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Calcineurin/NFAT signalling inhibits myeloid haematopoiesis.

Fric J, Lim CX, Koh EG, Hofmann B, Chen J, Tay HS, Mohammad Isa SA, Mortellaro A, Ruedl C, Ricciardi-Castagnoli P - EMBO Mol Med (2012)

Bottom Line: Reconstituting lethally irradiated mice with haematopoietic stem cells expressing an NFAT-inhibitory peptide resulted in enhanced development of the myeloid compartment.Global gene expression analysis of untreated DC and NFAT-inhibited DC revealed differential expression of transcripts that regulate cell cycle and apoptosis.In conclusion, these results provide evidence that calcineurin/NFAT signalling negatively regulates myeloid lineage development.

View Article: PubMed Central - PubMed

Affiliation: Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.

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NFAT1 is expressed in HSC lines and increases following stimulation with Flt3-LWestern blot analysis of un-stimulated and Flt3-L stimulated VIVIT expressing and control HSC lines. PMA treated splenic T cells were used as a positive control.qPCR analysis of NFAT1 mRNA expression in HSC lines after stimulation with Flt3-L.qPCR analysis of NFAT2 and NFAT1 expression in freshly isolated lineage (lin−) negative cells from BM stimulated with Flt3-L.Confocal images of VIVIT-eGFP and control-tdTomato expressing HSC lines stimulated for 48 h with Flt3-L. NFAT translocation was induced by treatment with ionomycin for 2 h (original magnification, ×200, scale bar 2 µm). Data are representative of three (A and C) and five (B) independent experiments (mean ± SEM). Three mice per group for each time-point were used (C).
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fig01: NFAT1 is expressed in HSC lines and increases following stimulation with Flt3-LWestern blot analysis of un-stimulated and Flt3-L stimulated VIVIT expressing and control HSC lines. PMA treated splenic T cells were used as a positive control.qPCR analysis of NFAT1 mRNA expression in HSC lines after stimulation with Flt3-L.qPCR analysis of NFAT2 and NFAT1 expression in freshly isolated lineage (lin−) negative cells from BM stimulated with Flt3-L.Confocal images of VIVIT-eGFP and control-tdTomato expressing HSC lines stimulated for 48 h with Flt3-L. NFAT translocation was induced by treatment with ionomycin for 2 h (original magnification, ×200, scale bar 2 µm). Data are representative of three (A and C) and five (B) independent experiments (mean ± SEM). Three mice per group for each time-point were used (C).

Mentions: Given that NFAT1 is expressed in stem cells more strongly than other NFAT proteins (Kiani et al, 2004) and that Flt3-L is a key growth factor in early myeloid development, we initially analysed total expression of NFAT1 protein in control-tdTomato and VIVIT-eGFP expressing HSC, both in the steady-state and in response to Flt3-L stimulation, to determine whether Flt3-L influences NFAT expression. By Western blot analysis, NFAT1 expression could be detected in untreated HSC lines, and stimulation with Flt3-L for 48 h moderately up-regulated expression levels (Fig 1A). Notably, NFAT1 expression was far lower than was observed in PMA-stimulated T cells (Fig 1A; positive control) and in un-stimulated T cells (unpublished observations). Up-regulation of NFAT1 protein in response to Flt3-L was also detected at the mRNA level (Fig 1B). In further experiments, it was possible to demonstrate enhanced expression of NFAT1 and NFAT2 mRNA in BM cells treated with Flt3-L. Sorted lineage negative BM cells (lin−; CD3e−, B220−, CD19− CD11b−, Gr-1−, TER-119−, NK1.1− and CD127−) were used to exclude any contribution from differentiated cells and lymphoid progenitors (Fig 1C).


Calcineurin/NFAT signalling inhibits myeloid haematopoiesis.

Fric J, Lim CX, Koh EG, Hofmann B, Chen J, Tay HS, Mohammad Isa SA, Mortellaro A, Ruedl C, Ricciardi-Castagnoli P - EMBO Mol Med (2012)

NFAT1 is expressed in HSC lines and increases following stimulation with Flt3-LWestern blot analysis of un-stimulated and Flt3-L stimulated VIVIT expressing and control HSC lines. PMA treated splenic T cells were used as a positive control.qPCR analysis of NFAT1 mRNA expression in HSC lines after stimulation with Flt3-L.qPCR analysis of NFAT2 and NFAT1 expression in freshly isolated lineage (lin−) negative cells from BM stimulated with Flt3-L.Confocal images of VIVIT-eGFP and control-tdTomato expressing HSC lines stimulated for 48 h with Flt3-L. NFAT translocation was induced by treatment with ionomycin for 2 h (original magnification, ×200, scale bar 2 µm). Data are representative of three (A and C) and five (B) independent experiments (mean ± SEM). Three mice per group for each time-point were used (C).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3376854&req=5

fig01: NFAT1 is expressed in HSC lines and increases following stimulation with Flt3-LWestern blot analysis of un-stimulated and Flt3-L stimulated VIVIT expressing and control HSC lines. PMA treated splenic T cells were used as a positive control.qPCR analysis of NFAT1 mRNA expression in HSC lines after stimulation with Flt3-L.qPCR analysis of NFAT2 and NFAT1 expression in freshly isolated lineage (lin−) negative cells from BM stimulated with Flt3-L.Confocal images of VIVIT-eGFP and control-tdTomato expressing HSC lines stimulated for 48 h with Flt3-L. NFAT translocation was induced by treatment with ionomycin for 2 h (original magnification, ×200, scale bar 2 µm). Data are representative of three (A and C) and five (B) independent experiments (mean ± SEM). Three mice per group for each time-point were used (C).
Mentions: Given that NFAT1 is expressed in stem cells more strongly than other NFAT proteins (Kiani et al, 2004) and that Flt3-L is a key growth factor in early myeloid development, we initially analysed total expression of NFAT1 protein in control-tdTomato and VIVIT-eGFP expressing HSC, both in the steady-state and in response to Flt3-L stimulation, to determine whether Flt3-L influences NFAT expression. By Western blot analysis, NFAT1 expression could be detected in untreated HSC lines, and stimulation with Flt3-L for 48 h moderately up-regulated expression levels (Fig 1A). Notably, NFAT1 expression was far lower than was observed in PMA-stimulated T cells (Fig 1A; positive control) and in un-stimulated T cells (unpublished observations). Up-regulation of NFAT1 protein in response to Flt3-L was also detected at the mRNA level (Fig 1B). In further experiments, it was possible to demonstrate enhanced expression of NFAT1 and NFAT2 mRNA in BM cells treated with Flt3-L. Sorted lineage negative BM cells (lin−; CD3e−, B220−, CD19− CD11b−, Gr-1−, TER-119−, NK1.1− and CD127−) were used to exclude any contribution from differentiated cells and lymphoid progenitors (Fig 1C).

Bottom Line: Reconstituting lethally irradiated mice with haematopoietic stem cells expressing an NFAT-inhibitory peptide resulted in enhanced development of the myeloid compartment.Global gene expression analysis of untreated DC and NFAT-inhibited DC revealed differential expression of transcripts that regulate cell cycle and apoptosis.In conclusion, these results provide evidence that calcineurin/NFAT signalling negatively regulates myeloid lineage development.

View Article: PubMed Central - PubMed

Affiliation: Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.

Show MeSH
Related in: MedlinePlus