Background: Oxygen sensing in mammalian cells is a conserved signaling pathway regulated by hypoxia inducible factor type 1 (HIF-1). Inadequate oxygen supply (hypoxia) is common to many pathological disorders where autophagy plays an import role. The aim of this study was the identification and characterization of novel HIF-1 target genes that promote autophagy during hypoxia.
Methods: Whole genome Chromatin Immune Precipitation from hypoxic HeLa cells was used to identify novel HIF-1 target genes. Hypoxia induced expression and transcription regulation was studied in wild type and HIF-deficient cells. siRNA silencing of candidate genes was used to establish their role during autophagy. Recombinant protein was used for screening immobilized glycosylated lipids to identify potential ligands.
Results: We identified the Nucleotide Oligomerization Domain 2 (NOD2/CARD15) as a novel HIF-1 target and 3-O-sulfo-galactoceramide (sulfatide) and Mycobacterium sp. specific sulfolipid-1 as the first NOD2 ligands that both compete for binding to NOD2. Loss of NOD2 function impaired autophagy upstream of the autophagy inhibitor chloroquine by reducing the number of acidic vesicles. Inhibition of sulfatide synthesis elicited defects in autophagy similar to the NOD2 loss of function but did not influence NOD2-mediated NF-kB signaling.
Conclusions: Our findings suggest that the interaction of NOD2 with sulfatide may mediate the balance between autophagy and inflammation in hypoxic cells.
General significance: These findings may lead to a better understanding of complex inflammatory pathologies like Crohn's disease and tuberculosis where both NOD2 and hypoxia are implicated.
Keywords: Autophagy; CQ; Chloroquine; Hypoxia; LRR; MDP; Mtb; Muramyl dipeptide; Mycobacterium tuberculosis; NOD2; Nucleotide-binding oligomerization domain-containing protein 2; PRR; SL-1; Sulfatide; V-ATPase; Vacuolar-type H(+)-ATPase; Vesicle; leucine rich repeats; mycobacteria specific Sulfolipid-1; pathogen recognition receptors.