Inducibility and role of mTORC1 signaling in intestinal epithelial cells as a result of cell differentiation
Authors: Kaur Harleen , He Bo , Moreau Regis 
Published: June 2021

Intestinal epithelium undergoes rapid turnover owing to its multiple functions and active metabolism. Stem cells (SE) residing at the base of the crypts give rise to the distinct lineages of specialized intestinal epithelial cells (IEC). In doing so, SE ensure epithelial renewal and maintain intestinal homeostasis. However, SE‐based tissue renewal is not the only means of maintaining intestinal homeostasis. Specialized IEC that become damaged by the repeated physical, chemical or microbial insults attempt to self‐repair by deploying a dedifferentiation program supported by mediators of growth and proliferation such as mechanistic target of rapamycin complex 1 (mTORC1); also contributing in tissue regeneration and repair. In an event of insult, physiological stimuli may reactivate mechanistic target of rapamycin complex 1 (mTORC1) signaling in differentiated IEC thereby, contributing to epithelial dysfunction, return to stemness or tissue damage. We investigated the inducibility of mTORC1 signaling in differentiated vs. undifferentiated Caco‐2 cells in response to growth factor (IGF‐1) or pro‐inflammatory cytokines. To ascertain the role of mTORC1 in the cell response to the mucosal inflammation, Caco‐2 cells were genetically altered resulting in high (Caco‐2shTSC2), low (Caco‐2shRaptor) or baseline (Caco‐2shScramble) mTORC1 activity. Results showed that, mTORC1 activity was several fold lower in differentiated vs. undifferentiated Caco‐2 cells. While IGF‐1 induced mTORC1 signaling in undifferentiated Caco‐2 cells (IGF‐1: +95%, P<0.05), the stimuli failed to do so in differentiated Caco‐2 cells. The cocktail of cytokines (IFNγ, TNFα, IL‐1β, LPS) prominently induced IL‐1β and IL‐8 mRNA abundance in either undifferentiated or differentiated Caco‐2 cells (IL‐1b, range +374–1213%, P<0.05; IL‐8, range +4180–3350%, P<0.05). Despite the overall low mTORC1 activity levels in differentiated Caco‐2 cells, the transepithelial electrical resistance (TEER) was markedly affected by the mTORC1 status of these cells. Caco‐2shRaptor cells developed higher TEER (+180%, P<0.05) than Caco‐2shScramble cells, while Caco‐2shTSC2 cells had a lower TEER (−60%, P<0.05) than Caco‐2shScramble cells. Media transfer studies, wherein differentiated stable Caco‐2 cells received the conditioned media from activated macrophages to model mucosal inflammation, revealed that cellular TEER dropped significantly irrespective of mTORC1 status. However, unlike Caco‐2shRaptor and Caco‐2shScramble cells, in which TEER recovered marginally, Caco‐2shTSC2 cells exhibited 100% TEER recovery within 3 days. In conclusion, the overall inducibility of mTORC1 in response to the tested exogenous stimuli in Caco‐2 was dependent upon the state of differentiation and mTORC1 activity in IEC. Henceforth, mTORC1 activity is downregulated and not readily inducible (by IGF‐1 or the cytokine cocktail) as Caco‐2 cells differentiate in culture. However, development of the highest TEER by Caco‐2shRaptor among the three cell lines; and highest rate of TEER recovery in Caco‐2shTSC2 suggests that mTORC1 may have a crucial role in maintaining barrier function and promoting epithelial recovery in response to inflammation in IEC.Support or Funding InformationUSDA‐NIFAThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.