{'fraction_non_alphanumeric': 0.029841467205470935, 'fraction_numerical': 0.01274479328566988, 'mean_word_length': 4.850909090909091, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Effect of cAMP elevation on the NPY gene transcription.

EFFECT OF cAMP ELEVATION ON THE NPY GENE TRANSCRIPTION. A. Pance, D. Balbi, N. Holliday and J.M. Allen. Physiological Laboratory, Downing St., Cambridge, CB2 3EG. Neuropeptide Y (NPY) is a 36 amino acid peptide which has widespread distribution in the mammalian central nervous system [ 11. This neuropeptide has been implicated in the regulation of many important physiological processes. A knowledge of the molecular mechanisms by which the expression of this peptide is regulated is important for understanding its role in neuronal differentiation and plasticity. NPY expression in adrenal medulla and post-ganglionic sympathetic nerves is induced by several factors, including vasoactive intestinal peptide (VIP). We have previously shown that the effect of VIP is almost entirely mediated via activation of adenylate cyclase in PC12 cells [2]. These cells are derived from a rat phaeochromocytoma and resemble immature chromaffin cells. The genomic structure of the rat NPY gene has been determined [ 3 ] and consensus sequences for various transcription factors have been found in the promoter region of the gene. Although no classic cAMP response elements are present, a number of sequences recognized by specific transcription factors have been identified, including an AP-I and two AP-2 sites [4]. A reporter function was used to study transcriptional control of the NPY gene. The promoter region of the NPY gene (700 bp) was fused to the chloramphenicol acetyl transferase (CAT) gene and transiently expressed in PC12 cells. Levels of CAT activity in cell extracts were determined to estimate promoter activity. Consensus sequences were specifically deleted using oligonucleotide directed mutagenesis in order to study their contribution to the transcriptional control of the gene, in particular that excerted by elevation of CAMP. In this way, three constructs were made in which either the AP-I, both AP-2 or all three sites were deleted. Specific activation of adenylate cyclase by forskolin in PC12 cells transfected with the wild type promoter produced a 7 fold activation (Fig. 1). A significant reduction of this effect was achieved after deletion of the two AP-2 sites, or all sites (4 fold rise in both cases). This coincides with studies in other systems, where AP-2 sites were found to mediate responses to cAMP [ 5 ] . In contrast, the effect of forskolin was only marginally affected by the removal of the AP-I sequence (6 fold induction). This indicates that the AP-1 site has minimal control of the NPY gene transcription in response to adenylate cyclase activation. The two AP-2 sites contribute significantly to this regulation, but additional sequences in the W Y 5' flanking region are involved. The contribution of two major signalling pathways to the forskolin response was assessed using the highly specific inhibitors Calphostin C and H-89, which target PKC and PKA respectively. In PC12 cells transfected with the wild type NPY promoter, theeffect of forskolin was markedly redwed (80%) by H-89, but only attenuated (30%) by Calphosiin C (Fig. 2). This indicated that the forskolin response is mediated mainly by PKA, although