CELL-TYPE-SPECIFIC REGULATION OF CREB GENE-EXPRESSION - MUTATIONAL ANALYSIS OF CREB PROMOTER ACTIVITY

Previous studies have shown that activation of the cyclic AMP (cAMP) p athway down-regulates CREB expression in CATH. a cells, an effect that appears to be mediated via inhibition of CREB gene transcription. In the current study, we compared this effect in CATH. a cells with regul ation of CREB expression in another cell line, C6 glioma cells, In con trast to the findings in CATH,a cells, activation of the cAMP pathway up-regulates CREB expression in C6 glioma cells. To determine whether these opposite effects can be explained by regulation of CREB promoter activity, chloramphenicol acetyltransferase (CAT) assays were perform ed in CATH,a and C6 glioma cells that were transiently transfected wit h a CREB promoter-CAT fusion plasmid, Activation of the cAMP pathway d ecreased levels of CAT activity in transfected CATH. a cells but incre ased CAT activity in transfected C6 glioma cells, We next investigated the effect of mutations in the CREB promoter on such regulation in th ese two cell lines. Mutations of single CRE or Spl binding sites in th e CREB promoter reduced basal levels of CAT activity but did not signi ficantly attenuate regulation of the promoter in CATH,a or C6 glioma c ells. However, mutation or deletion of two CRE sites in the CREB promo ter completely abolished up-regulation of CAT activity in the C6 gliom a cells and abolished basal levels of CAT activity in CATH. a cells. C REB promoter activity was also studied in cultured SHSY5Y cells and in primary cultures of striatal neurons as further comparisons. Activati on of the cAMP pathway was found to increase CAT activity in both cell types. In the striatal cultures, this effect was obliterated by mutat ion or deletion of either of the two CREs in the promoter. These findi ngs demonstrate cell type-specific effects of the cAMP pathway on CREB expression, which appear to be mediated via differential regulation o f the CREB promoter.