This project focuses on the transcriptional control of the dynorphin gene, which codes for the dynorphin family of opioid peptides. We have shown that, during peripheral inflammation, dynorphin gene expression is greatly increased in spinal cord neurons where it plays a role in modulating chronic pain. Transient transfection data using in vitro cell lines indicates that the dynorphin gene is activated via the cAMP second messenger system, less so by nerve growth factor, and minimally by the phosphatidyl inositol pathway. These results imply that a transmitter in primary afferent neurons stimulates adenylate cyclase in second order neurons. Thus, some of the neuronal hyperexcitability changes that accompany chronic pain may be maintained by cAMP-dependent events and may be counteracted by increased release of dynorphin peptides. We have functionally characterized the region surrounding the dynorphin AP-1-like (DAP) site with transient transfection assays. This site was previously characterized biochemically as a site that binds several transcription factor protein complexes, one of which is the Fos-containing AP-1 complex. A 41 bp synthetic oligonucleotide centered on the DAP element and one containing a 2 bp mutation, were placed directly upstream of the dynorphin minimal promoter. Cells stimulated with forskolin, produced a dose-related increased (up to 150-fold) in chloramphenacol acetyl transferase gene expression which was nearly eliminated by the mutation. The element is located at -1546 from the transcription start site. With longer constructs only those that contain the DAP element display forskolin induction. Through examination of additional control elements we cloned a protein (UreB1) which specifically binds at bases -208 to - 216. Phosphorylation of UreB1 at a tyrosine kinase consensus enhances binding and in vitro transcription. UreB1 is one of the few factors identified that utilize tyrosine phosphorylation to control activity. These studies elucidate the pivotal role of the spinal dynorphin system in pain mechanisms and may provide new avenues for the pharmacotherapy of pain and insights into chronic opioid use and tolerance.
