Egf Signaling Regulates Hematopoietic Regeneration Following Total Body Irradiation

VEGFR2+ sinusoidal endothelial cells are necessary for normal hematopoietic reconstitution following myelosuppressive chemo- or radiotherapy (Hooper et al, Cell Stem Cell 2009). However, the mechanisms through which BM endothelial cells promote HSC regeneration remain unknown. We developed a mouse model in which mice bearing cell-specific deletion of Bak and Bax in Tie2+ cells (Tie2Cre;Bak1-/-;BaxFl/- mice) were irradiated with sublethal and lethal doses of total body irradiation to assess whether protection of BM Tie2+ ECs from radiation-induced apoptosis could protect the hematopoietic compartment from myeloablative toxicity. Tie2Cre;Bak1-/-;BaxFL/- mice demonstrated protection of BM HSCs and 100% survival following lethal dose TBI (750 cGy), whereas mice that retained Bax expression in Tie2+ cells demonstrated depletion of BM HSCs and only 10% survival (p<0.0001). To determine the mechanism through which Tie2+ BM ECs regulate HSC regeneration, we performed a cytokine array screen of BM serum from Tie2Cre;Bak1-/-;BaxFl/- mice and compared with Tie2Cre;Bak1-/-;BaxFl/+ mice and C57Bl6 mice. Among several genes which were up- or down-regulated in the BaxFl/- mice, we found an 18-fold increase in the concentration of epidermal growth factor (EGF) compared to BaxFl/+ and C57Bl6 mice (p = 0.04). We then showed that BM ECs express EGFR and BM ckit+sca-1+lin- cells also express EGF in C57Bl6 mice. Interestingly, antibody blockade of EGF in vitro blocked the ability of BaxFl/- ECs to support HSC regeneration following 300 cGy irradiation. Furthermore, systemic administration of EGF to irradiated mice caused a profound recovery of BM HSC and progenitor cells compared to saline treated control mice. Similarly, administration of erlotinib, an EGFR antagonist, caused a significant delay in recovery of BM HSCs in mice following high dose irradiation. Mechanistic studies revealed that treatment of HSCs with EGF significantly increased EGFR phosphorylation and downstream activation of Akt signaling. Furthermore, inhibition of Akt signaling blocked the beneficial effect of EGF in mediating the recovery of HSCs following radiation exposure, suggesting that EGF action on HSC regeneration was mediated by Akt activation. These data demonstrate that EGF is an important regulator of HSC regeneration in vivo and a potential new target for therapies to accelerate hematopoietic reconstitution following chemotherapy and radiation exposure.