Identification of overlapping AP-2/NF-κB-responsive elements on the rat cholecystokinin gene promoter

In this study we evaluate both proximal and more distal transcriptional regulation of the 5' flanking region of the rat cholecystokinin gene in transfected GH3 (rat pituitary tumor) cells. Transcriptional activity was measured on the intact (-400 to +73) 5' flanking region of cholecystokinin (CCK), as well as with DNA constructs, which were deleted in both the conventional 5' to 3', as well as an unconventional 3' to 5' direction. Our in vivo studies indicate complex phorbol ester and forskolin interactions in the 10-base pair region between - 130 and -140. We conclude, there are at least two transcriptional factors involved in regulation of the rat CCK transcription in this region. In vitro studies utilizing heterologous nuclear (HeLa) extract, as well as purified transcription factors AP-2 and NF-KB, identify overlapped AP-2- and NF-KB-responsive elements within the 17-base pair sequence between -149 and -134 of the distal 5' flanking region. In this region complex transcriptional regulation occurs, which indicates inhibition of AP-2 CCK promoter complexing by NF-KB. Six-point mutations introduced into this sequence prevent AP-2 and NF-KB binding to CCK promoter, as well as its transcriptional activation by phorbol ester and forskolin in GH3 cells.