64 prior to nested PCR. Bands were visualized using DIG-labeled southern blotting. CD14, loading control. Results are representative of two independent experiments. (E) Southern blot of genomic DNA from Fr. B cells of WT and cKO mice subjected to the coding end assay. Genomic DNA was filled by Klenow, ligated with double-strand oligo linker of 5′-overhang, and subjected to nested PCR. Bands were visualized using DIG-labeled southern blotting. CD14, loading control. Ten mice per group were used in this experiment, and two independent experiments were performed.
Figure 4
Figure 4. PP4 deficiency causes excessive DNA damage and impairs B cell homeostasis.
(A) Microscopic images of comet assays of Fr. B and Fr. C cells from WT and cKO mice (n = 2/group). Results are representative of two independent experiments. (B) Enlarged microscopic images of the comet assays of the WT and cKO Fr. C cells in (A). (C) In vivo BrdU incorporation assay. WT and cKO mice (n = 3/group) were i.p. injected with BrdU at 16 h and 24 h before isolation of Fr. B and Fr. C cells by cell sorting. Cells were subjected to intracellular staining with 7AAD plus anti-BrdU and analyzed by flow cytometry. Numbers in quadrants represent the percentage of the indicated population relative to the total. Results are representative of three independent experiments. (D) Quantitation of the percentages of G1, G1/S, and S/G2 phase cells among the Fr. B and Fr. C cells in (C). Data are the mean ± SD (n = 3). (E) In vitro BrdU incorporation assay. Fr. B and Fr. C cells were sorted from WT and cKO mice (n = 2/group), cultured for 16 h in the presence of BrdU, and subjected to intracellular staining and flow cytometric analysis as for (C). Results are representative of two independent experiments. (F) In vivo apoptosis assay. Fr. B and Fr. C cells from WT and cKO mice (n = 4/group) were stained to detect AnnexinV plus 7AAD and analyzed by flow cytometry. Numbers in the lower right quadrant indicate the percentage of cells in early apoptosis, whereas the values in the upper right quadrant indicate the percentage of cells in late apoptosis. Results are representative of two independent experiments. (G) Quantitation of Fr. B and Fr. C cells in early or late apoptosis as calculated from the results in (F). Data are the mean ± SD (n = 4–6/group). (H) In vitro viability assay. Fr. B and Fr. cells were sorted from WT and cKO mice (n = 2/group) and cultured for 16 h under standard conditions. Cells were surface-stained with 7AAD and the percentage of viable cells was calculated. Data are the mean ± SD (n = 2). Results are representative of two independent experiments (*p≤0.05, **p≤0.005, ***p≤0.0005).
Figure 5
Figure 5. The defect in B cell development caused by PP4 deficiency can be rescued by Ig transgene expression.
(A) Flow cytometric profiles of: total BM lymphocytes from WT, cKO and cKO/IgHEL mice (n = 3–5/group) analyzed for B220 vs IgM (far left panel) or B220 vs CD43 (middle left panel); or B220+CD43+ BM lymphocytes analyzed for CD24 vs BP-1 (middle right panel); or B220+CD43 BM lymphocytes analyzed for IgM vs IgD (far right panel). (B) Quantitative cell count analysis of Frs. A to C cells from the mice in (A). (C) Quantitative cell count analysis of Frs. D to F cells from the mice in (A). For B and C, data are the mean ± SD (n = 3–5/group). Results are representative of two independent experiments (*p≤0.05, **p≤0.005; ns, not significant).

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References

    1. Hardy RR, Carmack CE, Shinton SA, Kemp JD, Hayakawa K (1991) Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J Exp Med 173: 1213–1225. - PMC - PubMed
    1. Li YS, Hayakawa K, Hardy RR (1993) The regulated expression of B lineage associated genes during B cell differentiation in bone marrow and fetal liver. J Exp Med 178: 951–960. - PMC - PubMed
    1. Alt FW, Yancopoulos GD, Blackwell TK, Wood C, Thomas E, et al. (1984) Ordered rearrangement of immunoglobulin heavy chain variable region segments. EMBO J 3: 1209–1219. - PMC - PubMed
    1. Pike KA, Ratcliffe MJ (2002) Cell surface immunoglobulin receptors in B cell development. Semin Immunol 14: 351–358. - PubMed
    1. Bassing CH, Swat W, Alt FW (2002) The mechanism and regulation of chromosomal V(D)J recombination. Cell 109 Suppl: S45–55 - PubMed

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Grant support

This work was supported by grants IM-102-PP-04 from the National Health Research Institutes (http://english.nhri.org.tw/NHRI_WEB/nhriw001Action.do) and NSC100-2320-B-400-003-MY3 from the National Science Council (http://web1.nsc.gov.tw/mp.aspx?mp=7), Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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