AP-1 DNA-BINDING ACTIVITY-INDUCED BY HYPEROSMOLALITY IN THE RAT HYPOTHALAMIC SUPRAOPTIC AND PARAVENTRICULAR NUCLEI

Immediate early gene products (c-fos, c-jun and their cognates) act as transcription factors coupling physiologically relevant stimuli to lo ng-term responses by binding to the AP-1 site in the promoter region o f tar et genes. The induction of c-Sos has been identified in the para ventricular (PVN) and supraoptic (SON) hypothalamic magnocellular nucl ei after hyperosmotic stimulation by using in situ hybridization and i mmunocytochemistry. In this study, AP-1 DNA binding activity, an indic ator of the functional form of the c-Sos transcription factor, was exa mined in nuclear extracts prepared from these brain regions using an e lectrophoretic mobility shift assay and a labeled oligonucleotide cont aining the AP-1 consensus sequence. Two hours after hypertonic saline injection (i.p.), rats were killed and nuclear proteins were extracted from tissue punches of brain regions to assess AP-1 binding activity. Hyperosmolality induced an increase of AP-1 binding activity in nucle ar protein from SON and PVN, but not striatum. This binding was compet itively displaced by excess unlabeled AP-1 oligonucleotide whereas add ition of increasing amounts of unlabeled SP-1 oligonucleotide (promote r site on housekeeping genes for the ubiquitous SP-I transcription fac tor) did not decrease the binding. The binding protein was shown to co ntain c-Fos/Fra and c-Jun since addition of c-Fos/Fra antiserum formed a supershift of the DNA, protein and antibody complex, and c-Jun anti body blocked the protein DNA binding. These data suggest that hyperosm olality leads to a selective and specific increase in AP-I DNA binding activity which may be responsible for regulating secondary target gen e expression in the hypothalamic SON and PVN.