The CCAAT-binding factor CBF/NF-Y regulates the human acetylcholinesterase promoter activity during calcium ionophore A23187-induced cell apoptosis

We previously reported that the expression of acetylcholinesterase during A23187-induced apoptosis of HeLa cells is regulated by Ca(2+) mobilization through the modulation of mRNA stability and acetylcholinesterase promoter activity. Transactivation of the human acetylcholinesterase promoter by A23187 was partially mediated by the distal CCAAT motif within the -1270 to -1248 fragment of the human acetylcholinesterase promoter, which was bound by the CCAAT binding factor (CBF/NF-Y). In the present study, we investigated the molecular mechanisms by which CBF/NF-Y regulates A23187-induced activation of the human acetylcholinesterase promoter. The results indicate that CBF/NF-Y binding to the distal CCAAT motif suppresses the promoter activity. Electrophoretic mobility shift assays (EMSAs) demonstrated that binding of CBF/NF-Y to the distal CCAAT motif decreased after A23187 treatment. Our results suggest that acetylcholinesterase promoter activation during A23187-induced HeLa cell apoptosis may result partly from the dissociation of CBF/NF-Y from the distal CCAAT motif in the acetylcholinesterase promoter, reversing this suppression.     

17beta-estradiol (E2) induces cdc25A gene expression in breast cancer cells by genomic and non-genomic pathways

Cdc25A is a potent tyrosine phosphatase that catalyzes specific dephosphorylation of cyclin/cyclin-dependent kinase (cdk) complexes to regulate G1 to S-phase cell cycle progression. Cdc25A mRNA levels are induced by 17beta-estradiol (E2) in ZR-75 breast cancer cells, and deletion analysis of the cdc25A promoter identified the -151 to -12 region as the minimal E2-responsive sequence. Subsequent mutation/deletion analysis showed that at least three different cis-elements were involved in activation of cdc25A by E2, namely, GC-rich Sp1 binding sites, CCAAT motifs that bind NF-Y, and E2F sites that bind DP/E2F1 proteins. Studies with inhibitors and dominant negative expression plasmids show that E2 activates cdc25A expression through activation of genomic ERalpha/Sp1 and E2F1 and cAMP-dependent activation of NF-YA. Thus, both genomic and non-genomic pathways of estrogen action are involved in induction of cdc25A in breast cancer cells.     

Regulation of acetylcholinesterase expression by calcium signaling during calcium ionophore A23187- and thapsigargin-induced apoptosis

We have recently reported that acetylcholinesterase expression was induced during apoptosis in various cell types. In the current study we provide evidence to suggest that the induction of acetylcholinesterase expression during apoptosis is regulated by the mobilization of intracellular Ca(2+). During apoptosis, treatment of HeLa and MDA-MB-435s cells with the calcium ionophore A23187 resulted in a significant increase in acetylcholinesterase mRNA and protein levels. Chelation of intracellular Ca(2+) by BAPTA-AM (1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester), an intracellular Ca(2+) chelator, inhibited acetylcholinesterase expression. A23187 also enhanced the stability of acetylcholinesterase mRNA and increased the activity of acetylcholinesterase promoter, effects that were blocked by BAPTA-AM. Perturbations of cellular Ca(2+) homeostasis by thapsigargin resulted in the increase of acetylcholinesterase expression as well as acetylcholinesterase promoter activity during thapsigargin induced apoptosis in HeLa and MDA-MB-435s cells, effects that were also inhibited by BAPTA-AM. We further demonstrated that the transactivation of the human acetylcholinesterase promoter by A23187 and thapsigargin was partially mediated by a CCAAT motif within the -1270 to -1248 fragment of the human acetylcholinesterase promoter. This motif was able to bind to CCAAT binding factor (CBF/NF-Y). These results strongly suggest that cytosolic Ca(2+) plays a key role in acetylcholinesterase regulation during apoptosis induced by A23187 and thapsigargin.