Bone remodeling is a balanced process between bone synthesis and degradation, maintaining homeostasis and a constant bone mass in adult life. Imbalance will lead to conditions such as osteoporosis or hyperostosis. Osteoblasts build bone, becoming embedded in bone matrix as mature osteocytes. Osteocytes have a role in sensing and translating mechanical loads into biochemical signals, regulating the differentiation and activity of osteoblasts residing at the bone surface through the secretion of Sclerostin (SOST), an inhibitor of WNT signaling. Excessive mechanical load can lead to activation of cellular stress responses altering cell behavior and differentiation. The unfolded protein response (UPR) is a shared pathway utilized by cells to cope with stress stimuli. We showed that in a transgenic mouse model, activation of the UPR in early differentiating osteocytes delays maturation, maintaining active bone synthesis. In addition, expression of SOST is delayed or suppressed; resulting in active WNT signaling and enhanced periosteal bone formation, and the combined outcome is generalized hyperostosis. A clear relationship between the activation of the unfolded protein response was established and the onset of hyperostosis that can be suppressed with a chemical chaperone, sodium 4-phenobutyrate (4-PBA). As the phenotype is highly consistent with craniodiaphyseal dysplasia (CDD; OMIM 122860), we propose activation of the UPR could be part of the disease mechanism for CDD patients as these patients are heterozygous for SOST mutations that impair protein folding and secretion. Thus, therapeutic agents ameliorating protein folding or the UPR can be considered as a potential therapeutic treatment.

Background The present study aimed to assess the perturbation in circular RNA (circRNA)/mRNA expression profiles and a circRNA-miRNA-mRNA coexpression network involved in the potential protective effect of diosgenin (DIO) on alveolar bone loss in rats subjected to ovariectomy (OVX). Methods The Wistar rats (female) manipulated with sham operation were classified as the SHAM group and the grouping of OVX rats administered with DIO, estradiol valerate or vehicle for 12 weeks was DIO group, EV group and OVX group respectively. Following treatments, the plasmatic levels of osteocalcin and tumor necrosis factor-alpha and the microstructure of alveolar bone were assayed. Based on microarray analyses, we identified differentially expressed (DE) circRNAs and mRNAs in alveolar bone of rats in both OVX and DIO group. The DE circRNAs and DE mRNAs involved in the bone metabolism pathway validated by RT-qPCR were considered key circRNAs/mRNAs. On the basis of these key circRNAs/mRNAs, we predicted the overlapping relative miRNAs of key circRNAs/mRNAs, and a circRNA-miRNA-mRNA network was built. Results DIO showed an anti-osteopenic effect on the rat alveolar bone loss induced by OVX. In total, we found 10 DE circRNAs (6 downregulated and 4 upregulated) and 614 DE mRNAs (314 downregulated and 300 upregulated) in samples of the DIO group compared with those of the OVX group. However, only one circRNA (rno_circRNA_016717) and seven mRNAs ( Sfrp1 , Csf1 , Il1rl1 , Nfatc4 , Tnfrsf1a , Pik3c2g , and Wnt9b ) were validated by qRT-PCR and therefore considered key circRNA/mRNAs. According to these key circRNA/mRNAs and overlapping predicted miRNAs, a coexpression network was constructed. After network analysis, one circRNA-miRNA-mRNA axis (circRNA_016717/miR-501-5p/ Sfrp1 ) was identified. Conclusion The mechanism of DIO inhibiting alveolar bone loss after OVX is possibly relevant to the simultaneous inhibition of osteogenesis and osteoclastogenesis by mediating the expression of important molecules in the Wnt, PI3K, RANK/RANKL or osteoclastogenic cytokine pathways. The circRNA_016717/miR-501-5p/ Sfrp1 axis may play important roles in these processes.

Osteoclasts are the only multinucleated cells in vivo responsible for bone resorption and are vital for regulating bone remodeling and maintaining bone mass. The RAW264.7 cell line is widely used to study osteoclastic differentiation and biological molecular mechanism. However, protocols for inducing osteoclast formation in RAW264.7 cells vary considerably between laboratories, hindering the replication of results. Therefore, we tested the influence of culture conditions on osteoclast differentiation, including cell density and receptor activator of nuclear factor kappa-B ligand (RANKL) concentrations with or without macrophage colony-stimulating factors (M-CSF). Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the morphology of osteoclasts. qPCR was used to detect gene expression of osteoclast-specific gene marker cathepsin K (CTSK), osteoclast transcription factors c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1). The bone resorption function was evaluated by a scanning electron microscope (SEM). RANKL treatment increased multinucleated osteoclasts formation and increased CTSK, c-Fos and NFATc1 gene expression. Compared with RANKL treatment, M-CSF significantly decreased multinucleated osteoclasts formation, reduced CTSK gene expression and had little effect on c-Fos and NFATc1 gene expression. Concerning bone resorption activity, RANKL treatment increased bone resorption pits on bovine bone slices. Significantly higher levels of osteoclastogenesis were observed with RAW264.7-cell density of 2×104 cells/well in 24-well plates. Our results suggest that the addition of 50 ng/ml M-CSF has no positive effect on osteoclastogenesis. RANKL treatment and cell density contribute to osteoclast formation, and the optimal conditions are beneficial when exploring osteoclast function and mechanism.

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The present study aimed to assess the changes in circulating microRNA (miRNA) expression profiles associated with the potential osteoprotective effect of diosgenin (DIO) in ovariectomized (OVX) rats. Wistar rats (female) were subjected to a sham operation (SHAM group) or ovariectomy. OVX rats were treated with DIO (DIO group) or vehicle (OVX group) for 12 weeks. Following treatment, the serum estradiol, bone turnover biomarker levels, and the microarchitecture of tibias were assayed. Based on miRNA microarray and qRT-PCR analyses, differentially expressed (DE) circulating miRNAs were identified between the OVX and SHAM groups (comparison A) and between the DIO and OVX groups (comparison B). Furthermore, putative target genes of shared DE miRNAs with opposite expression trends in the two comparisons were predicted by ingenuity pathway analysis (IPA). Finally, the expression levels of the putative target genes in serum and tibia were validated by qRT-PCR. The micro-CT results demonstrated that DIO had a substantial anti-osteopenic effect on the tibias of OVX rats. In total, we found 5 DE circulating miRNAs (four upregulated and one downregulated) in comparison A and 21 DE circulating miRNAs (15 upregulated and 6 downregulated) in comparison B. However, only one DE circulating miRNA (rno-miR-20a-5p) had opposite expression trends between the two comparisons. Including rno-miR-20a-5p, 7 of the 10 selected DE circulating miRNAs between the two comparisons passed qRT-PCR validation. Specifically, based on qRT-PCR validation, DIO upregulated the expression of rno-miR-20a-5p and downregulated that of three target genes (Tnf, Creb1, and Tgfbr2) of the "osteoclast differentiation" pathway in the tibias of OVX rats. Our results suggested that DIO could change the circulating miRNA profile of OVX rats and inhibited the downregulation of miR-20a-5p in serum and tibia. DIO might exert an antiosteoclastogenic effect on OVX rats by upregulating the expression of miR-20a-5p in circulation and bone tissue.