From the Center for Molecular Neurobiology and Alzheimer's Disease Research Group, University of Hamburg, Martinistrasse 52, D-20246 Hamburg, Germany
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ABSTRACT |
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In order to search for genes that are activated by muscarinic acetylcholine receptors (mAChRs), we used an mRNA differential display approach in HEK293 cells expressing m1AChR. The zinc-finger transcription factor genes Egr-1, Egr-2, and Egr-3 were identified. Northern blot analyses confirmed that mRNA levels of Egr-1, Egr-2, and Egr-3 increased readily after m1AChR stimulation and that a maximum was attained within 50 min. At that time, Egr-4 mRNA was also detectable. Western blots and electromobility shift assays demonstrated synthesis of EGR-1 and EGR-3, as well as binding to DNA recognition sites in response to m1AChR activation. Activation of m1AChR increased transcription from EGR-dependent promoters, including the acetylcholinesterase gene promoter. Activity-dependent regulation of Egr-1 mRNA expression and EGR-1 protein synthesis was also observed in cells expressing m2, m3, or m4AChR subtypes. Increased EGR-1 synthesis was mimicked by phorbol myristate acetate, but not by forskolin, and receptor-stimulated EGR-1 synthesis was partially inhibited by phorbol myristate acetate down-regulation. Together, our results demonstrate that muscarinic receptor signaling activates the EGR transcription factor family and that PKC may be involved in intracellular signaling. The data suggest that transcription of EGR-dependent target genes, including the AChE gene, can be under the control of extracellular and intracellular signals coupled to muscarinic receptors.
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INTRODUCTION |
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Muscarinic acetylcholine receptors (mAChRs)1 are members of a superfamily of G protein-coupled cell surface receptors with seven transmembrane domain topology. Five subtypes of mAChRs (m1-m5) can be divided according to their signaling mechanisms: m1, m3, and m5 AChRs preferentially couple to the pertussis toxin-insensitive Gq/G11 proteins that stimulate phosphoinositide hydrolysis, whereas m2 and m4 subtypes couple to the Gi/Go proteins that predominantly inhibit adenylyl cyclase (1, 2).
In brain, mAChRs are involved in such functions as attention, learning,
memory, and cognition (3, 4). m1 and m3 AChR subtypes are localized to
the somatodendritic cell surfaces of large pyramidal neurons throughout
the cortex and the hippocampus, as well as on small cholinergic
interneurons in the striatum. In contrast, m2 and m4 AChRs are
predominantly present on axons of the large basal forebrain projection
neurons that innervate cholinergic target cells throughout the cortex
and the hippocampus. Activation of the postsynaptic m1/m3/m5 AChR
family by acetylcholine triggers a large variety of distinct signaling
cascades including phospholipase D, adenylyl cyclase, phospholipase A2,
the generation of diacylglycerol which activates protein kinase C (PKC)
and couples mAChRs to the ERK-MAP-kinase signaling cascade, activation
of endoplasmic reticulum IP3 receptors, stimulation of ligand-operated cell-surface Ca2+ channels, and the activity of
voltage-gated potassium channels (5-12). Cellular responses of mAChRs
include the activation of neurite outgrowth, the fine-tuning of
membrane potentials, and the regulation of mitogenic growth responses
in cells that are not terminally differentiated (13). mAChRs are also
involved in the activity-dependent regulation of
posttranslational processing of the -amyloid precursor protein of
Alzheimer's disease (14-16) by an unidentified protease associated
with reduced generation of
-amyloid peptides (17-19), the principal
component of amyloid plaques in Alzheimer's disease brain (20).
mAChRs can activate the transcription of the immediate-early genes jun and fos (21, 22). In order to identify additional genes that are regulated by mAChRs, we used a differential mRNA display screen of cDNA populations derived from m1AChR expressing cells, with or without receptor stimulation. We identified several differentially expressed clones, including the Egr family of transcription factors. This gene family encodes proteins with a zinc-finger containing DNA binding domain, and includes Egr-1 (23) (also named Krox-24 (24), zif/268 (25), NGFI-A (26), or TIS8 (27)), Egr-2 (28) (also named Krox-20 (29)), Egr-3 (30), and Egr-4 (also named NGFI-C (31), or pAT 133 (32)), as well as the related Wilms' tumor suppressor gene WT1 (33, 34). EGR proteins bind DNA in a sequence-specific manner, recognizing the consensus element GCG(G/T)GGGCG (35, 36), and they can either activate or suppress the transcription of associated genes, depending on the respective promoter (37-42). We show here that the entire family of EGR transcription factors can be under the control of muscarinic acetylcholine receptors and their related signaling mechanisms.
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EXPERIMENTAL PROCEDURES |
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Cell Culture
Human HEK293 cells stably transfected with the muscarinic
acetylcholine receptor subtypes m1, m2, m3 or m4 were grown in
DMEM/F-12 medium supplemented with 10% fetal calf serum and 500 µg/ml geneticin (G418, Life Technologies, Inc.). The cells were
maintained in a 5% CO2 atmosphere at 37 °C. For
experiments, the cells were subcultured onto tissue culture dishes and
grown to an approximate density of 80-90%. 16 h before
experiments, growth media were replaced by serum-free DMEM/F-12 without
G418. Incubation of cells with test substances were performed by adding
concentrated stock solutions of carbachol (Sigma) at a final
concentration of 1 mM, carbachol together with atropine (10 µM, Sigma), phorbol 12-myristate 13-acetate (PMA) (1 µM, Sigma), forskolin (100 µM, Calbiochem), or 8-bromo-cAMP (100 µM, Calbiochem) to the culture
media. Identical cells generated in parallel from the same passage were
used as unstimulated controls. Media were frozen and were used to
ensure effective receptor responses by -amyloid precursor protein
ectodomain release assays (14).
Differential mRNA Display
Total RNA was prepared from the cells by using the RNeasy kit
(Qiagen) according to the manufacturer's instructions. The RNA preparations were subjected to DNaseI digestion (Boehringer Mannheim), in the presence of the RNase inhibitor RNasin (Promega) for 30 min. RNA
was extracted with phenol and precipitated in ethanol. Equal amounts of
0.2 µg of RNA each were transcribed to cDNA with ExpandTM Reverse Transcriptase (Boehringer Mannheim) by
using one-base-anchor primers HT11A, HT11G and
HT11C according to Liang et al. (43). The
obtained cDNAs were subjected to polymerase chain reaction (PCR)
employing the corresponding one-base-anchor oligonucleotides along with
HAP-1 (TGCCGAAGCTTGATTGCC), HAP-3C (TGCCGAAGCTTTGGTCAC), or HAP-3T
(TGCCGAAGCTTTGGTCAT) as random primers with a HindIII restriction site by using PCR conditions described by Zhao et al. (44). PCR was performed with AmpliTaq polymerase (Perkin-Elmer Corp.) in the presence of [-35S]dATP (2000 Ci/mmol,
NEN Life Science Products) along with dCTP, dTTP, and dGTP (Amersham
Pharmacia Biotech). The PCR products were separated on 6%
polyacrylamide urea sequencing gels and dried on Whatman 3MM paper, and
x-ray films (DuPont) were exposed for 8-16 h. Differential bands were
excised, and the contained cDNA was eluted in water, precipitated
with glycogen in ethanol, and dialyzed against 10% glycerol. The
obtained preparations were reamplified by 40 high-stringency cycles in
50-µl PCR mixtures containing the corresponding composite primer
pairs as described by Zhao et al. (44). Reamplified
cDNAs were purified by agarose gel electrophoreses, gel extraction
by using the QIAEX II kit (Qiagen, Hilden, Germany), and were ligated
into pBluescript IIKS+ (Stratagene) by using the HindIII
restriction sites. Cloned cDNA fragments were sequenced on an
automated ABI377 DNA sequencer (Perkin-Elmer Corp.) by using T3- and
T7-derived sequencing primers.
Northern Blot Analysis
5-20 µg of RNA each were separated by agarose
formaldehyde gel electrophoresis and blotted onto nylon membranes
(HybondTM-N+, Amersham Pharmacia Biotech) as described
(45). Membrane filters were probed with [32P]dCTP
random primed cDNA probes prepared from Egr-1, Egr-2, Egr-3, and
Egr-4 cDNA clones or from PCR products obtained in the differential display. Hybridized filters were washed under high stringency conditions and were subjected to autoradiography. Equal RNA loading was
confirmed by probing the identical blots with a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe.
Preparation of Nuclear Extracts
Cells were washed twice with PBS, scraped from the plates in hypotonic buffer, swollen on ice, and lysed with 1% Nonidet P-40. Nuclei were pelleted and extracted in 200 µl of ice-cold 10 mM HEPES (pH 7.9), 400 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 2 mM dithiothreitol, 1% Nonidet P-40 in the presence of pepstatin and aprotinin. Protein concentrations of nuclear extracts were determined by the Bradford protein assay (Bio-Rad).
Western Blot Analysis
50 µg of nuclear protein were separated by SDS-PAGE, transferred to nitrocellulose filters, blocked with 5% nonfat dry milk in PBST, and probed with polyclonal antisera against either EGR-1, EGR-2, or EGR-3 (Santa Cruz Biotechnology) at dilutions of 1:3000. Primary antisera were visualized with horseradish peroxidase conjugated to protein A and by enhanced chemiluminescence (Amersham Pharmacia Biotech) using x-ray films.
Electrophoretic Mobility Shift Assay
A radiolabeled double-stranded oligonucleotide containing two
copies of the consensus binding motif for EGR proteins
(5'-GATCCAGCGGGGGCGAGCGGGGGCGAGATC-3') was prepared with Klenow
polymerase and by using [32P]dCTP. 0.5 ng of the
32P-labeled DNA was incubated with 10 µg of nuclear
protein for 20 min at room temperature in a 20-µl reaction mixture
containing 10 mM HEPES (pH 8.0), 5% glycerol, 100 mM NaCl, 5 mM MgCl2, 2 mM dithiothreitol, 0.1 mM EDTA, 10 µM ZnCl, and 1 µg of poly(dI-dC) as nonspecific
competitor. In some cases, protein extracts were preincubated with
polyclonal antisera against EGR proteins (Santa Cruz Biotechnology).
Protein-DNA complexes were separated from free DNA on 4%
polyacrylamide gels using 0.5× TBE (Tris-borate-EDTA) as running
buffer.
Luciferase Assays
HEK293 cells stably expressing m1AChR, grown on poly-D-lysine, were transfected at 30-40% density with 4 µg of expression constructs precipitated by calcium phosphate. 24-48 h after transfection, cells were serum deprived and then treated with or without test substances; 19 h following stimulation, luciferase activity was quantitated with a luminometer (Lumat LB 9501, Berthold) (46).
EGR Assay--
Expression constructs pTATA-luc/EGR and
pTATA-luc/EGRmut encode an EGR responsive promoter fused to a
luciferase reporter gene. These plasmids were constructed by inserting
one copy of either the EGR binding site
5'-GATCCAGCGGGGGCGAGCGGGGGCGACTAG-3' or a
mutant EGR binding site 5'-
GATCCAGCTAGGGCGAGCTAGGGCGACTAG-3' into pTATA-luc that
includes the luciferase gene under control of the -globin minimal
promoter. pCMV/Krox24 together with either pTATA-luc/EGR or
pTATA-luc/EGRmut was used as a positive control for the
transfection.
AChE Assay-- Expression construct pAChE-luc (construct A) (37) consists of approximately 1.1 kb of DNA sequence upstream of the region similar to the mouse AChE gene exon 1 in addition to exon 1, and 1.5 kb of the putative intronic region between sequences corresponding to exons 1 and 2 in the AChE gene. The 3' end includes the first 16 bases of exon 2 upstream of the ATG start codon.
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RESULTS |
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m1AChR Stimulated Transcription of Egr-1, Egr-2, Egr-3, and Egr-4-- Cloning and sequence analysis of several clones derived from differential bands generated by the reverse transcription primer HT11G along with the PCR primer HAP1, and by the reverse transcription primer HT11A, along with the PCR primers HAP-3C and HAP-3T, revealed the zinc-finger domain transcription factor genes Egr-1 (48), Egr-2 (28), and Egr-3 (30). By using a probe generated from the reamplified differential band corresponding to Egr-1, we identified on Northern blots a message at 3.5 kb that was differentially up-regulated within 10-60 min of m1AChR stimulation. This message was undetectable in RNA preparations obtained from unstimulated cells. To verify that this message corresponded to the zinc-finger transcription factor Egr-1, Northern blot analyses of identical RNA preparations were done with a probe generated from a cDNA fragment of Egr-1. Again, we detected a major transcript at 3.5 kb (Fig. 1A) in the stimulated condition but not in the unstimulated control condition. Within 10 min of stimulation, a weak signal was detectable, and a maximum was attained after 50 min (Fig. 1B). The time course of m1-induced Egr-1 expression was biphasic, and a second peak was observed after 240 min and after a minimum was reached 100 min after receptor stimulation.
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Muscarinic Receptor Stimulation Increases Cellular Levels of Functional EGR-1 Protein-- Stimulation of m1AChR with carbachol readily increased the synthesis of EGR-1 protein within 120 min, and protein levels remained high until 180 min after stimulation (Fig. 3A). Receptor-induced EGR-1 synthesis was completely blocked by atropine, indicating a specific and receptor-mediated action of carbachol. EGR-1 synthesis was also stimulated with PMA, a potent activator of PKC, suggesting that PKC may couple m1AChR to EGR-1 synthesis (Fig. 3A). Down-regulation of PKC by 16 h of PMA pretreatment, however, was associated with only a partial block of the receptor-mediated induction, suggesting additional signaling mechanisms that are not responsive to PMA in the coupling of m1AChR to EGR-1 synthesis in HEK293 cells. To exclude cAMP as a signaling molecule for this response, we demonstrated that forskolin failed to influence EGR-1 synthesis (Fig. 3A). In agreement with the absence of Egr-1 message, no EGR-1 protein was detectable on Western blots of nuclear proteins extracted from unstimulated cells (Fig. 3A).
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m1AChR Activate Transcription via an EGR-Dependent Promoter-- Transfection experiments with luciferase reporter constructs fused to an EGR-dependent minimal promoter demonstrated that both m1AChR and PMA effectively induced transcription from this promoter, suggesting downstream activation of genes that contain EGR responsive promoters (Fig. 6A). Again, the m1-induced response was blocked by atropine, and it was completely absent when control constructs were assayed that contain a mutated EGR binding domain (Fig. 6B). 8-Bromo-cAMP failed to increase luciferase activity from the EGR-dependent promoter, confirming that cAMP is ineffective in its regulation.
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Transcriptional Activity of the AChE Gene Promoter Is Increased by m1AChR Stimulation-- Stimulation of m1AChR in cells transfected with the AChE gene promoter (37, 49) fused to a luciferase reporter gene resulted in an activation of the transcription via the AChE gene promoter, as indicated by increased luciferase activity (Fig. 7A). Atropine blocked the increase in luciferase activity, indicating that this effect was caused specifically by m1AChR activation. Again, PMA mimicked the m1AChR activation of the AChE gene promoter, whereas 8-bromo-cAMP failed to induce transcription. Co-transfection of the pAChE-luc construct together with Krox24 demonstrated that EGR effectively induced the AChE gene promoter in our experimental system (Fig. 7B).
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DISCUSSION |
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In brain, mAChRs are involved in long-term potentiation, synaptic plasticity, and higher cognitive functions, including learning and memory (50-52). Such plastic alterations in neuronal structure and function are associated with rapid and transient transcription of activity-dependent genes (4, 53, 54), such as the immediate-early genes c-fos, jun-B, Egr-1, and Egr-2 (21, 49, 55, 56). The results of our study show that mAChRs can increase the expression of the zinc-finger domain transcription factor genes Egr-1, Egr-2, Egr-3, and Egr-4, albeit to different extents.
Egr-1 message was detectable within 10 min of m1AChR stimulation, a maximum was reached within 50 min, and EGR-1 protein was present within 120 and 180 min of stimulation. m1AChR-induced EGR-1 protein bound to an EGR-specific DNA recognition domain, as evidenced by mobility shift assays. In addition, luciferase reporter assays showed that m1AChR induced transcription from EGR dependent promoters. The carbachol-induced effects on transcriptional regulation were m1AChR-specific, as evidenced by complete atropine blocks, and they were mimicked by direct activation of PKC with PMA. Down-regulation of PKC, however, was associated with only partial inhibitions of the receptor responses, indicating that activation of PMA-dependent forms of PKC can be sufficient, but is not necessary, for coupling m1AChR to transcriptional activation of the Egr-1 gene. After PKC inhibition in PC12 cells, the remaining induction of Egr-1 by mAChR could be blocked by chelation of extracellular calcium with EGTA (57), suggesting the involvement of calcium influx as an additional signaling mechanism. To exclude cAMP in the coupling of m1AChR to Egr-1 transcription, we used the potent adenylyl cyclase stimulators forskolin and 8-bromo-cAMP, and we found that they failed to increase Egr-1 expression.
Our data suggest Egr-1 as a major target among members of the Egr gene family of m1 receptor, because competition experiments with EGR-1-specific antibodies almost completely blocked the binding of nuclear extracts to the EGR recognition sequence that is known to interact with all members of the Egr family. Higher amounts of EGR-1 protein, corresponding to the much higher expression level than that found in Egr-2 and Egr-3, may account for this result. This interpretation is underscored by the failure of EGR-2- and EGR-3-specific antibodies to induce a detectable supershift with the above preparation. Nevertheless, EGR-3 protein was detected unequivocally on Western blots, indicating that the transcription of the Egr-3 gene led to the generation of translation products.
These results confirm and extend the observation that muscarinic AChRs increase Egr-1 message in PC12 (42, 57, 58), NG108-15 (59), and 3T3 cells transfected with m1 AChR cells (22). Our data show that in addition to Egr-1, the expression of Egr-2, Egr-3, and Egr-4 is under the control of muscarinic AChRs. Moreover, our results show binding to, and activation of, EGR promoter sequences followed by the synthesis of functional protein as a result of mAChR stimulation.
The muscarinic AChR subtypes used in our assays induced EGR-1 binding to EGR recognition domain with different efficacy: whereas m1, m2, and m3 AChRs caused a very clear induction of EGR-1 binding, m4AChR caused only subtle activation of EGR-1 binding in our cells. Stimulation of all analyzed mAChR receptor subtypes, including m4, increased EGR-1 protein synthesis, as verified by Western blot analyses of the same nuclear proteins used for the EGR binding assays. These results demonstrate that transcribed Egr-1 gene induced by mAChRs is followed by the synthesis of functional EGR-1 proteins. m2 and m4 AChRs efficiently inhibit adenylyl cyclase activity but activate phosphoinositide hydrolysis with only one-fifth of the efficacy of m1 and m3 AChRs (60). These levels of phosphoinositide turnover induced by m2 and m4 AChRs may be sufficient to mediate Egr-1 expression. Alternatively, other second messenger pathways, including the inhibition of adenylyl cyclase, may be involved in signaling.
The ability of different muscarinic AChR subtypes to stimulate Egr-1 expression suggests that similar genes are controlled by acetylcholine in both pre- and postsynaptic neuronal populations. It is possible that these use distinct signaling mechanisms in the coupling of the surface receptors to Egr expression. In vivo experiments with intact neuroanatomical structures, however, are required to test this hypothesis. Egr-1 expression is up-regulated also by nicotinic acetylcholine receptors in a mammalian skeletal muscle cell line (61), indicating that EGR transcription factors can be under the control of several distinct muscarinic and nicotinic surface receptor subtypes.
Together, our data suggest that target genes with EGR-responsible
promoters can be regulated by m1AChR. Candidate genes for EGR-mediated
regulation include the AChE gene (37), the platelet-derived growth
factor A and B chains (38, 39), the transforming growth factor 1
(40), luteinizing hormone (41), phenylethanolamine N-methyltransferase (42), and human interleukin 2 (62).
Thus, the regulation of multiple cellular functions, including
signaling, growth, and metabolism, may be coupled to muscarinic AChR
activity via EGR transcription factors.
EGR-1 increases the promoter activity of the AChE gene, a serine hydrolase that catalyzes the hydrolysis of acetylcholine (49). Our data, generated by using the AChE gene promoter fused to a luciferase reporter, show that stimulated m1AChR specifically increased AChE gene promoter activity. Even though basal AChE gene promoter activity was found in all cell types examined so far, AChE message could be determined in only a small faction of these cells. HEK293 cells, in particular, have a very low endogenous promoter activity and no detectable AChE enzyme activity (37). We were unable to detect AChE mRNA in either unstimulated or stimulated m1AChR cells, possibly reflecting the absence of AChE mRNA stabilization known to occur during neuronal differentiation (47). If confirmed for the subcortical cholinergic projection system in brain, EGR-dependent regulation of AChE gene transcription may be involved in a receptor-coupled feedback control of cholinergic transmission.
Cholinergic signaling in Alzheimer's disease brain is heavily impaired as a result of the early and massive degeneration of the long basal forebrain projection neurons to brain hippocampus and cortex. Inasmuch as EGR-dependent genes in postsynaptic cholinergic target cells are regulated by muscarinic AChR activity, expression of such genes may be decreased in Alzheimer's disease brains. Postmortem studies are required to test this hypothesis. Drugs designed to activate muscarinic AChRs, including AChE inhibitors and m1 agonists, that are currently being tested in clinical trials for the treatment of Alzheimer's disease may be expected to stimulate transcription of Egr genes along with EGR-dependent target genes. In vivo studies are required to test whether pharmacological treatments designed to stimulate brain muscarinic AChRs increase AChE gene expression, along with AChE enzyme activity and accelerated breakdown of acetylcholine.
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ACKNOWLEDGEMENTS |
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We thank Peter Zipfel for providing us with cDNA fragments of Egr-1, Egr-2 and Egr-4. We thank Palmer Taylor and Shelley Camp for the pAChE-luc construct.
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FOOTNOTES |
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* This study was funded by Bundesministerium für Bildung und Forschung, Deutsche Forschungsgemeinschaft, and Alzheimer Forschung International.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
49-40-4717-6273; Fax: 49-40-4717-6598; E-mail:
nitsch{at}plexus.uke.uni-hamburg.de.
1 The abbreviations used are: AChR, acetylcholine receptor; mAChR, muscarinic AChR; AChE, acetylcholinesterase; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; PCR, polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; luc, luciferase; kb, kilobase(s).
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REFERENCES |
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