Curcumin prevented the binding of AP-1 to the functionally important AP1-1 site carried in the S100A7 promoter region.

Biotin-labelled DNA fragments compatible with the sequence as -587 to -568 nucleotides of S100A7 promoter carrying either wild-type or mutated AP1-1 binding site were used to attach AP-1 complexes contained in the nuclear extracts prepared from HaCaT cells pre-incubated with PMA (lane(s) 2 and 6), curcumin (lane(s) 3 and 7) or combinations (lane(s) 4 and 8). The DNA-AP-1 complexes were immunoprecipitated by anti-c-Fos antibody than further processed for western blotting analysis since AP1-1 site acts as the predominant functional site for c-Jun/c-Fos to activate S100A7 promoter. (A) The presence of c-Fos at the wild-type AP1-1site was analyzed by western-blotting using anti-c-Fos antibody. Shown in lane(s) 1–4 are the 5% input of elute derived for each indicated condition. The amount of c-Fos recruited to the AP1-1 site is increased by the AP-1 stimulant PMA (comparing lane 6 to lane 1) but reduced by curcumin (compare lane 7 to lane 5). Moreover, the stimulatory effect derived from PMA treatment is also inhibited by curcumin (compare lane 8 to lane 6). (B) The presence of c-Fos at the mutated AP1-1site was also analyzed. Obviously, the recruitment of AP-1 is abolished by the mutagenesis performed on AP1-1 site (compare lane(s) 5–8 to lane 1~4). (C) The two PCR products from respective primer pairs covered the DNA fragment containing two AP-1 sites and indicated control region of endogenous S100A7 promoter were analyzed in the ChIP analysis. The different effect of curcumin were found using the respective antibody against different subunit of AP-1 complex, such as phosphorylated c-Fos and c-Jun. The results indicated more c-Jun-containing complexes will be recruited by curcumin for AP-1 sites occupancy in the endogenous S100A7 promoter. Results (A, B and C) are representative of two independent experiments.