Studies on regulation of versican gene expression by hypoxia in primary human macrophages

Hypoxia is a hallmark of many pathological tissues. Macrophages accumulate in hypoxic sites and up-regulate a number of hypoxia-inducible genes. The extracellular matrix glycoprotein versican has recently been identified as one such gene, but the mechanisms responsible for hypoxic induction are not well characterised. Here, hypoxic up-regulation of versican was investigated in primary human monocyte-derived macrophages. Flow cytometry of isolated peripheral blood mononuclear cells demonstrated a three-fold increase in versican protein in macrophages after 5 days incubation in hypoxia. Subset analysis showed that macrophages, and not lymphocytes, are the main peripheral blood mononuclear cells which express, and show hypoxic up-regulation of, versican protein and mRNA. This study showed that versican mRNA is up-regulated 34-fold after exposure of primary human macrophages to hypoxia for 18hrs. Further investigation showed that versican mRNA decay rates are not affected by hypoxia, indicating that hypoxic induction of versican mRNA is mediated by increased promoter activity rather than increased mRNA stability. Extensive deletion and transfection analysis of proximal versican promoter luciferase reporter constructs identified two regions which are required for high level activity of the promoter in hypoxic primary human macrophages. A recent publication suggested that hypoxic induction of versican mRNA in macrophages is mediated by the hypoxia inducible transcription factor HIF-1α. Here, bacterial lipopolysaccharide and the hypoxia mimetic agents desferrioxamine and cobalt chloride, three stimuli which are known to induce HIF-1α, were used to investigate the role of HIF-1 in the up-regulation of versican mRNA. Neither LPS nor cobalt chloride caused up-regulation of versican mRNA, although control HIF-1 regulated genes were up-regulated, suggesting that high-level transcription of the versican promoter in hypoxia occurs via a HIF-1 independent mechanism. Lastly, two specific inhibitors of PI3-kinase, LY294002 and Wortmannin, were shown to down-regulate hypoxic induction of versican mRNA, suggesting a possible role for PI3-kinase.