NF-kappa B Plays a Key Role in the Hematopoietic Niche Function of Osteoblast after Ionizing Radiation.

Abstract

Recent studies demonstrated that osteoblasts in the endosteal region of bone are a key cellular component of the hematopoietic niche that directly regulates hematopoietic stem cell (HSC) survival, proliferation and differentiation. In experiments with the human fetal osteoblast cell line hFOB1.19, we found survival of gamma-irradiated primary human hematopoietic CD34+ cells was significantly enhanced by co-culture with hFOB cells or by conditioned medium (CM) from hFOB cells, as shown by increased colony efficiency and colony size. In clonogenic assays of CD34+ cells starting with 5 × 103 cells/dish, co-culture with hFOB cells increased colony number from 428±53 (control) to 988±65 (co-culture) after 2 Gy (p<0.01), 301±44 to 739±56 after 4 Gy (p<0.01), and 45±15 to 72±10 after 6 Gy (p<0.05). Surprisingly, although radiation induces osteoblast cell damage, CM from 6 Gy-irradiated hFOB cells had a greater effect than CM from unirradiated hFOB cells in supporting CD34+ clonogenicity and resulted in colony number increase from 430±40 (CM from unirradiated hFOB cells) to 773±48 (CM from 6 Gy-irradiated hFOB cells) after 2 Gy IR (p<0.01). Using a cytokine array for 120 cytokines and chemokines, we detected 11 factors released from hFOB cells, and 4 of them (IL-6, G-CSF, GRO, and IL-8) were enhanced by ionizing radiation (IR). IR (2–8 Gy) caused hFOB cell cycle arrest in G2 phase after 24 h, and irreversible cell cycle block and apoptotic cell death were observed 72 h after IR in a radiation dose-dependent manner. IR induced NF-kappaB (NFkB) p65 phosphorylation and NFkB activity in hFOB cells. Inhibition of NFkB expression with siRNA blocked the ability of hFOB CM to support clonogenic survival of CD34+ cells, and abrogated the effect of IR on release of IL-6, G-CSF, GRO, and IL-8. Radiation upregulated p53 (ser15) phosphorylation within 4 h, and p21 expression after 24 h in hFOB cells. However, NFkB gene silencing accelerated p21 expression and induced a massive apoptosis of hFOB cells. We propose that IR induces NFkB and p53 activation simultaneously in hFOB cells. The fate of osteoblasts after irradiation depends on the amount and severity of injury, and on repair processes regulated by NFkB and p53. Silencing the NFkB gene disrupted this balance and resulted in hFOB cell apoptosis.