Metalloproteinase Action on the Tight Junction in Epithelial Cells.

Abstract

This study focuses on epithelial barrier function after exposure to recombinant MMP‐9 or proinflammatory cytokines TNFα and IFNγ in MDCK cells. The aim is to elucidate the mechanisms by which select metalloproteinases (those that are elevated after cytokine treatment) lead to modification of the tight junction. Sequences from tight junction proteins that contained putative MMP‐9 substrates were produced by solid‐phase peptide synthesis, treated with MMP‐9, and analyzed for fragmentation by HPLC. Limited proteolysis experiments were conducted for tight junction proteins to determine if MMP‐9 cleaved these substrates. These studies indicated increased fragmentation of occludin. Physiological measures including TER and paracellular flux confirm that MMP‐9 directly alters TJ function. Alteration of barrier function was not due to apoptosis; only modest caspase‐3 activation following cutokine exposure was observed. In addition to MMP‐9, MT1‐MMP may be involved in barrier dysfunction. Real‐time RT‐PCR and Western blotting demonstrated a dose dependent increase in MT1‐MMP expression following cytokine exposure. Based on these results, both MMP‐9 and MT1‐MMP expression are upregulated in response to inflammatory stressors. MT1‐MMP and MMP‐9 action likely modify epithelial barrier function by direct TJ cleavage. This work was supported by NIH Grant DK065652, HHMI Foundation and the TU‐SURF program.