Intestinal epithelial tight junctions (TJ) are the apical-most junctional complexes that act as functional and structural barrier against intestinal permeation of harmful luminal antigens which promote intestinal inflammation. The defective intestinal TJ barrier is a key pathogenic factor in inflammatory bowel disease (IBD). Clinical studies in IBD patients have shown that a persistent increase in intestinal permeability is predictive of poor clinical outcome, and that normalization of intestinal permeability correlates with sustained long-term clinical remission. It has been shown that matrix metalloproteinase-9 MMP-9 levels are markedly elevated in intestinal tissue, serum, and stool of patients with IBD and closely correlate with the disease activity and degree of inflammation. However, the effect of MMP-9 on intestinal epithelial TJ permeability and the intracellular mechanisms involved remain unclear. Our aims in these studies were to examine the effect of MMP-9 on intestinal epithelial TJ permeability and to elucidate the intracellular mechanisms involved.Methods:
Caco-2 monolayers were used as an in-vitro model system to examine our aims. Transepithelial resistance (TER) and inulin flux were measured to assess Caco-2 TJ permeability. Western blot analysis was performed for protein expression; real-time PCR for mRNA levels; Luciferase assay for promoter activity; molecular methods for micro-RNA and antisense transfections.Results:
(1) MMP-9 caused a dose and time-dependent drop in Caco-2 TER and an increase in permeability to paracellular marker inulin. (2) The MMP-9 induced increase in Caco-2 TJ permeability was associated with a decrease in occludin mRNA and protein expression. However, MMP-9 did not cause a decrease in occludin promoter activity, suggesting the possibility that the MMP-9 induced decrease in occludin mRNA may be due to a post-transcriptional degradation of occludin mRNA. (3) The possibility that the MMP-9 induced decrease in occludin mRNA and protein expression may be due to a miRNA induced degradation of occludin mRNA was examined. The bioinformatics analysis using Pic Tar software predicted that miR-122a, miR-200b and miR-200c had high probability (98, 96% and 92%, respectively) of having a regulatory role on occludin mRNA activity. (4) MMP-9 caused a marked increase (∼25-folds) in miR-200c and moderate increase (∼10-folds) in miR-122a and miR-200b expression in Caco-2 monolayers. (5) The effect of pre-miR-200c transfection on intestinal permeability and on occludin mRNA levels were tested. Transfection of pre-miR-200c caused a an increase in paracellular permeability to inulin in Caco-2 monolayers and a significant decrease in occludin mRNA levels as assessed by real-time PCR. (6) mMiR-200c, miR-122a or miR-200b expression were knocked-down by miRNA specific antisense oligoribonucleotide transfection. Antisense oligonucleotide inhibition of miR200c (but not miR-122a or miR-200b) inhibited the MMP-9 induced decrease in occludin mRNA, protein expression and increase in Caco-2 TJ permeability.Conclusions:
In Conclusion, our data show that MMP-9 induced increase in Caco-2 intestinal epithelial TJ permeability is mediated by occludin depletion via activation of miR-200c as a post-transcriptional degradation mechanism.