The circadian clock regulates various physiological processes, including bone metabolism. The nuclear receptors Reverbs, comprising Rev-erb alpha and Rev-erb beta, play a key role as transcriptional regulators of the circadian clock. In this study, we demonstrate that Rev-erbs negatively regulate differentiation of osteoclasts and osteoblasts. The knockdown of Rev-erb alpha in osteoclast precursor cells enhanced receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclast formation, as well as expression of nuclear factor of activated T cells 1 (NFATc1), osteoclast-associated receptor (OSCAR), and tartrate-resistant acid phosphatase (TRAP). The overexpression of Rev-erb alpha leads to attenuation of the NFATc1 expression via inhibition of recruitment of c-Fos to the NFATc1 promoter. The overexpression of Rev-erb alpha in osteoblast precursors attenuated the expression of osteoblast marker genes including Runx2, alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC). Rev-erb alpha interfered with the recruitment of Runx2 to the promoter region of the target genes. Conversely, knockdown of Reverb alpha in the osteoblast precursors enhanced the osteoblast differentiation and function. In addition, Rev-erb alpha negatively regulated osteoclast and osteoblast differentiation by suppressing the p38 MAPK pathway. Furthermore, intraperitoneal administration of GSK4112, a Rev-erb agonist, protects RANKL-induced bone loss via inhibition of osteoclast differentiation in vivo. Taken together, our results demonstrate a molecular mechanism of Rev-erbs in the bone remodeling, and provide a molecular basis for a potential therapeutic target for treatment of bone disease characterized by excessive bone resorption.

The non-receptor tyrosine kinase c-Src participates in bone metabolism by regulating the activities of both the bone-resorbing osteoclasts and bone-forming osteoblasts. In this study, we investigated whether megakaryocyte-associated tyrosine kinase (Matk), a potent inhibitor of c-Src, affects the functions of murine osteoclasts and osteoblasts. Results revealed that the formation of osteoclasts with actin rings was attenuated by Matk overexpression in osteoclast precursor cells but was enhanced by Matk knockdown. The inhibitory effect of Matk on osteoclasts was closely related with the inhibition of c-Src activity. Intriguingly, Matk overexpression in osteoblasts reduced bone nodule formation. Conversely, Matk knockdown increased osteoblast function. Most importantly, binding of Matk to Runx2 resulted in the inhibition of Runx2 translocation into the nucleus and downregulation of Runx2 target genes. Taken together, our findings demonstrated that Matk plays a critical role in bone metabolism by impairing the functions of osteoclasts and osteoblasts via distinct mechanisms involving inhibition of c-Src–dependent and –independent signaling pathways.

1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) indirectly stimulates bone formation, but little is known about its direct effect on bone formation. In this study, we observed that 1,25(OH)₂D₃ enhances adipocyte differentiation, but inhibits osteoblast differentiation during osteogenesis. The positive role of 1,25(OH)₂D₃ in adipocyte differentiation was confirmed when murine osteoblasts were cultured in adipogenic medium. Additionally, 1,25(OH)₂D₃ enhanced the expression of adipocyte marker genes, but inhibited the expression of osteoblast marker genes in osteoblasts. The inhibition of osteoblast differentiation and promotion of adipocyte differentiation mediated by 1,25(OH)₂D₃ were compensated by Runx2 overexpression. Our results suggest that 1,25(OH)₂D₃ induces the transdifferentiation of osteoblasts to adipocytes via Runx2 downregulation in osteoblasts.

The LIM-homeodomain transcription factor Lmx1b plays a key role in body pattern formation during development. Although Lmx1b is essential for the normal development of multiple tissues, its regulatory mechanism in bone cells remains unclear. Here, we demonstrated that Lmx1b negatively regulates bone morphogenic protein 2 (BMP2)-induced osteoblast differentiation. Overexpressed Lmx1b in the osteoblast precursor cells inhibited alkaline phosphatase (ALP) activity and nodule formation, as well as the expression of osteoblast maker genes, including runt-related transcription factor 2 (Runx2), alkaline phosphatase (Alpl), bone sialoprotein (Ibsp), and osteocalcin (Bglap). Conversely, the knockdown of Lmx1b in the osteoblast precursors enhanced the osteoblast differentiation and function. Lmx1b physically interacted with and repressed the transcriptional activity of Runx2 by reducing the recruitment of Runx2 to the promoter region of its target genes. In vivo analysis of BMP2-induced ectopic bone formation revealed that the knockdown of Lmx1b promoted osteogenic differentiation and bone regeneration. Our data demonstrate that Lmx1b negatively regulates osteoblast differentiation and function through regulation of Runx2 and provides a molecular basis for therapeutic targets for bone diseases.

Activating transcription factor 3 (ATF3) has been identified as a negative regulator of osteoblast differentiation in in vitro study. However, it was not associated with osteoblast differentiation in in vivo study. To provide an understanding of the discrepancy between the in vivo and in vitro findings regarding the function of ATF3 in osteoblasts, we investigated the unidentified roles of ATF3 in osteoblast biology. ATF3 enhanced osteoprotegerin (OPG) production, not only in osteoblast precursor cells, but also during osteoblast differentiation and osteoblastic adipocyte differentiation. In addition, ATF3 increased nodule formation in immature osteoblasts and decreased osteoblast-dependent osteoclast formation, as well as the transdifferentiation of osteoblasts to adipocytes. However, all these effects were reversed by the OPG neutralizing antibody. Taken together, these results suggest that ATF3 contributes to bone homeostasis by regulating the differentiation of various cell types in the bone microenvironment, including osteoblasts, osteoclasts, and adipocytes via inducing OPG production.