The HIF target ATG9A is essential for epithelial barrier function and tight junction biogenesis
| Autor: | Matthew S Goldberg, Louis E. Glover, Ian M. Cartwright, Tyler Ross, Rachael Kostelecky, Nichole Welch, Alexander S. Dowdell, Sean P. Colgan |
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| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Cell Physiology Vesicular Transport Proteins Autophagy-Related Proteins Biology Response Elements Cell Line Tight Junctions 03 medical and health sciences 0302 clinical medicine Humans Intestinal Mucosa Cytoskeleton Hypoxia Promoter Regions Genetic Molecular Biology Transcription factor Tight Junction Proteins Tight junction Autophagy Membrane Proteins Epithelial Cells Cell Biology Articles Actin cytoskeleton Hypoxia-Inducible Factor 1 alpha Subunit Transmembrane protein Cell Hypoxia Cell biology 030104 developmental biology Gene Expression Regulation 030220 oncology & carcinogenesis Caco-2 Cells Chromatin immunoprecipitation Biogenesis HeLa Cells Transcription Factors |
| Zdroj: | Molecular Biology of the Cell |
| ISSN: | 1939-4586 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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