Nicotine-Induced Expression of Low-Density Lipoprotein Receptor in Oral Epithelial Cells

Satoshi Ito,Kou Tsunoda,Ryosuke Koshi,Koichi Ito,Naoto Yoshinuma,Takahiro Gojoubori,Kazuo Komiyama,Eiji Goke,Naoyuki Sugano,Yoko Yamaguchi,Masatake Asano,Sompop Bencharit

doi:10.1371/journal.pone.0082563

Abstract

BackgroundNicotine use is one of the most important risk factors for the development of cardiovascular and periodontal diseases. Numerous reports have suggested the possible contribution of disturbed lipid metabolism for the development of both disease groups. Despite these observations, little is known about the relationship between tobacco smoking and the development of these diseases. Our previous microarray data revealed that nicotine induced low-density lipoprotein receptor (LDLR) expression in oral epithelial cells (OECs). The aim of the present study was to confirm nicotine-mediated LDLR induction and to elucidate the signaling mechanisms leading to the augmented expression of LDLR in OECs.Methods and ResultsLDLR and nicotinic acetylcholine receptor (nAChR) subunit expression was detected by real-time PCR. The production of LDLR was demonstrated by immunofluorescence staining. nAChR-mediated LDLR induction was examined by pre-incubation of the cells with its specific inhibitor, α-bungarotoxin (α-BTX). The functional importance of transcription factor specific protein 1 (Sp1) was examined by luciferase assay, mithramycin pre-incubation or by small interfering RNA (siRNA) transfection. The specific binding of Sp1 to R3 region of LDLR 5’-untranslated region was demonstrated with electrophoretic mobility shift assay (EMSA) and streptavidin-agarose precipitation assay followed by western blotting. The results confirmed that nicotine induced LDLR expression at the transcriptional level. Nicotine was sensed by nAChR and the signal was transduced by Sp1 which bound to the R3 region of LDLR gene. Augmented production of LDLR in the gingival epithelial cells was further demonstrated by immunofluorescence staining using the gingival tissues obtained from the smoking patients. ConclusionsTaken together, the results suggested that nicotine might contribute to the development of both cardiovascular and periodontal diseases by inducing the LDLR in OECs thereby disturbing lipid metabolism.

Highlights

Cigarette smoking is an important environmental risk factor for the development of several diseases, including obesity, atherosclerosis, Crohn’s disease, and periodontal disease [1,2,3]
We focused on the expression of the low-density lipoprotein receptor (LDLR) gene
Incubation with αBtx alone did not affect the LDLR expression level, indicating specific inhibition (Figure 2B). These results indicated that nicotinic acetylcholine receptor (nAChR) contributed to the increased expression of LDLR mRNA

Summary

Introduction

Cigarette smoking is an important environmental risk factor for the development of several diseases, including obesity, atherosclerosis, Crohn’s disease, and periodontal disease [1,2,3]. Nicotine contributes to the progression of plaque formation by angiogenic activity through the nicotinic acetylcholine receptor (nAChR), and can disturb lipid metabolism [4,5]. Numerous reports have suggested the possible contribution of disturbed lipid metabolism for the development of both disease groups. Augmented production of LDLR in the gingival epithelial cells was further demonstrated by immunofluorescence staining using the gingival tissues obtained from the smoking patients. Conclusions: Taken together, the results suggested that nicotine might contribute to the development of both cardiovascular and periodontal diseases by inducing the LDLR in OECs thereby disturbing lipid metabolism

Objectives

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Results

Conclusion