Positional Cloning: Regulation of Hematopoietic Stem and Progenitor Cell Mobilization through EGF Receptor Signaling.

There is significant individual variation in humans in their ability to mobilize hematopoietic stem and progenitor cells (HSPC) from bone marrow (BM) to peripheral blood upon G-CSF stimulation. Understanding the molecular mechanism underlying this variation may provide clinically significant targets to improve HSPC mobilization. Using forward genetics, we have previously demonstrated that a locus on chromosome 11 is responsible for murine inter-strain variation in G-CSF mediated HSPC mobilization (Geiger et. al., Exp. Hematol., 2004). Novel subcongenic animals were generated that further confined the locus to a 5 Mbp interval. Of the 12 genes in this interval, epidermal growth factor receptor (EGFR) was identified as a candidate gene for regulating inter-strain differences in mobilization. Using real-time PCR, we demonstrated that EGFR is expressed in both murine and human HSPC and that the level of EGFR expression is inversely correlated with GCSF-induced mobilization proficiency of murine HSPC. To further demonstrate a role of EGFR signaling in mobilization, C57BL/6 mice were treated with G-CSF and increasing concentrations of murine recombinant epidermal growth factor (EGF). Our results demonstrated that a single dose of EGF (0.8 ug/g) significantly inhibited mobilization efficiency (approx. 4-fold) (46.7±13 CFC/37.5ul PB with G-CSF treated mice vs. 12.67±1.5 CFC/37.5ul PB with G-CSF + EGF treated mice). To determine whether the reduction in mobilization efficiency is dependent upon EGFR activity, EGFR mutant mice (waved-2+/−) were treated with G-CSF and EGF. In contrast to control mice, EGF had no significant inhibitory effect on G-CSF-mediated mobilization in waved-2+/− mice indicating that EGFR signaling is necessary for inhibition of mobilization by EGF (46.7±13.0 vs. 12.67±1.5 CFC/37.5ul PB, G-CSF vs G-CSF+EGF treated control mice compared to 14.2±3.7 vs. 12.1±3.6 CFC/37.5ul PB with waved-2+/− mice). To determine if EGFR-signaling regulated mobilization is HSC intrinsic, we performed “competitive mobilization experiments” in which equal numbers of BM cells (4x106) from either control or waved-2−/− mice and competitor Ly5.1 cells were transplanted into lethally irradiated BoyJ mice and subsequently treated with G-CSF and EGF. Animals transplanted with control cells demonstrated a significant reduction in mobilization efficiency in response to EGF compared to animals transplanted with waved-2−/− cells (20.2±7.2 vs. 7.89±1.2 CFC/37.5ul PB, G-CSF vs G-CSF+EGF treated control transplanted mice compared to 20.3±4.5 vs. 18.67±1.2 CFC/37.5ul PB with waved-2−/− transplanted mice) indicating that EGFR activity in hematopoietic cells is necessary to confer inhibition of mobilization efficiency by EGF. Taken together, these findings identify EGFR signaling as a negative regulator and a genetic modifier of G-CSF induced mobilization proficiency and a potential novel target to enhance HSPC mobilization efficiency.