All-Trans Retinoic Acid Induces TGF-β(2) in Intestinal Epithelial Cells via RhoA- and p38α MAPK-Mediated Activation of the Transcription Factor ATF2

OBJECTIVE: We have shown previously that preterm infants are at risk of necrotizing enterocolitis (NEC), an inflammatory bowel necrosis typically seen in infants born prior to 32 weeks’ gestation, because of the developmental deficiency of transforming growth factor (TGF)-β(2) in the intestine. The present study was designed to investigate all-trans retinoic acid (atRA) as an inducer of TGF-β(2) in intestinal epithelial cells (IECs) and to elucidate the involved signaling mechanisms. METHODS: AtRA effects on intestinal epithelium were investigated using IEC6 cells. TGF-β(2) expression was measured using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and Western blots. Signaling pathways were investigated using Western blots, transiently-transfected/transduced cells, kinase arrays, chromatin immunoprecipitation, and selective small molecule inhibitors. RESULTS: AtRA-treatment of IEC6 cells selectively increased TGF-β(2) mRNA and protein expression in a time- and dose-dependent fashion, and increased the activity of the TGF-β(2) promoter. AtRA effects were mediated via RhoA GTPase, Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1), p38α MAPK, and activating transcription factor (ATF)-2. AtRA increased phospho-ATF2 binding to the TGF-β(2) promoter and increased histone H2B acetylation in the TGF-β(2) nucleosome, which is typically associated with transcriptional activation. CONCLUSIONS: AtRA induces TGF-β(2) expression in IECs via RhoA- and p38α MAPK-mediated activation of the transcription factor ATF2. Further studies are needed to investigate the role of atRA as a protective/therapeutic agent in gut mucosal inflammation.