Rapamycin does not reproduce the alterations to Caco‐2 transepithelial electrical resistance caused by the knockdown of Raptor

Abstract

Intestinal homeostasis is achieved through well‐coordinated interactions between the mucus layer, the epithelial layer and the immune cells of the lamina propria. Owing to its proximity with the luminal content, the intestinal epithelium is exposed to insults of chemical, physical or dysbiotic nature. These insults may perturb the epithelium permeability and trigger a localized immune response, which if not controlled may progress to damage the epithelium. Although the mechanistic target of rapamycin complex 1 (mTORC1) is deactivated in differentiated enterocytes, mTORC1 becomes active under repeated insult or injury to the epithelium and participates in epithelial cell repair. However, many aspects of mTORC1 re‐activation remain to be elucidated. To investigate the effects of graded levels of mTORC1 activity on epithelial barrier function, we knocked down (>90% down) mTORC1 signaling‐associated proteins, Raptor and TSC2, to create three Caco‐2 cell lines in which, after full differentiation in Transwell®, mTORC1 activity was either normal (shScramble cells), low (shRaptor cells, mTORC1 activity 50% lower than shScramble cells) or high (shTSC2 cells, mTORC1 activity 80% higher than shScramble cells). When compared with shScramble cells, shRaptor cells had higher abundance 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). 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. These mTORC1‐related changes were accompanied by significant alteration to the Caco‐2 transepithelial electrical resistance (TEER), which was 180% higher in shRaptor cells vs. shScramble cells, and conversely 60% lower in shTSC2 cells vs. shScramble cells. Although rapamycin suppressed mTORC1 activity in these cells, rapamycin caused a modest 34% (P<0.05) increase of TEER in shScramble cells, suggesting that factors other than mTORC1 ablation were responsible for the high TEER of shRaptor cells. The use of chloroquine (an inhibitor of autophagosome‐lysosome fusion) showed that autophagy alone could not explain the strong electrical resistance of shRaptor cells. We are currently investigating whether TSC2, whose expression increases as Caco‐2 cells differentiate concomitantly with mTORC1 silencing, might regulate paracellular permeability and tight junction assembly in the epithelium.Support or Funding InformationUSDA‐NIFA