Mice deficient for all PIM kinases display reduced body size and impaired responses to hematopoietic growth factors - PubMed (original) (raw)

Mice deficient for all PIM kinases display reduced body size and impaired responses to hematopoietic growth factors

Harald Mikkers et al. Mol Cell Biol. 2004 Jul.

Abstract

The Pim family of proto-oncogenes encodes a distinct class of serine/threonine kinases consisting of PIM1, PIM2, and PIM3. Although the Pim genes are evolutionarily highly conserved, the contribution of PIM proteins to mammalian development is unclear. PIM1-deficient mice were previously described but showed only minor phenotypic aberrations. To assess the role of PIM proteins in mammalian physiology, compound Pim knockout mice were generated. Mice lacking expression of Pim1, Pim2, and Pim3 are viable and fertile. However, PIM-deficient mice show a profound reduction in body size at birth and throughout postnatal life. In addition, the in vitro response of distinct hematopoietic cell populations to growth factors is severely impaired. In particular, PIM proteins are required for the efficient proliferation of peripheral T lymphocytes mediated by synergistic T-cell receptor and interleukin-2 signaling. These results indicate that members of the PIM family of proteins are important but dispensable factors for growth factor signaling.

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Figures

FIG. 1.

FIG. 1.

The generation of Pim1 Pim2 Pim3 compound KO mice. (a) Targeting constructs for Pim2 and Pim3. PGK-Php was introduced into the BamHI (B) sites of Pim2, thereby deleting exons 1, 2, and 3. For Pim3, a part of exon 3, exons 4 and 5, and part of exon 6 were deleted through introduction of the promoterless IRES-β_Geo_ cassette into the BstEII (Bs) sites. (b) Genotype analysis of Pim2 and Pim3 KO mice by Southern blotting using full-length Pim2 cDNA or the 3′ UTR of Pim3 as a probe. WT, wild type. (c) Comparison of the amino acid sequences of PIM1, PIM2, and PIM3. Identical residues are shown in white characters on a black background; similar residues are shown in white characters on a grey background. (d) Expression of Pim1, Pim2, and Pim3 mRNA. The results are shown for bone marrow-derived mast cells grown on IL-3 (BMMC), EL4 (T-cell line), and J558 (plasmacytoma). LPS, lipopolysaccharide. (e) RT-PCR analysis of Pim1, Pim2, and Pim3 expression in wild-type, Pim heterozygous, and Pim homozygous mutant splenocytes.

FIG. 2.

FIG. 2.

PIM-deficient mice display a growth defect. (a) Comparison of male neonatal and mature _Pim1_−/− _Pim2_− _Pim3_−/− mice with _Pim1_−/− _Pim2_− Pim3+/− littermates (upper and lower panel, respectively). (b) Growth curve of male Pim1+/− _Pim2_− Pim3+/−, _Pim1_−/− _Pim2_− Pim3+/−, Pim1+/− _Pim2_− _Pim3_−/−, and _Pim1_−/− _Pim2_− _Pim3_−/− littermates. The weight of Pim1+/− _Pim2_− Pim3+/− mice is similar to that of wild-type mice. (c) Cell numbers in spleens, bone marrows, and thymuses from 8-week-old wild-type (n = 9) and _Pim1_−/− _Pim2_−/− _Pim3_−/− (n = 9) mice.

FIG. 3.

FIG. 3.

B- but not T-lymphoid development is affected in Pim mutant mice. The first two columns represent PIM-deficient mice; the second series of two columns represents wild-type mice. FITC, fluorescein isothiocyanate; APC, antigen-presenting cells. (a) Flow cytometric analysis of wild-type and Pim mutant thymocytes by CD4/CD8 staining (panels 1 and 3) and CD25/CD44 staining on cells negative for CD4, CD8, B220, CD19, Mac1, Ter119, Gr-1, and NK1.1 (panels 2 and 4). (b) Mature B-cell and T-cell populations in Pim mutant and wild-type spleen. (c) Analysis of B-cell differentiation in the bone marrow. CD43-positive, B220-positive cells were gated and analyzed using BP1 and HSA markers to distinguish pre-pro-B, pro-B, and pro-B-early pre-B cells (panels 1 and 3). CD43-negative, B220-positive cells were gated and analyzed using IgM and IgD to distinguish late pre-B, IgM-positive immature B, and mature B cells (panels 2 and 4). (d) The number of cells belonging to distinct stages of B-cell development depicted as a percentage of total B220-positive cell numbers in the bone marrow of 6- to 8-week-old wild-type and Pim mutant mice. (e) Proliferation of Pim mutant bone marrow cells in response to IL-7. Bone marrow cells were cultured for 96 h in the presence of mIL-7. Proliferation was determined by [3H]thymidine incorporation. For the last 16 h, cells were cultured in the presence of [3H]thymidine. One representative of three independent experiments is shown. (f) Cell surface expression levels IL-7Rα and common-γ-chain on differentiating B cells in the bone marrow. The thin line represents wild-type cells; the thick line represents Pim-deficient cells.

FIG. 4.

FIG. 4.

The loss of PIM proteins affects [3H]thymidine incorporation induced by synergistic TCR and IL-2 signaling. Splenic T lymphocytes derived from wild-type mice (open squares) or PIM-deficient mice (closed triangles) were cultured for 64 h in the presence of different concentrations of coated αCD3 (a), a suboptimal concentration of coated αCD3 (0.3 μg/ml) and increasing concentrations of hIL-2 (b), constant hIL-2 (5 U/ml) and increasing amounts of coated αCD3 (c), or constant hIL-2 (200 U/ml) and increasing amounts of αCD3 (d). The proliferation of T lymphocytes was assayed by measuring [3H]thymidine incorporation for the last 16 h of culture. Each panel represents one of three independent experiments with similar results (n = 3 for each genotype). (e) STAT5 phosphorylation in response to a dilution series of 0.02, 2.0, and 200 U of IL-2 of wild-type and PIM-deficient spleen cells. (f) Cell surface expression of components of the IL-2R complex on splenic T cells of wild-type mice (open squares) and TKO mice (solid triangles). PE, phycoerythrin.

FIG. 5.

FIG. 5.

PIM proteins appear to facilitate cell cycle entry in response to TCR and IL-2 signaling. Splenic T lymphocytes derived from wild-type or PIM-deficient mice were cultured in the presence of αCD3 (0.3 μg/ml) and hIL-2 (200 U/ml). For all analyses the T-cell population was characterized by flow cytometry using αTCRβ or αCD3 antibodies. (a) The proportions of apoptotic and dead cells after 48 h of culturing, as determined by Annexin V and 7-AAD staining. (b to c) The number of cell divisions was determined after 48 h (b) or 72 h (c) of culture growth, with CFSE used as a tracking dye (black lines correspond to unstimulated cells). (d) Analysis of DNA synthesis after 24 h of culture growth in the presence of hIL-2 (200 U/ml) and αCD3 (0.3 μg/ml).

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