Paradoxical stimulation of cyclooxygenase-2 expression by glucocorticoids via a cyclic AMP response element in human amnion fibroblasts

Abstract

Human amnion fibroblasts produce abundant prostaglandins toward the end of gestation, which is one of the major events leading to parturition. In marked contrast to its well-described antiinflammatory effect, glucocorticoids have been shown to up-regulate cyclooxygenase-2 (COX-2) expression in human amnion fibroblasts. The mechanisms underlying this paradoxical induction of COX-2 by glucocorticoids have not been resolved. Using cultured human amnion fibroblasts, we found that the induction of COX-2 mRNA expression by cortisol was a glucocorticoid receptor (GR)-dependent process requiring ongoing transcription. Upon transfection of a COX-2 promoter-driven reporter gene into the amnion fibroblasts, cortisol stimulated the COX-2 promoter activity. This was abolished by mutagenesis of a cAMP response element (CRE) at -53 to approximately -59bp as well as by cotransfection of a plasmid expressing dominant-negative CRE-binding protein (CREB). The phosphorylation level of CREB-1 was significantly increased by cortisol treatment of the amnion fibroblasts, whereas the effect was attenuated either by the protein kinase A inhibitor H89 or the p38 -MAPK inhibitor SB203580. The induction of the COX-2 promoter activity and the phosphorylation of CREB-1 were also blocked by the GR antagonist RU486. Chromatin immunoprecipitation (ChIP) assay revealed that the binding of CREB-1 to the CRE of the COX-2 promoter was increased by cortisol treatment of the amnion fibroblasts. In conclusion, cortisol, via binding to GR, stimulated COX-2 expression by increasing phosphorylated CREB-1 binding to the CRE of the COX-2 gene. Cortisol may phosphorylate CREB-1 by activating either protein kinase A or p38-MAPK in the amnion fibroblasts.

Fig. 1.

A, Concentration-dependent induction of COX-2 mRNA expression by cortisol (F) treatment of human amnion fibroblasts for 24 h; B, stimulation of PGE₂ production by cortisol (1 μm) treatment of human amnion fibroblasts for 24 h; C, inhibition of cortisol-induced (1 μm) COX-2 mRNA expression by cotreatment of human amnion fibroblasts with GR antagonist RU486 (RU) (1 μm) for 24 h; D, inhibition of cortisol-induced (1 μm) COX-2 mRNA expression by pretreatment for 0.5 h and cotreatment with mRNA transcription inhibitor DRB (75 μm) for 24 h in human amnion fibroblasts; E, reversal of cortisol-induced (1 μm) COX-2 mRNA expression by pretreatment for 0.5 h and cotreatment with protein synthesis inhibitor CHX (10 μm) for 24 h in human amnion fibroblasts. n = 3–4; *, P < 0.05; **, P < 0.01 vs. respective control (ctr); ##, P < 0.01 vs. cortisol.

Fig. 2.

Inhibition of COX-2 mRNA expression (A) and PGE₂ production (B) by cortisol (F, 1 μm) treatment of human fetal lung fibroblasts (HFL-1) for 24 h. n = 3; **, P < 0.01 vs. control (ctr).

Fig. 3.

Sequence of the cloned COX-2 promoter spanning from −886 to +31 bp from human amnion fibroblasts. The arrows pointing right indicate the positions of 5′ primers used for progressive deletion of the promoter fragment with PCR, and the arrow pointing left indicates the position of the 3′ primer. The boxed bold letters indicate the putative transcription factor binding sites analyzed with TESS.

Fig. 4.

A, Concentration-dependent induction of COX-2 promoter (−886 bp) activity by cortisol (F) treatment of human amnion fibroblasts for 24 h; B, inhibition of cortisol-induced (1 μm) increase of COX-2 promoter (−886 bp) activity by cotreatment of human amnion fibroblasts with GR antagonist RU486 (RU) (1 μm) for 24 h; C, cortisol (1 μm) treatment of human amnion fibroblasts for 24 h increased the COX-2 promoter activities of −886, −694, −340, −201, −101, and −66 bp (top panel). The primer sequences for subcloning the COX-2 promoter fragments are listed in the bottom panel. The lowercase letters indicate the added nucleotides for the digestion with restriction enzymes (sense, KpnI; antisense, BglII). n = 3–4; **, P < 0.01 vs. control (ctr) without cortisol treatment; ##, P < 0.01 vs. cortisol. Luc, Luciferase.

Fig. 5.

A, Concentration-dependent inhibition of COX-2 promoter (−886 bp) activity by cortisol (F) treatment of human fetal lung fibroblasts (HFL-1) for 24 h; B, blockade of cortisol-induced (1 μm) inhibition of COX-2 promoter (−886 bp) activity by cotreatment of HFL-1 cells with GR antagonist RU486 (RU) (1 μm) for 24 h; C, inhibition of IL-1β-induced (10 ng/ml) increase of COX-2 promoter (−886 bp) activity by cotreatment of HFL-1 cells with cortisol (1 μm) for 24 h; D, cortisol (1 μm) treatment of HFL-1 cells for 24 h decreased the COX-2 promoter activities of −886, −694, and −453 but not −340, −201, or −101 bp (top panel). The primer sequences for subcloning the COX-2 promoter fragments are listed in the bottom panel. The lowercase letters indicate the added nucleotides for the digestion with restriction enzymes (sense, KpnI; antisense, BglII). n = 3; *, P < 0.05; **, P < 0.01 vs. control (ctr) without cortisol treatment; ##, P < 0.01 vs. cortisol. Luc, Luciferase.

Fig. 6.

Treatment of human amnion fibroblasts with forskolin (100 μm), an activator of adenylyl cyclase, for 24 h increased COX-2 promoter (−66 bp) activity (top panel) and COX-2 mRNA level (bottom panel). Introduction of nucleotide mutations into the CRE at −53 to −59 bp diminished the stimulation of COX-2 promoter (−66 bp) activity by both forskolin (100 μm) and cortisol (1 μm) in human amnion fibroblasts. The boxed nucleotide sequence shown on top is the CRE of the COX-2 promoter, and the bold letters represent the mutated nucleotides. n = 3; **, P < 0.01 vs. control without forskolin or cortisol treatment.

Fig. 7.

Introduction of nucleotide mutations into the NFκB binding site at −448 to −439 bp diminished the inhibition of COX-2 promoter (−453 bp) activity by cortisol (1 μm) in human fetal lung fibroblasts (HFL-1). The boxed nucleotide sequence shown on top is the NFκB binding site of the COX-2 promoter, and the bold letters represent the mutated nucleotides. n = 3; **, P < 0.01 vs. control without cortisol treatment.

Fig. 8.

Cortisol (F, 1 μm) treatment of human amnion fibroblasts for 12 h increased the level of p-CREB-1 but not the total CREB-1 protein level. Cotreatment of the amnion fibroblasts with cortisol (1 μm) and GR antagonist RU486 (RU, 1 μm) or PKA inhibitor H89 (20 μm) or p38-MAPK inhibitor SB203580 (SB, 10 μm) abolished the induction of phosphorylation of CREB-1 by cortisol. n = 3; **, P < 0.01 vs. vehicle control (ctr).

Fig. 9.

Cotreatment of human amnion fibroblasts with cortisol (1 μm) and PKA inhibitor H89 (20 μm) or p38-MAPK inhibitor SB203580 (SB, 10 μm) for 24 h abolished the induction of COX-2 mRNA (A and B) and COX-2 promoter (−886 bp) activity (C and D) by cortisol (1 μm). n = 3–4; **, P < 0.01 vs. vehicle control; #, P < 0.05; ##, P < 0.01 vs. vehicle control (cortisol without H89 or SB203580).

Fig. 10.

A, Cortisol (F, 1 μm) treatment of HFL-1 cells for 12 h decreased the level of p-CREB-1 but not the total CREB-1 protein level; B, time course of the effect of cortisol (1 μm) treatment on the level of p-CREB-1 in HFL-1 cells; C, effect of cortisol (1 μm, 12 h) treatment on PPP1, PPP2, and MKP-1 mRNA levels in HFL-1 cells. n = 3–4; *, P < 0.05 vs. respective vehicle controls (ctr).

Fig. 11.

A, ChIP demonstrated that cortisol (1 μm) stimulated the binding of CREB-1 to the CRE in the COX-2 promoter. Top panel is a representative gel of the PCR products amplified from the DNA fragments immunoprecipitated by CREB-1 antibody. B and C, Transfection of the plasmid expressing dnCREB into human amnion fibroblasts blocked the induction of COX-2 mRNA and promoter (−886 bp) activity by cortisol (1 μm, 24 h). n = 3; **, P < 0.01 vs. vehicle control; #, P < 0.05; ##, P < 0.01 vs. empty vector with cortisol. Ab, Antibody.

Fig. 12.

Simplified model illustrating the hypothesized mechanisms underlying the induction of COX-2 expression by cortisol in human amnion fibroblasts.