PPARγ ligands decreased IL-6 transcription and secretion largely by a PPARγ-dependent mechanism. (A) Adherent HS-5 cells were stimulated with KAS6/1 cells and treated with PPAR ligands 15-d-PGJ2, troglitazone, or WY14643 for 6 hours. KAS6/1 cells were washed out and collected. The remaining HS-5 cells were then harvested. The RNA samples, prepared from the above-harvested KAS6/1 cells or HS-5 cells, were hybridized with [³³P]-labeled RNA probes corresponding to transcripts for individual human IL-6 gene, according to the BD PharMingen protocol. RNase protected fragments were separated on 5% polyacrylamide gel electrophoresis (PAGE). (B) SiRNA-mediated knockdown of PPARγ alters its ligands' inhibition of IL-6 production. HS-5 cells were transfected with SiRNA-PPARγ or control vector and cultured for 96 hours. The above HS-5 cells were then treated with 1 μM 15-d-PGJ2 or 10 μM troglitazone and incubated with or without KAS6/1 cells for an additional 48 hours. IL-6 concentration was determined in supernatants. Insert figure shows knockdown of PPARγ expression in HS-5 cells transfected with SiRNA-PPARγ. HS-5 cells were transduced with SiRNA-PPARγ or control vector and incubated for 96 hours. Proteins were then extracted from the cells, and the levels of PPARγ expression were examined by Western blotting. The β-actin bands served as an internal control for equal total protein loading. (C) HS-5 cells were cotransfected with a PPARγ wild-type expression vector (pSG5-hPPARγ2) or control pSG5 plasmid, an IL-6 promoter luciferase reporter gene construct, and a pRL-TK control vector for 24 hours. Cells were then treated with 1 μM 15-d-PGJ2 or 10 μM troglitazone and incubated with or without KAS6/1 cells for an additional 48 hours. Luciferase activity of lysed cells was measured. The level of firefly luciferase activity was normalized to that of the Renilla luciferase activity. Insert figure shows the level of PPARγ expression in HS-5 cells transfected with PPARγ wild-type expression vector. (D) Effect of PPARγ ligands on transactivation of IL-6 promoter. HS-5 cells were transfected with the indicated IL-6 promoter luciferase reporter gene constructs. Twenty-four hours after transfection, KAS6/1 cells were added directly to the transfected confluent HS-5 BMSCs pretreated with 15-d-PGJ2 or troglitazone for 2 hours. The transfected HS-5 cells were also cultured alone as a control. The cocultured cells were incubated for an additional 24 hours. After washing out KAS6/1 cells, luciferase activity of adherent HS-5 cells was measured. Error bars represent the mean (± SE) from 3 independent experiments.