Abstract

Although the collagenase enzyme activity of matrix metalloproteinase-9 (MMP9) has been extensively explored, insights into its non-enzymatic activity remains unclear. While intracellular superoxide dismutase-1 (SOD1) downregulates MMP9, it is unknown how MMP9, a secretory protein, regulates extracellular antioxidant SOD3. To investigate the regulatory role of MMP9 on SOD3 levels, we transfected HEK293 cells with MMP9 overexpressing and catalytic (collagenase)-site mutant plasmids, and treated MMP9 overexpressing cells with an MMP9 activator or inhibitor. Cell lysates and culture medium were analyzed to determine intracellular and extracellular regulations. We performed in-silico analysis to determine protein-protein interactions of MMP9 with SOD3 and validated the results with proximal ligation assay and co-immunoprecipitation. We found that activated MMP9 upregulates SOD3, and this upregulation was not abated by MMP9 inhibitor treatment. Intriguingly, SOD3 was downregulated by catalytically inactive MMP9, plausibly due to MMP9 binding to SOD3, which potentially led to their subsequent proteolytic degradation. A similar pattern of MMP9 regulation on SOD3 was also observed in the cell culture medium, demonstrating that the intracellular and extracellular MMP9-SOD3 interactions are alike. In conclusion, we revealed a novel protein-protein interaction of MMP9 with SOD3 and uncovered a unique role of catalytically inactive MMP9 in downregulating SOD3, which otherwise upregulates SOD3.