Panoply™ Human MMP9 Over-expressing Stable Cell Line

Ferroptosis is a unique form of programmed cell death characterized by inhibition of glutathione peroxidase-4 (GPX4) and iron overload. Here, researchers discovered that matrix metalloproteinase-9 (MMP9) is a key regulator of ferroptosis by influencing GPX4 and iron homeostasis. Using a novel MMP9 construct lacking collagenase activity, they revealed that active MMP9 interacts with GPX4 and glutathione reductase, thereby reducing GPX4 expression and activity. Furthermore, MMP9 also inhibits key transcription factors (SP1, CREB1, NRF2, FOXO3, and ATF4) as well as GPX1 and ferroptosis inhibitor-1 (FSP1), thereby disrupting cellular redox homeostasis. MMP9 regulates iron import, storage, and export through a series of protein interactions, thus modulating iron metabolism. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 83 proteins that interact with MMP9 at the subcellular level, indicating their involvement in the regulation of ferroptosis. Integrated pathway analysis (IPA) highlighted the broad impact of MMP9 on the ferroptosis pathway, emphasizing its potential as a therapeutic target for redox homeostasis and iron metabolism disorders.

Since active MMP9 downregulates GPX4 expression, researchers sought to determine whether MMP9 regulates GPX4 transcription. They examined the expression of three key regulators of GPX4 transcription-nuclear factors SP1, CREB, and NRF2-as well as two nuclear factors that regulate GPX4 activity by modulating SLC7A11 (systemic xc−)-FOXO3 and ATF4 (Figure 1A). In both MMP9-overexpressing and mutant MMP9-expressing cells, SP1 and CREB expression were downregulated, indicating that upregulated MMP9 (regardless of its activity) can suppress SP1 and CREB expression (Figure 1B and 1C). However, MMP9 activity is crucial for downregulating NRF2 expression (Figure 1D). Notably, inactivated MMP9 failed to downregulate NRF2 expression (Figure 1D), indicating that MMP9 activity, rather than its expression level, is crucial for downregulating NRF2 expression. In both the MMP9-overexpressing and MMP9-mutant cell groups, the levels of ATF4 and FOXO3 were significantly downregulated (Figures 1E and F). This suggests that MMP9 exerts a strong negative regulatory effect on these two transcription factors, and that this effect is independent of its activity.

Figure 1. MMP9-driven regulatory network affecting GPX4 transcriptional control. (Gawargi F I, Mishra P K., 2024)