Nicotine Acts on Growth Plate Chondrocytes to Delay Skeletal Growth through the a7 Neuronal Nicotinic Acetylcholine Receptor Atsuo Kawakita1,2, Kazuki Sato2, Hatsune Makino 1 , Hiroyasu Ikegami2, Shinichiro Takayama 3 , Yoshiaki 2 1 Toyama , Akihiro Umezawa * 1 Department of Reproductive Biology, National Institute for Child Health and Development, Tokyo, Japan, 2 Department of Orthopaedic Surgery, Keio University School of Medicine, Tokyo, Japan, 3 Department of Orthopaedic Surgery, National Center for Child Health and Development, Tokyo, Japan Abstract Background: Cigarette smoking adversely affects endochondral ossification during the course of skeletal growth. Among a plethora of cigarette chemicals, nicotine is one of the primary candidate compounds responsible for the cause of smoking- induced delayed skeletal growth. However, the possible mechanism of delayed skeletal growth caused by nicotine remains unclarified. In the last decade, localization of neuronal nicotinic acetylcholine receptor (nAChR), a specific receptor of nicotine, has been widely detected in non-excitable cells. Therefore, we hypothesized that nicotine affect growth plate chondrocytes directly and specifically through nAChR to delay skeletal growth. Methodology/Principal Findings: We investigated the effect of nicotine on human growth plate chondrocytes, a major component of endochondral ossification. The chondrocytes were derived from extra human fingers. Nicotine inhibited matrix synthesis and hypertrophic differentiation in human growth plate chondrocytes in suspension culture in a concentration-dependent manner. Both human and murine growth plate chondrocytes expressed alpha7 nAChR, which constitutes functional homopentameric receptors. Methyllycaconitine (MLA), a specific antagonist of alpha7 nAChR, reversed the inhibition of matrix synthesis and functional calcium signal by nicotine in human growth plate chondrocytes in vitro. To study the effect of nicotine on growth plate in vivo, ovulation-controlled pregnant alpha7 nAChR +/2 mice were given drinking water with or without nicotine during pregnancy, and skeletal growth of their fetuses was observed. Maternal nicotine exposure resulted in delayed skeletal growth of alpha7 nAChR +/+ fetuses but not in alpha7 nAChR 2/2 fetuses, implying that skeletal growth retardation by nicotine is specifically mediated via fetal alpha7 nAChR. Conclusions/Significance: These results suggest that nicotine, from cigarette smoking, acts directly on growth plate chondrocytes to decrease matrix synthesis, suppress hypertrophic differentiation via alpha7 nAChR, leading to delayed skeletal growth. Citation: Kawakita A, Sato K, Makino H, Ikegami H, Takayama S, et al. (2008) Nicotine Acts on Growth Plate Chondrocytes to Delay Skeletal Growth through the a7 Neuronal Nicotinic Acetylcholine Receptor. PLoS ONE 3(12): e3945. doi:10.1371/journal.pone.0003945 Editor: Rory Edward Morty, University of Giessen Lung Center, Germany Received August 6, 2008; Accepted November 8, 2008; Published December 16, 2008 Copyright: 2008 Kawakita et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by a grant from the Smoking Research Foundation; by grants from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan; by the Ministry of Health, Labour and Welfare (MHLW) Sciences Research Grants; by a Research Grant on Health Science focusing on Drug Innovation from the Japan Health Science Foundation; by the program for promotion of Fundamental Studies in Health Science of the Pharmaceuticals and Medical Devices Agency; by a Research Grant for Cardiovascular Disease from the MHLW; and by a Grant for Child Health and Development from the MHLW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: umezawa@1985.jukuin.keio.ac.jp Introduction Though detrimental effects of cigarette smoking to the human body have been widely demonstrated, the effects on endochondral ossification are not well understood. Epidemiologically, maternal smoking reduces the height of newborns [1–5]. However, there are controversial views regarding the mechanisms behind delayed skeletal growth caused by cigarette smoking. The socioeconomic status of smoking mothers [6,7], deficient maternal diet [8], chronic hypoxia caused by carbon monoxide [9], impaired placental size and function, and decreased blood flow of placenta caused by nicotine [10] have all been reported as a possible causal factors responsible for reduction in height of newborns. Conversely, it has also been reported that socioeconomic status [11], maternal diet [12], and hypoxia are not responsible for the cause of delayed skeletal growth. Research suggests that smoking not only reduces body length but also brings ossification retardation in the rat smoking model [13]. Moreover, smoking delays chondrogenesis in a mouse model of fracture healing [14]. Cigarette smoking, thus, adversely affects endochondral ossification somehow during the course of skeletal growth and repair in animal models. Among a multitude of chemicals and physiological functions arising from cigarette smoking, nicotine is one of the leading candidates for causing small newborns. Epidemiologically, nicotine content in cigarette is related to reduced birth length in humans [15]. However, the possible mechanism of delayed skeletal growth caused by nicotine remains unclarified. In this study, we investigated the effect of nicotine on growth plate chondrocytes, the principle PLoS ONE | www.plosone.org 1 December 2008 | Volume 3 | Issue 12 | e3945