Role of Hypoxia and Toll-like Receptor Ligands in Matrix Metalloproteinase-7 Regulation in Primary Human Macrophages

Many solid tumours and other pathological sites such as infected wounds are characterised by hypoxic regions (defined by an O2 tension of <1%), which are often heavily infiltrated by macrophages. Macrophages respond to hypoxia by up-regulating a number of genes likely to promote tumour growth and spread, including MMP-7. MMP-7 has been shown to be up-regulated in various tumours and it also has important roles in protection against microbial infections including release of the pro-inflammatory cytokine TNF and activation of pro-defensins. The aim of this project was therefore to determine the mechanisms of hypoxic up-regulation of matrix metalloproteinase-7 (MMP-7) in primary human macrophages. An important aspect of this project was the analysis of the MMP-7 promoter in an attempt to identify the DNA elements required for hypoxic up-regulation, using wild-type and mutated luciferase reporter constructs transfected into primary human macrophages. A - 296 bp construct was shown to be up-regulated 3-fold in primary human macrophages exposed to 5 days of hypoxia. The luciferase expression from the constructs containing mutations in the Ets and AP-1 transcription factor binding sites was not detectable, suggesting that these sites were essential for basal MMP-7 gene expression. In this project, it was shown that MMP-7 mRNA was indeed up-regulated in severe hypoxia (0.2% O2 for 18 hrs) in primary human macrophages. However, further experiments produced the surprising finding that hypoxia alone was not able to up-regulate MMP-7 mRNA; rather, the gene was induced by co-stimulation with hypoxia and TLR ligands such as LPS. The use of Polymyxin B, which neutralises LPS, blocked MMP-7 hypoxic up-regulation. Therefore, my data indicate that the observed and previously published “hypoxic” up-regulation of MMP-7 mRNA is actually most likely to be due to the synergistic interaction of hypoxia with LPS or other TLR ligands. MMP-7 has previously been shown to be induced by TLR ligands, but my finding of synergy between these and hypoxia in up-regulation of MMP-7 mRNA and protein is novel, and challenges current opinion on MMP-7 regulation by hypoxia. Since the PI3K/Akt pathways is involved in TLR signaling and has been reported to be involved in hypoxic up-regulation of MMP-7, this pathway was investigated using two inhibitors, LY294002 and wortmannin. LY294002, and to a lesser extent wortmannin, inhibited LPS-induced MMP-7 up-regulation, linking MMP-7 LPS-regulation with the PI3K pathway. Another TLR signaling pathway, NF-κB, was investigated as a possible MMP-7 regulating pathway. NF-κB seems to be involved in MMP-7 up-regulation. Therefore, both PI3K and NF-κB pathways can be essential in MMP-7 up-regulation. These findings regarding the regulation MMP-7 expression will expand knowledge of its important role, especially in innate immunity in the context of hypoxia and infection.