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The clock system has been identified as one of the major mechanisms controlling cellular functions. Circadian clock gene oscillations also actively participate in the functions of various cell types including bone-related cells. Previous studies demonstrated that clock genes were expressed in bone tissue and also that their expression exhibited circadian rhythmicity. Recent findings have shown that sympathetic tone plays a central role in biological oscillations in bone. Adrenergic receptor (AR) signaling regulates the expression of clock genes in cancellous bone. Furthermore, α 1 -AR signaling in osteoblasts is known to negatively regulate the expression of bone morphogenetic protein-4 (Bmp4) by up-regulating nuclear factor IL-3 (Nfil3)/e4 promoter-binding protein 4 (E4BP4). The ablation of α 1B -AR signaling also increases the expression of the Bmp4 gene in bone. The findings of transient overexpression and siRNA experiments have supported the involvement of the transcription factor CCAAT/enhancer-binding protein delta (C/EBPδ, Cebpd) in Nfil3 and Bmp4 expression in MC3T3-E1 cells. These findings suggest that the effects of Cebpd are due to the circadian regulation of Bmp4 expression, at least in part, by the up-regulated expression of the clock gene Nfil3 in response to α 1B -AR signaling in osteoblasts. Therefore, AR signaling appears to modulate cellular functionality through the expression of clock genes that are circadian rhythm regulators in osteoblasts. The expression of clock genes regulated by the sympathetic nervous system and clock-controlled genes that affect bone metabolism are described herein.

Background and Purpose: The sympathetic nervous system regulates bone remodelling, in part, through ß2 -adrenoceptor signalling. However, the physiological role of α1 -adrenoceptor signalling in bone in vivo remains unclear. Therefore, to obtain a deeper understanding of bone remodelling by the sympathetic nervous system, we investigated the role of α1B -adrenoceptor signalling in bone metabolism., Experimental Approach: Prazosin, a nonspecific α1 -adrenoceptor antagonist, was administered for 2 weeks in C57BL6 mice, and efficacy was evaluated by bone microarchitecture using microcomputed tomography and determination of bone formation by fluorescent labelling of bone. We also compared the bone phenotype of α1B -adrenoceptor null mice (α1B (-/-) ) with that of wild-type littermates., Key Results: We demonstrated that the systemic administration of prazosin decreased bone formation. In addition, α1B -adrenoceptor-deficient mice had a lower bone mass due to decreased bone formation but did not exhibit any changes in bone-resorbing activity. Furthermore, stimulation with phenylephrine, a non-specific α1 -adrenoceptor agonist, increased the expression of the transcriptional factor CCAAT/enhancer-binding protein δ (Cebpd) in MC3T3-E1 osteoblastic cells. The overexpression of Cebpd induced cellular proliferation in MC3T3-E1 cells, whereas the silencing of Cebpd suppressed it., Conclusions and Implications: Taken together, these results suggested that α1B -adrenoceptor signalling is required for bone formation and regulated cellular proliferation through a mechanism relevant to the up-regulation of Cebpd in osteoblasts and, thus, provide new evidence for the physiological importance of α1B -adrenoceptor signalling in bone homeostasis., (© 2016 The British Pharmacological Society.)

Recent studies reported that serotonin (5-hydroxytryptamine, 5-HT) may be an endogenous paracrine and/or autocrine factor that is used for intercellular communication in bone cells and between multiple organs regulating bone homeostasis. In the present study, we showed that the administration of MDL11939, a selective 5-HT2A receptor antagonist, reduced bone mass in mice. The loss of bone mass in MDL11939-treated mice was associated with impaired bone formation in vivo, as demonstrated by the lower expression of osterix (Osx) and osteocalcin than that in vehicle-treated mice. On the other hand, no significant differences were observed in osteoclast numbers between MDL11939- and vehicle-treated mice. The pharmacological blockade of 5-HT2A receptor signaling significantly decreased alkaline phosphatase activity in osteoblastic cells. In addition, the knockdown of the 5-HT2A receptor by a siRNA treatment decreased Osx, but not Runx2 gene expression in MC3T3-E1 cells. These results suggest that 5-HT2A receptor signaling mediated bone mass by regulating osteoblast differentiation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The sympathetic nervous system modulates bone remodeling and mediates the expression of core clock genes in part through the β-adrenergic receptor (β-AR) in osteoblasts. In this study, we show that in MC3T3-E1 osteoblastic cells that isoproterenol (Iso), a non-selective β-AR agonist, upregulated the transcriptional factor Nfil3, and induced rhythmic mRNA expression of prostaglandin-endoperoxide synthase 2 (Ptgs2, also known as Cox2). The rhythmic effects of Iso on Ptgs2 expression were mediated by interplay between the Per2 and Bmal1 clock genes in osteoblasts. In addition, Ptgs2 was significantly decreased in bone after continuous Iso treatment. Overexpression of Nfil3 decreased Ptgs2 expression in MC3T3-E1 cells. Knockdown of Nfil3 upregulated the expression of Ptgs2 in MC3TC-E1 cells, indicating that Nfil3 negatively regulated Ptgs2 in osteoblasts. Furthermore, Iso acutely induced the expression Nfil3 and increased the binding of Nfil3 to the Ptgs2 promoter in MC3T3-E1 cells. These results suggest that Iso-mediated induction of Nfil3 in osteoblasts regulates the expression of Ptgs2 by driving the expression of circadian clock genes. These findings provide new evidence for a physiological role of circadian clockwork in bone metabolism., (© 2014. Published by The Company of Biologists Ltd.)

Several studies have demonstrated that the α1-adrenergic receptor (AR) plays an important role in regulating cell growth and function in osteoblasts. However, the physiological role of α1-AR signaling in bone metabolism is largely unknown. In this study, the stimulation of phenylephrine (PHE), a nonspecific α1-AR agonist, increased the transcriptional factor Nfil3/E4BP4 and led to the rhythmic expression of bone morphogenetic protein 4 (Bmp4) in MC3T3-E1 osteoblastic cells. We also showed that Bmp4 mRNA expression peaked in bone near zeitgeber time 8 in a 24-h rhythm. Furthermore, the expression of Nfil3 and Bmp4 displayed a circadian pattern with opposing phases, which suggested that Nfil3 repressed the expression of the Bmp4 gene during a circadian cycle. On a molecular level, both loss-of-function and gain-of-function experiments demonstrated that Nfil3/E4BP4 negatively regulated Bmp4 expression in osteoblasts. Furthermore, the systemic administration of PHE increased the expression of Nfil3 mRNA in bone, whereas it decreased that of Bmp4 mRNA. The expression of Bmp4 mRNA was decreased significantly by exposure to PHE, and this was concomitant with the increase in Nfil3 binding to the D-box-containing Bmp4 promoter region in MC3T3-E1 cells, which indicates that the expression of Nfil3 by α1-AR signaling can bind directly to the Bmp4 promoter and inhibit Bmp4 expression in osteoblasts. Our results suggest that α1-AR signaling regulates clock genes and Bmp4 expression in osteoblasts. Moreover, α1-AR signaling negatively regulated Bmp4 expression by up-regulating the transcriptional factor Nfil3/E4BP4 in osteoblasts., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

In the previous study, we reported the gene expression for proteins related to the function of 5-hydroxytryptamine (5-HT, serotonin) and elucidated the expression patterns of 5-HT(2) receptor subtypes in mouse osteoblasts. In the present study, we evaluated the possible involvement of 5-HT receptor subtypes and its inactivation system in MC3T3-E1 cells, an osteoblast cell line. DOI, a 5-HT(2A) and 5-HT(2C) receptor selective agonist, as well as 5-HT concentration-dependently increased proliferative activities of MC3T3-E1 cells in their premature period. This effect of 5-HT on cell proliferation were inhibited by ketanserin, a 5-HT(2A) receptor specific antagonist. Moreover, both DOI-induced cell proliferation and phosphorylation of ERK1 and 2 proteins were inhibited by PD98059 and U0126, selective inhibitors of MEK in a concentration-dependent manner. Furthermore, treatment with fluoxetine, a 5-HT specific re-uptake inhibitor which inactivate the function of extracellular 5-HT, significantly increased the proliferative activities of MC3T3-E1 cells in a concentration-dependent manner. Our data indicate that 5-HT fill the role for proliferation of osteoblast cells in their premature period. Notably, 5-HT(2A) receptor may be functionally expressed to regulate mechanisms underlying osteoblast cell proliferation, at least in part, through activation of ERK/MAPK pathways in MC3T3-E1 cells., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

We analyzed the expression of 5-HT(2) receptors and proteins related to inactivation of 5-HT in primary cultures of mouse osteoblasts. The mRNA for the 5-HT(2A) receptor was detectable in anaplastic osteoblasts as well as in differentiated and matured osteoblasts. The mRNA for the 5-HT(2B) receptor and 5-HT transporter was undetectable in anaplastic osteoblasts and became detectable in differentiated and matured osteoblasts. It was suggested that 5-HT might regulate the proliferation of anaplastic osteoblasts through the 5-HT(2A) receptor without control by 5-HT-inactivating mechanisms. The differentiation and maturation of osteoblasts might be regulated by the activation of the 5-HT(2B) receptor under the control of 5-HT inactivation.