Role of the AP‐1 binding site in regulating PKCε promoter activity in the fetal heart

Abstract

To determine whether the AP-1 binding site is involved in regulating PKCε promoter activity, the activities of 5′ deletion mutants of a 2000-bp PKCε promoter region were evaluated in the rat embryonic ventricular myocyte cell line H9C2. The levels of basal full-length promoter activity obtained with pPKCε2k-Luc was at least 15-fold greater than the vector alone in a high glucose medium. Basal promoter activity fell by 42% with a deletion from −2000 to −398, which contains the AP-1 binding site, and it fell 92% with a deletion from −2000 to −361, which lacks the AP-1 binding site. Electrophoretic mobility shift assays with oligonucleotide probes encompassing the putative AP-1 site showed a major DNA-protein complex using nuclear extracts from fetal rat hearts. Super-shift analyses showed that c-Jun, but not c-Fos or Fra-1, antibody caused a super-shifting of the AP-1 DNA-protein complex. In addition, chromatin immunoprecipitation assays demonstrated a binding of AP-1/c-Jun to the AP-1 binding site in the promoter region of the PKCε gene in intact chromatin in vivo in the fetal heart. The results indicate a strong stimulatory role of AP-1 (c-Jun homodimer) binding in PKCε promoter activity in the fetal heart. (Supported in part by NIH grant HL82779).