Breakdown of Mucin as Barrier to Digestive Enzymes in the Ischemic Rat Small Intestine

Abstract

Loss of integrity of the epithelial/mucosal barrier in the small intestine has been associated with different pathologies that originate and/or develop in the gastrointestinal tract. We showed recently that mucin, the main protein in the mucus layer, is disrupted during early periods of intestinal ischemia. This event is accompanied by entry of pancreatic digestive enzymes into the intestinal wall. We hypothesize that the mucin-containing mucus layer is the main barrier preventing digestive enzymes from contacting the epithelium. Mucin breakdown may render the epithelium accessible to pancreatic enzymes, causing its disruption and increased permeability. The objective of this study was to investigate the role of mucin as a protection for epithelial integrity and function. A rat model of 30 min splanchnic arterial occlusion (SAO) was used to study the degradation of two mucin isoforms (mucin 2 and 13) and two epithelial membrane proteins (E-cadherin and toll-like receptor 4, TLR4). In addition, the role of digestive enzymes in mucin breakdown was assessed in this model by luminal inhibition with acarbose, tranexamic acid, or nafamostat mesilate. Furthermore, the protective effect of the mucin layer against trypsin-mediated disruption of the intestinal epithelium was studied in vitro. Rats after SAO showed degradation of mucin 2 and fragmentation of mucin 13, which was not prevented by protease inhibition. Mucin breakdown was accompanied by increased intestinal permeability to FITC-dextran as well as degradation of E-cadherin and TLR4. Addition of mucin to intestinal epithelial cells in vitro protected against trypsin-mediated degradation of E-cadherin and TLR4 and reduced permeability of FITC-dextran across the monolayer. These results indicate that mucin plays an important role in the preservation of the mucosal barrier and that ischemia but not digestive enzymes disturbs mucin integrity, while digestive enzymes actively mediate epithelial cell disruption.

Cbl-b promotes cell detachment via ubiquitination of focal adhesion kinase

“…The present study demonstrated that FAK suppressed trypsin-induced cell detachment, and lysosome inhibitor NH 4 Cl suppressed cell detachment through mono-ubiquitination of FAK, while Cbl-b promoted cell detachment through ubiquitination of FAK. These results provide novel insights into the role of Cbl-b and FAK in cell detachment.…”

“…3A). In the NH 4 Cl-pretreated group, FAK degradation was suppressed compared with the control (Fig. 3B).…”

“…However, similar results were not observed following pretreatment with the proteasome inhibitor PS-341. Furthermore, ubiquitination of FAK was increased in cells pretreated with NH 4 Cl. These results indicate that the ubiquitin-lysosome system mediates trypsin-induced degradation of FAK.…”

“…3C). To further investigate whether the degradation of FAK occurs via the ubiquitin-lysosome system, MGC803 cells were transfected with plasmid expressing Flag-Ub for 48 h, in the absence or presence of 50 nM NH 4 Cl for 12 h. Mono-ubiquitination of FAK was enhanced in the NH 4 Cl pretreated group (Fig. 3D).…”

“…Normally, adhesions between normal epithelial cells and the cellular matrix are strong; cell detachment may be achieved via downregulation of cell adhesion molecules (2). It has been reported that loss of E-cadherin results in the detachment of adjacent cells (3,4). Focal adhesion kinase (FAK) is a central regulator of focal adhesion, promoting cell adhesion and metastasis (5).…”