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 long-term responses by binding to the AP-1 site in the promoter region of target genes. The induction of c-fos has been identified in the paraventricular (PVN) and supraoptic (SON) hypothalamic magnocellular nuclei after hyperosmotic stimulation by using in situ hybridization and immunocytochemistry. In this study, AP-1 DNA binding activity, an indicator of the functional form of the c-fos transcription factor, was examined in nuclear extracts prepared from these brain regions using an electrophoretic mobility shift assay and a labeled oligonucleotide containing 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 nuclear protein from SON and PVN, but not striatum. This binding was competitively displaced by excess unlabeled AP-1 oligonucleotide whereas addition of increasing amounts of unlabeled SP-1 oligonucleotide (promoter site on housekeeping genes for the ubiquitous SP-1 transcription factor) did not decrease the binding. The binding protein was shown to contain 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 antibody blocked the protein DNA binding. These data suggest that hyperosmolality leads to a selective and specific increase in AP-1 DNA binding activity which may be responsible for regulating secondary target gene expression in the hypothalamic SON and PVN.
Ying
Z
Z
Department of Physiology, School of Medicine, University of South Carolina, Columbia 29208, USA.
Reisman
D
D
Buggy
J
J
D000818:Animals; D017781:Genes, Immediate-Early / Q000235:genetics*; D007031:Hypothalamus / Q000378:metabolism*; D008297:Male; D009994:Osmolar Concentration; D010286:Paraventricular Hypothalamic Nucleus / Q000378:metabolism*; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018808:Transcription Factor AP-1 / Q000378:metabolism*
D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1