Journal: American Journal of Physiology - Cell Physiology

Article Title: Prostaglandin E 2 induces interleukin-6 expression in human chondrocytes via cAMP/protein kinase A- and phosphatidylinositol 3-kinase-dependent NF-?B activation

doi: 10.1152/ajpcell.00508.2009

Figure Lengend Snippet: PGE2 and forskolin induce NF-κB activation in human T/C28a2 chondrocytes via cAMP/PKA and PI3K signaling pathways. T/C-28a2 cells were treated with PGE2 (10 μM) (A) or forskolin (20 μM) (B) for the indicated time intervals. Nuclear extracts were then isolated, and NF-κB-specific DNA-protein complex formation was determined by EMSA. T/C-28a2 chondrocytes were treated for 2 h with either PGE2 (C, E, G, H) or forskolin (D, F, G, H) in the presence or absence of the PI3K inhibitors [LY-294002 (30 μM) or wortmannin (10 μM)], the PKA inhibitor H89 (10 μM), or the adenylyl cyclase inhibitor SQ-22536 (100 μM). Nuclear extracts were prepared for the determination of NF-κB-specific DNA-protein complex formation by EMSA (C, D). Supershift (E, F) assays using an anti-p65 Ab were carried out as outlined in materials and methods. Results of a competition experiment using 50-fold unlabeled NF-κB oligonucleotide (cold probe) are shown. Cross-linked chromatin was immunoprecipitated using an anti-p65 antibody (G, H). In ChIP assays, the anti-RNA polymerase II antibody was used as positive control, whereas the normal mouse IgG and anti-TLR4 antibodies were used as negative controls. DNA purified from both the immunoprecipitated (IP) and preimmune (input) specimens was subjected to PCR amplification using primers for the GAPDH (control) and p65 promoter genes. All experiments are representative of three independent experiments, all revealing similar results.

Article Snippet: PGE 2 , forskolin, the adenylate cyclase inhibitor SQ-22536, the PKA inhibitor H89, the specific activator of the exchange protein activated by cAMP (Epac) 8-(4-Chlorophenylthio)-2′- O -methyladenosine-3′,5′-cyclic monophosphate (8-pCPT-2′- O -Me-cAMP·Na or CPT), and the NF-κB inhibitor 6-amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ) were obtained from Enzo Life Sciences.

Techniques: Activation Assay, Isolation, Immunoprecipitation, Positive Control, Purification, Amplification

Journal: American Journal of Physiology - Cell Physiology

Article Title: Prostaglandin E 2 induces interleukin-6 expression in human chondrocytes via cAMP/protein kinase A- and phosphatidylinositol 3-kinase-dependent NF-?B activation

doi: 10.1152/ajpcell.00508.2009

Figure Lengend Snippet: Effect of NF-κB inhibition on IL-6 mRNA synthesis in human T/C-28a2 chondrocytes stimulated with PGE2 or forskolin. T/C-28a2 chondrocytes were treated with either PGE2 (10 μM) or forskolin (20 μM) for 2 h in the presence of an NF-κB-specific inhibitor QNZ (10 μM). IL-6 mRNA expression was determined by qRT-PCR (A, B). GAPDH served as internal control. Data are means ± SE of three independent experiments. *P < 0.05 with respect to QNZ treatment and no treatment control. Gel-shift (C) and super-shift (D) experiments using an anti-p65 Ab were carried out as described in materials and methods. These experiments are representative of three independent experiments, all revealing similar results.

Article Snippet: PGE 2 , forskolin, the adenylate cyclase inhibitor SQ-22536, the PKA inhibitor H89, the specific activator of the exchange protein activated by cAMP (Epac) 8-(4-Chlorophenylthio)-2′- O -methyladenosine-3′,5′-cyclic monophosphate (8-pCPT-2′- O -Me-cAMP·Na or CPT), and the NF-κB inhibitor 6-amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ) were obtained from Enzo Life Sciences.

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Electrophoretic Mobility Shift Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Prostaglandin E 2 induces interleukin-6 expression in human chondrocytes via cAMP/protein kinase A- and phosphatidylinositol 3-kinase-dependent NF-?B activation

doi: 10.1152/ajpcell.00508.2009

Figure Lengend Snippet: Proposed cascade of signaling events in human T/C-28a2 chondrocytes stimulated with PGE2 or forskolin. PGE2 stimulates cAMP formation, which in turn upregulates PI3K/Akt and PKA activities, leading to NF-κB activation. Binding of NF-κB to IL-6 promoter induces IL-6 synthesis in human T/C28a2 chondrocytes.

Article Snippet: PGE 2 , forskolin, the adenylate cyclase inhibitor SQ-22536, the PKA inhibitor H89, the specific activator of the exchange protein activated by cAMP (Epac) 8-(4-Chlorophenylthio)-2′- O -methyladenosine-3′,5′-cyclic monophosphate (8-pCPT-2′- O -Me-cAMP·Na or CPT), and the NF-κB inhibitor 6-amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ) were obtained from Enzo Life Sciences.

Techniques: Activation Assay, Binding Assay