Induction of Hypoxia Inducible Factor-1 in macrophages activates Wnt and Notch signalling pathway in epithelial colonic cells

D Ortiz-Masia1, J Cosin-Roger2, C Hernandez3, S Calatayud1,2, MD Barrachina1,2. 1CIBERehd, Gastrointestinal inflammation and Motility, Spain, 2Universidad de Valencia, Pharmacology, Spain, 3Fundacion Hospital Dr Peset, Pharmacology, Spain

Inflammatory bowel disease (IBD) is associated with impaired epithelial barrier function. The inflammatory microenvironment modulates the ability of epithelial cells to regenerate. We have recently reported presence of HIF-1 and HIF-2 in the lamina propria of the intestine of patients with IBD. Wnt and Notch signaling pathways play essential roles in proliferation of intestinal crypt cells and secretory cell differentiation. Considering the main influence that the inflammatory microenvironment may have in regulating epithelial cell behavior we aim to analyze the role that induction of HIFs in macrophages plays in the activation of Wnt and Notch signaling pathway and expression of βcatenin, Lgr5 and Hes1 in epithelial colonic cells.

MATERIAL&METHODS: U937 cells were differentiated to macrophages and were co-cultured in RPMI using Transwell® inserts. U937 cells were seeded on the inserts and subjected to hypoxia (3% O2, 16h). HIF-2α and HIF-1α proteins levels in these cells were analyzed by Western blotting (WB) and the expression of Wnt (Wnt1) and Notch (Jag1 and Dll4) ligands, by real time PCR. In some cases, U937 cells were subjected to transient transfection of HIF-1α, HIF-2α, Wnt1 or an empty vector with miRNA and the inserts were placed on top of confluent monolayers of Caco-2 cells for 16h. After that, the expression of Lgr5, βcatenin and Hes1 was analyzed in epithelial cells by real time PCR (Lgr5) or WB (βcatenin and Hes1).

RESULTS: Hypoxia induced in macrophages HIF-1α and HIF-2α stabilization and a time-dependent increase Wnt1, Jag1 and Dll4 mRNA expression in macrophages that peaked at 3h, 8h or 16h, respectively. Co-cultured of hypoxic macrophages with Caco-2 cells induced:
1) a significant increase in protein expression of βcatenin (1,3±0,10 times vs macrophages in normoxia) and the mRNA expression of Lgr5 (6,7±1,04 times of induction vs macrophages in normoxia) in epithelial cells. The increase in Lgr5 expression was significantly decreased when macrophages had been treated with miHIF-1α (4,1±0,6 times vs macrophages in normoxia) or miWnt1 (1,1±0,5 times vs macrophages in normoxia) and not with miHIF-2α (5,9±2,3 times vs macrophages in normoxia).

2) an increase in Hes1 expression (1,6± 0,3 times of induction vs macrophages in normoxia) in epithelial cells . The increase in Hes1 expression was prevented in macrophages that had been treated with miHIF-1α (0,6±0,1 times vs macrophages in normoxia).

CONCLUSION: HIF-1 and synthesis of Wnt1 induced by hypoxia in macrophages mediate an increase in the expression of Lgr5 in epithelial cells. Hypoxia induces the expression of several Notch ligands in macrophages. HIF-1 in macrophages mediates the increase in the expression of Hes1 in epithelial cells. Our results suggest that HIF expression in the lamina propria of the damaged mucosa of patients with IBD may play a role in the recovery of the mucosa through the induction of Wnt and Notch ligand genes.