The major CRE in the c-fos gene is necessary for its activation in response to parathyroid hormone (PTH) treatment in UMR 106-01 rat osteosarcoma cells as determined through transient transfection of mouse c-fos 5'-deletion constructs. This CRE binds protein(s) from these cells which include CREB. We now provide further evidence indicative of a role for phosphoCREB and the major CRE in PTH activation of c-fos. To directly assess the importance of the c-fos major CRE, we have mutated this element in the largest of our c-fos promoter constructs (-356/os-CAT). This construct was transiently transfected into UMR 106-01 ceils and treated with PTH (1fr1 M). The mutation reduced basal expression to 10% of wild type. Most significantly, PTH inducibility was substantially decreased from 2.7 to 1.5 fold stimulation. Gel mobility shift confirmed that this mutation prevents CREB binding to this CRE. CREB involvement in c-fos promoter activity is directly addressed by cotransfection of the dominant inhibitor KCREB with the c-fos deletion constructs. KCREB expression substantially (average 46%) inhibits induction of all PTH-activatable promoter constructs. Of primary importance, PTH treatment causes phosphorylation of CREB protein in our cells with a time course and PTH dose dependency that parallels previously measured protein kinase A Induction. These data support our hypothesis that PTH-induced phosphoCREB binds the major CRE in the c-fos 5' regulatory region then activates transcription of this gene in UMR 106-01 cells.
|Original language||English (US)|
|State||Published - 1996|
ASJC Scopus subject areas
- Molecular Biology