Epithelial growth factor like-7 (EGFL7) regulates hematopoiesis stem cell (HSC) activation via AP-1 suppression

Abstract

Abstract Limitations in the availability and expansion of hematopoietic stem cell (HSC) are a critical barrier for treating hematological disorders. Thus, understanding the regulatory mechanisms of HSC renewal, differentiation, and proliferation is crucial for developing techniques seeking to expand the HSC pool without compromising their multilineage potential. Epithelial Growth Factor Like-7 (EGFL7) is a ~30 kDa secreted protein highly expressed in adult bone marrow (BM) HSCs. Predicted gene expression profiling among tissues has revealed the highest level of protein expression in CD34+ hematopoietic cells (Hong et al., 2017); however, the precise role of EGFL7 in HSC regulation has not been thoroughly examined and remains poorly understood. To investigate the functional role of EGFL7 in HSC regulation, we used a germline Egfl7 knock-out (Egfl7 KO) mouse model. Egfl7 KO mice had 21% less BM cellularity and 36% lower numbers of long-term HSCs than WT controls (Lin-/Sca1+/cKit+/CD48-/CD150+/CD34-) (n=4-6, p<0.01 and p<0.05 respectively). To evaluate the function of EGFL7 in hematopoietic reconstitution, we performed competitive transplants of Egfl7 KO or WT sorted BM Lin-/Sca-1+/cKit+ (LSK) cells with WT LSK competitors (1:1, n=4-6). Egfl7 KO cells showed significantly reduced peripheral blood (PB) and BM engraftment 16 weeks post-transplant (32% vs 64%, 24% vs 45% respectively) and 19% less BM cellularity (p<0.05). We also performed serial competitive whole BM transplants of Egfl7 KO or WT with WT competitors (10:1 ratio, n=4-5) and found decreased reconstitution potential as evidenced by lower PB donor engraftment in the secondary transplants of Egfl7 KO group 38% vs 85%, p<0.001) as well as ~4-fold decrease in BM cellularity (p<0.05) and ~7-fold decrease in HSCs frequency (p<0.001), suggesting EGFL7 has a role in more primitive HSCs. To evaluate the role of EGFL7 in cell cycle regulation, we performed Bromodeoxyuridine (BrdU) incorporation in Egfl7 KO and WT mice and analyzed proliferating HSCs over 30 days while continuing BrdU treatment orally through water. Proliferating HSCs were lower in Egfl7 KO mice as early as day 3 (2.7% vs 4.8%, p<0.01). Furthermore, staining with Ki67 and DNA counterstain showed higher frequency of Egfl7 KO HSCs present in G0 phase (~84% vs ~72%, p<0.01), suggesting that EGFL7 is required for HSC activation and exit from quiescence. Next, we hypothesized that exogenous treatment with recombinant EGFL7 (rEGFL7) could expand HSCs in vivo and rescue the Egfl7 KO phenotype. We first treated WT mice with 10µg of rEGFL7 or vehicle daily for 10 days (n=3-6). rEGFL7-treated mice had 32% more HSCs (p<0.05), which led to an 18% increase in engrafting ability upon BM transplants (p<0.001). We then treated Egfl7 KO mice with rEGFL7; remarkably, treatment not only restored HSC numbers and BM cellularity, but also induced HSCs to leave quiescence and enter cell cycle, evidenced by ~2-fold decrease in G0 frequency and ~1.6-fold increase in proliferating HSCs (p<0.01). Taken together, these data show that EGFL7 enhances HSC functionality and is required for HSC proliferation. To examine molecular pathways regulated by EGFL7, we performed single-cell RNA sequencing (sc-RNA seq) on BM cKit+ cells from Egfl7 KO and WT mice. Consistent with our data, Egfl7 KO HSCsshowed higher percentage of cells in G1 (64% vs 54%) along with a reduction in their cell cycle score (-0.043 vs -0.035, p<0.05). Further analysis revealed overexpression of genes that regulate quiescence (Egr1, Btg2, Egf3, ler3), self-renewal (Klf2, Klf4, Klf6), and apoptotic markers (AP-1) (Fosb, Fos, Jun, Jund) in Egfl7 KO HSC. Given the potential of AP-1 factors to suppress cell cycle activity, we investigated phosphorylation levels of c-Fos by flow cytometry. We found higher levels of c-Fos phosphorylation (Ser32) in Egfl7 KO HSC, while exogenous rEGFL7 treatment reduced c-Fos phosphorylation levels in WT HSCs by ~45% (n=3, p<0.0001). AP-1 inhibition increased cycle progression in Egfl7 KO progenitor cells, as indicated by in vitro BrdU incorporation assay, suggesting that EGFL7 suppresses AP-1/c-Fos activity resulting in activation of quiescent HSCs. Our data uncovers the role of EGFL7 in the regulation of HSC cycle through inhibition of c-Fos to exit quiescence and subsequent entry into the cell cycle. Our findings provide a foundation for evaluating the potential therapeutic use of EGFL7 protein to expand the HSC pool while retaining functionality.