BASAL AND INDUCIBLE TRANSCRIPTIONAL ACTIVITY OF AN UPSTREAM AP-1 CRE ELEMENT (DYNCRE3) IN THE PRODYNORPHIN PROMOTER/

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 t hat Fos and non-Fos-containing complexes formed with oligonucleotides containing this element. To examine the functional significance of thi s site, constructs containing variable length regions of the prodynorp hin promoter were transiently transfected into PC12 or HeLa cells. Con structs containing the DYNCRE3 site consistently permitted higher leve ls of transcriptional activity than those lacking this site. Furthermo re, 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 transf ected with constructs containing the DYNCRE3 site responded to a far g reater degree to forskolin stimulation than those transfected with con structs that did not contain this site. Mutation of the DYNCRE3 site ( CTcgtca) markedly reduced forskolin-induced increases in transcription al activity. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate pr oduced little or no change in transcriptional activity. By examining s uccessively more isolated fragments of prodynorphin promoter and by mu tational analysis, we identify and characterize a 7-bp site, DYNCRE3, which, though largely unaffected by stimulations of the PKC pathway, d ramatically responds to stimulations via the PKA second messenger path way. (C) 1994 Academic Press, Inc.