Coordinated up-regulation of choline acetyltransferase and vesicular acetylcholine transporter gene expression by the retinoic acid receptor alpha, cAMP, and leukemia inhibitory factor/ciliary neurotrophic factor signaling pathways in a murine septal cell line.

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 α (RARα) agonist (Ro 40−6055) and prevented by a specific antagonist (Ro 41−5253), indicating that it was mediated by RARα. 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). 1 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 5Ј 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).
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.

EXPERIMENTAL PROCEDURES
Reagents-All-trans-retinoic acid (t-RA), dibutyryl cAMP (Bt 2 cAMP), and all molecular biology reagents (except as noted) were purchased from Sigma. The RAR␣-selective agonist Ro 40-6055 and the antagonist Ro 41-5253 were gifts from Drs. Arthur Levin and Michael Klaus of Hoffmann-La Roche. Manipulations involving retinoids were conducted under reduced light conditions. Recombinant mouse LIF and rat CNTF were purchased from R&D Systems. Oligonucleotides were custom synthesized by DNA International.
Cell Culture-Mouse septal neuron ϫ neuroblastoma hybrid SN56 cells (18,19) were maintained at 37°C in an atmosphere of 95% air, 5% CO 2 in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, and 50 g/ml gentamicin. The cells were subcultured by mechanically removing them from the dishes with squirts of fresh medium. Cells were grown to subconfluence in 35-mm culture dishes and then refed with fresh medium containing test compounds, e.g. retinoids or growth factors. The medium was then replaced every 24 h. Stock solutions of water-insoluble compounds were prepared in dimethyl sulfoxide (Me 2 SO); where those compounds were used, an equal volume of Me 2 SO (final concentration up to 0.1%) was added to all dishes. All treatments were performed in duplicate.
Extraction and Measurement of ACh-For the final 2 h of treatments, neostigmine (0.1 mM final concentration) was added to the culture dishes in order to inhibit acetylcholinesterase and improve the recovery of ACh. The cells were then washed once with ice-cold phosphatebuffered saline supplemented with 0.1 mM neostigmine, 1 ml of methanol was added, the cells were scraped into the methanol, and the suspension was transferred to a polypropylene tube. One ml of 0.1 N HCl was added followed by 2 ml of chloroform, the tubes were vortexed, and the extracts centrifuged to separate phases. The aqueous phase, containing ACh, and the interface/organic phase (containing cell protein) were collected and dried under a vacuum. The ACh pellets were resuspended in HPLC mobile phase (28 mM phosphate, pH 8.5, supplemented with Kathon CG Reagent) and filtered (0.2-m Nylon-66, Rainin). ACh was determined by HPLC with an enzymatic reactor containing acetylcholinesterase and choline oxidase in series with an electrochemical detector (Bioanalytical Systems) according to the method of Potter et al. (20). Protein content was determined by the method of Smith et al. (21).
PCR Amplification and DNA Probes-All recombinant DNA manipulations were performed according to Sambrook et al. (22) and Ausubel et al. (23). A 0.87-kb ChAT probe corresponding to the 3Ј end of the coding region was described previously (17). We isolated a portion of mouse VAChT cDNA by PCR of mouse brain cDNA (Clontech). PCR was performed in the DNA Thermal Cycler 480 from Perkin Elmer using the "hot start" technique according to the manufacturer's recommendations. Amplification was carried out for 35 cycles (1 min at 95°C, 2 min at 58°C, 2 min at 72°C), followed by 10 min at 72°C. PCR primers were designed and PCR conditions determined with the help of Oligo Primer Analysis software (National Biosciences).
We employed the following oligonucleotide primers, which were based on the published rat VAChT gene sequence (GenBank accession numbers X80395 and U09211): upper, 5Ј-AGC GGG CCT TTC ATT GAT CG-3Ј; lower, 5Ј-GGC GCA CGT CCA CCA GAA AGG-3Ј. 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. Extraction of RNA and Northern Blotting Analysis-Following the desired treatment, SN56 cells were washed twice with phosphate-buffered saline and total RNA was extracted using the guanidinium thiocyanate-phenol/chloroform method (24). After lysis and phenol/chloroform extraction, RNA was precipitated with isopropanol and the pellet was washed with 70% ethanol and stored as an ethanol precipitate at Ϫ80°C. For Northern analysis, RNA samples that had been equalized for ribosomal RNA content (20 g/lane) were size-fractionated on a 1% agarose gel containing 6% formaldehyde and transferred to a Biotrans Plus nylon membrane (ICN). DNA probes were labeled with [␣-32 P]dCTP (DuPont NEN) to a specific activity of 1-2 ϫ 10 9 cpm/g DNA with the random primer labeling kit (Pharmacia Biotech Inc.). The blots were probed successively with cDNAs for ChAT, VAChT, and a mouse glyceraldehyde-3-phosphate dehydrogenase as a control. Prehybridization and hybridization steps were performed at 65°C in Quick-Hyb solution (Stratagene) according to manufacturer's instructions. Final washes were carried out in 0.2 ϫ SSC (1 ϫ SSC ϭ 0.15 M NaCl, 0.015 M sodium citrate), 0.1% SDS at 55°C for ChAT probe and at 65°C for the VAChT and glyceraldehyde-3-phosphate dehydrogenase probes. The blots were exposed to Kodak XAR-2 film with an intensifying screen at Ϫ80°C for 1-2 days. Signal intensities were quantified directly from the blots with a PhosphorImager 400E and ImageQuant software (Molecular Dynamics).

RESULTS AND DISCUSSION
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 ef- Messenger RNA levels of ChAT and VAChT were increased by either agent (Fig. 2A). However, t-RA was more effective than Bt 2 cAMP in inducing ChAT, whereas Bt 2 cAMP was more effective than t-RA in inducing VAChT (Fig. 2A). The combination of Bt 2 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 2 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.
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 M) of Ro 41-5253 abolished the Ro 40-6055evoked increases in the abundance of both mRNAs. Treatment of the cells with Ro 41-5253 alone (10 M) 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.
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 Bt 2 cAMP (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 steadystate 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 2 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.
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 tissuespecific 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 5Ј of the ChAT first coding exon and 3Ј 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 Ϫ1242 to Ϫ1264 overlaps with a CRE-like sequence (Ϫ1242 to Ϫ1249). 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 -39), which bind to a cis-acting CNTF-RE (consensus sequence TTCC(N 3-4 )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.