Abstract
Intestinal barrier integrity dictates the intestinal homeostasis by stringently regulating the permeability of the luminal contents into the intestinal tissue of the human body. The protective role of a well‐formed epithelial barrier is substantiated by incidences of increased mucosal inflammation upon epithelial barrier disruption. The pathophysiology of many serious intestinal disorders such as inflammatory bowel disease, necrotizing enterocolitis, and celiac disease is associated with increased permeability of the intestinal wall to luminal contents. In order to identify new therapeutic strategies against these disorders, it is critical to better understand the underpinning molecular mechanisms regulating epithelial barrier permeability. The role of the mTORC1 (mechanistic target of rapamycin complex 1) signaling in the regulation of intestinal barrier function and permeability remains to be fully elucidated. To that end, we genetically manipulated human epithelial intestinal Caco‐2 cells to generate three stable cell lines with baseline (shScramble), low (shRaptor) or high (shTSC2) mTORC1 activity. The three cell lines maintained their intended mTORC1 activity upon differentiation on Transwell® inserts, i.e., shRaptor cells exhibited 50% lower mTORC1 activity vs. shScramble cells, while shTSC2 cells exhibited 80% higher mTORC1 activity vs. shScramble cells. Transepithelial electrical resistance (TEER), a measure of epithelial permeability was inversely proportional to mTORC1 activity (r2= 0.98, P<0.05). shRaptor cells had higher expression of p‐(Thr172)AMPK (+46%, P<0.05), lower abundance of p‐(Ser757)ULK‐1 (−50%, P>0.05), higher autophagic flux (+180%, P<0.05), and lower expression of pore‐forming tight junction protein Claudin‐2 (−65%, P<0.05) compared with shScramble cells. In contrast, shTSC2 cells had higher levels of p‐(Ser757)ULK‐1 (+100%, P<0.05) and Claudin‐2 (+250%, P<0.05) compared with shScramble cells. We treated shScramble cells with rapamycin in an attempt to pharmacologically reproduce the effects of Raptor knockdown on epithelial permeability and found that rapamycin modestly but statistically increased TEER (+34%, P<0.05) and decreased Claudin2 (−36%, P<0.05). The role of autophagy in rapamycin‐mediated increase of TEER was dismissed on the basis that chloroquine (inhibitor of autophagosome‐lysosome fusion) did not significantly reduce TEER in shScramble cells. Our results suggest that although mTORC1 regulates epithelial permeability during the early stages of Caco‐2 cell differentiation, TEER is not substantially affected by rapamycin and chloroquine in fully differentiated cells.Support or Funding InformationSupported by USDA‐NIFA
Published Version
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