Journal: Stem Cells (Dayton, Ohio)

Article Title: Calcium and Calcineurin-NFAT Signaling Regulate Granulocyte-Monocyte Progenitor Cell Cycle via Flt3-L

doi: 10.1002/stem.1813

Figure Lengend Snippet: Flt3-L triggers signaling leading to nuclear factor of activated T cells (NFAT) translocation in granulocyte–monocyte progenitors (GMPs). (A): Levels of intracellular Ca 2+ measured as Fluo4 fluorescence in sorted lin − Sca1 + cKIT + bone marrow (BM) cells (LSKs), common myeloid progenitors (CMPs), and GMPs. After 20 seconds of measurements cells were triggered with FLT3-L, ionomycin, thapsigargin, or Hanks' balanced salt solution (HBSS), and Ca 2+ levels were assessed for another 5 minutes. (B): Intracellular Ca 2+ chelator BAPTA was used to block Ca 2+ release induced by FLT3-L in sorted GMPs. (C): Flt3-L induces Ca 2+ release in GMPs sorted as lin − , cKit + , Sca-1 − , CD34 + , CD16/32 high . Graph of Ca 2+ release analysis in GMPs from lineage-depleted BM cells. Flt3-L (1μg/ml) was added after 1 minute of measurement followed by 4 minutes Ca 2+ release measurement. Representative of three independent experiments, where BM cells from five mice were pooled. (D): Different levels of phospholipase Cγ (PLCγ 2 ) phosphorylation (pPLCγ 2 ) in LSKs (pool of hematopoietic stem cell [HSCs] and multipotent progenitors [MPPs]; lin − , cKit + , Sca-1 + ), CMPs, and GMPs in freshly isolated BM. (E–G): Different levels of pPLCγ 1 in progenitor populations before and after Flt3-L administration. Lineage-depleted BM cells were cultured for 4 hours in HSC medium, Flt3-L (1 μg/ml) was added 15 minutes before fixing and labeling with antibodies for progenitor markers, PLCγ 1 and pPLCγ 2 . Cells were gated as LSKs (pool of HSCs and MPPs; lin − , cKit + , Sca-1 + ), CMPs (lin − , cKit + , Sca-1 − , CD34 + , CD16/32 int ), and GMPs (lin − , cKit + , Sca-1 − , CD34 + , CD16/32 high ). (E, F): Percentage of pPLCγ 1 cells and Gm of fluorescence of pPLCγ 1 and double labeling of total PLCγ 1 with pPLCγ 1 upon trigger with Flt3-L are shown. Data are presented as mean ± SE, *, p < .05 and **, p < .01 in an unpaired Student's t -test. (G): Histogram overlay of pPLCγ 1 intensity in GMPs after the Flt3-L trigger. Representative experiment from three independent experiments is shown. (H): Flt3-L induces NFAT translocation in cKIT + -enriched BM cells. BM cells were depleted of lineaged cells and enriched with cKIT + beads, maintained in HSC medium and transduced with NFAT reporter constructs. Transfected cells were kept in HSC medium for 48 hours, and 1 μg/ml of Flt3-L was added for last 4 hours of culture before nuclear translocation of NFAT reflecting activity of luciferase reporter gene was measured. Data are presented as mean ± SE from one representative of three independent experiments, *, p < .05 in an unpaired Student's t -test. BM pooled from 10 mice was used for each experiment. (I): Proposed graphical scheme of cell cycle regulation in GMPs. Flt3-L activates PLCγ 1 and induce increase in intracellular Ca 2+ levels, this further activates calcineurin and NFAT translocation. When calcineurin-NFAT interaction is blocked, expression of cell cycle regulation genes in GMPs is changed to promote further proliferation. Abbreviations: CMP, common myeloid progenitors; CsA, Cyclosporine A; FITC, fluorescein isothiocyanate; GMP, granulocyte–monocyte progenitor; HBSS, Hanks' balanced salt solution; LSK, lin − Sca1 + cKIT + bone marrow cells; NFAT, nuclear factor of activated T cells; NT, non-treated; PLC, phospholipase Cγ.

Article Snippet: Alternatively, cells were fixed and incubated with anti-pPLCγ 2 -FITC (BD Phosphoflow, BD Bioscience, Mississauga ON, www.bdbiosciences.com ), according to the kit instructions, using Phosphoflow Perm Buffer III.

Techniques: Translocation Assay, Fluorescence, Blocking Assay, Isolation, Cell Culture, Labeling, Transduction, Construct, Transfection, Activity Assay, Luciferase, Expressing