Journal: Arthritis Research & Therapy

Article Title: Human articular chondrocytes express 15-lipoxygenase-1 and -2: potential role in osteoarthritis

doi: 10.1186/ar2652

Figure Lengend Snippet: 13-HODE and 15-HETE suppressed IL-1β-induced MMP-1/MMP-13 production in a PPARγ dependent manner. (a) 13-HODE and 15-HETE activate endogenous PPARγ in human chondrocytes. Chondrocytes were transiently transfected with a reporter construct containing three copies of a consensus PPRE placed upstream from the Tk-luciferase reporter (PPRE 3 -Tk-Luc) along with the internal control pSV40-β-gal using FuGene 6 transfection reagent. Six hours later, the cells were washed and changed to medium containing 0.5% fetal calf serum for an additional 18 hours. Transfected cells were then treated with the control vehicle dimethyl sulfoxide or increasing concentrations of 13-HODE or 15-HETE for 18 hours. Luciferase activity values were determined and normalized to β-galactosidase activity. Results are expressed as fold changes, considering 1 as the value of unstimulated cells, and are the mean ± standard deviation of three independent experiments. * P < 0.05 versus unstimulated cells. (b) PPARγ antagonist (GW9662) prevented the suppressive effect of 13-HODE and 15-HETE on IL-1β-induced MMP-1 and MMP-13 release. Chondrocytes were pretreated with increasing concentrations (1, 5, and 10 μmol/l) of GW9662 for 30 minutes. Then, the cells were treated with or without IL-1β (100 pg/ml) for 24 hours in the absence or the presence of 50 μmol/l 13-HODE (panel a) or 50 μmol/l 15-HETE (panel b). The levels of MMP-1 and MMP-13 proteins in conditioned media were measured using ELISA. Results are expressed as the percentage of control, considering 100% as the value of cells treated with IL-1β alone, and are the mean ± standard deviation of four independent experiments. * P < 0.05 versus cells treated with IL-1β and 13-HODE or 15-HETE. HETE, hydroxyeicosatetraenoic acid; HODE, hydroxy octadecadienoic acid; MMP, matrix metalloproteinase; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator-activated receptor-responsive element.

Article Snippet: GW9662, 13(S)-HODE, 15(S)-HETE, anti-15-LOX-1 and 15-LOX-2 antibodies were from Cayman Chemical Co. (Ann Arbor, MI, USA).

Techniques: Transfection, Construct, Luciferase, Control, Activity Assay, Standard Deviation, Enzyme-linked Immunosorbent Assay