FIG. 6.

A nongenomic, PI3K-dependent pathway is necessary for the action of E₂. (A) Western blot analysis of phosphorylated ERK (P-ERK) and ERK proteins in total extracts from RAW 264.7 cells treated with 1 nM E₂ for 10 min before treatment with LPS at 50 μg/ml for 30 min. ctrl, control. (B) Subcellular localization of p65. Immunocytochemical analysis with anti-p65 antibody was performed on RAW 264.7 cells treated with PD98059 (50 μM) (PD) or LY294002 (50 μM) (LY) for 1 h, with 1 nM E₂ for 10 min, and with LPS at 50 μg/ml for 30 min as indicated. (C) Quantification of E₂ activity shown in panel B. The percentage of cells with cytoplasmic p65 is plotted relative to the total cell number. Black bars, LPS; hatched bars, E₂ plus LPS. Actinomycin D (5 μg/ml) (ActD) was added 1 h before E₂ and LPS. Values are the means ± SDs of a single experiment performed in triplicate. Single and double asterisks indicate P values of <0.05 in a comparison with LPS and in a comparison with E₂ plus LPS, respectively. (D) PI3K activity. RAW 264.7 cells were treated in the presence or absence of 50 μM LY294002 for 1 h and then with 1 nM E₂ for 5, 10, or 20 min. Cell lysates were immunoprecipitated with anti-p85 antibody (lower panel), and PI3K activity was assayed. The upper panel shows a representative autoradiogram of a thin-layer chromatography analysis of the migration of labeled PIP3. (E) Gene transcription inhibition by actinomycin D. MIP-2 mRNA levels in RAW 264.7 cells treated with actinomycin D (5 μg/ml) for 1 h and then with LPS for 1 h were assessed by RT-PCR.