Background: Studies on ulcerative colitis (UC) patients show an excessive induction of endoplasmic reticulum stress (ERS) in intestinal epithelial cells (IEC) of the colonic mucosa, leading to intestinal barrier disruption and inflammation. Moreover, the secretion of serine proteases from IEC is enhanced in UC patients. Herein, we hypothesized that intestinal barrier (IB) disruption and mucosal inflammation associated to ERS in UC are mediated by an increased release of serine proteases. Therefore, we aimed to study the link between ERS and proteolytic activity and its impact on IB functions
Methods: Monoloayers of differentiated Caco-2 cells grown in a transwell system were stimulated with thapsigargin or tunicamycin to induce ERS. After 6 and 24 hours, supernatants were collected to quantify trypsin-like activity. Paracellular permeability to dextran-FITC, ELISA to IL-8 and gene expression of antimicrobial peptides (AMC) were assessed to evaluate the impact of proteases on IB, by treating cells with the irreversible serine protease inhibitor (AEBSF). Moreover, antagonists of protease-activated receptors (PAR-1, -2 and -4) were used to characterize the mechanism of action.
Results: ERS activation increased trypsin-like activity in supernatants from the apical side of Caco-2 monolayers, at 6 hours, while this effect is lost at 24 hours, probably due to store depletion. After ERS induction, we observed increased paracellular permeability, enhanced IL-8 release and upregulated AMP (DEFB1 (HBD1), DEFB4 (HBD2), MUC2, MUC5 and TTF3). In contrast, AEBSF recovered paracellular permeability, diminished IL-8 release, and at 24h, AMP mRNA levels were stabilized in ERS-induced cells. F2LR1 (PAR-2) and F2LR3 (PAR4s) mRNA levels increased after ERS induction. Furthermore, the increase of paracellular permeability associated to ERS activation was reduced 24h after treatment with PARs antagonists.
Conclusions: IEC under ERS increases TLA, which in turn disturbs IB by promoting paracellular permeability, chemokine induction and deregulation of AMP expression. Besides, the disturbance of IB and inflammation seems to be mediated by PAR activation. In conclusion, our data suggest a crosstalk between ERS and proteolytic activity, two fundamental features of UC.