MOLECULAR MECHANISMS OF STRESS-INDUCED PROENKEPHALIN GENE-REGULATION - CREB INTERACTS WITH THE PROENKEPHALIN GENE IN THE MOUSE HYPOTHALAMUSAND IS PHOSPHORYLATED IN RESPONSE TO HYPEROSMOLAR STRESS

We have established a transgenic model to facilitate the study of stre ss-induced gene regulation in the hypothalamus. This model, which uses a human proenkephalin-beta-galactosidase fusion gene, readily permits anatomic and cellular colocalization of stress-regulated immediate ea rly gene products (e.g. Fos) and other transcription factors [e.g. cAM P response element-binding protein (CREB)] with the product of a poten tial target gene. Moreover, Fos provides a marker of cellular activati on that is independent of the transgene. Hypertonic saline stress indu ced Fos in almost all cells in the PVN that exhibited basal expression of the proenkephalin transgene; however, all cells in which the trans gene was activated by stress also expressed Fos. CREB was found in ess entially all neurons. Gel shift analysis with and without antisera to Fos and CREB showed that AP-1 binding activity, containing Fos protein , was induced by hyperosmotic stress. However, Fos was not detected bi nding to the proenkephalin second messenger-inducible enhancer even in hypothalamic cell extracts from stressed animals. In contrast, CREB f ormed specific complexes with both the proenkephalin enhancer and a cA MP- and calcium-regulated element (CaRE) within the c-fos gene. Moreov er, we found that hypertonic saline induced CREB phosphorylation in ce lls that express the transgene within the paraventricular nucleus and supraoptic nucleus. These results suggest a model in which proenkephal in gene expression in the paraventricular nucleus is regulated by CREB in response to hypertonic stress.