MPA modulates cyclin D1 expression via AP-1. (A) Cells were transfected with a cyclin D1 promoter luciferase construct containing the −954 TRE and with a construct with a point mutation in the TRE (−963 mut AP-1). When indicated, cells were co-transfected with TAM-67 or A-Fos and were then treated with MPA or pretreated with RU or U0 before MPA stimulation. As control of PR transcriptional activity cells were transfected with a PRE-Luc plasmid and stimulated with MPA. Results are presented as fold induction of luciferase activity with respect to cells untreated with MPA. Data represent the mean of three independent experiments for each cell type ± SEM. For b vs. a, and c vs. b: P <0.001. pA3 Luc, empty vector. MPA induces cyclin D1 expression at protein and mRNA levels via AP-1. Cells were transfected with TAM-67 and A-Fos vectors (B) and with c-Jun and c-Fos siRNAs (C) and then treated with MPA. Cyclin D1 protein expression was analyzed by WB. (D) Cyclin D1 mRNA expression levels were determined by RT-qPCR. The fold change of mRNA levels upon MPA treatment was calculated by normalizing the absolute levels of cyclin D1 mRNA to GAPDH levels, which was used as internal control, and setting the value of untreated cells as 1. Experiments shown were repeated three times with similar results. MPA, Medroxyprogesterone acetate.