GADD45B promotes apoptosis in intestinal ischemia/reperfusion through DNA demethylation of MST1/Hippo

Intestinal ischemia/reperfusion (I/R) injury is a prevalent pathophysiological occurrence that results in significant morbidity and mortality. Growth arrest and DNA damage-inducible β (GADD45B) mediates various cellular responses and is engaged in apoptosis, DNA demethylation and repair, and cell survival. Nonetheless, the role of GADD45B in intestinal I/R injury remains inadequately defined. This study aimed to elucidate whether GADD45B contributes to intestinal I/R injury through the regulation of apoptosis and DNA demethylation.

In this study, we established a mouse intestinal I/R model using mesenteric artery occlusion, and Caco-2 cells were used to create the in vitro hypoxia/reoxygenation (H/R) model. Using these models, the function of GADD45B in intestinal I/R injury was investigated by knockdown and overexpression approaches, combined with histological staining, immunohistochemistry, and Western blotting. Transcriptomic analyses were performed to identify downstream signaling pathways and molecular targets of GADD45B. The specific molecular mechanisms by which GADD45B regulates intestinal I/R injury were further elucidated using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), methylation-specific polymerase chain reaction (MSP), co-immunoprecipitation (Co-IP), and chromatin immunoprecipitation (ChIP) assays.

Our research revealed a notable increase in GADD45B in both H/R-induced Caco-2 cells and mouse models of intestinal I/R. GADD45B knockdown significantly mitigated intestinal barrier dysfunction and apoptosis resulting from H/R, whereas its overexpression had the opposite effect in vitro . GADD45B -knockdown mice were generated via the adeno-associated virus (AAV)-short hairpin (sh)-GADD45B and subjected to I/R. These findings indicated that GADD45B knockdown alleviated intestinal injury and reduced apoptosis triggered by intestinal I/R. Transcriptomic Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that GADD45B regulated apoptosis during intestinal I/R by activating the Hippo pathway, with mammalian sterile 20-like kinase 1 ( MST1 ) recognized as a downstream target gene of GADD45B. ChIP confirmed that GADD45B bound to the MST1 promoter and that ten-eleven-translocation 1 (TET1) occupied the MST1 promoter region following GADD45B overexpression. Co-IP confirmed that GADD45B interacts with TET1. MSP confirmed that TET1 knockdown impaired GADD45B-induced MST1 promoter DNA demethylation. Mechanistically, GADD45B interacted with TET1 to enhance MST1 expression through DNA demethylation, which subsequently activated the Hippo pathway and exacerbated apoptosis following intestinal I/R.

GADD45B represents a pivotal regulatory factor in intestinal I/R injury, and targeting the GADD45B/TET1/MST1 axis might serve as a feasible treatment strategy.