Le, Anne

ABSTRACT Macrophages play a central role in determining the outcomes of healing, coordinating regeneration in some injuries and scar formation in others. In both cases, this coordination involves the cross-talk between macrophages and surrounding cells. But what drives the different cross-communication pathways to determine healing outcomes is not well known. In this study, we make use of the mouse digit tip amputation model, in which an amputation through the third phalangeal element (P3) is able to completely regenerate whereas an amputation through the second phalangeal element (P2) forms a scar. We identify a population of macrophages that is specific to the P3 regenerating digit. By integrating single-cell RNAseq, spatial transcriptomics, and metabolomic analyses, we show that this population localizes specifically to the growing bone front, express BMP ligands that drive downstream BMP activation in neighboring osteoblasts and is governed by a two-part metabolic switch involving increased fatty acid oxidation coupled with reduced glycolytic activity. This spatially restricted, BMP-expressing macrophage population is entirely absent in the scar-forming P2 injury, and our data indicate that environmental conditions unique to the regenerating digit are responsible for its emergence. Together these findings identify a regeneration-specific macrophage signaling center for patterned bone formation and suggest that targeting the metabolic conditions that drive this population could improve the efficacy of regenerative therapies.

Ectopic Myc expression plays a key role in human tumorigenesis, and Myc dose-dependent tumorigenesis has been well established in transgenic mice, but the Myc target genes that are dependent on Myc levels have not been well characterized. In this regard, we used the human P493-6 B cells, which have a preneoplastic state dependent on the Epstein–Barr viral EBNA2 protein and a neoplastic state with ectopic inducible Myc, to identify putative ectopic Myc target genes. Among the ectopic targets, JAG2 that encodes a Notch receptor ligand Jagged2, was directly induced by Myc. Inhibition of Notch signaling through RNAi targeting JAG2 or the γ-secretase Notch inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester (DAPT) preferentially inhibited the neoplastic state in vitro. Furthermore, P493-6 tumorigenesis was inhibited by DAPT in vivo. Ectopic expression of JAG2 did not enhance aerobic cell proliferation, but increased proliferation of hypoxic cells in vitro and significantly increased in vivo tumorigenesis. Furthermore, the expression of Jagged2 in P493-6 tumors often overlapped with regions of hypoxia. These observations suggest that Notch signaling downstream of Myc enables cells to adapt in the tumor hypoxic microenvironment.