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Osteocytes the most abundant cells in an
Osteocytes, the most abundant cells in an adult skeleton, are essentially mature osteoblasts that are surrounded by the products they secreted (Noble and Reeve, 2000). Osteocytes serve as mechanosensors that direct bone remodeling and that regulate the activities of osteoblasts and osteoclasts (Bonewald, 2006). Interestingly, there are several neurogenic markers and neurotransmitter-related receptors found in osteocytes (Bliziotes et al., 2006, Paic et al., 2009). We have recently verified that corticosterone regulates the expression of two Paxilline neurogenic markers, namely neuropeptide Y (NPY) and reelin, in osteocytic MLO-Y4 cells (Ma et al., 2012). Although the presence of the mRNA of nAChRs (including α1, β, γ and α4 subtypes) has been reported in osteocytes (Paic et al., 2009), there are no reports about the presence of osteocytic mAChRs mRNA. The available evidence has suggested that osteoblasts and mesenchymal stem cells express some of the n- and mAChR subtypes, which act as modulators to cell differentiation (Hoogduijn et al., 2009, Liu et al., 2011, Paic et al., 2009, Sato et al., 2010). Acetylcholine, nicotine and muscarine, which depend on nAChRs and/or mAChRs, have been reported to be associated with not only general biological processes but also osteogenic-related events within osteoblasts (Rothem et al., 2011, Rothem et al., 2009, Sato et al., 2010). It is unknown whether the cholinergic pathway has the potential to control osteocytes. In the present study, we have identified the gene expression of osteocytic m- and nAChRs as well as the influence of ACh in osteocytes via AChRs.
Materials and methods
Results
Discussion
Bone is a dynamic tissue that is constantly renewed under several stimuli including hormones, cytokines, mechanical and transcriptional signals (Chen et al., 2010, Robling et al., 2006) that target bone cells at various stage of their lifespan. It is intriguing that bone cells express a broad range of neurotransmitters and neurotransimitter receptors or transporters (Spencer et al., 2004). Evidence of their functional effects by pharmacological manipulation (Bliziotes et al., 2001, Hodge et al., 2013, Spencer et al., 2004, Westbroek et al., 2001) support the existence of a complex and functionally significant neurotransmitter-mediated signaling network in bone. These results also suggest distinctive osteo-neuromediators that are involved in bone remodeling. The cholinergic pathway is predominant in the nervous system, and the presence of cholinergic receptors was mentioned in bone cells (Paic et al., 2009). In the present paper, we provide evidence for the expression of cholinergic receptor subtypes in osteocytes and ACh receptor-mediated control of cell activity and gene expression.
Acetylcholine represents the most exemplary prototype of a neurotransmitter. When acetylcholine binds to acetylcholine receptors, it activates the related signal pathways and causes biological functions (Wessler and Kirkpatrick, 2008). There are two classes of cholinergic recepors, nAChR and mAChR. In the present study, we detected the expression of AChRs in mouse bone and osteocytic MLO-Y4 cells using brain tissue as the positive control. As shown in Fig. 1, most of m- and nAChRs subunits mRNAs were detected in the mouse brain and increased between 3 and 6weeks of postnatal development. Reports in the literature about the expression of AChRs in brain are not completely consistent (Shideler and Yan, 2010), which may be associated with the processes of development and the differences in the animal models and samples. Within the past 60years, considerable evidence has accumulated that suggests ACh independent of neurons (non-neuronal ACh) is widely expressed in nature and is involved in the regulation of basic cell functions (Wessler et al., 1999). We found that AChRs gene had a high expression level in the tibia of postnatal 6-week-old mice but with a low or zero expression level in the tibia of 3-week-old mice (Fig. 1). Interestingly, the tibia tissue can match the brain in 6-week-old mice in terms of the gene expression of AChRs which include the M1, M2, M4, M5, α1, α4, α5,β2, β4, γ subunits. It implies that the cholinergic pathway likely plays an important role as a neuroskeletal factor in the process of the development of the young bone. Coincidently, recent studies implied distinct actions of AChRs affecting the remodeling of the adult bone. Mice deficient of α2 (Bajayo et al., 2012) or M3 (Shi et al., 2010) subunit exhibited increased bone resorption due to different cellular reactions. Quantitative analyses of the bone composition of mice (16-week-old) indicated that the α7 subtype is not involved in the regulation of bone collagen synthesis, whereas the M3 subunit exerts stimulatory effects on cancellous bone microarchitecture, flexural rigidity, and bone matrix synthesis (Kliemann et al., 2012). However, we barely detected the mRNA of the α2, α7 or M3 subunits in the tibia of 3- or 6-week-old mice. We thought that the young bone might be the reason why we could not find them and the division of AChRs possibly was clear-cut, each one or some together being charged with specific responsibilities for bone development or bone remodeling. It is necessary to do the further study to identify their potential functions in the different periods of bone development.