P071 - MECHANISM OF MMP-9 ACTIVATION OF MYOSIN LIGHT CHAIN KINASE (MLCK) MEDIATED DISRUPTION OF INTESTINAL TIGHT JUNCTION BARRIER
(Room Poster Hall)
19 Jan 18
5:30 PM
-
7:00 PM
Tracks:
Clinical and Research Challenges
Background: Matrix Metalloproteinases 9 (MMP-9) has been implicated to have a key pathogenic factor in inflammatory bowel disease (IBD). MMP-9 is elevated in intestinal tissue of patients with inflammatory bowel disease (IBD). IBD patients have a defective intestinal tight junction (TJ) barrier manifested by an increase in intestinal permeability. The role of MMP-9 in intestinal barrier function remains unclear. Aims: The purpose of this study was to delineate the molecular mechanisms involved in MMP-9 induced increase in intestinal epithelial TJ barrier. 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 induced increase in Caco-2 TJ permeability was associated with an increase in myosin light chain kinase (MLCK) mRNA and protein expression. 2.MMP-9 induced increase in MLCK gene and protein expression was regulated in part by p38 kinase signaling pathway. 3. Inhibition of p38 kinase by pharmacologic inhibitor (SB203580) or by siRNA silencing prevented the MMP-9 induced increase in MLCK promoter, mRNA and protein expressio and increase in Caco-2 TJ permeability. 4. our data show that MMP-9 caused activation of p38 kinase-dependent transcription factor ATF-2. 5. SiRNA induced silencing of ATF-2 prevented the MMP-9 mediated increase in MLCK promoter, mRNA and protein expression and the increase in Caco-2 TJ permeability. Conclusion: Our data demonstrated the involvement of p38 kinase signaling pathway in mediating the MMP-9 induced activation of MLCK gene activity and subsequent increase in Caco-2 TJ permeability.