Osteoblasts have been thought to be
Osteoblasts have been thought to be the major cell type that expresses RANKL (Suda et al., 1999) which is a ligand for osteoprotegerin (OPG) and which functions as a key factor for osteoclast differentiation and activation. Recently, Nakashima and his colleagues (Nakashima et al., 2011) demonstrated that purified osteocytes express a much higher amount of RANKL and have a greater capacity to support osteoclastogenesis in vitro than osteoblasts and bone marrow stromal cells. Their remarkable work also indicated that osteocytes are the major source of RANKL in bone remodeling in vivo. The present study showed a reciprocal regulation of RANKL and OPG mRNA caused by ACh via the cholinergic receptors, which resulted in a significant increase in the mRNA ratio of RANKL:OPG in osteocytes (Fig. 3B). It indicates that the cholinergic pathway possibly plays a role in the RANKL-mediated osteocytic modulation to osteoclasts.
NPY and reelin, two Peptone neurogenic markers, have been identified having higher mRNA levels in osteocytes than osteoblasts (Paic et al., 2009), which was also demonstrated by using the MLO-Y4 and MC3T3-E1 cell lines in the present paper (Fig. 4). We further revealed the presence of both proteins in both cell lines by immunofluorescence (Fig. 5). NPY exerts hypothalamic actions on the bone and adipose tissues and locally affects osteoblasts and adipocytes (Karsenty, 2000, Zengin et al., 2010). It is thought to be a potential modulator of bone remodeling. There is little evidence (Rawlinson et al., 2009, Schrauwen et al., 2009, van der Zande et al., 2010) that reelin has a possible physiological effect on bone, and this effect remains unclear. In our previous work, we demonstrated that the expression of reelin and NPY can be modulated by corticosterone, a catabolic factor for bone, in the MLO-Y4 cells (Ma et al., 2012). In this study, we found that ACh could regulate the mRNA expression of osteocytic NPY via m- and n-AChRs without changing the protein levels (Fig. 6). Additionally, the treatment of ACh simultaneously activated the m- and n-AChRs, which had an opposite effect that resulted in a decrease in the protein expression of reelin and no change in the gene expression (Fig. 6B and E). The antagonism among the cholinergic receptor subtypes indicates that the specific activation or inhibition of one receptor subunit may destroy a certain metabolic balance in healthy cells.
Taken together, our study provided more details regarding the expression of cholinergic receptors in osteocytes and ACh-induced cell proliferation and gene expression via the cholinergic receptors in vitro. Acetylcholine can play an intermediary role in the interactions of non-neuronal cells with the external environment, hormones, growth factors, cytokines and the neural system (Wessler and Kirkpatrick, 2008). In addition, In vivo studies (Kliemann et al., 2012, Shi et al., 2010) have shown that the inhibition of cholinergic activity favors bone loss, whereas its stimulation favors the accrual of bone mass. Our observations and the previously published findings are helpful in understanding cholinergic activity and its association with bone and osteocytes. These results may support the discovery of promising therapies for bone diseases based on the cholinergic pathway.
Acknowledgments We thank Prof. L.F. Bonewald from the Department of Oral Biology, University of Missouri in Kansas City, Missouri, USA, for the gift of the MLO-Y4 cells. This work was supported by the National Natural Science Foundation of China (Nos. 81170941 and 81300856), and the Science & Technology Support Project, Science and Technology Department of the Sichuan Province (No. 2011SZ0157).
Introduction Vascular endothelial growth factor (VEGF) is involved in angiogenesis and vasculogenesis and plays an important role in pathological and physiological phenomena such as wound healing, the menstrual cycle, and permeability of the blood brain barrier (Adini et al., 2017; Detmar et al., 1995; Ferrara, 2004; Greenberg and Jin, 2005; Shifren et al., 1996; Zhang et al., 2000). VEGF is also a key mediator of angiogenesis and neovascularization, which are required for cancer cell proliferation. However, recent evidence indicates that VEGF plays a critical role in neuroprotection and neurogenesis (Greenberg and Jin, 2005). VEGF was also shown to protect HN33 cells, an immortalized hippocampal neuronal cell line, from hypoxia and glucose deprivation, suggesting that its neuroprotective effects are due to the stimulation of VEGF receptor 2 (VEGFR2), which was led by the phosphorylation of the receptor (Jin et al., 2000). In addition, VEGF from astrocytes enhanced cell survival signaling in neurons through the activation of VEGFR2 after oxygen and glucose deprivation (Zhang et al., 2017).