Mechanisms of Hypoxic Up-Regulation of Versican Gene Expression in Macrophages

RESEARCH ARTICLE Mechanisms of Hypoxic Up-Regulation of Versican Gene Expression in Macrophages Fattah Sotoodehnejadnematalahi1, Karl J. Staples2, Elvina Chrysanthou3, Helen Pearson3, Loems Ziegler-Heitbrock4, Bernard Burke5* 1 Department of Biology, School of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, Iran, 2 Clinical & Experimental Sciences, University of Southampton Faculty of Medicine, Sir Henry Wellcome Laboratories, Southampton General Hospital, Southampton, United Kingdom, 3 Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom, 4 EvA Study Center, Asklepios Fachkliniken and Helmholtz-Zentrum Muenchen, Gauting, Germany, 5 Dental School, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom * Abstract OPEN ACCESS Citation: Sotoodehnejadnematalahi F, Staples KJ, Chrysanthou E, Pearson H, Ziegler-Heitbrock L, Burke B (2015) Mechanisms of Hypoxic UpRegulation of Versican Gene Expression in Macrophages. PLoS ONE 10(6): e0125799. doi:10.1371/journal.pone.0125799 Academic Editor: Sonia Rocha, University of Dundee, UNITED KINGDOM Received: November 9, 2014 Accepted: March 26, 2015 Published: June 9, 2015 Copyright: © 2015 Sotoodehnejadnematalahi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. 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 upregulation 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. Introduction 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]. PLOS ONE | DOI:10.1371/journal.pone.0125799 June 9, 2015 1 / 19 Hypoxic Up-Regulation of Versican Gene Expression in Macrophages Cells of the monocyte/macrophage lineage are involved in all of the above pathologies. Monocytes are derived from myeloid stem cells, and following release from the bone marrow circulate in the bloodstream for 1–3 days before migrating into tissues where they differentiate into macrophages [8, 9]. Macrophages are phagocytic, and can take up and destroy microorganisms or inhaled microscopic foreign bodies such as smoke, diesel exhaust, and pollen particles, and also have important roles in innate and adaptive immunity and tissue repair [10, 11] It has been known for many years that macrophages accumulate in poorly vascularized, hypoxic sites [12]. Accumulation of macrophages has been reported in avascular, hypoxic and necrotic sites in breast [13] and ovarian carcinomas [14], wounds [15], atherosclerotic plaques [16] and arthritic joints [17]. Hypoxic macrophages up-regulate a number of hypoxia-inducible transcription factors, the most important of which is Hypoxia-inducible factor 1 (HIF-1) [18]. Macrophages are unusual in that they rely heavily on HIFs for energy generation and activity even under normal oxygen tensions [19], meaning that they are able to respond rapidly and effectively to the challenges posed by the need to function in hypoxic sites. Previous studies have shown that many genes are up-regulated in hypoxic macrophages [20, 21, 22, 23, 24]. The extra cellular matrix (ECM) proteoglycan versican has been identified as one such hypoxia-inducible gene [25]. Versican is a large aggregating chondroitin sulphate proteoglycan, and occurs in at least four isoforms [26]. It is found in various sites including the brain [27], and skin [28], and increased expression is observed in sites of tissue injury [29] and in cancers including breast [30], cervical [31], gastrointestinal tract, prostate [32], brain [33], and melanoma [34]. Several reports have also highlighted the role of versican in wound healing [35, 36] and in vascular disease, especially atherosclerosis [37, 38]. Versican binds low-density lipoprotein particles, and accumulation of versican in blood vessel walls is believed to promote extracellular lipoprotein retention and uptake leading to foam cell formation [39]. In the study which first reported hypoxic induction of versican [25], it was suggested to be regulated, at least in part, by Hypoxia-Inducible Factor 1 (HIF-1), the most important hypoxia-inducible transcription factor, which has been described as the “master regulator” of the transcriptional response to hypoxia. The aims achieved in this study were to increase understanding of the mechanisms responsible for the up-regulation of versican by hypoxia in primary human macrophages, using promoter reporter deletion constructs, transcription factor over-expression, and gene expression quantification. Results 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. All 13 donors tested showed substantial hypoxic induction of total versican mRNA (using PCR primers which amplify all mRNA splice variants), however there was considerable variability (...truncated)