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. 2007 Jan;27(1):79-91.
doi: 10.1128/MCB.00799-06. Epub 2006 Oct 23.

Conditional Knockout Mice Reveal an Essential Role of Protein Phosphatase 4 in Thymocyte Development and pre-T-cell Receptor Signaling

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Conditional Knockout Mice Reveal an Essential Role of Protein Phosphatase 4 in Thymocyte Development and pre-T-cell Receptor Signaling

Jr-Wen Shui et al. Mol Cell Biol. .
Free PMC article

Abstract

Okadaic acid-sensitive serine/threonine phosphatases have been shown to regulate interleukin-2 transcription and T-cell activation. Okadaic acid inhibits protein phosphatase 4 (PP4), a novel PP2A-related serine/threonine phosphatase, at a 50% inhibitory concentration (IC(50)) comparable to that for PP2A. This raises the possibility that some cellular functions of PP2A, determined in T cells by using okadaic acid, may in fact be those of PP4. To investigate the in vivo roles of PP4 in T cells, we generated conventional and T-cell-specific PP4 conditional knockout mice. We found that the ablation of PP4 led to the embryonic lethality of mice. PP4 gene deletion in the T-cell lineage resulted in aberrant thymocyte development, including T-cell arrest at the double-negative 3 stage (CD4(-) CD8(-) CD25(+) CD44(-)), abnormal thymocyte maturation, and lower efficacy of positive selection. PP4-deficient thymocytes showed decreased proliferation and enhanced apoptosis in vivo. Analysis of pre-T-cell receptor (pre-TCR) signaling further revealed impaired calcium flux and phospholipase C-gamma1-extracellular signal-regulated kinase activation in the absence of PP4. Anti-CD3 injection in PP4-deficient mice led to enhanced thymocyte apoptosis, accompanied by increased proapoptotic Bim but decreased antiapoptotic Bcl-xL protein levels. In the periphery, antigen-specific T-cell proliferation and T-cell-mediated immune responses in PP4-deficient mice were dramatically compromised. Thus, our results indicate that PP4 is essential for thymocyte development and pre-TCR signaling.

Figures

FIG. 1.
FIG. 1.
PP4 gene conditional deletion in the T-cell lineage. (A) Generation of neo-free PP4-floxed mice by the two-loxP, two-frt strategy. The frt-flanked neo marker was removed from PP4+/flox-frt-neo mice by crossing them with FLPe deletion mice. Lck-Cre transgenic mice were used to generate T-cell-specific PP4 conditional knockout mice. Genomic DNA fragments after EcoRI digestion are indicated. E, EcoRI; H, HindIII; A, ApaI; EV, EcoRV; Nh, NheI; N, NdeI; P, PstI; DTA, diphtheria toxin A. (B) Efficient Cre-mediated PP4 gene deletion in thymuses of T-cell-specific conditional knockout mice. Genomic DNA was prepared and subjected to Southern blotting with EcoRI digestion. Wild-type alleles (WT; 6.2 kb), null alleles (deleted; 3.6 kb), and floxed alleles (flox; 2.8 kb) are indicated. (C) Loss of PP4 protein expression in PP4-deficient thymocytes. Lysates were prepared from thymocytes and analyzed by Western blotting using the PP4-specific antibody. WB, Western blotting. (D) Generation of a reading frame shift and a stop codon in exon 4 of PP4 after exon 3 deletion mediated by loxP-mediated DNA recombination. aa, amino acid.
FIG. 2.
FIG. 2.
Aberrant thymocyte development in T-cell PP4 conditional knockout mice. (A) Thymocytes were prepared from mice (>15 per group), stained with anti-CD4 and anti-CD8 antibodies, and analyzed by flow cytometry. Absolute cell numbers were calculated to determine cellularity. (B) Developmental arrest of thymocytes at the DN3 stage in T-cell PP4 conditional knockout mice. DN thymocytes were gated and analyzed by CD25/CD44 expression. Absolute cell numbers and flow cytometry results are shown. PP4 ablation led to developmental arrest at DN3 (CD4 CD8 CD25+ CD44).
FIG. 3.
FIG. 3.
DN3 developmental block and aberrant thymocyte maturation in T-cell-specific PP4 conditional knockout mice. (A) Cell arrest at E DN3 and L DN3 in T-cell PP4 conditional knockout mice. The CD44low CD25+ cells (DN3) in the CD4 CD8 population were gated to show their forward versus side scatter for 20,000 events. E represents the small DN3 thymocytes and L represents the large DN3 thymocytes. (B) A bar graph comparing cell numbers (knockout/wild type [KO/WT]) of different thymocyte subpopulations is shown to indicate cell arrest at DN3. (C) Impaired thymocyte maturation in the absence of PP4. Thymocytes were stained by anti-CD3 and anti-TCRβ antibodies and analyzed by flow cytometry. Mature thymocytes (TCRβhi CD3hi) are indicated. (D) TCR surface expression levels of each thymocyte subpopulation. Thymocytes were isolated from mice and stained for the TCRβ chain and CD4 and CD8 expression. TCRβ expression levels are indicated as mean fluorescence intensity (MFI).
FIG. 4.
FIG. 4.
PP4-deficient thymocytes were less proliferative in vivo. (BrdU panels) Mice (4 per group) received one intraperitoneal injection of BrdU (1 mg) and were sacrificed 1 h later, and thymocytes were stained with anti-BrdU, anti-CD4, and anti-CD8 antibodies. Numbers are the percentages of BrdU-positive cells in gated DN, DP, CD4 SP, and CD8 SP subpopulations. (Annexin V panels) PP4-deficient thymocytes were more apoptotic in vivo. Thymocytes were isolated and stained with anti-annexin V, anti-CD4, and anti-CD8 antibodies. Numbers are the percentages of annexin V-positive cells in gated DN, DP, CD4 SP, and CD8 SP subpopulations. Representative data are shown. FC, fluorescein isothiocyanate.
FIG. 5.
FIG. 5.
PP4 gene deletion leads to impaired positive selection. (A) Decreased CD69+ TCRβhi cell populations in T-cell-specific PP4 conditional knockout mice. Thymocytes were stained with anti-CD69 and anti-TCRβ antibodies and analyzed by flow cytometry. (B) Decreased numbers of mature CD4+ TCRβhi and CD8+ TCRβhi thymocytes in T-cell-specific PP4 conditional knockout mice. Thymocytes were stained with anti-CD4, anti-CD8, and anti-TCRβ antibodies, and the percentages of CD4+ TCRβhi and CD8+ TCRβhi cell populations are shown. Absolute CD4+ TCRβhi and CD8+ TCRβhi cell numbers as well as CD4/CD8 ratios were calculated. FC, fluorescein isothiocyanate; PE, phycoerythrin; TC, tricolor or PE-cyanine dye 5.
FIG. 6.
FIG. 6.
Analysis of positive selection in OT-II PP4-deficient mice. (A) Decreased cellularity of thymuses in OT-II PP4-deficient mice. (B) Decreased efficacy of positive selection of OT-II thymocytes in OT-II T-cell-specific PP4 conditional knockout mice. Thymocytes from indicated mice were stained with anti-CD4 and anti-CD8 antibodies and analyzed by flow cytometry. The ratio of CD4 SP cells to DP cells (with normalization of CD4 SP cell numbers to DP cell numbers) was calculated to determine the efficacy of positive selection. (C) OT-II (Vβ5+) thymocytes are less-positively selected in OT-II T-cell-specific PP4 conditional knockout mice. Thymocytes from indicated mice were stained with anti-CD3, anti-CD4, anti-CD8, and anti-Vβ5+ TCR antibodies and analyzed. The ratio of Vβ5 to CD3 cells was calculated to determine the efficacy of positive selection for Vβ5+ cell populations, with normalization to total DP cell numbers. PE, phycoerythrin; FC, flucytosine.
FIG. 7.
FIG. 7.
PP4 gene deletion leads to impaired pre-TCR signaling. Tyrosine phosphorylation of Lck (A) and ZAP-70 (B) was not affected in PP4-deficient thymocytes stimulated with anti-CD3 and anti-CD4. Cell lysates from stimulated thymocytes were analyzed by Western blotting, and the membranes were probed with the anti-phospho-Lck and anti-phospho-ZAP-70 antibodies, respectively. (C) Decreased tyrosine phosphorylation of PLC-γ1 in PP4-deficient thymocytes. Lysates prepared from stimulated thymocytes were immunoprecipitated with the anti-PLC-γ1 antibody, and tyrosine phosphorylation was measured by probing the membrane with antiphosphotyrosine antibody. IP, immunoprecipation. (D) Impaired calcium mobilization in response to TCR cross-linking in PP4-deficient thymocytes. Thymocytes were labeled with calcium chelators, and calcium flux was measured after anti-CD3 and anti-CD4 cross-linking. (E) Decreased ERK activation in stimulated PP4-deficient thymocytes. Cell lysates were analyzed by Western blotting, and the membranes were probed with the anti-phospho-ERK antibody, followed by reprobing of the membrane with the anti-total ERK antibody. p, phospho; WB, Western blotting.
FIG. 8.
FIG. 8.
PP4 regulates thymocyte apoptosis. (A) Susceptibility of thymocytes to cell death induced by different stimuli. Thymocytes were treated with the indicated concentration of anti-Fas antibody, plate-bound anti-CD3 antibody, or dexamethasone (Dex) for 24 h. Cell viability was calculated based on the percentages of the annexin V and 7-amino-actinomycin D double-negative populations. The results are representative of two independent experiments. (B) In vivo susceptibility of DP thymocytes to cell depletion induced by anti-CD3 antibody administration. Mice (three per group) were injected intraperitoneally with PBS or the indicated amount of the anti-CD3 antibody. After 20 h, CD4+ CD8+ populations were analyzed by flow cytometry to determine their viability. (C) Upregulation of the proapoptotic Bim protein levels in PP4-deficient thymocytes isolated from anti-CD3-injected mice. Thymocyte lysates were prepared from anti-CD3-injected mice and subjected to Western blotting with different proapoptotic antibodies, including anti-Bim, anti-Bad, and anti-Bax antibodies. BimEL, extralong Bim; BimL, long Bim; BimS, short Bim. (D) Decreased antiapoptotic Bcl-xL protein levels in PP4-deficient thymocytes isolated from anti-CD3-injected mice. Lysates were prepared and analyzed as described for panel C but with different antiapoptotic antibodies, including anti-Bcl-xL, anti-Bcl-2, and anti-A1 antibodies. α, anti.
FIG. 9.
FIG. 9.
PP4 is essential for maintenance of peripheral T-cell cellularity and T-cell-dependent immune functions. (A) Significant reductions in CD3+, CD4+, and CD8+ T cells were detected in peripheral T-cell compartments of Lck-Cre; F/F mice (representative results for 6 mice). SP, spleen; LN, lymph node. (B and C) Analysis of anti-CD3-induced cell proliferation with [3H]thymidine incorporation assay and CFSE labeling. For CFSE analysis, splenic T cells were labeled with CFSE before stimulation. CD4+ T cells were gated and analyzed for fluorescence intensity after 48 h. (D) Decreased antigen-specific T-cell proliferation in PP4-deficient mice. Mice were immunized subcutaneously with KLH-CFA. Sensitized draining lymph node T cells were restimulated with KLH, and proliferation was determined by the [3H]thymidine incorporation assay. (E) Decreased humoral immune responses in PP4-deficient mice. Primary immune response was evaluated by measuring various nitrophenol (NP)-specific antibody titers at 14 days after immunization with alum-precipitated DNP-KLH. The results are representative of two independent experiments.

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