Four NPY receptor subtypes have been cloned, and shown to be coupled to both Ca2+ and cAMP. However, very little is known about the downstream elements mediating NPY actions. It has recently been demonstrated in our laboratory that intrahypothalamic (i.h.t.) 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 Ca2+ 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 NPY-induced reporter gene regulation in SK-N-BE2 cells is mediated by intracellular Ca2+ and
CREB protein.