Abstract
Hypoxia is a hallmark of many pathological tissues. Macrophages accumulate in hypoxic sites and up-regulate a range of hypoxia-inducible genes. The matrix proteoglycan versican has been identified as one such gene, but the mechanisms responsible for hypoxic induction are not fully characterised. Here we investigate the up-regulation of versican by hypoxia in primary human monocyte-derived macrophages (HMDM), and, intriguingly, show that versican mRNA is up-regulated much more highly (>600 fold) by long term hypoxia (5 days) than by 1 day of hypoxia (48 fold). We report that versican mRNA decay rates are not affected by hypoxia, demonstrating that hypoxic induction of versican mRNA is mediated by increased transcription. Deletion analysis of the promoter identified two regions required for high level promoter activity of luciferase reporter constructs in human macrophages. The hypoxia-inducible transcription factor HIF-1 has previously been implicated as a key potential regulator of versican expression in hypoxia, however our data suggest that HIF-1 up-regulation is unlikely to be principally responsible for the high levels of induction observed in HMDM. Treatment of HMDM with two distinct specific inhibitors of Phosphoinositide 3-kinase (PI3K), LY290042 and wortmannin, significantly reduced induction of versican mRNA by hypoxia and provides evidence of a role for PI3K in hypoxic up-regulation of versican expression.
Highlights
Hypoxia is a feature of many pathological tissues
Hypoxia induces versican gene expression in primary human monocytederived macrophages We investigated the effect of 18h hypoxia (0.2% O2 [1.5 mmHg]) on versican gene expression in 5-day differentiated primary human monocyte-derived macrophages (HMDM) using RealTime RT-PCR
While most studies on hypoxia are done with relatively short term exposure, we wanted to model the behaviour of monocytes entering hypoxic tissue and undergoing differentiation into macrophages under hypoxia, since this is biologically relevant to pathological conditions in which chronic hypoxia is a feature and we have shown previously that this produces considerably higher fold induction of the Vascular Endothelial Growth Factor (VEGF) gene than short term hypoxia [40]
Summary
Hypoxia (low oxygen tension) is a feature of many pathological tissues. The median oxygen tension in normal tissues is usually between 20 and 70 mmHg, but in ischemic pathological sites can be as low as zero mmHg [1]. Such hypoxic areas are found in tumours [2], wounds [3], atherosclerotic plaques [4], arthritic joints [5], and the retina [6] and ischemic limbs of diabetics [7]. Macrophages are phagocytic, and can take up and destroy microorganisms or inhaled microscopic foreign bodies such as smoke, diesel exhaust, and pollen particles, and have important roles in innate and adaptive immunity and tissue repair [10, 11]
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