Mutated C-terminal fragments of Clostridium perfringens enterotoxin have increased affinity to claudin-4 and reversibly modulate tight junctions in vitro.
Takahashi Azusa,Kondoh Masuo,Uchida Hiroshi,Kakamu Yohei,Hamakubo Takao,Yagi Kiyohito
Biochemical and biophysical research communications
Passage across epithelial cell sheets is the first step in drug absorption. Tight junctions (TJs) are located between adjacent epithelial cells and seal the intercellular space preventing leakage of solutes. Claudin, a tetra-transmembrane protein family, is a pivotal functional and structural component of the TJ barrier. Modulation of the claudin-based TJ seal is a strategy for mucosal drug absorption. We previously found that a claudin-4 binder, a C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE194), was a modulator of the TJ seal and a potent mucosal absorption enhancer. In the present study, we attempted to improve claudin-4 binders by modification of C-CPE194. Substitution of Asn at position 309 and Ser at position 313 with Ala increased the affinity to claudin-4 by 9.9-fold as compared to C-CPE194. Deletion of 10 amino acids in the N-terminal domain of the double-alanine-substituted mutant increased affinity to claudin-4 by 23.9-fold as compared to C-CPE194. These C-CPE194 mutants reversibly modulated the TJ seal in human intestinal epithelial cell sheets. The N-terminal-truncated mutant was the most potent modulator of the TJ seal. These findings indicate that the C-CPE mutant may be a promising lead for the development of a clinical TJ modulator.
Molecular mechanisms of somatostatin-mediated intestinal epithelial barrier function restoration by upregulating claudin-4 in mice with DSS-induced colitis.
Cai Lin,Li Xiao,Geng Chong,Lei Xuelian,Wang Chunhui
American journal of physiology. Cell physiology
Intestinal barrier dysfunction plays a crucial role in the pathogenesis of ulcerative colitis (UC). Previous studies have shown somatostatin (SST) can protect intestinal barrier structure possibly through upregulating tight junction (TJ) protein expression, but the mechanisms of this upregulation remain undefined. This study aimed to investigate the molecular mechanisms of interaction of SST with its downstream regulatory elements in DSS-induced colitis mice. In DSS-induced colitis mice, exogenous SST supplement (octreotide) effectively ameliorated disease progression, restored colonic barrier structure and function, and stimulated claudin-4 expression. Similar effects were also observed for SST on Caco-2 cells intervened by TNF-α. SST receptor 5 (SSTR5) agonist L-817,818 upregulated the claudin-4 expression whereas the SSTR2 agonist seglitide could not reverse TNF-α-induced reduction of claudin-4. SST treatment significantly decreased the phosphorylation levels of ERK1/2 and p38 induced by TNF-α. PD-98059 (ERK1/2 pathway inhibitor) but not SB-202190 (p38 pathway inhibitor) could reverse TNF-α-induced suppression of claudin-4 expression. Both inhibitors could improve the TJ barrier function damaged by TNF-α. Our studies suggest that the protective effect of SST on intestinal barrier achieved by upregulating claudin-4 expression through activation of SSTR5 and suppression of the ERK1/2 pathways. These findings will benefit the development of novel treatment regimens for UC.