The ATF3–OPG Axis Contributes to Bone Formation by Regulating the Differentiation of Osteoclasts, Osteoblasts, and Adipocytes

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

Downregulation of Runx2 by 1,25-Dihydroxyvitamin D3 Induces the Transdifferentiation of Osteoblasts to Adipocytes

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

Overexpression of Neurogenin 1 Negatively Regulates Osteoclast and Osteoblast Differentiation

Neurogenin 1 (Ngn1) belongs to the basic helix–loop–helix (bHLH) transcription factor family and plays important roles in specifying neuronal differentiation. The present study aimed to determine whether forced Ngn1 expression contributes to bone homeostasis. Ngn1 inhibited the p300/CREB-binding protein-associated factor (PCAF)-induced acetylation of nuclear factor of activated T cells 1 (NFATc1) and runt-related transcription factor 2 (Runx2) through binding to PCAF, which led to the inhibition of osteoclast and osteoblast differentiation, respectively. In addition, Ngn1 overexpression inhibited the TNF-α- and IL-17A-mediated enhancement of osteoclast differentiation and IL-17A-induced osteoblast differentiation. These findings indicate that Ngn1 can serve as a novel therapeutic agent for treating ankylosing spondylitis with abnormally increased bone formation and resorption

The ATF3–OPG Axis Contributes to Bone Formation by Regulating the Differentiation of Osteoclasts, Osteoblasts, and Adipocytes

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

Bifunctional Role of CrkL during Bone Remodeling

Coupled signaling between bone-forming osteoblasts and bone-resorbing osteoclasts is crucial to the maintenance of bone homeostasis. We previously reported that v-crk avian sarcoma virus CT10 oncogene homolog-like (CrkL), which belongs to the Crk family of adaptors, inhibits bone morphogenetic protein 2 (BMP2)-mediated osteoblast differentiation, while enhancing receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation. In this study, we investigated whether CrkL can also regulate the coupling signals between osteoblasts and osteoclasts, facilitating bone homeostasis. Osteoblastic CrkL strongly decreased RANKL expression through its inhibition of runt-related transcription factor 2 (Runx2) transcription. Reduction in RANKL expression by CrkL in osteoblasts resulted in the inhibition of not only osteoblast-dependent osteoclast differentiation but also osteoclast-dependent osteoblast differentiation, suggesting that CrkL participates in the coupling signals between osteoblasts and osteoclasts via its regulation of RANKL expression. Therefore, CrkL bifunctionally regulates osteoclast differentiation through both a direct and indirect mechanism while it inhibits osteoblast differentiation through its blockade of both BMP2 and RANKL reverse signaling pathways. Collectively, these data suggest that CrkL is involved in bone homeostasis, where it helps to regulate the complex interactions of the osteoblasts, osteoclasts, and their coupling signals

Modulation of Androgen Receptor Transactivation by the SWI3-Related Gene Product (SRG3) in Multiple Ways

The SWI3-related gene product (SRG3), a component of the mouse SWI/SNF complex, has been suggested to have an alternative function. Here, we demonstrate that in the prostate transactivation of the androgen receptor (AR) is modulated by SRG3 in multiple ways. The expression of SRG3, which is developmentally regulated in the prostate, is induced by androgen through AR. SRG3 in turn enhances the transactivation of AR, providing a positive feedback regulatory loop. The SRG3 coactivation of AR transactivation is achieved through the recruitment of coactivator SRC-1, the protein level of which is upregulated by SRG3, providing another pathway of positive regulation. Interestingly, SRG3 coactivation of AR transactivation is fully functional in BRG1/BRM-deficient C33A cells and the AR/SRG3/SRC-1 complex formed in vivo contains neither BRG1 nor BRM protein, suggesting the possibility of an SRG3 function independent of the SWI/SNF complex. Importantly, the AR/SRG3/SRC-1 complex occupies androgen response elements on the endogenous SRG3 and PSA promoter in an androgen-dependent manner in mouse prostate and LNCaP cells, respectively, inducing gene expression. These results suggest that the multiple positive regulatory mechanisms of AR transactivation by SRG3 may be important for the rapid proliferation of prostate cells during prostate development and regeneration

Transcription Factor Lmx1b Negatively Regulates Osteoblast Differentiation and Bone Formation

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