PURPOSE:Gelatinases impact the pathogenesis of constrictive bronchiolitis obliterans (CBO), the pathologic correlate of chronic allograft rejection which limits lung transplant survival. We have shown elevation of MMP-9 in tissue and BAL of inbred rodent models with CBO pathology. We hypothesize that inhibition of phosphorylation pathways will reduce MMP-9 and attenuate CBO. We tested three inhibitor chemicals acting on phosphorylation pathways between TGFβ and MMP-9 for 1) cellular toxicity and 2) inhibition of MMP-9 in vitro.
METHODS:Inhibitors of MAP kinase SB203580 (#1), JUN kinase SP600125 (#2) and SMAD-ERK PD98059 (#3) were tested on human pulmonary mesothelial cells (MeT5A), which produce high levels of MMP-9. Inhibitor levels were determined by testing 1 and 10 αM inhibitor solutions in cell culture. Toxicity was determined by adding MTT to the inhibited cells. Inhibition of MMP-9 was determined on cell supernatants removed at 24 hours of culture and measured for MMP-9 by ELISA (R&D associates).
RESULTS:Toxicity assays: Color intensity at 550 nm, which increases with cell viability, produced the following: 1.726 ± 0.063 in DMSO controls; inhibitor #1 and #3 showed toxicity at 1x dose (1.529 ± 0.12 and 1.556 ± 0.179). Inhibitor #2 was nontoxic at 1x dose, 1.725 ± 0.057. MMP-9 inhibitor amounts were calculated from toxicity data. ELISA: MeT5A cells were cultured in the presence of inhibitors, and supernatant was analyzed for MMP-9. Cells alone produced 54.6 ± 3 pg/mL MMP-9. Inhibitors 1 and 3 failed to inhibit MMP-9 (56.5 ± 0.7 pg/mL and 57.8 ± 1.5 pg/mL, ANOVA and post tests, p=0.84 and 0.44 respectively). At a nontoxic dose, the JUN kinase inhibitor, #2, significantly inhibited production of MMP-9 (44.5 ± 3.4 pg/mL; p<0.001 ELISA and post tests).
CONCLUSION:We show that the JUN kinase pathway from TGFβ to MMP-9 is inhibited without cellular toxicity in human lung cells.
CLINICAL IMPLICATIONS:Further in vivo evaluation in the CBO animal model, will demonstrate if blocking MMP-9 phosphorylation pathways attenuates disease progression and offers future CBO therapy.
DISCLOSURE:Jennifer Svetlecic, No Financial Disclosure Information; No Product/Research Disclosure Information