Angiopoietin-Like Proteins Stimulate HSC Development Through Direct Interaction With Notch

The angiopoietin-like proteins (angptls) are novel growth factors that are capable of stimulating expansion of mouse and human hematopoietic stem and progenitor cells (HSPCs) ex vivo. Although the receptor for some of the angptl family members is known, their molecular mechanism of action is undefined. Here, we show that overexpression of angptl2 in a stable heatshock inducible transgenic zebrafish line, Tg(hsp70:zangptl2), is sufficient to increase cmyb- and runx1-positive HSPCs in the aorta-gonad-mesonephros (AGM) region, the site of definitive hematopoiesis. Anti-sense morpholino knockdown of angptl1 and 2 resulted in a significant decrease in cmyb- and runx1-positive HSPCs in the AGM, suggesting that angptls are required for definitive hematopoiesis. These double morphants also displayed severe disruption in vascular development and differentiation prior to the defects observed in the AGM, indicating that angptl regulation of HSPC development occurs through an early specification of the hemogenic endothelium. The loss of function phenotypes in the developing aorta is reminiscent of mutant fish with defective notch signaling (mindbomb) and a tight genetic interaction occurs between angptl and notch signaling. Knocking down angptl1 and 2 decreased notch signaling in a transgenic notch reporter line while overexpression of a constitutively active intracellular notch rescues the angptl double morphant phenotype. These data imply that angptls function upstream of notch signaling. Interestingly, the absence of HSPCs and notch signaling in the mindbomb mutants are rescued by overexpression of angptl2 in Tg(hsp70:zangptl2), suggesting that angptls can regulate notch signaling. To examine the molecular mechanism of Angptl-mediated Notch activation, we stimulated cultured human CD34+ cells or endothelial cells with purified Angptl2 and observed a rapid increase in Notch receptor cleavage indicating that Angptl2 can induce Notch activation. Furthermore, we found through endogenous co-immunoprecipitation experiments that the Angptl receptor, LILRB2, interacted with Notch receptor. This strongly points to a direct regulation of Notch activation/signaling by Angptls through physical interactions between Notch and Angptl receptors. Previously, we found that angptl-mediated akt activation is important for HSPC formation in the AGM. To examine the role of Akt during Notch activation, we immunoprecipitated all Akt-phosphorylated substrates in Angptl-stimulated cells and found the presence of ADAM17/TACE, one of the cleavage enzymes that is crucial to Notch receptor activation. Together these results suggest a model in which Angptl-binding of the LILRB2 receptor enables recruitment of downstream molecules such as Akt proximal to Notch, allowing for subsequent cleavage and activation of Notch receptor. Finally, to examine downstream signaling of Angptl-mediated Notch activation, we performed chromatin immunoprecipitation for Notch followed by sequencing in Angptl2-stimulated CD34+ cells and found enrichment for Myc binding elements. Independent microarrays also revealed a strong Myc signature through gene set enrichment analysis. Thus, to confirm results from our bioinformatics analyses, we overexpressed zebrafish myc in angptl1 and 2 double morphants or mindbomb mutants, and found a significant rescue in HSPCs formation in the AGM. Collectively with these results, we propose that angptl can regulate notch signaling through receptor interaction, leading to activation of myc target genes during definitive hematopoiesis. Our data provide new insights to the previously uncharacterized Angptl signaling during HSPC development and present a novel mechanism of action for Notch activation.