Bone morphogenetic proteins (BMPs) induce osteogenesis in various environments. However, when BMPs are used alone in the bone marrow environment, the maintenance of new bone formation is difficult owing to vigorous bone resorption. This is because BMPs stimulate the differentiation of not only osteoblast precursor cells but also osteoclast precursor cells. The present study aimed to induce and maintain new bone formation using the topical co-administration of recombinant human BMP-2 (rh-BMP-2) and zoledronate (ZOL) on beta-tricalcium phosphate (β-TCP) composite.

Developmental dysplasia of the hip (DDH) is characterized by anatomical abnormalities of the hip joint, ranging from mild acetabular dysplasia to hip subluxation and eventually dislocation. The mechanism underlying the cartilage degeneration of the hip joints exposed to reduced dynamic loads due to hip dislocation remains unknown. We established a rodent hip dislocation (disarticulation; DA) model of DDH (DA-DDH rats and mice) by swaddling. Expression levels of periostin (Postn) and catabolic factors, such as interleukin-6 (IL-6) and matrix metalloproteinase 3 (Mmp3), increased and those of chondrogenic markers decreased in the acetabular cartilage of the DA-DDH models. Postn induced IL-6 and Mmp3 expression in chondrocytes through integrin αVβ3, focal adhesion kinase, Src, and nuclear factor-κB (NF-κB) signaling. The microgravity environment created by a random positioning machine induced Postn expression in chondrocytes through signal transducer and activator of transcription 3 (STAT3) signaling. IL-6 stimulated Postn expression via STAT3 signaling. Furthermore, cartilage degeneration was suppressed in the acetabulum of Postn-/- DA-DDH mice compared with that in the acetabulum of wild type DA-DDH mice. In summary, reduced dynamic loads due to hip dislocation induced acetabular cartilage degeneration via IL-6 and MMP3 through STAT3/periostin/NF-κB signaling in the rodent DA-DDH models.

Resistance training (RT) has been known to be effective in maintaining and improving bone strength, which is based on bone mineral density (BMD) and bone quality. However, it is not clear whether RT is effective in improving bone strength in patients with type-2 diabetes mellitus (T2DM), who have a high risk of fracture. Therefore, we tested the effects of a 6-week RT regimen using percutaneous electrical stimulation in T2DM model rats, male Otsuka Long-Evans Tokushima Fatty (OLETF), and its control, Long-Evans Tokushima Otsuka (LETO). After 6 weeks of RT, tibial BMD in RT legs was significantly higher than that in control (CON) legs in both groups. In diaphyseal cortical bone, bone area/tissue area, and cortical thickness was significantly increased in RT legs compared with CON legs in both groups. Cortical porosity was highly observed in OLETF compared with LETO, but RT improved cortical porosity in both groups. Interestingly, trabecular number, trabecular thickness and trabecular space as well as BMD and bone volume/tissue volume in proximal tibial metaphyseal trabecular bone were significantly improved in RT legs compared with CON legs in both groups. In contrast, connectivity density and structural model index were not affected by RT. These results indicate that the 6-week RT regimen effectively increased BMD and improved bone quality in T2DM model rats as well as control rats. Therefore, RT may have the potential to improve bone strength and reduce fracture risk, even in patients with T2DM.

TGF-ß is a multifunctional cytokine that is involved in cell proliferation, differentiation and function. We previously reported an essential role of the TGF-ß -Smad2/3 pathways in RANKL-induced osteoclastogenesis. Using chromatin immunoprecipitation followed by sequencing, we comprehensively identified Smad2/3 target genes in bone marrow macrophages. These genes were enriched in the gene population upregulated by TGF-ß and downregulated by RANKL. Recent studies have revealed that histone modifications, such as trimethylation of histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3), critically regulate key developmental steps. We identified Nedd9 as a Smad2/3 target gene whose histone modification pattern was converted from H3K4me3(+)/H3K4me27(+) to H3K4me3(+)/H3K4me27(-) by TGF-ß. Nedd9 expression was increased by TGF-ß and suppressed by RANKL. Overexpression of Nedd9 partially rescued an inhibitory effect of a TGF-ß inhibitor, while gene silencing of Nedd9 suppressed RANKL-induced osteoclastogenesis. RANKL-induced osteoclastogenesis were reduced and stimulatory effects of TGF-ß on RANKL-induced osteoclastogenesis were partially abrogated in cells from Nedd9-deficient mice although knockout mice did not show abnormal skeletal phenotypes. These results suggest that Nedd9 is a Smad2/3 target gene implicated in RANKL-induced osteoclastogenesis.

Epigenetic transcriptional regulation is essential for the differentiation of various types of cells, including skeletal muscle cells. DNA methyltransferase 1 (Dnmt1) is responsible for maintenance of DNA methylation patterns via cell division. Here, we investigated the relationship between Dnmt1 and skeletal muscle regeneration. We found that Dnmt1 is upregulated in muscles during regeneration. To assess the role of Dnmt1 in satellite cells during regeneration, we performed conditional knockout (cKO) of Dnmt1 specifically in skeletal muscle satellite cells using Pax7CreERT2 mice and Dnmt1 flox mice. Muscle weight and the cross-sectional area after injury were significantly lower in Dnmt1 cKO mice than in control mice. RNA sequencing analysis revealed upregulation of genes involved in cell adhesion and apoptosis in satellite cells from cKO mice. Moreover, satellite cells cultured from cKO mice exhibited a reduced number of cells. These results suggest that Dnmt1 is an essential factor for muscle regeneration and is involved in positive regulation of satellite cell number.

Macrophages are progenitors of osteoclasts as well as regulators of bone metabolism. Macrophages mediate not only bone formation by osteoblasts under physiological conditions, but also bone regeneration after fracture. The mechanisms of macrophages regulation of bone formation and regeneration remain unclear, however. Here, we demonstrate that the liposome-encapsulated Clodronate (Clod-lip) injected mouse model with cortical bone defect induced by drill-hole injury and targeted depletion of phagocytic macrophages exhibits impaired angiogenesis of type H vessels that couple angiogenesis and osteogenesis. Moreover, we identify Tgfbi (encoding TGFBI), Plau (encoding uPA) and Tgfb1 (encoding TGF-β1), through RNA-seq analysis, as genes of macrophage-secreted factors mediating angiogenesis and wound healing. The relevant mRNA was highly expressed in bone marrow-derived macrophages among bone cells, as determined through qRT-PCR. Finally, we disclose that treatment with uPA inhibitor or TGF-β receptor I, receptor II inhibitor impairs bone regeneration after injury, confirming the importance of uPA and TGF-β1 during bone regeneration. Our findings reveal a novel mechanism of bone regeneration mediated by macrophages.

Lenalidomide, an immunomodulatory drug (IMiD), is commonly used as a first-line therapy in many haematological cancers, such as multiple myeloma (MM) and 5q myelodysplastic syndromes (5q MDS), and it functions as a molecular glue for the protein degradation of neosubstrates by CRL4CRBN. Proteolysis-targeting chimeras (PROTACs) using IMiDs with a target protein binder also induce the degradation of target proteins. The targeted protein degradation (TPD) of neosubstrates is crucial for IMiD therapy. However, current IMiDs and IMiD-based PROTACs also break down neosubstrates involved in embryonic development and disease progression. Here, we show that 6-position modifications of lenalidomide are essential for controlling neosubstrate selectivity; 6-fluoro lenalidomide induced the selective degradation of IKZF1, IKZF3, and CK1α, which are involved in anti-haematological cancer activity, and showed stronger anti-proliferative effects on MM and 5q MDS cell lines than lenalidomide. PROTACs using these lenalidomide derivatives for BET proteins induce the selective degradation of BET proteins with the same neosubstrate selectivity. PROTACs also exert anti-proliferative effects in all examined cell lines. Thus, 6-position-modified lenalidomide is a key molecule for selective TPD using thalidomide derivatives and PROTACs.

Osteophyte formation is attracting attention as an early-stage pathology of knee osteoarthritis (OA). Although osteophyte formation is understood as a defense response to joint instability, its role and impact on OA remain largely unknown. Many studies have been conducted using the surgical destabilization of the medial meniscus (DMM) mouse model, but there are few standard evaluation methods, especially in the histological evaluation of early-stage osteophytes. The purpose of this study was to establish a reproducible and uniform method for histological evaluation of characteristics of early osteophyte formation in the DMM mouse model.

The bulbocavernosus (BC) is a sexually dimorphic muscle observed only in males. Androgen receptor knockout mouse studies show the loss of BC formation. This suggests that androgen signaling plays a vital role in its development. Androgen has been known to induce muscle hypertrophy through satellite cell activation and myonuclei accretion during muscle regeneration and growth. Whether the same mechanism is present during embryonic development is not yet elucidated. To identify the mechanism of sexual dimorphism during BC development, the timing of morphological differences was first established. It was revealed that the BC was morphologically different between male and female mice at embryonic day (E) 16.5. Differences in the myogenic process were detected at E15.5. The male BC possesses a higher number of proliferating undifferentiated myoblasts. To identify the role of androgen signaling in this process, muscle-specific androgen receptor (AR) mutation was introduced, which resulted in no observable phenotypes. Hence, the expression of AR in the BC was examined and found that the AR did not colocalize with any muscle markers such as Myogenic differentiation 1, Myogenin, and paired box transcription factor 7. It was revealed that the mesenchyme surrounding the BC expressed AR and the BC started to express AR at E15.5. AR mutation on the nonmyocytic cells using spalt-like transcription factor 1 (Sall1) Cre driver mouse was performed, which resulted in defective BC formation. It was revealed that the number of proliferating undifferentiated myoblasts was reduced in the Sall1 Cre:AR(L-/Y) mutant embryos, and the adult mutants were devoid of BC. The transition of myoblasts from proliferation to differentiation is mediated by cyclin-dependent kinase inhibitors. An increased expression of p21 was observed in the BC myoblast of the Sall1 Cre:AR(L-/Y) mutant and wild-type female. Altogether this study suggests that the nonmyocytic AR may paracrinely regulate the proliferation of myoblast possibly through inhibiting p21 expression in myoblasts of the BC.

DNA methylation is an essential form of epigenetic regulation responsible for cellular identity. In muscle stem cells, termed satellite cells, DNA methylation patterns are tightly regulated during differentiation. However, it is unclear how these DNA methylation patterns affect the function of satellite cells. We demonstrate that a key epigenetic regulator, ubiquitin like with PHD and RING finger domains 1 (Uhrf1), is activated in proliferating myogenic cells but not expressed in quiescent satellite cells or differentiated myogenic cells in mice. Ablation of Uhrf1 in mouse satellite cells impairs their proliferation and differentiation, leading to failed muscle regeneration. Uhrf1-deficient myogenic cells exhibited aberrant upregulation of transcripts, including Sox9, with the reduction of DNA methylation level of their promoter and enhancer region. These findings show that Uhrf1 is a critical epigenetic regulator of proliferation and differentiation in satellite cells, by controlling cell-type-specific gene expression via maintenance of DNA methylation.

Osteocytes are master regulators of skeletal homeostasis. However, little is known about the molecular mechanism of their differentiation. Epigenetic regulations, especially H3K27me3 modification, play critical roles in cell differentiation. Here, we found that H3K27me3 in the loci of osteocyte-expressing genes decreased during osteocyte differentiation and that H3K27me3 demethylase, Utx, was bound to the loci of those genes. To investigate the physiological functions of Utx in vivo, we generated late osteoblast-to-osteocyte specific Utx knockout mice using Dmp1-cre mice (UtxΔOcy/ΔOcy). Micro CT analyses showed that UtxΔOcy/ΔOcy displayed osteopenic phenotypes with lower bone volume and trabecular number, and greater trabecular separation. Bone histomorphometric analysis showed that bone mineralization and formation were significantly lower in UtxΔOcy/ΔOcy. Furthermore, Dmp1 expression and the number of osteocytes were significantly decreased in UtxΔOcy/ΔOcy. These results suggest that Utx in Dmp1-expressing osteoblast/osteocyte positively regulates osteoblast-to-osteocyte differentiation through H3K27me3 modifications in osteocyte genes. Our results provide new insight into the molecular mechanism of osteocyte differentiation.

Transcriptional regulation can be tightly orchestrated by epigenetic regulators. Among these, ubiquitin-like with PHD and RING finger domains 1 (Uhrf1) is reported to have diverse epigenetic functions, including regulation of DNA methylation. However, the physiological functions of Uhrf1 in skeletal tissues remain unclear. Here, we show that limb mesenchymal cell-specific Uhrf1 conditional knockout mice (Uhrf1ΔLimb/ΔLimb ) exhibit remarkably shortened long bones that have morphological deformities due to dysregulated chondrocyte differentiation and proliferation. RNA-seq performed on primary cultured chondrocytes obtained from Uhrf1ΔLimb/ΔLimb mice showed abnormal chondrocyte differentiation. In addition, integrative analyses using RNA-seq and MBD-seq revealed that Uhrf1 deficiency decreased genome-wide DNA methylation and increased gene expression through reduced DNA methylation in the promoter regions of 28 genes, including Hspb1, which is reported to be an IL1-related gene and to affect chondrocyte differentiation. Hspb1 knockdown in cKO chondrocytes can normalize abnormal expression of genes involved in chondrocyte differentiation, such as Mmp13 These results indicate that Uhrf1 governs cell type-specific transcriptional regulation by controlling the genome-wide DNA methylation status and regulating consequent cell differentiation and skeletal maturation.

Macrophages adapt to microenvironments, and change metabolic status and functions to regulate inflammation and/or maintain homeostasis. In joint cavities, synovial macrophages (SM) and synovial fibroblasts (SF) maintain homeostasis. However, under inflammatory conditions such as rheumatoid arthritis (RA), crosstalk between SM and SF remains largely unclear.

While menin plays an important role in preventing T-cell dysfunction, such as senescence and exhaustion, the regulatory mechanisms remain unclear. We found that menin prevents the induction of dysfunction in activated CD8 T cells by restricting the cellular metabolism. mTOR complex 1 (mTORC1) signaling, glycolysis, and glutaminolysis are augmented by menin deficiency. Rapamycin treatment prevents CD8 T-cell dysfunction in menin-deficient CD8 T cells. Limited glutamine availability also prevents CD8 T-cell dysfunction induced by menin deficiency, and its inhibitory effect is antagonized by α-ketoglutarate (α-KG), an intermediate metabolite of glutaminolysis. α-KG-dependent histone H3K27 demethylation seems to be involved in the dysfunction in menin-deficient CD8 T cells. We also found that α-KG activates mTORC1-dependent central carbon metabolism. These findings suggest that menin maintains the T-cell functions by limiting mTORC 1 activity and subsequent cellular metabolism.

Ligamentum flavum (LF) hypertrophy is a major cause of lumbar spinal canal stenosis. Although mechanical stress is thought to be a major factor involved in LF hypertrophy, the exact mechanism by which it causes hypertrophy has not yet been fully elucidated. Here, changes in gene expression due to long-term mechanical stress were analyzed using RNA-seq in a rabbit LF hypertrophy model. In combination with previously reported analysis results, periostin was identified as a molecule whose expression fluctuates due to mechanical stress. The expression and function of periostin were further investigated using human LF tissues and primary LF cell cultures. Periostin was abundantly expressed in human hypertrophied LF tissues, and periostin gene expression was significantly correlated with LF thickness. In vitro, mechanical stress increased gene expressions of periostin, transforming growth factor-β1, α-smooth muscle actin, collagen type 1 alpha 1, and interleukin-6 (IL-6) in LF cells. Periostin blockade suppressed the mechanical stress-induced gene expression of IL-6 while periostin treatment increased IL-6 gene expression. Our results suggest that periostin is upregulated by mechanical stress and promotes inflammation by upregulating IL-6 expression, which leads to LF degeneration and hypertrophy. Periostin may be a pivotal molecule for LF hypertrophy and a promising therapeutic target for lumbar spinal stenosis.

The incidence of endometrial cancer (EC) has been increasing worldwide. However, because there are limited chemotherapeutic options for the treatment of EC, the prognosis of advanced-stage EC is poor.

Vascular endothelial cell growth factor receptor 2 (VEGFR2) is an essential receptor for the homeostasis of endothelial cells. In this study, we showed that NEDD8-conjugated Cullin3 (CUL3)-based ubiquitin E3 (UbE3) ligase plays a crucial role in VEGFR2 mRNA expression. Human umbilical vein endothelial cells treated with MLN4924, an inhibitor of NEDD8-activating enzyme, or with CUL3 siRNA drastically lost their response to VEGF due to the intense decrease in VEGFR2 expression. Moreover, speckle-type POZ protein (SPOP) and death-domain associated protein (DAXX) were involved in the CUL3 UbE3 ligase complex as a substrate adaptor and a substrate, respectively. Knockdown of SPOP and CUL3 led to the upregulation of DAXX protein and downregulation of VEGFR2 levels. These levels were inversely correlated with one another. In addition, simultaneous knockdown of SPOP and DAXX completely reversed the downregulation of VEGFR2 levels. Moreover, the CUL3-SPOP-DAXX axis had the same effects on NOTCH1, DLL4 and NRP1 expression. Taken together, these findings suggest that the CUL3-SPOP-DAXX axis plays a very important role in endothelial cell function by targeting key angiogenic regulators.

Zinc finger and SCAN domain containing 10 (Zscan10) was identified as a novel transcription factor that is involved in osteoclast differentiation in our previous report. However, the biological functions of Zscan10 are not fully understood except its roles in the maintenance of genome stability and pluripotency of embryonic stem cells. Therefore, the purpose of this study was to clarify the function of Zscan10 in somatic cells, especially during osteoclast differentiation. First, Zscan10 KO RAW264 (KO) cells were established by genome editing using CRISPR/Cas9 and single cell sorting. Then, control (Ctrl) and KO cells were differentiated into osteoclasts by RANKL stimulation. We observed that TRAP activity and the expression levels of differentiation marker genes, such as Nfatc1, were significantly increased and the expression of inhibitory factors, such as Irf8, was decreased in KO cells compared to Ctrl cells. These results suggest that Zscan10 might regulate transcription of the genes that negatively control osteoclastogenesis. To understand gene expression profiles controlled by Zscan10, RNA-seq was performed and stringent analyses identified the haptoglobin gene (Hp) as a possible target of Zscan10. In addition, ChIP against Zscan10 revealed that Zscan10 could interact with its binding motif located near the Hp gene locus as well as the transcription start site of Hp, suggesting that Zscan10 can directly regulate transcription of Hp. Finally, to examine the effects of Hp on osteoclastogenesis, KO cells were treated with recombinant Hp (rHp). rHp treatment suppressed TRAP activity of KO cells without affecting cell viability. Furthermore, it has been reported that Hp KO mice exhibit decreased bone mass and increased osteoclast number. Importantly, hemolytic disease patients exhibited decreased serum level of Hp as well as low bone mineral density. Taken together, this study suggests that Zscan10 negatively regulates osteoclast differentiation through transcription of Hp.

Nuclear factor-κB (NF-κB) proteins are transcription factors that play key roles in regulating most immune responses and cell death. Constitutively active NF-κB has been shown to exhibit chemoresistance by inducing anti-apoptosis in tumor cells. Multiple myeloma is known as a constitutive NF-κB activating disease, and the proteasome inhibitor bortezomib is used to treat multiple myeloma and mantle cell lymphoma. We demonstrate here that DANFIN (N,N'-bis-(2,4-dimethyl-phenyl)-ethane-1,2-diamine) functions as an inhibitor of the p65 family proteins and induces chemosensitization to bortezomib in multiple myeloma. DANFIN was found to be an inhibitor of interactions between p65 and IκBα without the inhibition of the DNA binding activity of the p65 protein. In addition, DANFIN affected the IκBα binding region in Rel Homology Domain (RHD) and suppressed the nuclear translocalization of the p65 protein in cells. Furthermore, in multiple myeloma cells, DANFIN suppressed the expression level of NF-κB target genes and induced apoptosis. The combination therapy of DANFIN with bortezomib dramatically enhanced the apoptosis of multiple myeloma cells and indicated a remarkable anti-tumor effect in a multiple-myeloma xenograft mouse model.

PHF2 is a JmjC family histone demethylase that removes the methyl group from H3K9me2 and works as a coactivator for several metabolism-related transcription factors. In this study, we examined the in vivo role of PHF2 in mice. We generated Phf2 floxed mice, systemic Phf2 null mice by crossing Phf2 floxed mice with CMV-Cre transgenic mice, and tamoxifen-inducible Phf2 knockout mice by crossing Phf2 floxed mice with Cre-ERT2 transgenic mice. Systemic Phf2 null mice had partial neonatal death and growth retardation and exhibited less adipose tissue and reduced adipocyte numbers compared with control littermates. Tamoxifen-induced conditional knockout of PHF2 resulted in impaired adipogenesis in stromal vascular cells from the adipose tissue of tamoxifen-inducible Phf2 knockout mice as well as of Phf2 knocked-down 3T3-L1 cells. PHF2 interacts with CEBPA and demethylates H3K9me2 in the promoters of CEBPA-regulated adipogenic genes. These findings suggest that PHF2 histone demethylase potentiates adipogenesis through interaction with CEBPA in vivo. Taken together, PHF2 may be a novel therapeutic target in the treatment of obesity and the metabolic syndrome.