Nicotinic acetylcholine receptor α7 and β4 subunits contribute nicotine-induced apoptosis in periodontal ligament stem cells

Abstract

Nicotine, a major component of cigarette smoking, is the important risk factor for the development of periodontal disease. However, the mechanisms that underlie the cytotoxicity of nicotine in human periodontal ligament stem cells (PDLSCs) are largely unknown. Thus, the purpose of this study was to determine the cytotoxic effect of nicotine by means of nicotinic acetylcholine receptor (nAChR) activation in PDLSCs. We first detected α7 and β4 nAChRs in PDLSCs. The gene expressions of α7 and β4 nAChR were increased by nicotine administration. Nicotine significantly decreased cell viability at a concentration higher than 10(-5) M. DNA fragmentation was also detected at high doses of nicotine treatment. Moreover, the detection of sub G1 phase and TUNEL assay demonstrated that nicotine significantly induced apoptotic cell death at 10(-2) M concentration. Western blot analysis confirmed that p53 proteins were phosphorylated by nicotine. Under various doses of nicotine, a decrease in the anti-apoptotic protein Bcl-2, but an increase in p53 and cleaved caspase-3 protein levels, was detected in a dose-dependent manner. However, the apoptotic effect of nicotine was inhibited by the pretreatment of α-bungarotoxin, a selective α7 nAChR antagonist or mecamylamine, a non-selective nAChR antagonist. Finally, increases in the subG1 phase and DNA fragmentation by nicotine was attenuated by each nAChR antagonist. Collectively, the presence of α7 and β4 nAChRs in PDLSCs supports a key role of nAChRs in the modulation of nicotine-induced apoptosis.

Fig. 1.

Effects of different concentrations of nicotine on the mRNA expression of nAChRs. (A) The mRNA expression of α1–7 and β1–4 nAChRs in PDLSCs was analyzed using RT-PCR. Cells were incubated with different concentrations of nicotine (0–10⁻² M) and the mRNA levels of (B) α7 and (C) β4 nAChRs were then determined by real time RT-PCR technique. The values reported are the mean ± S.D. of three independent experiments. *P < 0.05 or ^#P < 0.001 vs. control value.

Fig. 2.

Effects of nicotine on cell viability. Cells were incubated with different concentrations of nicotine (0–10⁻² M) for (A) 24 or (B) 48 h each then cell viability was assessed as described in “Materials and Methods”. (C) Cells were treated with various concentrations of nicotine (0–10⁻² M) for 48 h and the level of DNA fragmentation of apoptotic cells was then determined. The values reported are the mean ± S.D. of seven independent experiments. *P < 0.05 or ^#P < 0.001 vs. control value.

Fig. 3.

Evaluation of apoptosis by Apo-BrdU TUNEL assay. Cells were subjected to APO-BrdU TUNEL staining after 10⁻² M nicotine exposure for 48 h. The nuclei were counterstained with DAPI. Each image shown is a representative of five separate experiments. The size bars on the panels represent 500 μm.

Fig. 4.

Effect of nicotine on apoptotic cell death in PDLSCs. (A) Cells were treated with various concentrations of nicotine (0–10⁻² M) for 24 h and the DNA contents of cells were then measured by flow cytometry. (B) The bars denote the percentage of cells in the subG1 phase. The values reported are the mean ± S.D. of five independent experiments. *P < 0.05 or ^#P < 0.001 vs. control value.

Fig. 5.

Effect of nicotine on Bcl-2, p53, and caspase-3 protein levels. (A) Cells were subjected to Western blot analysis after 10⁻² M nicotine treatment for various amounts time (0–120 min) and then phosphorylation of p53 was determined. (B) Cells were treated with nicotine (0–10⁻² M) for 48 h then the protein levels of Bcl-2, p53, and cleaved caspase-3 were assessed using total protein lysates. The panels (bars) denote the mean ± S.D. of five experiments for each condition determined from densitometry relative to β-actin. *P < 0.05 or ^#P < 0.001 vs. control value. (C) Cells were pretreated with each α-BTX or mecamylamine for 1 h before the addition of 10⁻² M nicotine. After 48 h of incubation, each apoptotic signal protein was analyzed.

Fig. 6.

Effect of nAChR antagonists on nicotine-induced apoptosis in PDLSCs. Cells were incubated with each α-BTX or mecamylamine for 1 h before 10⁻² M nicotine treatment and (A) the DNA contents and (C) the level of DNA fragmentation of apoptotic cells were then determined. (B) The data shows the percentage of cells in the subG1 phase. The values reported are the mean ± S.D. of four independent experiments. *P < 0.05 vs. control value, **P < 0.05 vs. nicotine treatment.