The Signal Transduction Pathway Underlying Ion Channel Gene Regulation by Sp1-c-Jun Interactions

Irena N Melnikova,Paul D Gardner

doi:10.1074/jbc.m010735200

Abstract

During neuronal differentiation, an exquisitely controlled program of signal transduction events takes place, leading to the temporally and spatially regulated expression of genes associated with the differentiated phenotype. A critical class of genes involved in this phenomenon is that made up of genes encoding neurotransmitter-gated ion channels that play a central role in signal generation and propagation within the nervous system. We used the well established PC12 cell line to investigate the molecular details underlying the expression of the neuronal nicotinic acetylcholine receptor class of ion channels. Neuronal differentiation of PC12 cells can be induced by nerve growth factor, leading to an increase in neuronal nicotinic acetylcholine receptor gene expression. Nerve growth factor initiates several signal transduction cascades. Here, we show that the Ras-dependent mitogen-activated protein kinase and phosphoinositide 3-kinase pathways are critical for the nerve growth factor-mediated increase in the transcriptional activity of a neuronal nicotinic acetylcholine receptor gene promoter. In addition, we show that a component of the Ras-dependent mitogen-activated protein kinase pathway, nerve growth factor-inducible c-Jun, exerts its effects on receptor gene promoter activity most likely through protein-protein interactions with Sp1. Finally, we demonstrate that the target for nerve growth factor signaling is an Sp1-binding site within the neuronal nicotinic acetylcholine receptor gene promoter.

Highlights

Nerve growth factor (NGF)1 is critical for the survival and differentiation of sensory and sympathetic neurons in the peripheral nervous system [1] and of basal forebrain and hippocampal cholinergic neurons in the central nervous system [2]
To investigate which of the NGF-activated pathways is involved in the transcriptional regulation of neuronal nicotinic acetylcholine receptors (nAChRs) gene expression in PC12 cells, we examined the effects of blocking several of these pathways on the transcriptional activity of the ␤4 gene promoter
Inhibitors of phospholipase C, U-73122, and protein kinase C, calphostin C, and bisinodylmalemide, had little effect on the activity of the ␤4 promoter in transfected cells in response to NGF (Fig. 1). These results suggest that activation of the ␤4 promoter in response to NGF is mediated in part via the mitogen-activated protein kinase (MAPK)- and phosphoinositide 3-kinase (PI3-K)-dependent pathways in PC12 cells

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Transfections—SN17 cells [26] and Drosophila melanogaster Schneider SL2 cells were maintained as described [27, 28]. PC12 cells [29] were cultured and differentiated with NGF (Upstate Biotechnology, Lake Placid, NY) as previously described [25]. Transfections were performed by a calcium phosphate method using a commercially available kit (Eppendorf-5 Prime, Inc., Boulder, CO). SL2 cells were transfected as previously described [28], except that 100 ng of the effector DNAs (pActSp1 or pPacJun) were used. All transfections were done a minimum of two times with two different preparations of plasmid DNAs. To correct for differences in transfection efficiencies between dishes, the luciferase activity in each sample was normalized to the ␤-galactosidase activity in the same sample, which was measured using a commercially available kit (Galacto-Light; Tropix, Inc., Bedford, MA). Nuclear extracts were prepared by the method of Dignam et al [37] as previously described [38], except that nuclear extracts were dialyzed against RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate)

RESULTS

Regulation of Acetylcholine Receptor Gene Expression

DISCUSSION