The HIF target ATG9A is essential for epithelial barrier function and tight junction biogenesis

Abstract

Intestinal epithelial cells (IECs) exist in a metabolic state of low oxygen tension termed “physiologic hypoxia.” An important factor in maintaining intestinal homeostasis is the transcription factor hypoxia-inducible factor (HIF), which is stabilized under hypoxic conditions and mediates IEC homeostatic responses to low oxygen tension. To identify HIF transcriptional targets in IEC, chromatin immunoprecipitation (ChIP) was performed in Caco-2 IECs using HIF-1α or HIF-2α specific antibodies. ChIP-enriched DNA was hybridized to a custom promoter microarray (termed ChIP-chip). This unbiased approach identified autophagy as a major HIF-1 targeted pathway in IEC. Binding of HIF-1 to the ATG9A promoter, the only transmembrane component within the autophagy pathway, was particularly enriched by exposure of IEC to hypoxia. Validation of this ChIP-chip revealed prominent induction of ATG9A, and luciferase promoter assays identified a functional hypoxia response element upstream of the TSS. Hypoxia-mediated induction of ATG9A was lost in cells lacking HIF-1. Strikingly, we found that lentiviral-mediated knockdown of ATG9A in IECs prevents epithelial barrier formation by >95% and results in significant mislocalization of multiple tight junction (TJ) proteins. Extensions of these findings showed that ATG9A knockdown cells have intrinsic abnormalities in the actin cytoskeleton, including mislocalization of the TJ binding protein vasodilator-stimulated phosphoprotein (VASP). These results implicate ATG9A as essential for multiple steps of epithelial TJ biogenesis and actin cytoskeletal regulation. Our findings have novel applicability for disorders that involve a compromised epithelial barrier and suggest that targeting ATG9A may be a rational strategy for future therapeutic intervention.

Microbial‐derived indoles inhibit neutrophil myeloperoxidase to diminish bystander tissue damage

“…T84 colorectal adenocarcinoma cells (ATCC# CCL-248) are a well-characterized in vitro model of the colonic intestinal epithelium and were grown as previously described. [38][39][40][41][42][43][44][45] Briefly, cells were cultured at 37°C, 5% CO 2 in 1:1 DMEM/ F-12 (Thermo Fisher# 11330-032) supplemented with heatinactivated bovine calf serum (HyClone# SH30072.03), GlutaMAX (Thermo Fisher# 35050-061), and penicillin/ streptomycin (Thermo Fisher# 15140-122). Cells were passaged weekly using trypsin (Thermo Fisher# 25200-056) dissociation and sub-cultured into 6-well plates.…”

Essential role for epithelial HIF-mediated xenophagy in control of Salmonella infection and dissemination

“…Previous work suggests that HIF drives autophagy in a variety of cell types, including epithelial cell lineages ( Bellot et al, 2009 ; Dowdell et al, 2020 ). Using our previously reported chromatin immunoprecipitation (ChIP)-chip screen ( Glover et al, 2013 ) in which Caco-2 IECs were subjected to hypoxia, immunoprecipitated DNA was obtained using either anti-HIF-1α or anti-HIF-2α antibodies following specific fixation protocols.…”

“…C2BBe1 is a colorectal adenocarcinoma cell line described previously by our group ( Alexeev et al, 2021 ; Dowdell et al, 2020 ; Glover et al, 2013 ). HeLa is a cervical adenocarcinoma cell line with high transfection efficiency.…”

“…Recent work has also shed light on the importance of HIF signaling in the regulation of autophagy. HIF-1α has been shown to regulate hypoxia-induced autophagy in mouse embryonic fibroblasts through BNIP3 and BNIP3L/NIX; likewise, the essential autophagy protein ATG9A, necessary for intestinal barrier/tight junction biogenesis, was found to regulated by HIF/hypoxia in intestinal epithelial cells (IECs) ( Bellot et al, 2009 ; Dowdell et al, 2020 ; Saitoh et al, 2009 ).…”

A short chain fatty acid–centric view of Clostridioides difficile pathogenesis

“…SCFAs, most notably butyrate, promote mucus production [ 30 , 31 ]. Additionally, SCFAs improve intestinal barrier function by promoting tight junction (TJ) formation [ 32 ] and by stabilizing hypoxia-inducible factor (HIF), which is implicated in promoting TJ biogenesis and actin cytoskeletal regulation, to enhance epithelial barrier function [ 33 – 35 ].…”