Granulocyte-monocyte progenitor cell cycle regulation occurs through calcium flux and calcineurin-NFAT signaling via Flt3-L

Complex regulatory mechanisms control continuous maintenance of myeloid progenitors and renewal of differentiated cells. Transcription factors play a important role in these processes. Here we report that the activation the calcineurin-NFAT signaling pathway inhibit the proliferation of myeloid granulocyte-monocyte progenitor (GMP). Myeloid progenitor subtypes possessed different susceptibilities to Ca2+ flux induction and consequently differential engagement of the calcineurin-NFAT pathway. This study show that inhibition of the calcineurin-NFAT pathway enhanced proliferation of GMPs both in vivo and in vitro. The calcineurin-NFAT signaling in GMPs is initiated through Flt3-L. The inhibition of the calcineurin-NFAT pathway altered the expression of the cell cycle regulation genes CDK4, CDK6, and CDKN1A, thus enabling faster cell cycle progression. The extensive use of NFAT inhibitors in the clinic should take into account that, in addition to the immunosuppression role in lymphoid cells, these NFAT inhibitors also affect the maintenance of the myeloid compartment. Microarray technology was used to understand the effects of NFAT inhibitors on C-kit enriched lineage negative cells. Bone marrow from C57BL/6 mouse depleted for lineage positive differentiated cells (lin-, CD3e-, B220-, CD19- CD11b-, CD11c-, Gr-1-, TER-119-, NK1.1- with addition of CD127-) and enriched for c-kit. lineage negative, cKIT positive progenitors were cultured in IMDM (containing 200ng/ml mrFLT3-L (Stem cells), 10% heat-inactivated FCS (Life Technologies), streptomycin 100mg/ml and penicillin 100 U/ml) at 3x106 cells/ml. For culture of sorted progenitors, stem cell factor (SCF; 50ng/ml), IL-6 (20ng/ml) and IL-3 (10ng/ml; all R&D Systems) were added (referred to as HSC medium). 2µg/ml CsA or 0.2µg/ml FK506 (Cell signalling Technology) were added to the cell culture for 30 mins preceding the addition of Flt3-L were maintained throughout the culture.