Reciprocal regulation of 11β-hydroxysteroid dehydrogenase 1 and glucocorticoid receptor expression by dexamethasone inhibits human coronary artery smooth muscle cell proliferation in vitro

Abstract

The actions of glucocorticoids are mediated, in part, by 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), which amplifies their effects at the pre-receptor level by converting cortisone to cortisol. Glucocorticoids, such as dexamethasone, inhibit vascular smooth muscle cell proliferation; however, the role of 11β-HSD1 in this response remains unknown. Accordingly, we treated human coronary artery smooth muscle cells (HCSMC) with dexamethasone (10(-9)-10(-6) mol/l) and found that after 72 h dexamethasone increased 11β-HSD1 expression (14.16 ± 1.6-fold, P < 0.001) and activity (6.21 ± 1.2-fold, P < 0.001) in a dose- and time-dependent manner, which was dependent upon glucocorticoid receptor (GR) activation and C/EBPβ and C/EBPδ signaling. As glucocorticoids are known to negatively regulate GR expression, we examined the effect of decreasing 11β-HSD1 expression on GR expression. In HCSMC transfected with 11β-HSD1 siRNA, GR expression was increased; this effect was associated with protein kinase A activation and CREB phosphorylation. To examine the role of 11β-HSD1 in HCSMC proliferation, we decreased 11β-HSD1 expression and stimulated cells with platelet-derived growth factor (PDGF) (10 ng/ml). Decreased 11β-HSD1 expression was associated with increased cell proliferation in the absence of PDGF compared to scrambled control-transfected cells (236.10 ± 13.11%, n = 4, P < 0.001) and this effect was augmented by PDGF. Furthermore, the inhibitory effect of dexamethasone on cellular proliferation was abrogated in 11β-HSD1 siRNA-transfected HCSMC. Downregulation of 11β-HSD1 was associated with decreased p27(kip1) expression and increased phosphorylated retinoblastoma protein, consistent with a proliferative response. These findings suggest that 11β-HSD1 plays a role in the effects of glucocorticoids on vascular smooth muscle cell phenotype.

Fig. 1. Dexamethasone increases 11β-HSD1 expression and activity

Human coronary artery smooth muscle cells (HCSMC) (a) were treated with increasing concentrations of dexamethasone and 11β-HSD1 mRNA levels were determined by qRT-PCR after 18 h (n=3). HCSMC were treated with dexamethasone (10⁻⁷ mol/L) for 0–72 h and 11β-HSD1 (b) mRNA levels were determined by qRT-PCR (n=3), (c) protein expression was examined by Western immunoblotting (n=3), and (d) 11β-HSD1 activity was measured. (n=3). Data are presented as mean ± SEM. A representative blot is shown.*p<0.01 vs. UN, 0. UN, treated; Dex, dexamethasone

Fig. 2. The effect of dexamethasone on glucocorticoid receptor, C/EBPβ and C/EBPδ expression

Human coronary artery smooth muscle cells (HCSMC) were treated with 10⁻⁷ mol/L dexamethasone for 0–72 h and expression of (a) the glucocorticoid receptor (GR) (n=3) and (b) the phospho-GR (n=3) were determined by Western immunoblotting. At the same timepoints, (c) C/EBPβ and (d) C/EBPδ mRNA levels were determined by qRT-PCR (n=3) and corresponding (e) C/EBPβ and (f) C/EBPδ protein levels were assessed by Western blotting (n=3). Data are presented as mean ± SEM. Representative blots are shown. *p<0.05 vs. 0

Fig. 3. The glucocorticoid receptor, C/EBPβ, and C/EBPδ regulate the dexamethasone-mediated increase in 11β-HSD1 expression

Human coronary artery smooth muscle cells (HCSMC) were transfected with an siRNA to decrease glucocorticoid receptor (GR) expression or a scrambled control (SS), treated with dexamethasone (10⁻⁷ mol/L), and (a) 11β-HSD1 mRNA levels were determined by qRT-PCR (n=3) and (b) protein expression by Western immunoblotting (n=3). HCSMC were transfected with an siRNA to decrease C/EBPβ or C/EBPδ expression, respectively, or a scrambled control, treated with dexamethasone (10⁻⁷ mol/L), and (c) 11β-HSD1 mRNA levels were determined by qRT-PCR (n=3) and (d) protein expression by Western immunoblotting (n=3). mRNA and protein levels were determined. Data are presented as mean ± SEM and representative blots are shown. *p<0.001 vs. SS, **p<0.001 vs. SS + Dex

Fig. 4. 11β-HSD1 regulates glucocorticoid receptor expression

HCSMC were transfected with an siRNA to decrease 11β-HSD1 expression, treated with dexamethasone (10⁻⁷ mol/L), and glucocorticoid receptor (GR) (a) protein expression was determined by Western immunoblotting (n=3). (b) Protein kinase A (PKA) and phospho-PKA expression were assessed by Western blotting (n=3), and (c) PKA activity was measured (n=4). (d) Expression of the cyclic AMP response element binding protein (CREB) and phospho-CREB were assayed by Western blotting (n=3). Data are presented as mean ± SEM and representative blots are shown. *p<0.001 vs. SS; **p<0.001 vs. HSD1 siRNA; #p<0.05 vs. SS

Fig. 5. 11β-HSD1 mediates the inhibitory effects of dexamethasone on PDGF-stimulated HCSMC

Human coronary artery smooth muscle cells (HCSMC) were transfected with an siRNA to decrease 11β-HSD1 expression, synchronized by serum starvation (0.2% serum) for 48 h, and stimulated with 5% fetal calf serum and platelet-derived growth factor-BB (PDGF) (10 ng/ml) in the presence or absence of dexamethasone (10⁻⁷ mol/L). Cell proliferation was determined by measuring BrdU incorporation (n=4). Data are presented as mean ± SEM and results are corrected for cell number. *p<0.001 vs. SS; **p<0.001 vs. SS + PDGF; ^#p<0.001 vs. HSD1 siRNA; ^##p<0.001 vs. HSD1 siRNA + PDGF

Fig. 6. 11β-HSD1 mediates the inhibitory effects of dexamethasone on proliferation in PDGF-stimulated murine aortas

Thoracic aortas from C57Bl/6 male mice (n=12) were isolated, sectioned into 5 mm rings, and transfected with 80 nmol/L Stealth RNAi^™ to 11β-HSD1 mRNA or scrambled control (SS) added to smooth muscle basal medium. (a) After 48 h, 11β-HSD1 expression was examined by immunohistochemistry of paraffin embedded sections. (b) The influence of 11β-HSD1 on platelet-derived growth factor-BB (PDGF) (10 ng/ml)-stimulated proliferation, in the presence or absence of dexamethasone (10⁻⁷ mol/L), was examined by immunofluorescence labeling of Ki67. Representative hematoxylin & eosin stained sections are provided for comparison (top) and corresponding fluorescent sections are shown (bottom). Images are magnified 200X.

Fig. 7. 11β-HSD1 influences the expression of cell cycle regulators in response to PDGF and dexamethasone

Human coronary artery smooth muscle cells (HCSMC) were transfected with an siRNA to decrease 11β-HSD1 expression, synchronized by serum starvation (0.2% serum) for 48 h, and stimulated with 5% fetal calf serum and platelet-derived growth factor-BB (PDGF) (10 ng/ml) in the presence or absence of dexamethasone (10⁻⁷ mol/L). Expression of (a) p27^kip1 (n=3) and (b) phosphorylation of retinoblastoma protein (phospho-Rb) at Ser608 (n=3) were determined by Western immunoblotting. Representative blots are shown