11β-hydroxysteroid dehydrogenase type 1 deficiency in bone marrow-derived cells reduces atherosclerosis

11β-Hydroxysteroid dehydrogenase type-1 (11β-HSD1) converts inert cortisone into active cortisol, amplifying intracellular glucocorticoid action. 11β-HSD1 deficiency improves cardiovascular risk factors in obesity but exacerbates acute inflammation. To determine the effects of 11β-HSD1 deficiency on atherosclerosis and its inflammation, atherosclerosis-prone apolipoprotein E-knockout (ApoE-KO) mice were treated with a selective 11β-HSD1 inhibitor or crossed with 11β-HSD1-KO mice to generate double knockouts (DKOs) and challenged with an atherogenic Western diet. 11β-HSD1 inhibition or deficiency attenuated atherosclerosis (74–76%) without deleterious effects on plaque structure. This occurred without affecting plasma lipids or glucose, suggesting independence from classical metabolic risk factors. KO plaques were not more inflamed and indeed had 36% less T-cell infiltration, associated with 38% reduced circulating monocyte chemoattractant protein-1 (MCP-1) and 36% lower lesional vascular cell adhesion molecule-1 (VCAM-1). Bone marrow (BM) cells are key to the atheroprotection, since transplantation of DKO BM to irradiated ApoE-KO mice reduced atherosclerosis by 51%. 11β-HSD1-null macrophages show 76% enhanced cholesterol ester export. Thus, 11β-HSD1 deficiency reduces atherosclerosis without exaggerated lesional inflammation independent of metabolic risk factors. Selective 11β-HSD1 inhibitors promise novel antiatherosclerosis effects over and above their benefits for metabolic risk factors via effects on BM cells, plausibly macrophages.—Kipari, T., Hadoke, P. W. F., Iqbal, J., Man, T. Y., Miller, E., Coutinho, A. E., Zhang, Z., Sullivan, K. M., Mitic, T., Livingstone, D. E. W., Schrecker, C., Samuel, K., White, C. I., Bouhlel, M. A., Chinetti-Gbaguidi, G., Staels, B., Andrew, R., Walker, B. R., Savill, J. S., Chapman, K. E., Seckl, J. R. 11β-hydroxysteroid dehydrogenase type 1 deficiency in bone marrow-derived cells reduces atherosclerosis

The function of the conserved regulatory element within the second intron of the mammalian Csf1r locus

The gene encoding the receptor for macrophage colony-stimulating factor (CSF-1R) is expressed exclusively in cells of the myeloid lineages as well as trophoblasts. A conserved element in the second intron, Fms-Intronic Regulatory Element (FIRE), is essential for macrophage-specific transcription of the gene. However, the molecular details of how FIRE activity is regulated and how it impacts the Csf1r promoter have not been characterised. Here we show that agents that down-modulate Csf1r mRNA transcription regulated promoter activity altered the occupancy of key FIRE cis-acting elements including RUNX1, AP1, and Sp1 binding sites. We demonstrate that FIRE acts as an anti-sense promoter in macrophages and reversal of FIRE orientation within its native context greatly reduced enhancer activity in macrophages. Mutation of transcription initiation sites within FIRE also reduced transcription. These results demonstrate that FIRE is an orientation-specific transcribed enhancer element

Effect of CSF-1 and LPS on <i>Csf1r</i> primary and antisense transcripts.

<p>(<b>A</b>) Macrophages were differentiated from mouse bone marrow under the influence of CSF-1. Cells were starved of CSF-1 for 24 hours, and then re-stimulated with a combination of CSF-1 and LPS. Primary RNA levels containing intronic sequences were measured by Real-Time PCR assays using primers downstream of the promoter, FIRE or intron 3. Normalisation was performed using rRNA-specific primers. Error bars represent the mean value of triplicate PCRs. (<b>B</b>) Cells and treatments were identical to (A). ChIP assays measuring the recruitment of Serine 5 phosphorylated RNA Pol II using primers covering the indicated cis-regulatory elements. Prom – 1.5 kb refers to a region upstream of the transcription start site which served as an internal negative control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054935#pone.0054935-Tagoh3" target="_blank">[29]</a>. Values were normalised against an internal negative control as described in materials and methods. Measurements are representative of at least two independent experiments and error bars represent the mean value of three different measurements. (<b>C</b>) Primers were produced that contain sequence from the positive strand of <i>Csf1r</i> upstream of the FIRE region within intron 2 (Asp1 or Asp2). These primers prime negative strand transcripts by reverse transcriptase reaction (+). DNAse treated RNA was used for the reaction and as a control RNA was primed in the absence of reverse transcriptase (−). cDNA products were detected by PCR with nested forward (NF1, NF2, or NF3) and reverse (NR) primers. (<b>D</b>) Cells and treatments were identical to (A). Antisense RNA-expression was assayed by real-time quantitative PCR exactly as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054935#pone.0054935-Tagoh2" target="_blank">[20]</a>.</p

The enhancer activity of FIRE is orientation dependent and requires the transcription start sites.

<p>(<b>A</b>) Schematic of FIRE constructs: the entire <i>Csf1r</i> regulatory region plasmid (pGL-7.2 fms), pGL-7.2 fms with FIRE subcloned into the reverse orientation (pGL-7.2 fms FIRE-), pGL-7.2 fms with FIRE deleted (pGL-7.2 fms ΔFIRE), and pGL-7.2 fms with intron 2 deleted leaving 3.5 Kb of the <i>Csf1r</i> promoter (pGL2-3.5 fms). (<b>B</b>) RAW264.7 cells were transfected with pGL-7.2 fms, pGL-7.2 fms FIRE-, or pGL-7.2 fms ΔFIRE constructs and luciferase activity was assessed. Data is shown as a percentage of pGL2-7.2 fms (100%) and error bars represent the SEM. Statistically significant differences versus pGL-7.2 fms are indicated (t-test; ***p<0.001). (<b>C</b>) RAW264.7 cells were transfected with pGL2-3.5 fms, pGL-7.2 fms, or pGL-7.2 fms FIRE- constructs. Following treatment with LPS, luciferase activity was assessed. Data for each construct are shown normalised to the same construct untreated and error bars represent the SEM. Statistically significant differences between the LPS treated construct versus the same construct untreated are indicated (t-test; **p<0.01). (<b>D</b>) RAW264.7 cells were transfected with linearized pGL-7.2 fms or a linearized pGL-7.2 fms construct containing one of the four 6 bp deletions in FIRE shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054935#pone-0054935-g004" target="_blank">Figure 4D</a>. Forty-eight hours post transfection, luciferase activity was assessed. The data represents six separate experiments performed in triplicate and error bars represent the SEM. Data are shown as a percentage of pGL2-7.2 fms (100%). Statistically significant differences versus pGL-7.2 fms are indicated (t-test; *** p<0.001, ** p<0.01, and * p<0.05).</p

The FIRE region acts as an inducible promoter.

<p>(<b>A</b>) Schematic of FIRE constructs: the entire <i>Csf1r</i> regulatory region plasmid (pGL-7.2 fms), for reference, the <i>Csf1r</i> promoter cloned into the pGL2B vector upstream of a luciferase reporter (pGL0.5 fms), FIRE in reverse orientation was cloned into the pGL2B vector upstream of a luciferase reporter (pGLFIRE-). (<b>B</b>) RAW264.7 cells were transfected with pGL0.5 fms or pGLFIRE-. Transfected cells were treated (+) with either LPS or bacterial DNA or left untreated (−) for 8 hours before assay of luciferase activity. (<b>C</b>) Naïve RAW264.7 cells (CSF-1R −) or RAW264.7 cells stable transfected with a CSF-1R expression plasmid (CSF-1R +) were transfected with reporter constructs as above and treated (+) with CSF-1 for 20 hours, PMA for 8 hours, or both CSF-1 & PMA or left untreated (−) before assay of luciferase activity. Columns in B & C represent the mean RLU/µg protein and error bars the SEM of three independent assays which each showed the same pattern.</p

<i>In vivo</i> DMS footprinting of the <i>Csf1r</i> promoter and FIRE in stimulated BMM.

<p>Macrophages were differentiated from mouse bone marrow under the influence of CSF-1 (+) and subjected to DMS footprinting after either starving cells of CSF-1 (−) or restimulation with CSF-1 (<b>A</b>), LPS (<b>B</b>), or CSF-1 & LPS (<b>C</b>) for the indicated time points. G: Maxam-Gilbert reaction followed by LM-PCR with purified genomic DNA. ES: <i>In vivo</i> footprinting performed with ES cells. Putative transcription factor binding sites in chromatin showing alterations in methylation compared to a Maxam-Gilbert G-reaction of naked genomic DNA are shown as vertical bars on the right hand side of the gel images. Nucleotide positions relative to the ATG start are designated by numbers on the left. Macrophage specific footprints are indicated as circles (black: enhancement, white: inhibition) while LPS responsive footprints are indicated as squares (black: enhancement, white: inhibition). L-shaped arrows are the position of antisense RNA start sites at FIRE.</p

Mapping of transcription factor motifs in active chromatin identifies IRF5 as key regulator in classical Hodgkin lymphoma

Deregulated transcription factor (TF) activities are commonly observed in hematopoietic malignancies. Understanding tumorigenesis therefore requires determining the function and hierarchical role of individual TFs. To identify TFs central to lymphomagenesis, we identified lymphoma type-specific accessible chromatin by global mapping of DNaseI hypersensitive sites and analyzed enriched TF-binding motifs in these regions. Applying this unbiased approach to classical Hodgkin lymphoma (HL), a common B-cell-derived lymphoma with a complex pattern of deregulated TFs, we discovered interferon regulatory factor (IRF) sites among the top enriched motifs. High-level expression of the proinflammatory TF IRF5 was specific to HL cells and crucial for their survival. Furthermore, IRF5 initiated a regulatory cascade in human non-Hodgkin B-cell lines and primary murine B cells by inducing the TF AP-1 and cooperating with NF-{kappa}B to activate essential characteristic features of HL. Our strategy efficiently identified a lymphoma type-specific key regulator and uncovered a tumor promoting role of IRF5

Derepression of an endogenous long terminal repeat activates the CSF1R proto-oncogene in human lymphoma

Mammalian genomes contain many repetitive elements, including long terminal repeats (LTRs), which have long been suspected to have a role in tumorigenesis. Here we present evidence that aberrant LTR activation contributes to lineage-inappropriate gene expression in transformed human cells and that such gene expression is central for tumor cell survival. We show that B cell–derived Hodgkin's lymphoma cells depend on the activity of the non-B, myeloid-specific proto-oncogene colony-stimulating factor 1 receptor (CSF1R). In these cells, CSF1R transcription initiates at an aberrantly activated endogenous LTR of the MaLR family (THE1B). Derepression of the THE1 subfamily of MaLR LTRs is widespread in the genome of Hodgkin's lymphoma cells and is associated with impaired epigenetic control due to loss of expression of the corepressor CBFA2T3. Furthermore, we detect LTR-driven CSF1R transcripts in anaplastic large cell lymphoma, in which CSF1R is known to be expressed aberrantly. We conclude that LTR derepression is involved in the pathogenesis of human lymphomas, a finding that might have diagnostic, prognostic and therapeutic implications.Björn Lamprecht... Raman Kumar... David F. Callen... et al