GMI, a Ganoderma Immunomodulatory Protein, Down-regulates Tumor Necrosis Factor α-Induced Expression of Matrix Metalloproteinase 9 via NF-κB Pathway in Human Alveolar Epithelial A549 Cells

Matrix metalloproteinase 9 (MMP-9) has been implicated in airway injury in chronic obstructive pulmonary disease (COPD), lung inflammation, and lung cancer and plays a major role in tumor necrosis factor-alpha (TNF-alpha)-stimulated tumor invasion and lung inflammation. MMP-9 activity is promoted by the pro-inflammatory cytokine TNF-alpha. GMI, cloned from Ganoderma microsporum and purified, is one of the recombinant fungal immunomodulatory proteins. To understand the molecular mechanisms involved in the suppression of TNF-alpha-mediated tumor invasion and inflammation, GMI modulation of this pathway was investigated in human alveolar epithelial A549 cells in this study. GMI exhibited an inhibitory effect on TNF-alpha-induced invasion, with GMI treatment and TNF-alpha exposure presenting the most anti-invasive properties on Boyden chamber assay. GMI reduced TNF-alpha-induced MMP-9 activities on gelatin zymography assay through inhibition of MMP-9 transcriptional activity. RT-PCR and MMP-9 promoter luciferase analysis revealed that GMI inhibits the transcription of MMP-9 mRNA. Moreover, in vitro and in vivo binding experiments, an electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation assay (ChIP) demonstrated that GMI suppresses DNA binding of nuclear factor (NF)-kappa B transcription factors to MMP-9 promoter. Western blot analysis indicated that GMI blocks the phosphorylation and degradation of I kappa B alpha, which in turn leads to suppression of the phosphorylation and nuclear translocation of p65. Thus, overall, our results indicated that GMI mediates antitumor invasion and anti-inflammatory effects through modulation of NF-kappa B/MMP-9 pathways.