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. 2012 Feb 24;287(9):6454-68.
doi: 10.1074/jbc.M111.327874. Epub 2012 Jan 4.

Induction of COX-2 enzyme and down-regulation of COX-1 expression by lipopolysaccharide (LPS) control prostaglandin E2 production in astrocytes

Miriam Font-Nieves  1 , M Glòria Sans-Fons, Roser Gorina, Ester Bonfill-Teixidor, Angélica Salas-Pérdomo, Leonardo Márquez-Kisinousky, Tomàs Santalucia, Anna M Planas
Affiliations

Induction of COX-2 enzyme and down-regulation of COX-1 expression by lipopolysaccharide (LPS) control prostaglandin E2 production in astrocytes

Miriam Font-Nieves et al. J Biol Chem. .

Abstract

Pathological conditions and pro-inflammatory stimuli in the brain induce cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism mediating the production of prostanoids that, among other actions, have strong vasoactive properties. Although low basal cerebral COX-2 expression has been reported, COX-2 is strongly induced by pro-inflammatory challenges, whereas COX-1 is constitutively expressed. However, the contribution of these enzymes in prostanoid formation varies depending on the stimuli and cell type. Astrocyte feet surround cerebral microvessels and release molecules that can trigger vascular responses. Here, we investigate the regulation of COX-2 induction and its role in prostanoid generation after a pro-inflammatory challenge with the bacterial lipopolysaccharide (LPS) in astroglia. Intracerebral administration of LPS in rodents induced strong COX-2 expression mainly in astroglia and microglia, whereas COX-1 expression was predominant in microglia and did not increase. In cultured astrocytes, LPS strongly induced COX-2 and microsomal prostaglandin-E(2) (PGE(2)) synthase-1, mediated by the MyD88-dependent NFκB pathway and influenced by mitogen-activated protein kinase pathways. Studies in COX-deficient cells and using COX inhibitors demonstrated that COX-2 mediated the high production of PGE(2) and, to a lesser extent, other prostanoids after LPS. In contrast, LPS down-regulated COX-1 in an MyD88-dependent fashion, and COX-1 deficiency increased PGE(2) production after LPS. The results show that astrocytes respond to LPS by a COX-2-dependent production of prostanoids, mainly vasoactive PGE(2), and suggest that the coordinated down-regulation of COX-1 facilitates PGE(2) production after TLR-4 activation. These effects might induce cerebral blood flow responses to brain inflammation.

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Figures

FIGURE 1.
FIGURE 1.
LPS administration to the rat brain induced COX-2 in glial cells. Rats received an intrastriatal injection of LPS (5 μl, 1 μg/μl) or the vehicle (PBS), and the brain tissue was obtained 8 h later to study mRNA and protein expression. A and B, rats injected with LPS show a very pronounced increase in the expression of Tnf-α and Cox-2 mRNA in the ipsilateral hemisphere compared with that in animals receiving the vehicle or in the contralateral hemisphere (n = 3–5 rats per group). C, COX-2 protein was detected by Western blotting in the ipsilateral hemisphere 8 h after LPS. D, semi-quantification of COX-2 band intensity indicated a significant increase of COX-2 expression after LPS (n = 3 rats per group). E and F, expression of Tnf-α and Cox-2 mRNA in the ipsilateral hemisphere of mice 8 h after striatal injection of LPS (0.7 μl, 1 μg/μl) is powerfully attenuated in MyD88-deficient mice (MyD88 KO) compared with the WT (n = 3–4 per group). Control animals received intrastriatal injection of the vehicle (PBS). One symbol, p < 0.05; two symbols, p < 0.01; three symbols, p < 0.001. Symbols indicate comparison versus either (*) control or (&) LPS WT.
FIGURE 2.
FIGURE 2.
COX-2 is induced in astrocytes and microglia after LPS in the rat brain. Immunohistochemistry against COX-2 (brown in A–G) and COX-1 (brown in H–O) in control rat brain (A, C, E, H, J, L, and N) and after LPS (B, D, F, G, I, K, M, and O) shows co-localization of COX-2 with markers of microglia (Iba-1) (B and D) and astroglia (GFAP) (arrow in F and G) (dark blue/purple), whereas COX-1 is predominantly expressed in microglia (H–J) and to a lower extent in astroglia (L–N), and it is not up-regulated after LPS (I, K, M, and O). The areas indicated with rectangles in L and M are magnified in N and O, respectively. Bar scale, 30 μm (A–G, J–K, N, and O); 60 μm (L and M); 120 μm (H and I). P and Q, quantification of the proportion of microglia and astroglia cells expressing COX-2. Values are expressed as % of total Iba-1+ microglia (P) or GFAP+ astroglia (Q). n = 4–5 LPS-treated mice and n = 3 mice injected with PBS. Controls are taken as the contralateral nonaffected hemispheres, n = 7. One-way analysis of variance, and *** indicates p < 0.001.
FIGURE 3.
FIGURE 3.
COX-2 induction after LPS is dependent on NFκB and MyD88 pathways. LPS (10 ng/ml) was added to rat (A, B, and E) or mouse (C–D and F–I) astrocyte cultures, and mRNA/proteins were studied at different time points (A–D) or at 4 h. A and B, time course expression of Tnf-α and Cox-2 mRNA after LPS. C, time course of COX-2 protein expression after LPS was assessed by Western blotting. D, semi-quantification of COX-2 band intensity in C. Data were fit to the curve with nonlinear regression analysis (one-phase exponential association). The goodness of the fit was assessed by r2. E, cells were transfected with Oligofectamine carrying small interference RNA (siRNA) against either the p65 subunit of NFκB (si-p65) or the gp91 subunit of NADPH (si-gp91). A nonsilencing (ns) scramble double-stranded RNA was used as control. In these cells, the induction of Cox-2 mRNA is dependent on NFκB. F, NFκB inhibitor PDTC also prevents Cox-2 mRNA and protein induction after LPS. G–I, induction of Tnf-α (G) and COX-2 (H) mRNA is strongly reduced in astrocytes deficient in MyD88 (MyD88−/−) versus the wild type (MyD88+/+). Likewise, COX-2 protein is detected by Western blotting in WT cells but not in MyD88-deficient cells (I). β-Tubulin is shown in the Western blots as a loading control. Values are expressed as the mean ± S.E. of n = 3 per condition. One symbol, p < 0.05; two symbols, p < 0.01; three symbols, p < 0.001. Symbols indicate comparison versus either (*) control or (&) LPS (WT or treatment control).
FIGURE 4.
FIGURE 4.
LPS-induced COX-2 is dependent on p38 and JNK MAPK. LPS (10 ng/ml) was added to mouse astrocyte cultures, and mRNA/proteins were studied at 4 or 8 h. A, astrocytes were treated with LPS in the presence or absence (−) of the indicated doses of MAPK inhibitors (or the vehicle): SB239063 (SB), SP600125 (SP), and U0126 (U), which inhibit the p38, JNK, and MEK pathways, respectively, and Cox-2 mRNA was studied 4 h after LPS. The results show the involvement of p38 and JNK in LPS-induced Cox-2 mRNA. B–D, COX-2 protein expression 8 h after LPS was inhibited by SB239063 (from 1 to 25 μm) (B), and SP600125 (from 10 to 25 μm) (C), but not by MEK inhibitors (up to 25 μm U0126 and up to 40 μm PD98059) (D). E, COX-2 protein 4 h after LPS was also inhibited by p38 and JNK inhibitors but not after MEK inhibition (U0126). F, accordingly, SB239063 and SP600125, but not U0126, inhibit the production of PGE2, as assessed by ELISA in the culture medium 8 h after LPS. G, silencing MAPK with siRNA effectively reduces the expression of the corresponding target proteins by 65–70%. H–J, silencing MAPK10 (JNK3) or MAPK14 (p38), but not MAPK1 (ERK2), reduces Cox-2 mRNA (H) and protein (I and J) versus treatment with nonsilencing (ns) RNA 4 h after LPS. β-Tubulin is shown in the Western blots as a loading control. Values are expressed as the mean ± S.E. of n = 3 per condition in at least three independent experiments. One symbol, p < 0.05; two symbols, p < 0.01; three symbols, p < 0.001.
FIGURE 5.
FIGURE 5.
LPS down-regulates COX-1 expression in astrocytes. Cultured mouse astrocytes were treated with LPS (10 ng/ml) for 4, 8, or 24 h. A, Cox-1 mRNA expression is down-regulated 8 h after LPS in WT (+/+) and Cox-2-deficient cells (−/−). B–D, lack of expression of Cox-2 mRNA and protein in Cox-2 KO cells is shown compared with WT. E and F, COX-1 protein expression decreases from 8 h after LPS but not at 4 h. G–J, down-regulation of COX-1 after LPS is not MAPK-dependent because it is not altered by MAPK inhibitors (G and H) or by silencing the indicated MAPK (I and J) (ns indicates treatment with control nonsilencing RNA). K, LPS-induced down-regulation of Cox-1 is not observed in MyD88-deficient cells, suggesting that it is MyD88-dependent. L, intracerebral administration of LPS to mice also induces a reduction of Cox-1 mRNA expression in the ipsilateral (ipsi) hemisphere 8 h after LPS, and this effect is strongly attenuated in MyD88-deficient mice. contra, contralateral. M, NFκB inhibition with PDTC (10 μm) significantly attenuates the reduction of Cox-1 mRNA induced by LPS at 4 h in cultured astrocytes. N, compared with WT cells, low levels of Cox-1 mRNA are found in Cox-1-deficient cells, and COX-1 protein is not detected in these cells by Western blotting (inset). O, Cox-1 KO cells produce Cox-2 mRNA and protein 4 and 8 h after LPS, respectively. β-Tubulin is shown as a loading control. n = 3 per condition in each result. One symbol, p < 0.05; two symbols, p < 0.01; three symbols, p < 0.001. Symbols indicate comparison versus either (*) control or (&) LPS (WT or untreated).
FIGURE 6.
FIGURE 6.
LPS induces expression of mPges-1 mRNA but not of the enzymes that synthesize other prostanoids. Astrocytes of Cox-1 or Cox-2 KO mice (−/−) and their respective wild type (+/+) astrocytes were treated with LPS (10 ng/ml), and mRNA was extracted at 8 h. A, LPS strongly induces microsomal PGE2 synthase-1 (mPGES1) mRNA in the different genotypes. c, control. B, induction of mPges1 mRNA after LPS is dependent on the MyD88 pathway, as shown by lack of mPges-1 mRNA up-regulation in MyD88-deficient (MyD88−/−) cells after LPS. C and D, expression of prostacyclin synthase (PGIS) mRNA (C) and that of TS mRNA (D) is reduced after LPS in all genotypes. E and F, accordingly, in astrocytes from C57 WT mice, LPS significantly up-regulates the expression of mPGES1 protein (E), although TS protein shows a nonsignificant tendency to progressively decrease with time versus controls (F). n = 3 for each genotype. One symbol, p < 0.05; two symbols, p < 0.01; three symbols, p < 0.001. * indicates comparison versus control; & indicates comparison versus LPS in WT.
FIGURE 7.
FIGURE 7.
LPS induces secretion of prostanoids to the culture medium. Purified cultures of rat astrocytes were exposed to LPS (10 ng/ml) for different times, and the medium was collected and studied by ELISA. ELISAs were carried out in five independent experiments, and curves from representative experiments are shown. A, time course of PGE2 concentrations in the culture medium. B, LPS does not induce PGE2 in the presence of the Cox-2 inhibitor NS-398 (3 μm), and PGE2 concentration is lower in the presence of the Cox-1 inhibitor SC-560 (10 nm). C, time course of TxB2 as an indicator of the generation of TxA2. D, NS-398 strongly reduces the production of TxB2 induced by LPS, whereas SC-560 attenuates the effect of LPS. E, time course of PGF-1α concentration to indirectly assess the formation of prostacyclin. F, NS-398 strongly reduces the concentration of PGF1-α in the medium, whereas some reduction is observed with the Cox-1 inhibitor (inh) SC-560. G–I, cPLA2 inhibitor arachidonyltrifluoromethyl ketone (2 μm) completely abrogates LPS-induced prostanoid formation as assessed at 8 h after LPS exposure. AA, arachidonic acid. One symbol, p < 0.05; two symbols, p < 0.01; three symbols, p < 0.001. * indicates comparison versus control. & indicates comparison versus LPS alone. Data (mean ± S.D.) were fit to the curve with nonlinear regression analysis (one-phase exponential association) in A and C, and with linear regression in E. The goodness of the fit was assessed by r2.
FIGURE 8.
FIGURE 8.
LPS-induced prostanoid production is dependent on COX-2 although COX-1 exerts selective regulatory effects. Purified cultures of mouse astrocytes were treated with LPS (10 ng/ml), and the concentration of prostanoids in the culture medium was studied by ELISA. Cells were collected at 4 and 8 h (A–C), or 8 h (D–I) after LPS. A–C, production of prostanoids is strongly attenuated in MyD88-deficient cells. D–F, LPS does not induce PGE2 (G), PGF1α (H), or TxB2 (I) in Cox-2 KO cells (−/−). G–I, production of PGE2 induced by LPS is enhanced in Cox-1-deficient astrocytes (−/−) (G). In contrast, the production of PGF1α (H) and TxB2 (I), as indirect assessments of PG and TxA2, respectively, is smaller in Cox-1 KO cells (−/−) compared with the wild type (+/+), both in the presence or absence of LPS. J–L, silencing COX-1 expression with siRNA slightly enhances the production of PGE2 (J) but does not modify the production of PGI2 (K) or TxA2 (L). n = 3 in at least two independent experiments. One symbol, p < 0.05; two symbols, p < 0.01; three symbols, p < 0.001. * indicates comparison versus control. & indicates comparison versus LPS in WT or after treatment with nonsilencing (ns) RNA. # indicates comparison versus control KO cells.
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References

    1. Hata A. N., Breyer R. M. (2004) Pharmacology and signaling of prostaglandin receptors. Multiple roles in inflammation and immune modulation. Pharmacol. Ther. 103, 147–166 - PubMed
    1. Iadecola C., Niwa K., Nogawa S., Zhao X., Nagayama M., Araki E., Morham S., Ross M. E. (2001) Reduced susceptibility to ischemic brain injury and N-methyl-d-aspartate-mediated neurotoxicity in cyclooxygenase-2-deficient mice. Proc. Natl. Acad. Sci. U.S.A. 98, 1294–1299 - PMC - PubMed
    1. Nagayama M., Niwa K., Nagayama T., Ross M. E., Iadecola C. (1999) The cyclooxygenase-2 inhibitor NS-398 ameliorates ischemic brain injury in wild-type mice but not in mice with deletion of the inducible nitric-oxide synthase gene. J. Cereb. Blood Flow Metab. 19, 1213–1219 - PubMed
    1. Araki E., Forster C., Dubinsky J. M., Ross M. E., Iadecola C. (2001) Cyclooxygenase-2 inhibitor ns-398 protects neuronal cultures from lipopolysaccharide-induced neurotoxicity. Stroke 32, 2370–2375 - PubMed
    1. Candelario-Jalil E., González-Falcón A., García-Cabrera M., León O. S., Fiebich B. L. (2007) Post-ischemic treatment with the cyclooxygenase-2 inhibitor nimesulide reduces blood-brain barrier disruption and leukocyte infiltration following transient focal cerebral ischemia in rats. J. Neurochem. 100, 1108–1120 - PubMed

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