IDENTIFICATION OF A FUNCTIONAL CAMP RESPONSE ELEMENT IN THE SECRETOGRANIN-II GENE

Secretogranin II is an acidic secretory protein with a widespread dist ribution in secretory granules of neuronal and endocrine cells. The se cretogranin II gene contains, like other members of the granin family, a cAMP response element (CRE) in its upstream region. To investigate the functional significance of this motif, intracellular cAMP levels w ere increased in a neuronal cell line derived from the septal region o f the brain and the level of secretogranin II gene expression was anal ysed. It was found that increased cAMP levels did, in fact, induce sec retogranin II gene expression. To analyse the cis-acting sequence resp onsible for this induction, a hybrid gene containing the upstream regi on of the mouse secretogranin II gene fused to beta-globin as a report er was constructed. Transfection analysis revealed that cAMP-induced t ranscription of the secretogranin II promoter/beta-globin gene in sept al and insulinoma cells. DNA-protein binding assays showed that recomb inant CRE-binding protein (CREB), produced in bacteria or human cells, bound in a sequence-specific manner to the secretogranin II promoter CRE. Moreover, deletion mutagenesis revealed that the CRE motif is a b ifunctional genetic regulatory element in that it mediates basal as we ll as cAMP-stimulated transcription. Interestingly, cAMP had no effect upon secretogranin II gene transcription in PC12 and neuroblastoma ce lls. An increase in the intracellular cAMP concentration activated a G ALA-CREB fusion protein upon transcription in neuroblastoma cells indi cating the integrity of the cAMP signaling pathway to the nucleus. Bas al as well as cAMP-stimulated transcription, directed from the secreto granin II promoter was, however, impaired in insulinoma cells by overe xpression of CREB-2, a negative-acting CRE-binding protein. These resu lts indicate that competitive effects are likely to occur between CRE- bound transcriptional activators and repressors. We conclude that cAMP -stimulated induction of secretogranin II gene transcription is mediat ed by the CRE motif in a cell-type-specific manner, and is likely to d epend on the balance between positive and negative CRE-binding protein s in a particular cell type.