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    • There are several reports about the expression of AChRs

    2024-03-22

    There are several reports about the expression of AChRs and their modulation to osteogenic factors in osteoblasts (Rothem et al., 2011, Sato et al., 2010, Walker et al., 2001). The normal osteocytic MLO-Y4 22(R)-hydroxy Cholesterol expressed multiple subtypes, which was demonstrated by the detection of M1, M2, M5, β2, β4 and γ mRNAs by RT-PCR (Fig. 1). The MLO-Y4 cells also have the potential to express the M4, α4, α5 and δ subunits under the given chemical stimulations in the present study (Fig. 1). The wide range of osteocytic AChRs suggested that the response to receptor activation might initiate diverse intracellular signaling pathways. Muscarinic receptors are members of the G protein-coupled receptors (GPCRs) family, while nicotinic ones are pentameric ligand-gated cation channels (Brown, 2010, Taly et al., 2009). The activation of muscarinic and nicotinic subtypes is linked to diverse signaling molecules, such as Ras-mitogen-activated protein kinase (MAPK), extracellular regulated protein kinases (ERK), phosphatidylinositol 3-kinase (PI3k)-Akt and protein kinase C (PKC). They are involved in cell survival, proliferation, apoptosis or DNA synthesis (Resende and Adhikari, 2009) and regarded as regulators of skeletogenesis and bone homeostasis. (Greenblatt et al., 2010, Kono et al., 2007, Xia et al., 2010). Explorations into the relationship between the cholinergic signaling pathway and osteocytes need to be performed in future studies, and the cholinergic receptors may become a potential therapeutic targets of bone diseases. ACh treatment could affect the gene expression of osteocytic M1, M4, β4 and γ subunits (Fig. 1). Mec and d-TC not only reversed the effects of ACh, but also acted similar to ACh alone in some instances (Fig. 1). In other instances, d-TC and Mec did not inhibit the actions of ACh instead of altering the mRNA expression of a certain subunit similarly to ACh (Fig. 1). It is well-known that Mec and d-TC are two different nAChR antagonists. To characterize a drug candidate as an AChR antagonist, it must, by definition, have no intrinsic activity, i.e., no agonist efficacy at the targeted AChR (Dwoskin and Crooks, 2001). However, gene expression was also reported to be modulated by d-TC and Mec alone in osteoblasts (Rothem et al., 2011), for which the explanation can be the changes in the conformation of the AChR caused by the binding of antagonists. In the present study, we think that the major contributor to the outcome can be ACh-induced activation of other subtypes than those subtypes that were competitively inhibited by Mec or d-TC. AChE plays cholinergic roles in the hydrolysis and inactivation of acetylcholine, as well as noncholinergic roles during development and tissue morphogenesis (Inkson et al., 2004). BChE is known as serum cholinesterase or pseudo-cholinesterase with less functions than AChE (Soreq and Seidman, 2001). AChE and BChE have emerged as novel mediators in bone (En-Nosse et al., 2009, Genever et al., 1999). Previous studies identified AChE expression at sites of new bone formation which suggests its role as a noncollagenous bone matrix protein (Genever et al., 1999) that could mediate cell–matrix interactions and could be regulated by mechanical strain (Inkson et al., 2004). Moreover, there are many binding sites for osteogenic factors, which include Cbfa-1, vitamin D3 and 17β-estradiol (Grisaru et al., 1999) in the extended human AChE promoter. In the present paper, the gene expression of AChE was present in normal MLO-Y4 cells, but was absent after the application of ACh (Fig. 1). Perhaps AChE could hydrolyze ACh to regulate the concentration of this neurohormone which simultaneously mediated AChE gene expression via m- or nAChRs. It needs additional studies on the functional role of AChE in osteocytes and the relation among AChE, AChRs and ACh. The osteocytic MLO-Y4 cells treated by ACh showed a significant increase in cell proliferation and viability on day three compared with the control, which was reversed by the non-specific nAChR antagonists, Mec and d-TC (Fig. 2D and E). These results provided evidence for the involvement of nAChR in ACh-induced cell proliferation and viability. Mecamylamine, a relatively nonspecific nAChR antagonist, blocks the effects of α7 homomeric nAChR as well as more complex heteromeric nAChRs which contain α3, α5, β2, andβ4 subunits (Song et al., 2003). d-Tubocurarine competitively inhibits ACh-evoked response by binding toα7 nAChR and heteromeric nAChRs which contain α2, α3, α4, β2 and β4 subunits (Dwoskin and Crooks, 2001). According to the quantity of gene expression shown in Fig. 2, the α4, α5, β2 and β4 subunits might play roles in MLO-Y4 proliferation.