Reversible Suppression of Cyclooxygenase 2 (COX-2) Expression <i>In Vivo</i> by Inducible RNA Interference

<div><p>Prostaglandin-endoperoxide synthase 2 (PTGS2), also known as cyclooxygenase 2 (COX-2), plays a critical role in many normal physiological functions and modulates a variety of pathological conditions. The ability to turn endogenous COX-2 on and off in a reversible fashion, at specific times and in specific cell types, would be a powerful tool in determining its role in many contexts. To achieve this goal, we took advantage of a recently developed RNA interference system in mice. An shRNA targeting the <i>Cox2</i> mRNA 3′untranslated region was inserted into a microRNA expression cassette, under the control of a tetracycline response element (TRE) promoter. Transgenic mice containing the COX-2-shRNA were crossed with mice encoding a CAG promoter-driven reverse tetracycline transactivator, which activates the TRE promoter in the presence of tetracycline/doxycycline. To facilitate testing the system, we generated a knockin reporter mouse in which the firefly luciferase gene replaces the <i>Cox2</i> coding region. <i>Cox2</i> promoter activation in cultured cells from triple transgenic mice containing the luciferase allele, the shRNA and the transactivator transgene resulted in robust luciferase and COX-2 expression that was reversibly down-regulated by doxycycline administration. <i>In vivo</i>, using a skin inflammation-model, both luciferase and COX-2 expression were inhibited over 80% in mice that received doxycycline in their diet, leading to a significant reduction of infiltrating leukocytes. In summary, using inducible RNA interference to target COX-2 expression, we demonstrate potent, reversible <i>Cox2</i> gene silencing <i>in vivo</i>. This system should provide a valuable tool to analyze cell type-specific roles for COX-2.</p></div

DOX-dependent suppression of TPA-induced COX-2 expression by shCox2 in mice homozygous for the <i>Cox2</i> gene.

<p>(<b>A</b>) Double transgenic shCox2/C3 mice, which have two wild type <i>Cox2</i> alleles, were maintained on either a control (no DOX) or a DOX-supplemented diet (+DOX) for 12 days, then painted with TPA on the back. 24 hours after TPA administration the mice were euthanized and skin extracts were assayed for GFP and COX-2 protein by Western blotting. Quantification is from three independent experiments. The COX-2 signal was normalized to the GAPDH loading control. Data are means +/− S.D. (***p<0.001). (<b>B</b>) COX-2 (upper panels) and GFP (lower panels) immunohistochemistry in skin of untreated shCox2/C3 mice (left panels), 24 hours after TPA administration to shCox2/C3 mice maintained on a DOX-free diet (center panels) and 24 hours after TPA treatment of shCox2/C3 mice maintained for 12 days on a DOX-supplemented diet (right panels). COX-2 staining is visible as brown patches (white arrow) in the basal epithelium. (<b>C</b>) H&E stain to visualize leukocytes in skin sections from double transgenic shCox2/C3 mice treated with TPA and/or DOX as in B. The graph depicts quantification of leukocytes in the dermis. Quantification is from two independent experiments. Data are means +/− S.D. (**p<0.01). The scale bar indicates 50 µm.</p

Construction of <i>Cox2^tm2Luc/+</i> a mouse strain in which firefly luciferase replaces the <i>Cox2</i> coding region.

<p>(<b>A</b>) Schematic representation of the wild-type <i>Cox2</i> allele and the targeting strategy to create the <i>Cox2^tm2Luc</i> knockin allele. The firefly luciferase coding region (ffLuc), PGK-neo (neo) selection cassette, and PGK-DT (DT) selection cassette in the targeting vector are shown as open boxes. Grey triangles depict loxP sites. Homologous recombination was confirmed by PCR (black arrows) and Southern blot analysis in ES cells. The neomycin-resistance cassette was deleted by Cre recombinase expression, resulting in the ‘neo-deleted’ allele. Deletion was confirmed by PCR (grey arrows). <i>Cox2</i> gene sequences are replaced by the firefly luciferase coding region between the ATG translational start site located at the end of exon 1 (e1) and the TAA <i>Cox2</i> stop codon located on exon 10. There are no modifications of the untranslated 5′UTR and 3′UTR either upstream of the ATG or downstream from the TAA. (<b>B</b>) Unstimulated luciferase activity in isolated <i>Cox2^tm2Luc/+</i> tissues. Luciferase activity was quantified by <i>ex vivo</i> bioluminescent imaging. Data are means +/− SD (n = 4). (<b>C</b>) COX-2 and luciferase induction in primary cells isolated from <i>Cox2^tm2Luc/+</i> mice. Bone marrow macrophage cultures were stimulated with LPS (50 ng/mL) for four hours. Lung fibroblast cultures were stimulated with 20% serum for six hours. Cell extracts were analyzed for luciferase enzymatic activity and COX-2 protein. Luciferase activity is displayed as relative light units (RLU) per microgram protein. Data are means +/− SD (**, <i>p</i><0.01, ***, <i>p</i><0.001, n = 3). (<b>D</b>) Interferon gamma and endotoxin (IFNγ/LPS) COX-2 and luciferase induction in the spleens of heterozygous <i>Cox2^tm2Luc/+</i> mice. Four mice were injected i.p. with IFNγ, (1 µg/mouse) and two hours later with LPS (3 mg/kg) or saline. After 6 hours, mice were euthanized, spleens were excised and luciferase bioluminescence was quantified by bioluminescent imaging (left panel). Luciferase enzymatic activity and COX-2 protein levels were measured in extracts (right panel). Data are means +/− SD (**, p<0.01).</p

Inducible COX-2 shRNA expression suppresses <i>Cox2-</i>driven gene expression in cells cultured from triple transgenic mice.

<p>(<b>A</b>) Upper panel: The diagram shows the pCol-TGM targeting construct encoding GFP and COX-2 shRNA, expressed from a TRE promoter. Co-electroporation, with pCAGS-Flpe recombinase, into KH2 ES cells results in integration of the construct into the <i>ColA1</i> locus. ShCox2 mice are crossed with tet-transactivator mice (CAG-rtTA3/C3) to create double trangenics. Lower panel; in triple transgenic shCox2/C3/Luc+ mice, DOX-rtTA3 activates the TRE promoter, driving GFP and shCox2 expression; shCox2 blocks COX-2 and luciferase expression. Without DOX the TRE promoter is quiescent; COX-2 and luciferase are expressed normally. (<b>B</b>) Inducible suppression of <i>Cox2</i> gene expression in fibroblasts. Skin fibroblasts from triple transgenic shCox2/C3/Luc+ and Luc+ mice were cultured for four days in the absence or presence of DOX (1 µg/mL), shifted to 1% serum overnight, then stimulated with 20% serum for 6 hours. Luciferase activity was measured in extracts, COX-2 and GFP protein were analyzed by Western blot. Luciferase activity was normalized to protein content. (<b>C</b>) Reversible suppression of <i>Cox2</i> gene expression in skin fibroblasts. Cells were untreated (no DOX), treated for 4 days with DOX (D4 DOX), or treated for 4 days with DOX followed by 4 days without DOX (D4 DOX, D4 no DOX). Cells were stimulated with medium containing 20% FBS; luciferase activity, COX-2 expression and GFP expression were analyzed. (<b>D</b>) Bone marrow macrophages from shCox2/C3/Luc+ and control Luc+ mice were cultured in the indicated concentrations of DOX overnight, then stimulated for 4 hours with LPS (50 ng/mL) to induce COX-2. Cell extracts were analyzed for luciferase activity and COX-2 protein. Luciferase activities are normalized to LPS stimulation in the absence of DOX. Data are means +/− SD. Statistics compare DOX-treated cultures with cells not receiving DOX (*, p<0.05; **, p<0.01; ***, p<0.001).</p

Distribution of luciferin-dependent bioluminescence in cardiovascular tissue from <i>Cox2</i>^{<i>fLuc/+</i>} mice.

<p>(a) Quantification of basal expression from the aortic tree, vena cava, chambers of the heart and, for comparison, brain from <i>Cox2</i>^{<i>fLuc/+</i>} mice and (b) and representative images of bioluminescence. Arteries, veins and chambers of the heart were essentially devoid of expression from the <i>Cox2</i> gene, in comparison with the brain as a reference tissue. The only exception to this was weak, but detectable, expression in the region of the aortic arch. n=3.</p

6-keto-PGF_1α production in isolated mouse aorta; measurement by enzyme immunoassay, radio immunoassay, and liquid chromatography tandem mass spectrometry (LC-MS/MS).

<p>Prostacyclin release by isolated rings of mouse aorta stimulated with Ca²⁺ ionophore A23187 (50µM), measured as the stable breakdown product 6-keto-PGF_1α, was not altered by <i>Cox2</i> gene deletion, but was reduced >10-fold by <i>Cox1</i> gene deletion. The pattern and level of 6-keto-PGF_1α accumulation was similar whether measured by (a) enzyme immunoassay, (b) radio immunoassay or (c) LC-MS/MS. Representative LC-MS/MS chromatograms show the presence or absence of 6-keto PGF_1α in all sample types (retention time 2.81 min; transition ion <i>m</i>/<i>z</i> 369>163). n=4-7. *, p<0.05 by 1-way ANOVA with Bonferonni’s post-test.</p

Bradykinin-stimulated prostanoid accumulation in the circulation <i>in</i><i>vivo</i> in wild-type, <i>Cox1</i>^<i>-/-</i>, and <i>Cox2</i>^<i>-/-</i> mice.

<p>Accumulation of the stable prostacyclin breakdown product, 6-keto-PGF_1α in plasma after bradykinin administration (100nmol/kg i.v.) is dependent on COX-1 but not COX-2 when measured by LC-MS/MS (a). Representative LC-MS/MS chromatograms show the presence or absence of 6-keto PGF_1α in all sample types (retention time 2.81 min; transition ion <i>m</i>/<i>z</i> 369>163). Similar data were obtained for plasma levels of PGE₂ (b), 13,14-dihydro-15-keto-PGE₂ (c), PGD₂ (d), TXB₂ (e) and (f) PGF_2α. Plasma 6-keto-PGF_1α levels in all genotypes compare well with those previously published using enzyme immunoassay measurements. n=6. *, p<0.05 by 1-way ANOVA with Bonferonni’s post-hoc test.</p

Distribution of luciferin-dependent bioluminescence in tissues from <i>Cox2</i>^{<i>fLuc/+</i>} mice.

<p>(a) Basal expression from organs of the <i>Cox2</i>^{<i>fLuc/+</i>} mice was visualized by bioluminescent imaging of tissues dissected from <i>Cox2</i>^{<i>fLuc/+</i>} reporter mice after injection of D-luciferin in vivo (125mg/kg i.p.). (b) Imaging data are expressed as maximum luminescent emission from each tissue. Basal <i>Cox2</i> gene driven luciferase expression was present in many tissues including the vas deferens, brain, intestine, and thymus but was notably low to absent in the aorta (highlighted with red circles). Sub-division of the (c) brain, (d) intestine, (e) kidney and (f) stomach revealed regional expression patterns within each tissue. n=5.</p

COX-2-dependent prostanoid production by aorta versus other mouse tissues in <i>Cox1</i>^<i>-/-</i> mice.

<p>(a) PGE₂ formation, normalized to tissue mass, was measured by immunoassay in supernatants of Ca²⁺ ionophore A23187 (50µM)-stimulated tissue segments from <i>Cox1</i>^<i>-/-</i> mice. Cox1^-/- tissues released a variable amount of PGE₂ with low levels in the aorta (highlighted in red), and substantially higher levels in the thymus, intestines, renal medulla, brain and vas deferens. This distribution correlates well with luciferase expression in organs of the <i>Cox2</i>^{<i>fLuc/+</i>} mouse, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069524#pone-0069524-g003" target="_blank">Figures 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069524#pone-0069524-g004" target="_blank">4</a>. n=6.</p