Figure 4

Regulation of Cyp7b mRNA expression and activity in FLS. (a) Human fibroblast-like synoviocytes (FLS; STSF.388, passages 8–10) were incubated for 6 hours with medium control (-), 0.5 ng/ml tumour necrosis factor (TNF)-α, TNF-α combined with the proteasome inhibitor (PSI) 1 × 10E^-6 mol/l, SN50 200 μg/ml, SP600125 (SP) 1 × 10E^-5 mol/l, PD98059 (PD) 1 × 10E^-5 mol/l or SB23580 (SB) 1 × 10E^-5 mol/l, as described in Materials and methods. RNA was isolated and cDNA was made and used for reverse transcription with GAPDH and cytochrome p450 enzyme 7b (Cyp7b) specific primers. The ratio of Cyp7b to GAPDH mRNA expression was 1.4 (TNF-α alone), 8 × 10^-6 (PSI + TNF-α), 1.3 × 10^-6 (SN50 + TNF-α), 0.5 × 10^-6 (SP + TNF-α), 0.3 (PD + TNF-α) and 0.2 (SB + TNF-α). Data are representative of two independent experiments. (b) FLS (STSF.388, passages 8–10) were preincubated in the presence or absence (medium control [-]) of SN50 for 2 hours or SP600125 (SP), PD98059 (PD) or SB23580 (SB) for 1 hour. Thereafter, FLS were incubated in the presence or absence of 0.5 ng/ml TNF-α plus 1.5 × 10^-8 mol/l dehydroepiandrosterone (DHEA) for 24 hours and processed for radioimmunoassay detection of 7α-hydroxy-dehydroepiandrosterone (7α-OH-DHEA). Results are expressed as the mean ± standard error of the mean of triplicate samples. Data are representative for two independent experiments. *P < 0.005 versus TNF-α (Student's t-test).