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, 96 (9), 5292-7

Suppression of Inducible Cyclooxygenase 2 Gene Transcription by Aspirin and Sodium Salicylate

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Suppression of Inducible Cyclooxygenase 2 Gene Transcription by Aspirin and Sodium Salicylate

X M Xu et al. Proc Natl Acad Sci U S A.

Abstract

The pharmacological action of salicylate cannot be explained by its inhibition of cyclooxygenase (COX) activity. In this report, the effects of aspirin and sodium salicylate on COX-2 expressions in human umbilical vein endothelial cells and foreskin fibroblasts were evaluated. Aspirin and sodium salicylate at therapeutic concentrations equipotently blocked COX-2 mRNA and protein levels induced by interleukin-1beta and phorbol 12-myristate 13-acetate. The suppressing effect was more pronounced in cultured cells deprived of fetal bovine serum for 24 h, suggesting that it may be cell cycle related. Salicylate inhibited nascent COX-2 transcript synthesis but had no effect on COX-2 mRNA stability. It inhibited COX-2 promoter activity in a concentration-dependent manner. In mice pretreated with aspirin (10 and 30 mg/kg), followed by challenge with lipopolysaccharide, COX-2 mRNA expression in peritoneal macrophages was markedly suppressed. These findings suggest that salicylate exerts its antiinflammatory action in part by suppressing COX-2 induction, thereby reducing the synthesis of proinflammatory prostaglandins.

Figures

Figure 1
Figure 1
Effects of aspirin and sodium salicylate on induced HUVEC COX-2 mRNA levels measured by competitive PCR by using a gRNA generated from a human genomic COX-2 DNA fragment as the competitor in that assay. (a) A representative autoradiograph illustrating the calibration (lanes 1–7) using increasing concentrations of gRNA (lanes 1–7, 20–1280 amol in a 2-fold escalation) competing against a standard concentration (256 nmol/tube) of COX-2 mRNA. In lanes 8–16, each tube contained in a RT-PCR mixture 640 amol of gRNA and 0.5 μg of total RNA prepared from HUVEC stimulated with IL-1β (1 ng/ml, 4 h) in the absence of (lanes 8 and 13) or in the presence of aspirin (lanes 9–12) or sodium salicylate (lanes 14–16). gDNA and cDNA were clearly separated as indicated. (b) An autoradiograph illustrating the suppressing effect of sodium salicylate on PMA-induced COX-2 mRNA by competitive PCR. Lanes 1–7 serve as the calibration for quantifying COX-2 mRNA levels. Increasing concentrations of gRNA (lanes 1–7, 160–10240 amol per tube in a 2-fold escalation) were competing against a standard COX-2 mRNA concentration. Lanes 8–13 denote mRNA levels under PMA (100 nM, 4 h) stimulation in the absence (lane 8) or presence (lanes 9–13) of sodium salicylate. (c) COX-2 mRNA levels derived from the calibration curves of a and b.
Figure 2
Figure 2
Analysis of human COX-2 mRNA in cultured cells (a and b) by Northern blotting and murine COX-2 mRNA (c) by RT-PCR. a shows the effect of sodium salicylate at concentrations up to 10−5 M on HUVEC COX-2 mRNA induced by PMA. Concurrent glyceraldehyde 3-phosphate dehydroxygenase (GAPDH) mRNA blotting was included as the control. b shows the effect of aspirin on HFF COX-2 mRNA induced by PMA. c shows the in vivo effects of aspirin at dosages of 10 and 30 mg/kg on murine macrophage COX-2 mRNA expression induced by LPS. Lanes: 1–3, LPS-treated mice in the absence (lane 1) and presence (lanes 2 and 3) of aspirin; 4, normal mouse peritoneal macrophages. Glyceraldehyde 3-phosphate dehydroxygenase (GAPDH) mRNAs, which were included to serve as internal controls, are shown in the lower panel.
Figure 3
Figure 3
Effect of salicylate on COX-2 gene transcription. (a) Nuclear run-off experiments showing concentration-dependent suppression of PMA-induced COX-2 transcripts. The circles show the dot blot of COX-2 transcripts. C denotes non-PMA-induced basal control. (b) Effect of sodium salicylate on COX-2 promoter activity transiently expressed in HFF. Each bar represents mean of ±SD of five experiments. The data are statistically significant when analyzed by ANOVA (P < 0.05).
Figure 4
Figure 4
(ad) Western blot analysis of COX-2 proteins in cultured HFF. (a and b) The effect of sodium salicylate on PMA-induced COX-2 proteins with (a) and without (b) FBS in the medium. (c and d) The effects of indomethacin (c) and NS398 (d) on COX-2 proteins in HFF. Std., purified COX-2 proteins as standards. (ef) COX-2 activity in cultured HFF. (e) The effect of sodium salicylate (10−5 M) on basal and PMA-induced PGE2 levels. Each bar represents mean ± SD of four determinations. (f) The effect of sodium salicylate (10−5 M) on eicosanoid production in response to [1-14C] arachidonate treatment in HFF stimulated with PMA. The data are representative of two experiments with similar results. ♦ denotes PMA plus vehicle, ● denotes PMA plus sodium salicylate, and ▴ denotes PMA plus NS398.

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