EGFR Signaling in Osteoblasts Regulates Circadian Rhythm of HSPC in Circulation

EGFR signaling regulates growth, differentiation, proliferation and migration in multiple organ systems. We previously demonstrated that inhibition of EGFR signaling on hematopoietic stem and progenitor cells (HSPCs) enhances G-CSF induced stem cell mobilization, while preliminary data suggested that inhibition of EGFR signaling in the stem cell niche actually had the opposite effect of inhibiting mobilization (Ryan et al., Nat Med, 2010). We thus tested the novel hypothesis that there is a role for EGFR signaling in the bone marrow (BM) niche with respect to regulating hematopoiesis. We utilized a set of mouse strains that express the recombinase Cre under distinct promoters to specifically delete EGFR in various types of stroma cells including Col-Cre; EGFR^f/f (deletion in osteoprogenitor/osteoblasts (OBs)), Dermo-Cre; EGFR^f/f (mesenchymal stem cells (MSCs) including chondrocytes and OB), Tie2-Cre; EGFR^f/f (Endothelial cells) and Nestin-Cre; EGFR^f/f (Schwann/neural cells) and compared them to no Cre-EGFR^f/f mice as control wild type for EGFR.

Basic parameters of steady-state hematopoiesis were not altered in mice devoid of EGFR signaling in the various types of stroma cells listed above. We further investigated HSPC mobilization in EGFR^f/f mice and interestingly, Col-cre and Dermo-cre EGFR^f/f mice exhibited a lower number of circulating HSPC in blood in comparison to wild type mice, as determined by colony forming units (CFUs) or flow cytometry. Deletion of the EGFR in endothelial (Tie2-Cre) and neuronal (Nestin-Cre) compartments did not result in a decline in the number of circulating HSPCs. Upon G-CSF challenge, Col-cre and Dermo-cre EGFR^f/f mice mobilized HSPCs similar to controls, suggesting that EGFR signaling in OBs/MSCs is dispensable for G-CSF induced mobilization. HSPCs circulation under steady state follows a circadian rhythm. We next tested whether EGFR signaling in OBs might play a role in circadian rhythm driven HSPC circulation. After 5 hours of light cycle i.e. Zeitgeber time-5 (ZT-5), when the number of circulating HSPCs is high, Col-Cre EGFR^f/f and Dermo-Cre EGFR^f/f mice presented with low numbers of circulating HSPCs. After 13 hours of light cycle (ZT-13) (low number of circulating HSPCs), the number of HSPCs in blood in Col-Cre EGFR^f/f and Dermo-Cre EGFR^f/f mice were similar to controls. Together, this suggests that EGFR signaling in OBs is essential for the rhythmic increase in circulating HSPC in blood at ZT5. Currently, we are investigating molecular mechanisms driven by EGFR signaling in OBs that regulate HSPC retention in BM and circadian regulation of EGFR signaling. In summary, our data suggest an important and also very specific (no other phenotype altered) role of EGFR signaling for regulating the circadian rhythm of HSPC circulation in peripheral blood.