Fig 1.
Time-course effect of MMP-9 on Caco-2 intestinal epithelial TJ permeability.
(A) Time-course effect of MMP-9 (400 ng/ml) on Caco-2 TER and (B) mucosal-to-serosal flux of paracellular marker dextran 10 kDa (n = 4). *** P < 0.001 vs control. The effect of MMP-9 on Caco-2 TER and paracellular permeability were measured over a 72-hr experimental period. (C) Effect of recovery after MMP-9 treatment cessation on Caco-2 TER (n = 4). *** P < 0.001 vs control; * P < 0.05 vs control. Filter-grown Caco-2 monolayers were refreshed with control media after MMP-9 experimental period ended.
Fig 2.
Effect of MMP-9 on NF-κB p65 activation in Caco-2 monolayers.
(A) Phospho-NF-κB p65 and IκB-α expression were determined in filter-grown Caco-2 monolayers treated with MMP-9 (400 ng/ml) for increasing time periods (0–6 hrs). (B) NF-κB p65 expression in the cytoplasmic and nuclear fractions was assayed by Western blot analysis after MMP-9 treatment (1-hr experimental period). (C) NF-κB p65 cytoplasmic-to-nuclear translocation was determined by immunostaining. (Yellow, NF-kB p65; Blue, DAPI (nuclei)). Original magnification, ×40. (D) NF-κB p65 binding to the oligonucleotide probe containing the κB-binding site was determined by ELISA-binding assay. MMP-9 caused a significant increase in NF-κB p65 binding (1-hr experimental period). The oligonucleotide containing a mutated NF-κB-binding (mut) motif did not inhibit the NF-κB p65 binding to the DNA probe; however, the addition of wild-type (WT) oligonucleotide containing the consensus NF-κB p65-binding site as a competitive inhibitor prevented the binding of NF-κB. *** P < 0.001 vs control. (E) NF-κB p65 siRNA transfection resulted in a marked depletion in NF-κB p65 protein expression. Caco-2 monolayers were transfected with NF-κB p65 siRNA for a 72-hr time period, (NT; not-target siRNA). (F) NF-κB p65 siRNA knock-down prevented the MMP-9 induced increase in NF-κB p65 binding to the oligonucleotide probe containing the κB-binding site. *** P < 0.0001 vs control; ### P < 0.001 vs MMP-9 treatment.
Fig 3.
Effect of NF-κB inhibition on MMP-9 induced increase in Caco-2 TJ permeability and increase in MLCK expression.
(A) NF-κB p65 siRNA transfection prevented the MMP-9-induced drop in Caco-2 TER and (B) increase in dextran 10 kDa flux (n = 4). *** P < 0.001 vs control; ## P < 0.01 vs MMP-9 treatment; **** P < 0.0001 vs control; #### P < 0.0001 vs MMP-9 treatment. (C) MMP-9 treatment resulted in a significant increase in NF-κB target gene IL-8. NF-κB p65 siRNA prevented the IL-8 production by Caco-2 monolayers after MMP-9 treatment (24 hrs). IL-8 secretion was determined by collecting and centrifuging the media and then assayed by ELISA-based kit. *** P < 0.0001 vs control; ### P < 0.001 vs. MMP-9 treatment. (D) Knocking-down NF-κB p65 inhibited the MMP-9 induced increase in MLCK protein expression as assessed by western blot analysis. (NT; non-target siRNA). (E) Silencing p38 kinase by siRNA transfection prevented the MMP-9-induced activation of NF-κB p65 as assessed by phosphorylation of p65. Caco-2 monolayers were transfected with p38 kinase siRNA for a 72-hr time period and then treated with MMP-9 for 1 hr, (NT; not-target siRNA).
Fig 4.
Effect of MMP-9 on Caco-2 cell death.
(A) Dual-color dot plots representing cellular apoptosis. Upper left panel, Necrotic cells; lower right panel, apoptotic cells. Caco-2 monolayers were treated with MMP-9 for 24 and 48 hrs. Caco-2 monolayers were labeled with Annexin VFITC (apoptosis) or 7-AAD (necrosis). MMP-9 did not induce apoptosis or cell necrosis in Caco-2 monolayers. (B) % of live cell detection by trypan blue exclusion method. MMP-9 treatment did not affect total number of dead Caco-2 cells compared to control.
Fig 5.
Schematic diagram of intracellular mechanism involved in MMP-9 regulation of Caco-2 TJ barrier function.