Nodal negatively regulates osteoclast differentiation by inducing STAT1 phosphorylation.

Several members of the transforming growth factor beta (TGF-ß) superfamily regulate the proliferation, differentiation, and function of bone-forming osteoblasts and bone-resorbing osteoclasts. However, it is still unknown whether Nodal, a member of the TGF-ß superfamily, serves a function in bone cells. In this study, we found that Nodal did not have any function in osteoblasts but instead negatively regulated osteoclast differentiation. Nodal inhibited RANKL-induced osteoclast differentiation by downregulating the expression of pro-osteoclastogenic genes, including c-fos, Nfatc1, and Blimp1, and upregulating the expression of antiosteoclastogenic genes, including Bcl6 and Irf8. Nodal activated STAT1 in osteoclast precursor cells, and STAT1 downregulation significantly reduced the inhibitory effect of Nodal on osteoclast differentiation. These findings indicate that Nodal activates STAT1 to downregulate or upregulate the expression of pro-osteoclastogenic or antiosteoclastogenic genes, respectively, leading to the inhibition of osteoclast differentiation. Moreover, the inhibitory effect of Nodal on osteoclast differentiation contributed to the reduction of RANKL-induced bone loss in vivo.

MCP-5 suppresses osteoclast differentiation through Ccr5 upregulation.

Human monocyte chemoattractant protein-1 (MCP-1) in mice has two orthologs, MCP-1 and MCP-5. MCP-1, which is highly expressed in osteoclasts rather than in osteoclast precursor cells, is an important factor in osteoclast differentiation. However, the roles of MCP-5 in osteoclasts are completely unknown. In this study, contrary to MCP-1, MCP-5 was downregulated during receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation and was considered an inhibitory factor in osteoclast differentiation. The inhibitory role of MCP-5 in osteoclast differentiation was closely related to the increase in Ccr5 expression and the inhibition of IκB degradation by RANKL. Transgenic mice expressing MCP-5 controlled by Mx-1 promoter exhibited an increased bone mass because of a decrease in osteoclasts. This result strongly supported that MCP-5 negatively regulated osteoclast differentiation. MCP-5 also prevented severe bone loss caused by RANKL.

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.

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.

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.

c-Src-Dependent and -Independent Functions of Matk in Osteoclasts and Osteoblasts.

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.

Endoplasmic Reticulum-Bound Transcription Factor CREBH Stimulates RANKL-Induced Osteoclastogenesis.

Endoplasmic reticulum (ER) stress is triggered by various metabolic factors, such as cholesterol and proinflammatory cytokines. Recent studies have revealed that ER stress is closely related to skeletal disorders, such as osteoporosis. However, the precise mechanism by which ER stress regulates osteoclast differentiation has not been elucidated. In this study, we identified an ER-bound transcription factor, cAMP response element-binding protein H (CREBH), as a downstream effector of ER stress during RANKL-induced osteoclast differentiation. RANKL induced mild ER stress and the simultaneous accumulation of active nuclear CREBH (CREBH-N) in the nucleus during osteoclastogenesis. Overexpression of CREBH-N in osteoclast precursors enhanced RANKL-induced osteoclast formation through NFATc1 upregulation. Inhibiting ER stress using a specific inhibitor attenuated the expression of osteoclast-related genes and CREBH activation. In addition, inhibition of reactive oxygen species using N-acetylcysteine attenuated ER stress, expression of osteoclast-specific marker genes, and RANKL-induced CREBH activation. Furthermore, inhibition of ER stress and CREBH signaling pathways using an ER stress-specific inhibitor or CREBH small interfering RNAs prevented RANKL-induced bone destruction in vivo. Taken together, our results suggest that reactive oxygen species/ER stress signaling-dependent CREBH activation plays an important role in RANKL-induced osteoclastogenesis. Therefore, inactivation of ER stress and CREBH signaling pathways may represent a new treatment strategy for osteoporosis.

Role of CrkII Signaling in RANKL-Induced Osteoclast Differentiation and Function.

Rac1, a member of small GTPases, is a key regulator of osteoclast differentiation and function. The Crk family adaptor proteins, consisting of Src homology (SH) 2 and SH3 protein-binding domains, regulate cell proliferation, migration, and invasion through Rac1 activation. In this study, we examined the role of CrkII in osteoclast differentiation and function. Retroviral overexpression of CrkII in osteoclast precursors enhanced osteoclast differentiation and resorptive function through Rac1 activation. The knockdown of CrkII in osteoclast precursors using small interfering RNA inhibited osteoclast differentiation and its resorption activity. Unlike wild-type CrkII, overexpression of the three SH domains in mutant forms of CrkII did not enhance either osteoclast differentiation or function. Phosphorylation of p130 Crk-associated substrate (p130Cas) by osteoclastogenic cytokines in preosteoclasts increased the interaction between p130Cas and CrkII, which is known to be involved in Rac1 activation. Furthermore, transgenic mice overexpressing CrkII under control of a tartrate-resistant acid phosphatase promoter exhibited a low bone mass phenotype, associated with increased resorptive function of osteoclasts in vivo. Taken together, our data suggest that the p130Cas/CrkII/Rac1 signaling pathway plays an important role in osteoclast differentiation and function, both in vitro and in vivo.

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.

Akt induces osteoclast differentiation through regulating the GSK3ß/NFATc1 signaling cascade.

SHIP is an SH2-containing inositol-5-phosphatase expressed in hematopoietic cells. It hydrolyzes the PI3K product PI(3,4,5)P(3) and blunts the PI3K-initiated signaling pathway. Although the PI3K/Akt pathway has been shown to be important for osteoclastogenesis, the molecular events involved in osteoclast differentiation have not been revealed. We demonstrate that Akt induces osteoclast differentiation through regulating the GSK3ß/NFATc1 signaling cascade. Inhibition of the PI3K by LY294002 reduces formation of osteoclasts and attenuates the expression of NFATc1, but not that of c-Fos. Conversely, overexpression of Akt in bone marrow-derived macrophages (BMMs) strongly induced NFATc1 expression without affecting c-Fos expression, suggesting that PI3K/Akt-mediated NFATc1 induction is independent of c-Fos during RANKL-induced osteoclastogenesis. In addition, we found that overexpression of Akt enhances formation of an inactive form of GSK3ß (phospho-GSK3ß) and nuclear localization of NFATc1, and that overexpression of a constitutively active form of GSK3ß attenuates osteoclast formation through downregulation of NFATc1. Furthermore, BMMs from SHIP knockout mice show the increased expression levels of phospho-Akt and phospho-GSK3ß, as well as the enhanced osteoclastogenesis, compared with wild type. However, overexpression of a constitutively active form of GSK3ß attenuates RANKL-induced osteoclast differentiation from SHIP-deficient BMMs. Our data suggest that the PI3K/Akt/GSK3ß/NFATc1 signaling axis plays an important role in RANKL-induced osteoclastogenesis.

Sestrin2 inhibits RANKL-induced osteoclastogenesis through AMPK activation and ROS inhibition.

Sestrins are stress-responsive proteins with antioxidant properties. They participate in cellular redox balance and protect against oxidative damage. This study investigated the effects of Sestrin2 (Sesn2) on osteoclast differentiation and function. Overexpressing Sesn2 in osteoclast precursor cells significantly inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis. This was assessed as reduced expression of various osteoclast markers, including c-Fos, nuclear factor of activated T cells 1 (NFATc1), osteoclast-associated receptor, tartrate-resistant acid phosphatase, and cathepsin K. Conversely, downregulation of Sesn2 produced the opposite effect. Mechanistically, Sesn2 overexpression enhanced AMPK activation and the nuclear translocation of nuclear factor erythroid-derived factor 2-related factor 2 (Nrf2), promoting antioxidant enzymes. Moreover, azithromycin (Azm) induced Sesn2 expression, which suppressed RANKL-induced osteoclast differentiation. Specifically, Azm treatment reduced RANKL-induced production of reactive oxygen species in osteoclasts. Furthermore, intraperitoneal administration of Azm ameliorated RANKL-induced bone loss by reducing osteoclast activity in mice. Taken together, our results suggested that Azm-induced Sesn2 act as a negative regulator of RANKL-induced osteoclast differentiation through the AMPK/NFATc1 signaling pathway. Concisely, targeting Sesn2 can be a potential pharmacological intervention in osteoporosis.

Nano-formulations for bone-specific delivery of siRNA for CrkII silencing-induced regulation of bone formation and resorption to maximize therapeutic potential for bone-related diseases.

CrkII, a member of the adaptor protein family, is known to participate in bone homeostasis via the regulation of osteoclasts and osteoblasts. Therefore, silencing CrkII would beneficially impact the bone microenvironment. In this study, CrkII siRNA encapsulated by a bone-targeting peptide (AspSerSer)6-liposome was evaluated for its therapeutic applications using a receptor activator of nuclear factor kappa-B ligand (RANKL)-induced bone loss model. (AspSerSer)6-liposome-siCrkII maintained its gene-silencing ability in both osteoclasts and osteoblasts in vitro and significantly reduced osteoclast formation while increasing osteoblast differentiation in vitro. Fluorescence image analyses showed that the (AspSerSer)6-liposome-siCrkII was present largely in bone, where it remained present for up to 24 hours and was cleared by 48 hours, even when systemically administrated. Importantly, microcomputed-tomography revealed that bone loss induced by RANKL administration was recovered by systemic administration of (AspSerSer)6-liposome-siCrkII. Collectively, the findings of this study suggest that (AspSerSer)6-liposome-siCrkII is a promising therapeutic strategy for the development of treatments for bone diseases, as it overcomes the adverse effects derived from ubiquitous expression via bone-specific delivery of siRNA.

Pim-1 regulates RANKL-induced osteoclastogenesis via NF-κB activation and NFATc1 induction.

Pim kinases are emerging as important mediators of cytokine signaling pathways in hematopoietic cells. In this study, we demonstrate that Pim-1 positively regulates RANKL-induced osteoclastogenesis and that Pim-1 expression can be upregulated by RANKL signaling during osteoclast differentiation. The silencing of Pim-1 by RNA interference or overexpression of a dominant negative form of Pim-1 (Pim-1 DN) in bone marrow-derived macrophage cells attenuates RANKL-induced osteoclast formation. Overexpression of Pim-1 DN blocks RANKL-induced activation of TGF-ß-activated kinase 1 (TAK1) and NF-κB as well as expression of NFATc1 during osteoclastogenesis. However, we found that overexpression of TAK1 in the presence of Pim-1 DN rescues NF-κB activation. Additionally, Pim-1 interacts with RANK as well as TAK1, indicating that Pim-1 is involved in RANKL-induced NF-κB activation via TAK1. Furthermore, we demonstrate that Pim-1 also regulates NFATc1 transcription activity and subsequently induces osteoclast-associated receptor expression, an osteoclast-specific gene. Taken together, our results reveal that Pim-1 positively regulates RANKL-induced osteoclastogenesis.

RANKL induces NFATc1 acetylation and stability via histone acetyltransferases during osteoclast differentiation.

NFATc1 (nuclear factor of activated T-cells c1), a key transcription factor, plays a role in regulating expression of osteoclast-specific downstream target genes such as TRAP (tartrate-resistant acid phosphatase) and OSCAR (osteoclast-associated receptor). It has been shown that RANKL [receptor activator of NF-κB (nuclear factor κB) ligand] induces NFATc1 expression during osteoclastogenesis at a transcriptional level. In the present study, we demonstrate that RANKL increases NFATc1 protein levels by post-translational modification. RANKL stimulates NFATc1 acetylation via HATs (histone acetyltransferases), such as p300 and PCAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor], thereby stabilizing NFATc1 proteins. PCAF physically interacts with NFATc1 and directly induces NFATc1 acetylation and stability, subsequently increasing the transcriptional activity of NFATc1. In addition, RANKL-mediated NFATc1 acetylation is increased by the HDAC (histone deacetylase) inhibitors sodium butyrate and scriptaid. Overexpression of HDAC5 reduces RANKL- or PCAF-mediated NFATc1 acetylation, stability and transactivation activity, suggesting that the balance between HAT and HDAC activities might play a role in the regulation of NFATc1 levels. Furthermore, RANKL and p300 induce PCAF acetylation and stability, thereby enhancing the transcriptional activity of NFATc1. Down-regulation of PCAF by siRNA (small interfering RNA) decreases NFATc1 acetylation and stability, as well as RANKL-induced osteoclastogenesis. Taken together, the results of the present study demonstrate that RANKL induces HAT-mediated NFATc1 acetylation and stability, and subsequently increases the transcriptional activity of NFATc1 during osteoclast differentiation.

LIM Homeobox Transcription Factor 1-ß Expression is Upregulated in Patients with Osteolysis after Total Ankle Arthroplasty and Inhibits Receptor Activator of Nuclear Factor-κB Ligand-Induced Osteoclast Differentiation in Vitro.

BACKGROUND: Osteolysis is one of the most common problems that occurs after total hip and knee arthroplasty and has recently become a significant problem after total ankle arthroplasty (TAA). In this study, we investigated the role of LIM homeobox transcription factor 1-ß (Lmx1b) in osteoclast differentiation. By evaluating the expression profiles associated with osteolysis following TAA treatment, Lmx1b was found to be differentially expressed in patients with osteolysis after TAA. METHODS: To identify the important genes associated with osteolysis after TAA, RNA sequencing was performed by analyzing 8 patient samples: 5 primary TAA samples (control group) and 3 TAA samples revised for flexion instability (osteolysis group). By analyzing the differentially expressed genes and gene ontologies, Lmx1b expression was found to be upregulated in the osteolysis group compared to that in the control group. Focusing on the role of Lmx1b in bone cells, Lmx1b was overexpressed by a retrovirus in osteoclast precursor cells. The cultured cells were stained with tartrate-resistant acid phosphatase, and the expression of osteoclast-related genes was analyzed using real-time polymerase chain reaction. RESULTS: Lmx1b overexpression in osteoclast precursors suppresses osteoclast formation and resorptive activity. The expression of osteoclast marker genes was significantly reduced during osteoclast differentiation by Lmx1b overexpression. Furthermore, Lmx1b is associated with nuclear factor of activated T cells 1 (NFATc1) and inhibited NFATc1 translocation into the nucleus. CONCLUSIONS: These results provide novel insights into the anti-bone resorptive effect of Lmx1b on osteolysis after TAA and may lead to the development of effective preventative and therapeutic strategies for peri-implant osteolysis.

Negative feedback control of osteoclast formation through ubiquitin-mediated down-regulation of NFATc1.

The regulation of NFATc1 expression is important for osteoclast differentiation and function. Herein, we demonstrate that macrophage-colony-stimulating factor induces NFATc1 degradation via Cbl proteins in a Src kinase-dependent manner. NFATc1 proteins are ubiquitinated and rapidly degraded during late stage osteoclastogenesis, and this degradation is mediated by Cbl-b and c-Cbl ubiquitin ligases in a Src-dependent manner. In addition, NFATc1 interacts endogenously with c-Src, c-Cbl, and Cbl-b in osteoclasts. Overexpression of c-Src induces down-regulation of NFATc1, and depletion of Cbl proteins blocks NFATc1 degradation during late stage osteoclastogenesis. Taken together, our data provide a negative regulatory mechanism by which macrophage-colony-stimulating factor activates Src family kinases and Cbl proteins, and subsequently, induces NFATc1 degradation during osteoclast differentiation.

The mechanism of osteoclast differentiation induced by IL-1.

IL-1 is a potent cytokine that can induce bone erosion in inflammatory sites such as rheumatoid joint regions via activation of osteoclasts. Not only is IL-1 capable of activating osteoclasts, but it is also a key cytokine involved in the differentiation, multinucleation, and survival of osteoclasts. Herein, we show that IL-1 has the potential to drive osteoclast differentiation via a receptor activator of NF-kappaB ligand (RANKL)/RANK-independent mechanism. Although IL-1 has a synergistic effect on RANKL-induced osteoclast formation, IL-1 alone cannot induce osteoclast differentiation from osteoclast precursors (bone marrow-derived macrophages (BMMs)) due to a lack of IL-1 signaling potential in these cells. However, we demonstrate that overexpression of the IL-1RI receptor in BMMs or induction of IL-1RI by c-Fos overexpression enables IL-1 alone to induce the formation of authentic osteoclasts by a RANKL/RANK-independent mechanism. The expression of IL-1RI is up-regulated by RANKL via c-Fos and NFATc1. Furthermore, the addition of IL-1 to IL-1RI overexpressing BMMs (IL-1/IL-1RI) strongly activates NF-kappaB, JNK, p38, and ERK which is a hallmark gene activation profile of osteoclastogenesis. Interestingly, IL-1/IL-1RI does not induce expression of c-Fos or NFATc1 during osteoclast differentiation, although basal levels of c-Fos and NFATc1 seem to be required. Rather, IL-1/IL-1RI strongly activates MITF, which subsequently induces osteoclast-specific genes such as osteoclast-associated receptor and tartrate-resistant acid phosphatase. Together, these results reveal that IL-1 has the potential to induce osteoclast differentiation via activation of microphthalmia transcription factor under specific microenvironmental conditions.

IRF2 enhances RANKL-induced osteoclast differentiation via regulating NF-κB/NFATc1 signaling.

Interferon regulatory factors (IRFs) play roles in various biological processes including cytokine signaling, cell growth regulation and hematopoietic development. Although it has been reported that several IRFs are involved in bone metabolism, the role of IRF2 in bone cells has not been elucidated. Here, we investigated the involvement of IRF2 in RANKL-induced osteoclast differentiation. IRF2 overexpression in osteoclast precursor cells enhanced osteoclast differentiation by regulating the expression of NFATc1, a master regulator of osteoclastogenesis. Conversely, IRF2 knockdown inhibited osteoclast differentiation and decreased the NFATc1 expression. Moreover, IRF2 increased the translocation of NF-κB subunit p65 to the nucleus in response to RANKL and subsequently induced the expression of NFATc1. IRF2 plays an important role in RANKL-induced osteoclast differentiation by regulating NF-κB/NFATc1 signaling pathway. Taken together, we demonstrated the molecular mechanism of IRF2 in osteoclast differentiation, and provide a molecular basis for potential therapeutic targets for the treatment of bone diseases characterized by excessive bone resorption. [BMB Reports 2021; 54(9): 482-487].

Alternative regulatory mechanism for the maintenance of bone homeostasis via STAT5-mediated regulation of the differentiation of BMSCs into adipocytes.

STAT5 is a transcription factor that is activated by various cytokines, hormones, and growth factors. Activated STAT5 is then translocated to the nucleus and regulates the transcription of target genes, affecting several biological processes. Several studies have investigated the role of STAT5 in adipogenesis, but unfortunately, its role in adipogenesis remains controversial. In the present study, we generated adipocyte-specific Stat5 conditional knockout (cKO) (Stat5fl/fl;Apn-cre) mice to investigate the role of STAT5 in the adipogenesis of bone marrow mesenchymal stem cells (BMSCs). BMSC adipogenesis was significantly inhibited upon overexpression of constitutively active STAT5A, while it was enhanced in the absence of Stat5 in vitro. In vivo adipose staining and histological analyses revealed increased adipose volume in the bone marrow of Stat5 cKO mice. ATF3 is the target of STAT5 during STAT5-mediated inhibition of adipogenesis, and its transcription is regulated by the binding of STAT5 to the Atf3 promoter. ATF3 overexpression was sufficient to suppress the enhanced adipogenesis of Stat5-deficient adipocytes, and Atf3 silencing abolished the STAT5-mediated inhibition of adipogenesis. Stat5 cKO mice exhibited reduced bone volume due to an increase in the osteoclast number, and coculture of bone marrow-derived macrophages with Stat5 cKO adipocytes resulted in enhanced osteoclastogenesis, suggesting that an increase in the adipocyte number may contribute to bone loss. In summary, this study shows that STAT5 is a negative regulator of BMSC adipogenesis and contributes to bone homeostasis via direct and indirect regulation of osteoclast differentiation; therefore, it may be a leading target for the treatment of both obesity and bone loss-related diseases.