NPY upregulates genes containing cyclic AMP response element in human neuroblastoma cell lines bearing Y1 and Y2 receptors: involvement of CREB

Four NPY receptor subtypes have been cloned, and shown to be coupled to both Ca 2+ and cAMP. However, very little is known about the downstream elements mediating NPY actions. It has recently been demonstrated in our laboratory that intrahypothalamic (IHT) administration of NPY induces hypothalamic CaM kinase activity, cyclic AMP response element binding protein (CREB) phosphorylation and cyclic AMP response element (CRE) binding activity in rat hypothalamic nuclear proteins. In the present study, we have investigated whether these changes in CRE binding transcriptional factors activated by NPY results in gene regulation using a human neuroblastoma cell line (SK-N-BE2). This cell line which expresses the Y2 subtype of NPY receptors was transfected with a fusion gene containing 1.305 kb of human CRF 5′ flanking region with a perfect palindromic CRE site linked to firefly luciferase gene. NPY treatment increased CaM kinase II activity, CREB phosphorylation and CRE binding in these cells. In transfected cells, luciferase activity was also increased by NPY (1.8–4-fold) within 4 h of treatment. Moreover, forskolin (7–30-fold), which stimulates cAMP production, and thapsigargin (6–8-fold), which mobilizes intracellular calcium, also increased luciferase activity within 4 h of treatment. PMA (phorbol-12-myristate-13-acetate), an activator of protein kinase-C, induced luciferase activity by 1.8-fold. NPY augmented forskolin-stimulated luciferase activity from 11- to 15-fold, but had no significant effect on thapsigargin-induced luciferase activity. These findings suggest that activation of protein kinase A (PKA) or CaM kinase leads to the induction of fusion gene. NPY treatment upregulated fusion gene expression through Ca 2+ pathway in SK-N-BE2 cell line. Pretreatment with CREB antisense, but not the sense oligodeoxynucleotides, inhibited forskolin-, thapsigargin- and NPY-stimulated luciferase activity. However, CREB sense or antisense oligodeoxynucleotide treatment had no effect on PMA-stimulated luciferase activity. Furthermore, NPY induced CRE binding activity and the expression of CRE containing Y1 receptor gene in SK-N-MC cell line. These findings suggest that NPY can upregulate CRE containing reporter gene including Y1 receptor gene and NPYinduced reporter gene regulation in SK-N-BE2 cells is mediated by intracellular Ca 2+ and CREB protein.