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

Real Time RT-PCR analysis of the effect of PI3K inhibitors on induction of versican and GLUT-1 mRNAs by 18h of exposure to hypoxia (0.2% O₂).

<p>LY290042 was used at 2μM and wortmannin at 300μM. ^^; p<0.05 compared to DMSO control, *; p<0.05 compared to untreated control, ratio paired t test, one tailed. Data from 5 independent experiments using HMDM from different donors, expressed as means ± SEM.</p

Activity of versican promoter luciferase reporter constructs in normoxia and hypoxia in HMDM.

<p><b>(A)</b> Putative transcription binding sites for hypoxia inducible factor (HIF), cAMP responsive element binding (CREB), activator Protein 1 (AP1), SP1, nuclear factor 1 (NF-1) and E2F within the 240 bp (-56+184) versican promoter sequence. The -56 to -26 and +54 to +104 sequences which are important for high level expression are in bold. <b>(B)</b> Schematic diagram of versican promoter or random 26mer luciferase reporter constructs used. <b>(C)</b> activity of versican promoter or random 26mer luciferase reporter constructs in HMDM after 5d incubation in normoxia (20.9% O₂), or hypoxia (0.2% O₂). Data from an average of 6 independent experiments with each construct, minimum n = 3 for each construct, are expressed as means ± SEM. Luciferase data were normalized to protein levels. Data assessed for significant increase in hypoxia compared to random construct control using two-tailed t tests, *** = p <0.001 ** p < 0.01,* = p <0.05, ns = not significant.</p

Immunoblotting shows lack of correlation between HIF-1α protein level and versican mRNA up-regulation by hypoxia.

<p><b>(A)</b> After incubation under the conditions indicated, cell lysates were prepared from HMDM and immunoblotted for HIF-1α and actin. A blot representative of 3 independent experiments is shown. <b>(B and C)</b> Real time-PCR analyses show versican and GLUT-1 mRNA fold induction after 5 days of normoxia, 4 days of normoxia followed by 1 day of hypoxia, or 5 days of hypoxia. N: normoxia (20.9% O₂), H: hypoxia (0.2% O₂). Data were normalized to 2MG mRNA levels. Data from 3 independent experiments are expressed as means ± SEM. The normoxic value in each experiment was assigned an arbitrary value of 1. Data were further analyzed using two-tailed, paired t-tests. *** p< 0.001</p

Investigation of the role of Hypoxia Inducible Factor 1 (HIF-1) in versican up-regulation.

<p><b>(A)</b> Effect of over-expression of HIF-1α on the 240 bp (-56+184) versican promoter construct in HMDM. PGK was used as a positive control and pGL4 basic as a negative control. <b>(B and C)</b> Real time-PCR analyses showing VEGF and versican mRNA fold induction after treatment with two different preparations of LPS (MINN LPS, Salmonella Minnesota LPS; SAE LPS, Salmonella abortus equii LPS) in comparison with hypoxia. <b>(D)</b> Real time-PCR analyses show versican, VEGF, and GLUT-1 mRNA fold induction in hypoxic and normoxic HMDM. <b>(E)</b> Real time-PCR analyses of versican, VEGF, and GLUT-1 mRNA fold induction in HMDM treated with cobalt chloride (COB). N: normoxia 20.9% O₂, H: hypoxia 0.2% O₂. All incubations, with or without hypoxia, were for 18hrs. Data were normalized to 2MG mRNA levels. Data from 3 (A, B, and C) or 5 (D and E) or 8 (F) independent experiments are expressed as means ± SEM. Data were further analyzed using paired two-tailed t-tests. *** = p <0.001 ** p < 0.01,* = p <0.05</p

Quantitation of versican protein expression in monocytes / macrophages by flow cytometry.

<p><b>(A and D)</b> Dot plot analysis of PBMC after 5 days in normoxia (20.9% O₂; A) and hypoxia (0.2% O₂; D). Monocyte/macrophages are subdivided into 3 regions R3-R1 in respect of increasing cell size (forward scatter). Lymphocytes are included in region 4. Region 5 encompasses all monocyte macrophages in Regions 1, 2, and 3. A representative example of 5 independent experiments is shown. <b>(B and E)</b> Percentage of the total monocyte/macrophage population (R5) present in regions R1, R2, and R3 in normoxia (B) and hypoxia (E). Data from 5 independent experiments are expressed as means ± SEM. <b>(C and F)</b> Versican mean fluorescent intensity in regions 1, 2, 3 and 4 in Normoxia (C) and Hypoxia (F). Data from 5 independent experiments are expressed as means ± SEM. <b>(G)</b> Histogram of the fluorescent intensity with a versican specific antibody (black fill) compared to the isotype control antibody (white line) in region R1 cells in Normoxia and Hypoxia. A representative example of 5 independent experiments is shown. <b>(H)</b> Histogram analysis of the versican fluorescent intensity in region R1 cells in Normoxia (shaded) and Hypoxia (clear). A representative example of 5 independent experiments is shown. <b>(I)</b> Versican protein fold induction in cells region R1 in normoxia and hypoxia. Data from 5 independent experiments are expressed as means ± SEM. For panels B, E, C, F, and I, the normoxic value in each experiment was assigned an arbitrary value of 1. Data were further analyzed using two-tailed, paired t-tests. ** p < 0.01,* = p <0.05.</p

Assessment of versican mRNA decay in HMDM in normoxia and hypoxia by real-time RT-PCR after addition of Actinomycin D.

<p>N: normoxia 20.9% O₂, H: hypoxia 0.2% O₂. Data were normalized to 2MG mRNA levels. Data from 5 independent experiments are expressed as means ± SEM.</p

Up-regulation of versican gene expression by hypoxia in primary human macrophages.

<p><b>(A)</b> Real Time RT-PCR quantification of the effect of 18hrs hypoxia (0.2% O₂) on versican mRNA in 5-day differentiated HMDM from 13 different donors. Values are hypoxic fold induction relative to normoxia. <b>(B)</b> Changes in versican mRNA fold induction levels in response to 18hrs of hypoxia (0.2% O₂) were quantified by real-time RT-PCR in HMDM, CD14⁺ magnetic bead purified monocyte-macrophages and CD14^- cells, all incubated for 5d after isolation before being exposed to a further 18h of either normoxia or hypoxia, in 3 independent experiments using different donors. Values are hypoxic fold induction relative to normoxia. <b>(C)</b> Real-time RT-PCR quantification of versican mRNA isoforms in HMDM after differentiation either 5d in normoxia (20.9% O₂), 4d in normoxia followed by 1d in hypoxia, or 5d in hypoxia (0.2% O₂), in 4 independent experiments using different donors. All data were normalized to 2MG mRNA levels determined by separate PCRs, and are expressed as mean fold induction (relative to the equivalent normoxic culture) ± SEM, and were analyzed for significance using paired t-tests. **** = p <0.0001, *** = p <0.001, * = p <0.05.</p