Basal and Inducible Transcriptional Activity of an Upstream AP-1/CRE Element (DYNCRE3)in the Prodynorphin Promoter

Abstract

During chronic pain and inflammation, prodynorphin gene expression is elevated in the spinal cord. To characterize the molecular regulation of prodynorphin gene expression, we examined an AP-1/CRE-like element, TGCGTCA, located at -1545 in the prodynorphin gene (the DYNCRE3 site). Previous work in our laboratory demonstrated by gel shift analysis that Fos and non-Fos-containing complexes formed with oligonucleotides containing this element. To examine the functional significance of this site, constructs containing variable length regions of the prodynorphin promoter were transiently transfected into PC12 or HeLa cells. Constructs containing the DYNCRE3 site consistently permitted higher levels of transcriptional activity than those lacking this site. Furthermore, placement of upstream regions containing the DYNCRE3 site adjacent to the minimal promoter yielded transcriptional activity much greater than that in the presence of the native constructs. PC12 cells transfected with constructs containing the DYNCRE3 site responded to a far greater degree to forskolin stimulation than those transfected with constructs that did not contain this site. Mutation of the DYNCRE3 site (CTcgtca) markedly reduced forskolin-induced increases in transcriptional activity. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate produced little or no change in transcriptional activity. By examining successively more isolated fragments of prodynorphin promoter and by mutational analysis, we identify and characterize a 7-bp site, DYNCRE3, which, though largely unaffected by stimulations of the PKC pathway, dramatically responds to stimulations via the PKA second messenger pathway.