(Received for publication, June 5, 1995; and in revised form, July 11, 1995)
From the
The proteins responsible for acetylcholine (ACh) synthesis
(choline acetyltransferase, ChAT) and storage (vesicular ACh
transporter, VAChT) are encoded by two closely linked genes in
vertebrates, with the VAChT coding sequence contained within the first
intron of the ChAT gene. This unusual genomic organization suggests
that the transcription of these two genes is coordinately regulated.
Using Northern analysis we studied the modulation of ChAT and VAChT
expression in a murine septal cell line (SN56) by three groups of
agents: retinoids, trophic factors belonging to the leukemia inhibitory
factor/ciliary neurotrophic factor (LIF/CNTF) family, and cAMP.
All-trans-retinoic acid increased both ChAT and VAChT mRNA
levels in SN56 cells up to 3.5-fold, and elevated intracellular ACh
levels by 2.5-fold. This effect was mimicked by a retinoic acid
receptor (RAR
) agonist (Ro 40-6055) and prevented by a
specific antagonist (Ro 41-5253), indicating that it was mediated by
RAR
. ChAT- and VAChT-specific transcripts were also induced (up to
3-fold) by treatment with CNTF or LIF (20 ng/ml, 48 h), as well as by
dibutyryl cAMP (1 mM). All these agents increased the ACh
level in the cells (up to 2.5-fold). Dibutyryl cAMP had a greater
effect on the level of VAChT mRNA (4-fold induction) than on the level
of ChAT mRNA (2-fold induction), suggesting a quantitatively
differential transcriptional regulation of the two genes by the cAMP
pathway. The effects of the three groups of agents studied on ChAT and
VAChT mRNA levels were additive, pointing to several independent
mechanisms by which the cholinergic properties of septal neurons can be
modulated.
Cholinergic neurotransmission depends on coexpression of
proteins involved in the synthesis, storage, and release of
acetylcholine (ACh). Collectively, these proteins make up
the cholinergic phenotype of a variety of neuronal populations,
including certain basal forebrain cells that may function in processes
underlying memory(1, 2) . Of the proteins contributing
to the cholinergic phenotype the best studied so far has been the
ACh-synthesizing enzyme, choline acetyltransferase (ChAT, EC 2.3.1.6) (3) . ChAT activity and expression have been used as markers
for cholinergic neurons and as indices for the actions of trophic
factors on those neurons, and previous studies have shown that ChAT
activity and/or expression can be up-regulated by a variety of
extracellular signals, including cholinergic differentiation factor (4) shown to be identical to leukemia inhibitory factor (LIF) (5) and ciliary neurotrophic factor
(CNTF)(6, 7) . In addition, pharmacologic treatments
that cause increases in cAMP concentrations(8) , and
retinoids(9, 10) , have been used to increase ChAT
activity in a variety of experimental systems(3) . Studies of
the ChAT gene have shown that differential promoter use and alternative
RNA splicing contribute to the formation of several ChAT mRNA variants,
which differ at the 5` end(3) . The promoter region of this
gene is rich in putative regulatory nucleotide sequences (Fig. 1), including ones identical or homologous to cAMP
response element (CRE), retinoic acid response element (RARE), and CNTF
response element (CNTF-RE), but only a few have been demonstrated to
serve as cis-acting regulatory elements in reporter gene
assays(11, 12) .
Figure 1: Schematic representation of the structure of the 5` region of the rodent cholinergic locus. The diagram shows only the features directly relevant to the current studies. For simplicity, the information from rat and mouse genomic sequence has been combined. The intron/exon boundaries are not conserved between rat and mouse; however, all of the indicated types of non-coding exons are present in both species, and the rat and mouse genomic sequences show a high degree of homology (for review see (3) ). The 5` region contains the R exon of ChAT, which is followed by the intronless open reading frame encoding VAChT, and then exons N and M and a number of ChAT coding exons. Potential transcription starts for ChAT and VAChT are indicated by horizontalarrows. Differential use of these sites and alternative splicing yield several ChAT transcripts that differ at the 5` end, but all code for the same protein. The transcription start site at the 5` end of exon R may be shared between certain ChAT and VAChT transcripts. Verticalarrows indicate initiation codons for VAChT and ChAT. Some of the relevant putative cis-acting regulatory elements are shown. Using the nomenclature proposed by Wu and Hersh (3) in which the +1 nucleotide within the gene sequence corresponds to the ``A'' of the ATG translation start codon of ChAT, the positions of these sequences are as follows: RARE-like at -274, -517, -1264, -1385, -2629, and -3691; CNTF-RE-like at -377, -2721, and -3117; CRE-like at -1249. In the rat gene, the region upstream exon R contains a sequence homologous to a CRE (TGAGCTCA) and a CNTF-RE consensus sequence TTCCTGAAA.
Recently, the gene for rat and
human vesicular ACh transporter (VAChT), a protein catalyzing the
uptake of ACh into secretory vesicles, was
cloned(13, 14, 15) . The entire VAChT coding
sequence was shown to be contained within the first intron of the ChAT
gene (Fig. 1), prompting Erickson et al.(14) to coin the term ``cholinergic gene
locus.'' The mechanisms regulating ChAT expression are partially
understood, while the regulation of VAChT expression has not yet been
explored. However, the unusual (for mammals) organization of the ChAT
and VAChT genes strongly suggests that they may share some
transcriptional signals and that their expression may be regulated in a
coordinated fashion by extracellular factors(16) . Using the
SN56 neuronal cell line derived from the basal forebrain (septum), we
have previously shown that ChAT expression, activity, and intracellular
ACh levels are increased by activation of the retinoic acid receptor
(RAR
) (17) and by elevations of intracellular cAMP
concentrations (17) . Consistent with the prediction that ChAT
and VAChT expression are coordinately regulated, we report that mRNA
levels for both ChAT and VAChT are increased by three groups of agents
in SN56 cells: retinoids, growth factors of the CNTF/LIF family, and
cAMP. Significantly, this up-regulation results in proportional
increases in the steady-state levels of intracellular ACh. The effects
of these agents are additive, pointing to several independent
mechanisms by which the cholinergic properties of septal neurons can be
modulated.
We employed the following oligonucleotide primers, which were based on the published rat VAChT gene sequence (GenBank accession numbers X80395 and U09211): upper, 5`-AGC GGG CCT TTC ATT GAT CG-3`; lower, 5`-GGC GCA CGT CCA CCA GAA AGG-3`. The primers flank an 814-base pair fragment within the rat VAChT coding sequence. The mouse PCR product was of the same size, as assessed by agarose gel electrophoresis.
In order to compare the expression of VAChT and ChAT in SN56 cells, we prepared mouse cDNA probes for these genes. The ChAT probe was described previously(17) , and the VAChT probe was obtained by PCR amplification of mouse brain cDNA, using primers based on the published rat VAChT gene sequence. In Northern analysis of RNA prepared from SN56 cells, the VAChT probe hybridized to a band of approximately 3 kb, consistent with the findings of Erickson et al.(14) and Roghani et al.(15) , who identified a single 3-kb VAChT message in various rat brain regions containing cholinergic neurons and in rat pheochromocytoma PC12 cells.
We have previously shown that retinoids and an adenylate cyclase
activator, forskolin, increase the abundance of ChAT mRNA in SN56
cells(17) . Prior to those studies, others reported that ChAT
expression is transcriptionally regulated by agents that increase
intracellular cAMP levels in several experimental
systems(11, 25, 26, 27) . We
directly compared the effects of cAMP and t-RA on ChAT and VAChT mRNA
by probing the same blots in succession with ChAT and VAChT cDNA
following a 48 h treatment of the cells with maximally effective
concentrations of BtcAMP (1 mM) and t-RA (1
µM). Messenger RNA levels of ChAT and VAChT were increased
by either agent (Fig. 2A). However, t-RA was more
effective than Bt
cAMP in inducing ChAT, whereas
Bt
cAMP was more effective than t-RA in inducing VAChT (Fig. 2A). The combination of Bt
cAMP and
t-RA resulted in an additive increase of ChAT and VAChT mRNA levels,
indicating that the two agents operate through two independent
mechanisms. Cyclic AMP- and t-RA-evoked induction of ChAT and VAChT
mRNA were accompanied by roughly proportional increase in intracellular
ACh levels (Fig. 2B). A combined treatment with t-RA
and Bt
cAMP resulted in an additive increase in ACh content.
Thus the up-regulation of the expression of cholinergic genes is an
effective mechanism for increasing the amounts of stored ACh in SN56
cells.
Figure 2:
The effects of BtcAMP on ChAT
and VAChT mRNA levels are additive with those of t-RA. SN56 cells were
grown for 2 days in the presence or absence of 1 µM t-RA,
1 mM Bt
cAMP, or both compounds. A, ChAT
and VAChT mRNA levels were determined by Northern blot analysis. Total
RNA preparations (20 µg/lane) were size-fractionated, transferred
to a nylon membrane, and probed successively with cDNAs for ChAT,
VAChT, and glyceraldehyde-3-phosphate dehydrogenase as described under
``Experimental Procedures.'' Similar results were obtained in
three additional experiments. B, intracellular ACh levels were
measured in cell extracts as described under ``Experimental
Procedures.'' The results are presented as means ±
S.D.
Using synthetic retinoids, one selective agonist (Ro
40-6055), and one antagonist (Ro 41-5253) of the RAR(28) ,
we have previously shown that activation of this receptor increases ACh
levels in SN56 cells (17) . We have now used these compounds to
determine whether they similarly affect the expression of ChAT and
VAChT. Both ChAT and VAChT mRNA levels were markedly increased
(3-4-fold) upon treatment of SN56 cells with 100 nM Ro
40-6055 for 48 h (Fig. 3). Moreover, a 100-fold molar excess (10
µM) of Ro 41-5253 abolished the Ro 40-6055-evoked
increases in the abundance of both mRNAs. Treatment of the cells with
Ro 41-5253 alone (10 µM) slightly reduced the basal levels
of ChAT and VAChT mRNA, indicating that retinoids, present in serum,
may participate in maintaining the cholinergic phenotype of SN56 cells
under our culture conditions (Fig. 3). These results constitute
the first demonstration that retinoids modulate ChAT and VAChT
expression, and that RAR
mediates this process.
Figure 3:
The
effects of retinoids on ChAT (A) and VAChT (B) mRNA
levels are mediated by RAR. The cells were treated for 2 days with
a RAR
agonist (Ro 40-6055, 0.1 µM), a RAR
antagonist (Ro 41-5253, 10 µM), 1 µM t-RA, or
combinations as indicated, and ChAT and VAChT mRNA levels were
determined by Northern blot analysis as described under
``Experimental Procedures'' and in Fig. 2. Data in A and B are derived from two independent
experiments.
ChAT activity (29) and expression (30) can be increased in cultured
primary septal neurons by treatments with nerve growth factor (NGF),
and it would be of interest to examine the effects of NGF on VAChT mRNA
levels in septal cells. However, SN56 cells do not respond to
NGF(19) . Another neurotrophin, LIF, is also of particular
interest because it may be important in maintaining the cholinergic
phenotype of certain neuronal populations after
injury(31, 32) . In order to determine whether the
cholinergic phenotype of the septal cell line could also be
up-regulated by LIF, we treated SN56 cells with this protein and with a
functionally related neurotrophin, CNTF(33) . Both trophic
factors increased intracellular ACh content of SN56 cells (Fig. 4B) in a dose-dependent and saturable fashion
(with a maximally effective concentration of 10 ng/ml; data not shown).
Northern analysis of RNA from cells grown for 2 days in the presence of
20 ng/ml LIF or 20 ng/ml CNTF showed that these growth factors
up-regulate both ChAT and VAChT expression, albeit to a lesser extent
than do retinoids or BtcAMP (up to 2-fold induction, Fig. 4A). The effects of LIF and CNTF on ChAT and VAChT
mRNA levels were nearly additive with those of t-RA (Fig. 4A). However, no additivity with the effects of
t-RA was observed when steady-state ACh levels were used as an index of
the neurotrophin action (Fig. 4B). The reason for this
lack of additivity is presently unclear; however, this result could
indicate that cells treated with t-RA and neurotrophins are
characterized by disproportionately accelerated release of ACh. In a
previous study we showed that ACh release can be enhanced by treating
SN56 cells with Bt
cAMP(34) . Whether the released
ACh derives entirely from the vesicular pool of the transmitter is
currently not known. It will be interesting to determine if the
up-regulation of VAChT by the agents described here correlates with the
amounts of ACh stored in secretory vesicles, and with the ability of
SN56 cells to secrete ACh.
Figure 4: Effects of LIF, CNTF, and t-RA on ChAT and VAChT mRNA. The cells were grown for 2 days in the presence or absence of 1 µM t-RA, 20 ng/ml LIF, 20 ng/ml CNTF, or combinations as indicated. A, ChAT and VAChT mRNA levels were determined by Northern blotting as in Fig. 2. Similar results were obtained in two additional experiments. B, intracellular ACh levels were measured as described under ``Experimental Procedures.'' The results are presented as means ± S.D.
Taken together, these data suggest a coordinated up-regulation of ChAT and VAChT gene expression by cAMP, retinoid, and CNTF/LIF signaling pathways. However, subtle differences exist in the regulation of expression of these two genes, e.g. cAMP increases VAChT mRNA level more efficiently than that of ChAT (Fig. 2). In addition, the fact that these pathways exert additive effects on ChAT and VAChT mRNA levels suggests that they are independent from each other.
The observation that these two closely linked genes, both essential components of the cholinergic phenotype, are expressed coordinately is consistent with the observations that the tissue distributions of the ChAT and VAChT transcripts are virtually identical(13, 14, 15) . This coexpression of VAChT and ChAT suggests that both are regulated by the same tissue-specific transcriptional signals. It is worth noting that a region upstream of exon R (Fig. 1), which would be expected to direct the expression of both VAChT and ChAT in the appropriate tissues, has been shown to confer cholinergic tissue-specific expression of a reporter gene(12) . Although we did not measure the rates of formation of the mRNA for VAChT and ChAT, the available data suggest that retinoids, cAMP, and LIF/CNTF directly stimulate transcription of these genes. Analysis of the ChAT/VAChT genomic sequence reveals numerous putative cis-acting regulatory sequences that may take part in this process (Fig. 1). There are six sequences with high homology to the RARE (35) in the N/M promoter region of the ChAT gene(17) , i.e. positioned 5` of the ChAT first coding exon and 3` of the VAChT open reading frame (Fig. 1). It is possible that some of them confer the retinoic acid response to both ChAT and VAChT promoters. Interestingly, in the ChAT gene, the RARE present in inverted orientation in position -1242 to -1264 overlaps with a CRE-like sequence (-1242 to -1249). Using DNA constructs containing a relatively long region of the ChAT gene (2.7 kb) linked to a reporter, Misawa et al.(11) showed that the region downstream from exon M is responsible for the induction of ChAT by cAMP, and suggested that this putative CRE conferred the effect.
The
transcriptional effects of CNTF and LIF are known to be mediated by the
members of the STAT family of
proteins(36, 37, 38, 39) , which
bind to a cis-acting CNTF-RE (consensus sequence
TTCC(N)AA)(36, 40) . Two perfect
matches of the CNTF-RE and two sequences homologous to CNTF-RE are
present in the ChAT/VAChT locus (7) (Fig. 1).
Additionally, recent studies demonstrated that, in addition to STATs,
the C/EBP transcription factors are necessary for CNTF/LIF inducibility
of the gene encoding vasoactive intestinal peptide(41) , a
neuropeptide induced by CNTF and LIF in sympathetic neurons. There are
four perfect matches of the C/EBP consensus binding site
(T(T/G)NNGNAA(T/G)) clustered in the murine M promoter region, and the
rat R promoter also contains four perfect matches of this sequence.
Moreover, although the CRE-binding protein is generally credited for
activating transcription via the CRE sites, it has been reported that
C/EBP
can also bind to CRE and mediate the effects of cAMP on
transcription(42, 43, 44) . Thus, it is
possible that modulation of ChAT/VAChT gene expression could be a
result of complex cross-talk among STAT, C/EBP, cAMP, and retinoic acid
regulatory pathways.
The coordinated up-regulation of ChAT and VAChT mRNA levels indicates that common signaling pathways control both genes. A better understanding of the mechanisms that regulate transcription of those genes awaits the detailed characterization of their promoters, and determination of the functional activity of the putative cis-acting elements and of the transcription factors interacting with these elements. This information will be useful in understanding the mechanisms of diseases characterized by malfunction of cholinergic neurons, e.g. Alzheimer's disease or amyotrophic lateral sclerosis, and may help in designing treatment strategies directed toward repair of those defects.
Addendum-While this paper was under review, similar results were reported by Berrard et al.(45) .