Antagonizing Retinoic Acid Receptors Increases Myeloid Cell Production by Cultured Human Hematopoietic Stem Cells

Abstract

Activities of the retinoic acid receptor (RAR)α and RARγ are important to hematopoiesis. Here, we have investigated the effects of receptor selective agonists and antagonists on the primitive human hematopoietic cell lines KG1 and NB-4 and purified normal human hematopoietic stem cells (HSCs). Agonizing RARα (by AGN195183) was effective in driving neutrophil differentiation of NB-4 cells and this agonist synergized with a low amount (10 nM) of 1α,25-dihydroxyvitamin D₃ to drive monocyte differentiation of NB-4 and KG1 cells. Treatment of cultures of human HSCs (supplemented with stem cell factor ± interleukin 3) with an antagonist of all RARs (AGN194310) or of RARα (AGN196996) prolonged the lifespan of cultures, up to 55 days, and increased the production of neutrophils and monocytes. Slowing down of cell differentiation was not observed, and instead, hematopoietic stem and progenitor cells had expanded in number. Antagonism of RARγ (by AGN205728) did not affect cultures of HSCs. Studies of CV-1 and LNCaP cells transfected with RAR expression vectors and a reporter vector revealed that RARγ and RARβ are activated by sub-nM all-trans retinoic acid (EC₅₀-0.3 nM): ~50-fold more is required for activation of RARα (EC₅₀-16 nM). These findings further support the notion that the balance of expression and activity of RARα and RARγ are important to hematopoietic stem and progenitor cell expansion and differentiation.

Keywords: Agonist; All-trans retinoic acid; Antagonist; Hematopoiesis; Monocytes; Neutrophils; Retinoic acid receptor.

Fig. 1

Agonism of RARα is sufficient to drive differentiation of NB-4 and KG1 cells. The expression of RAR isoforms was measured in NB-4 and KG1 cells as to mRNA (a) and protein (b) levels. The expressions RARA, RARB, and RARG were examined in NB-4 and KG1 cells by Real-time PCR and relative to GAPDH expression levels. The expression level obtained for RARA in NB-4 cells was calculated as 1. The bar charts show mean values (±SD) of the relative quantity (RQ). The levels of RARα protein were determined in NB-4 and KG1 cells by Western blots. The cytosolic (C) and nuclear (N) extracts were separated by SDS-PAGE, transferred to PVDF membranes, and the proteins were revealed using anti-RARα, anti-actin, and anti-HDAC1 antibodies. NB-4 (c) and KG1 (d) cells were exposed to 1 µM ATRA or to 100 nM synthetic retinoids with or without 10 nM 1,25D for 96 h. The synthetic retinoids were the α agonist AGN195183, pan-antagonist AGN194310, α antagonist AGN196996, and the γ antagonist AGN205728. Cell surface expression of the CD11b and CD14 differentiation markers was detected by flow cytometry to enumerate cells that had differentiated towards neutrophils (CD11b^+veCD14^−ve) and monocytes (CD11b^+veCD14^+ve). The bar charts show the mean ± SEM of the values obtained from triplicate cultures. *Values that are significantly higher than control sample; ^#values that are significantly higher than ATRA-treated sample and ^&values that are significantly higher than 1,25D-treated sample. Representative histograms are presented for NB-4 (e) and KG1 (f) cells. The grey area represents the expression of a given cell surface marker in untreated cells, the black line in α agonist-treated cells, the blue line in 1,25D-treated cells, and the red line in cells treated with combination of both agents

Fig. 2

Antagonism of RARs increases neutrophil production in cultures of human haematopoietic stem cells. Purified human haematopoietic stem cells (CD133^+ve/CD34^+ve) were cultured from a starting density of 2.5 × 10⁵ cells/ml in RPMI1640 medium with 10 % FBS, 100 ng/ml SCF, and 20 ng/ml IL-3, and these conditions together with 10 nM of the pan-RAR antagonist AGN194310. Cells were cultured in 10 % FBS, 100 ng/ml SCF, 5 ng/ml IL-3, and 30 ng/ml G-CSF to promote the optimal production of neutrophils and monocytes. Cultures were fed with fresh medium plus agents as required and expanded when the cell density reached 1 × 10⁶/ml. a Shows the cumulative number of cells and the fraction of mature cells as measured by the FACS analysis of surface expression of CD11b (neutrophils and monocytes). b Shows the cumulative number of neutrophils (CD11b^+ve/CD65^+ve) and monocytes (CD11b^+ve/CD14^+ve). Data are mean ± SEM of the values obtained from triplicate cultures

Fig. 3

Antagonising RARα enhances ex vivo expansion of human hematopoietic stem cells. a Purified lineage^−ve/CD133^+ve/CD34^+vecells were cultured in HPGM supplemented with 10 % human serum and 100 ng/ml SCF and in the presence of 100 nM of either a pan or α or γ RAR antagonist. Lineage^−ve/CD133^+ve/CD34^+vecells were measured by the multi-color FACS analysis. The data shown are from seven separate experiments using seven different donors. Data are mean ± SEM. b At 2 weeks, cells harvested from cultures of control cells (shaded bars), and cells grown in the presence of the pan-RAR antagonist (cross-hatched bars) and absence of antagonist (solid bars) were plated into a standard methylcellulose assay to determine numbers of various colony-forming cells. The numbers on the graph show the fold level of expansion of colony-forming units as compared to control cultures. CFU colony-forming unit, CFU-GEMM granulocyte/erythroid/megakaryocyte/macrophage colony-forming unit, CFU-GM granulocyte/macrophage colony-forming unit, BFU-E burst-forming unit-erythroid, CFU-E colony-forming unit-erythroid

Fig. 4

ATRA stimulates transactivation via RARγ and RARβ at sub-nanomolar concentrations, whilst much higher concentrations are required for RARα-mediated transactivation. The effect of increasing concentrations of ATRA on RARα-, RARβ-, and RARγ-mediated transactivation in a CV-1 cells and b serum-free-adapted LNCaP cells. Cells were transfected as described in the experimental section and were treated with 10⁻¹⁰ to 10⁻⁶ M ATRA for 24 h. Results shown are from a typical experiment performed in triplicate and are expressed as RLU (arbitrary units) ± SEM. To correct for differences in transfection efficiency between samples values were normalized with respect to β-galactosidase activity