Nicotine regulates mRNA level of tyrosine hydroxylase gene but not that of nicotinic acetylcholine receptor genes in PC12 cells

Neuroscience Letters 228 (1997) 37–40

Nicotine regulates mRNA level of tyrosine hydroxylase gene but not that of nicotinic acetylcholine receptor genes in PC12 cells Hiroshi Ishiguro, Naohiro Ichino, Kouji Yamada, Toshiharu Nagatsu* Institute for Comprehensive Medical Science, School of Medicine, Fujita Health University, Toyoake, Aichi 470-11, Japan Received 17 April 1997; revised version received 8 May 1997; accepted 8 May 1997

Abstract To understand the molecular mechanism of nicotine addiction, we examined the mRNA level of the tyrosine hydroxylase (TH) gene and that of the nicotinic acetylcholine receptor (nAChR) genes by long-term nicotine treatment. The transcript levels of the four subunit genes of the nAChR (a3, a5, a7, and b4) were down-regulated by the treatment with forskolin, whereas the mRNA levels of the TH gene was increased in PC12 cells. By long-term nicotine treatment, the mRNA level of the nAChR genes did not change, but transcript levels of a3, a5, a7, and b4 nAChR genes were still negatively regulated by forskolin. However, the mRNA level of TH gene did not change by forskolin under long-term nicotine treatment. The TH gene may be regulated by a nicotine-related signaling pathway, whereas a3, a5, a7, and b4 nAChR genes may be further regulated by a protein kinase A (PKA) pathway under long-term nicotine treatment.  1997 Elsevier Science Ireland Ltd. Keywords: PC12 cells; Nicotine; Tyrosine hydroxylase; Nicotinic acetylcholine receptor; Protein kinase A; Signal transduction

Nicotine is one of the major components of cigarettes, and smokers are chronically exposed to it. Appropriate doses of nicotine produce many physiological changes such as excitation of respiration and increases in heart rate and blood pressure. Nicotine is a ligand of nAChRs, and nicotine binding to these receptors increases the influx of calcium ions into nerve cells [20,25]. Calcium ion may stimulate the calcium-related secondary signal pathways including kinases such as protein kinase A (PKA) [4], Ca2+/calmodulin kinase II [3,14] and Ca2+- and phospholipid-dependent protein kinase (PKC) [24]. As a part of these kinase pathways, the transcription of the tyrosine hydroxylase (TH) gene might be enhanced in catecholaminergic cells [8,13,23,26]. Nicotine and nicotinic agonists such as dimethylphenylpiperazine increase not only the protein level of TH but also the mRNA level of the TH gene in rat brain [22] and bovine adrenal chromaffin cells [5,6]. Recently, Hiremagalur et al. [10] reported that the mRNA level of TH and DBH genes in PC12 cells was increased by nicotine treatment and that the cyclic AMP response ele-

* Corresponding author. Tel.: +81 562 939391; fax: +81 562 938831.

ment (CRE; TGACGTCA) plays a key role in long-term nicotine-induced activation of the TH gene. Thirteen nicotinic acetylcholine receptor (nAChR) subunit genes classified as a and b were found in central nerve system (CNS) and peripheral nerve system (PNS) [7,21], and modulation of neuronal nAChRs in the CNS may cause many physiological changes [19]. Except for the muscle-type nAChRs a1 and b1, eight a subunits (a2–a9) and three b subunits (b2–b4) are distributed in the cholinergic neurons in many ganglionic neuronal cell populations and in the chromaffin tumor cells, PC12 [17]. The PKA pathway decreased the mRNA levels of a3, a5 and b4 subunit genes of nAChRs, but the mRNA level of the b2 subunit gene was increased by the addition of dibutyryl cAMP (dbcAMP) to PC12 cells [15]. Nicotine treatment up-regulated the protein level of nAChR subunits a4 and b2 in clonal cell lines and in primary cultures of fetal rat brain, but their mRNA levels did not change [2]. Long-term treatment with nicotine, which might induce calcium influx into the cells, enhanced the transcript level of the b2 subunit gene; but that of the a3 subunit gene was down-regulated [16]. PKA-deficient PC12 cells treated with nicotine did not show any change in nicotine binding or mRNA level of nAChR subunit genes. These

0304-3940/97/$17.00  1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0360-1

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H. Ishiguro et al. / Neuroscience Letters 228 (1997) 37–40

reports suggest that PKA is required to regulate mRNA level of nAChR subunit genes a3 and b2. In this paper, we examined the mRNA level of the TH gene and that of the nAChR genes by long-term nicotine treatment. PC12 cells (RIKEN Cell Bank; Tsukuba Science City, Japan) were maintained in Dulbecco’s modified Eagle medium (Gibco BRL; Grand Island, NY, USA) supplemented with penicillin-streptomycin (Gibco), 10% heat-inactivated horse serum, and 10% heat-inactivated fetal bovine serum (Gibco) in a 5% CO2 atmosphere at 37°C. Cells were treated with 200 mM nicotine([−]-1-methyl-2-[3-pyridyl]-pyrrolidine; SIGMA) or 10 mM forskolin (RBI; Natick, MA, USA), with fresh reagent added each time. Complementary DNA (cDNA) was synthesized from poly(A) + RNA by random hexamer priming using M–MLV reverse transcriptase (Gibco). The cDNA probes of nAChR subunit genes were prepared by polymerase chain reaction (PCR) with an LAPCR kit (TAKARA, Japan), and sequences were confirmed by the use of a 373A DNA sequencer (Applied Biosystems). Northern blot analysis of poly(A) + RNA level was performed based on a modification of the method of Badley et al. [1]. One microgram of poly(A) + RNA was applied to a 1.0% agarose gel containing formaldehyde, subjected to electrophoresis at 100 V for 2 h, and transferred to a HybondN + (Amersham) membrane by capillary diffusion. Hybridized membranes were brought into contact with a BAS1000 plate (Type BAS-IIIs) and analyzed with a BAS1000 (Fuji Photo Film, Japan). To find the TH gene and six nAChR subunit genes expressed in PC12 cells, we examined the expression of RNA transcripts by Northern blot analysis (Fig. 1). Although each transcript of TH gene, and that of b3 and b4 subunit genes of nAChRs was found as a single major band, the mRNAs of a3, a5, a7, and b2 subunits were detected as multiple transcripts. As shown in Fig. 2, poly (A)+ RNA from forskolin-treated PC12 cells was analyzed by the Northern blot technique using full-length cDNA probes for TH, a3, a5, a7, b3, and b4. The transcripts of four of the nAChR subunit genes (a3, a5, a7, and b4) were down-regulated by the addition of forskolin. In contrast, the transcript level of the b3 subunit gene was unaffected by the

Fig. 1. Northern blot hybridization analyses for TH and nAChR genes in PC12 cells. Closed triangles show major transcripts. At right side of the line, the positions of rat ribosomal RNA (28S and 18S) are shown as size markers.

Fig. 2. Forskolin treatment changes the mRNA level of TH and nAChR genes in PC12 cells. PC12 cells had been treated with forskolin for 3, 6, or 24 h. Each band shown by closed triangles in Fig. 1 is calculated by the BAS station. The data are calculated total magnitudes in each lane, and are presented as the means ± SD of three independent experiments for TH and each nAChR subunit. Densities are significantly different from control (0 h); *P , 0.05, §P , 0.025, and †P , 0.004. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as a standard.

addition of forskolin. To analyze the changes in the mRNA level of nAChR subunits (a3, a5, a7, b3, and b4) affected by the addition of nicotine, we treated PC12 cells with nicotine for 3 weeks (Table 1). The mRNA level of nAChR subunits (a3, a5, a7, b3, and b4) in PC12 cells failed to change during 3 weeks of nicotine treatment, but approximately 2-fold increase was observed in steady state level of TH mRNA. To analyze further the effects of longterm nicotine treatment on transcription of the TH gene and nAChR genes, we additionally treated the nicotine-treated PC12 cells with forskolin. TH mRNA level failed to increase by the addition of forskolin (Fig. 3). Even though the PC12 cells had been subjected to the long-term nicotine treatment, the mRNA levels of nAChR subunits (a3, a5, a7, and b4) were also affected in similar fashion by the addition of forskolin. PC12 cells are cholinergic and catecholaminergic cells, and many investigators have used these cells to analyze the mechanism of the transcription and regulation of genes related to catecholamines and acetylcholine. We showed that our PC12 cells expressed six nAChR subunits, a3,

H. Ishiguro et al. / Neuroscience Letters 228 (1997) 37–40 Table 1 Comparison of mRNA levels of TH and nAChR subunits after 3 weeks nicotine treatment % Ratio (nicotine-treated/nicotine-untreated × 100) TH nAChR

a3 a5 a7 b3 b4

220 118 110 127 121 114

± ± ± ± ± ±

91* 18 18 54 31 23

Data are presented as the mean ± SD of three independent experiments. *Density is significantly greater than that at day 0 (P = 0.05).

a5, a7, b2, b3, and b4, by Northern blot analysis. These results agree with other reports on PC12 cells [9,18]. The nAChR subunit genes are reported to be regulated by dbcAMP, nerve growth factor (NGF), and nicotine [2,9,11,15,16,18]. Madhok et al. [15] showed that dbcAMP up-regulated the transcription of the b2 subunit gene of nAChR, and down-regulated that of a3, a5, and b4 subunit genes. We also showed that forskolin decreased the mRNA levels of a3, a5, a7, and b4, but did not change the mRNA level of the b3 subunit in PC12 cells (Fig. 2). In addition, we confirmed that the mRNA level of the b2 subunit gene was up-regulated by the treatment of forskolin (data not shown). Our data obtained with forskolin confirm that the PKA pathway down-regulates the transcriptions of a3, a5, a7, and b4 nAChR genes. In addition, each signal strength of the major and minor transcripts of nAChR genes showed similar changes by the treatment with forskolin. It was also reported that the addition of nicotine to PC12 cells increased the mRNA level of the b2 nAChR subunit and decreased the mRNA level of the a3 subunit [16]. Furthermore, using in vitro cultures of rat fetal cerebral cortex, Bencherif et al. [2] reported that the mRNA levels of the a4 and b2 subunits were increased by the addition of nicotine but that their mRNA levels did not change in these fetal rat brain cortical cells. We showed here that the mRNA levels of the a3, a5, a7, b3 and b4 subunits in our PC12 cells were not affected by nicotine treatment for 3 weeks (Table 1). The discrepancies between our results and those by Bencherif et al. [2] may be caused by the differences in the culture conditions or characterizations of PC12 cells. In addition, the mRNA levels of a3, a5, a7, and b4 nAChR subunits genes were decreased by the addition of forskolin after 3 weeks of nicotine treatment (Fig. 3). These data suggest that although PKA regulates a3, a5, a7, and b4 nAChR subunit genes, the signal transduction pathway related to the increase in intracellular calcium may not affect the transcription of these nAChR subunit genes. Furthermore, the main transcriptional machinery of the TH gene is CRE in the promoter region (−45 bp from the CAP site) which is regulated by the PKA pathway [12,13]. Using PKA deficient PC12 cells, Hiremagalur et al. [10] found that PKA is required for the induction of the TH gene by nicotine. We showed that the mRNA level of TH

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did not change by the addition of forskolin after 3 weeks of nicotine treatment (Fig. 3). Nicotine treatment may indirectly affect the pathway related to the phosphorylation of CRE binding protein (CREB), and forskolin may not further affect the phosphorylation of CREB after long-term nicotine treatment. The b3 gene has a unique characteristics. There are no reports on the Northern blot analysis of the b3 mRNA in PC12 cells. We confirmed the PCR products of the b3 subunit mRNA by sequencing and using poly(A)+ RNA-blotted membranes (Fig. 1). We found an extremely smaller mRNA band with a radioisotope-labeled probe of b3 as compared with the mRNA bands of a3, a5, a7, and b4 nAChRs. In addition, the mRNA level of b3 subunit did not change by the treatment with forskolin or nicotine, or both of them. These data suggest that transcription of b3 may be regulated by different mechanisms. In summary, we have shown that the PKA pathway negatively regulated the nAChR transcripts of the a3, a5, a7, and b4 subunit genes in PC12 cells but had no effect on the b3 subunit gene. Long-term treatment of PC12 cells with nicotine did not change the mRNA level of the nAChR genes, but did increase the mRNA level of TH. Furthermore, forskolin treatment after the long-term nicotine treatment also decreased the mRNA levels of nAChR, a3, a5, a7, and b4 subunit genes, but did not affect the mRNA level of TH gene. These results may indicate that the transcription of the TH gene may be regulated under nicotine-dependent manner, and that PKA pathway may still regulate the

Fig. 3. Northern blot analysis of mRNAs of TH and nAChR subunits in PC12 cells treated with forskolin after 3 weeks of nicotine treatment (200 mM). GAPDH mRNA was used as a standard. This experiment was performed three times with similar results.

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