Creatine Transporter, Reduced in Colon Tissues From Patients With Inflammatory Bowel Diseases, Regulates Energy Balance in Intestinal Epithelial Cells, Epithelial Integrity, and Barrier Function.

BACKGROUND & AIMS: Patients with inflammatory bowel diseases (IBDs) have intestinal barrier dysfunction. Creatine regulates energy distribution within cells and reduces the severity of colitis in mice. We studied the functions of the creatine transporter solute carrier family 6 member 8 (SLC6A8, also called CRT) in intestinal epithelial cells (IECs) and mice, and we measured levels in mucosal biopsies from patients with IBD. METHODS: Colon biopsy specimens from patients with IBD (30 with Crohn's disease and 27 with ulcerative colitis) and 30 patients without IBD (control individuals) and colon tissues from mice (with and without disruption of Crt) were analyzed by immunofluorescence, immunoblots, and/or quantitative reverse-transcription polymerase chain reaction (qRT-PCR). CRT was knocked down or overexpressed in T84 cells, which were analyzed by immunofluorescence, immunoblots, high-performance liquid chromatography (to measure creatine levels), qRT-PCR, transepithelial electrical resistance, barrier function, actin localization, wound healing, mitochondrial oxygen consumption, and glycolysis extracellular acidification rate assays. Organoids from colon cells of CRT-knockout mice and control mice were analyzed by qRT-PCR, immunoblot, and transepithelial electrical resistance. RESULTS: CRT localized around tight junctions (TJs) of T84 IECs. In analyses of IECs with CRT knockdown or overexpression, we found that CRT regulates intracellular creatine, barrier formation, and wound healing. CRT-knockout organoids also had diminished barrier formation. In the absence of adequate creatine, IECs transition toward a stressed, glycolysis-predominant form of metabolism; this resulted in leaky TJs and mislocalization of actin and TJ proteins. Colon tissues from patients with IBD had reduced levels of CRT messenger RNA compared with those from control individuals. CONCLUSIONS: In an analysis of IEC cell lines and colonoids derived from CRT-knockout mice, we found that CRT regulates energy balance in IECs and thereby epithelial integrity and barrier function. Mucosal biopsy specimens from patients with ulcerative colitis and inactive Crohn's disease have lower levels of CRT, which might contribute to the reduced barrier function observed in patients with IBD.

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

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 (KD) 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 KD cells have intrinsic abnormalities in the actin cytoskeleton, including mislocalization of the TJ binding protein vasodilator-stimulated phosphoprotein. 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.