Ca²⁺/Calmodulin‐Dependent Transcriptional Activation of Neuropeptide Y Gene Induced by Membrane Depolarization: Determination of Ca²⁺‐ and Cyclic AMP/Phorbol 12‐Myristate 13‐Acetate‐Responsive Elements

Abstract

Membrane depolarization stimuli (high potassium concentration and veratridine) increased neuropeptide V (NPY) mRNA abundance time-dependently, without a change in~-actin mRNA level, in NG1O8-15 and PC12 cells. Although the induction by veratridine was blocked completely by tetrodotoxin, the induction by potassium was suppressed minimally. Voltage-dependent Ca channel blockers and calmodulin antagonists inhibited the increases by both depolarization stimuli completely, suggesting involvement of Ca2Vcalmodulin-dependent kinases (CaM kinases). Transient assay using chioramphenicol acetyltransferase reporter genes containing the rat NPY gene promoter indicated that membrane depolarization and Ca entry stimulate transcription of the NPY gene. The depolarization-induced transactivation was also blocked by CaM kinase inhibitors. The 200-bp 5'upstream region (-344/-145) was localized as a calmodulin-responsive element (CaMRE), which confers depolarization-induced transactivation. It is interesting that this CaMRE did not contain the canonical Ca-responsive elements such as CRE, SRE, NF-AT, or the C/EBP~3binding site and was separated from a 64-bp cyclic AMP! phorbol 1 2-myristate 13-acetate-responsive element (-1441-81). These findings suggested that membrane depolarization regulates the NPY gene transcription positively through the unique CaMRE by activation of CaM kinases following Ca entry through L-type Ca channels.

Transcriptional Regulation of Neuronal Genes and Its Effect on Neural Functions: Transcriptional Regulation of Neuropeptide Y Gene by Leptin and Its Effect on Feeding

“…To confirm the leptin-induced transactivation of NPY gene in neural cell lines, we used mouse N18TG2 neuroblastoma cells for transient transfection with NPYluc reporter containing the 2.1 kilobase (kb) 5'-flanking region of rat NPY gene promoter (−2.1 k/ +18) that confers the neural tissue-specific expression (20). Long form of leptin receptor (OB-Rb) expression plasmid was co-transfected because OB-Rb was not expressed in N18TG2 cells by RT-PCR (data not shown).…”

“…Leptin-induced transactivation is a discrete phenomenon from depolarization-induced transactivation Because the NPY gene is transactivated by membrane depolarization following increase in neural activity (20), leptin-induced transactivation of the NPY gene might be due to membrane depolarization that was induced by activation of ATP-sensitive potassium channels by leptin (24,25). To investigate whether the activation of NPY gene promoter by leptin was due to the increase in membrane potential, the treatment with KCl was studied.…”

“…The NPY gene is regulated by many signals at the transcriptional level (19,20). In contrast to the prediction that leptin might reduce the NPY gene transcription, it has been reported that leptin transactivates the NPY gene and induces production of NPY peptides in rat pheochromocytoma PC12 cells and human neuroblastoma SH-SY5Y cells (21,22).…”

Activated Transcription of the Human Neuropeptide Y Gene in Differentiating SH‐SY5Y Neuroblastoma Cells Is Dependent on Transcription Factors AP‐1, AP‐2α, and NGFI

“…transcribed in response to membrane depolarization in PC12 cells (Higuchi et al, 1996). Both transcriptional and posttranscriptional mechanisms appear to be operative for maximal NPY gene expression in PC12 cells in response to combined treatment with phorbol ester and forskolin.…”

Feeding behavior and gene expression of appetite-related neuropeptides in mice lacking for neuropeptide Y Y5 receptor subclass

“…We have been studying the regulatory mechanism of NPY gene expression in hypothalamus, so as to elucidate the mechanism of feeding-related regulation of NPY gene expression [8][9][10][11] . Leptin is an anti-obesity hormone derived from adipose tissue and reduces the NPY gene expression in hypothalamus.…”