The complex role of Rcor2: Regulates mesenchymal stromal cell differentiation in vitro but is dispensable in vivo.

Recent research has revealed several important pathways of epigenetic regulation leading to transcriptional changes in bone cells. Rest Corepressor 2 (Rcor2) is a coregulator of Lysine-specific histone demethylase 1 (Lsd1), a demethylase linked to osteoblast activity, hematopoietic stem cell differentiation and malignancy of different neoplasms. However, the role of Rcor2 in osteoblast differentiation has not yet been examined in detail. We have previously shown that Rcor2 is highly expressed in mesenchymal stromal cells (MSC) and particularly in the osteoblastic lineage. The role of Rcor2 in osteoblastic differentiation in vitro was further characterized and we demonstrate here that lentiviral silencing of Rcor2 in MC3T3-E1 cells led to a decrease in osteoblast differentiation. This was indicated by decreased alkaline phosphatase and von Kossa stainings as well as by decreased expression of several osteoblast-related marker genes. RNA-sequencing of the Rcor2-downregulated MC3T3-E1 cells showed decreased repression of Rcor2 target genes, as well as significant upregulation of majority of the differentially expressed genes. While the heterozygous, global loss of Rcor2 in vivo did not lead to a detectable bone phenotype, conditional deletion of Rcor2 in limb-bud mesenchymal cells led to a moderate decrease in cortical bone volume. These findings were not accentuated by challenging bone formation by ovariectomy or tibial fracture. Furthermore, a global deletion of Rcor2 led to decreased white adipose tissue in vivo and decreased the capacity of primary cells to differentiate into adipocytes in vitro. The conditional deletion of Rcor2 led to decreased adiposity in fracture callus. Taken together, these results suggest that epigenetic regulation of mesenchymal stromal cell differentiation is mediated by Rcor2, which could thus play an important role in defining the MSC fate.

Distinctive effects of SGLT2 inhibitors on angiogenesis in zebrafish embryos.

Sodium glucose cotransporter 2 (SGLT2) inhibitor canagliflozin has been found to increase the risk for lower-limb amputations in type 2 diabetics about two-fold. Conversely, empagliflozin and dapagliflozin do not display a similar effect. A question arises whether the increased risk for minor amputations is associated only with canagliflozin or whether it is a class effect of SGLT2 inhibitors. Defective angiogenesis has a role in amputations. We compared the effects of empagliflozin, dapagliflozin and canagliflozin on angiogenesis in vivo using zebrafish model, and in vitro using human umbilical vein endothelial cells (HUVECs). The effects of SGLT2 inhibitors on the formation of intersegmental blood vessels (ISVs) of the zebrafish embryos were clarified. Additionally, transcriptome analysis was performed to explore whether putative angiogenesis-associated genes are differentially regulated by SGLT2 inhibitors. The effects of SGLT2 inhibitors on the viability of HUVECs were examined. We noticed that especially empagliflozin and also dapagliflozin significantly accelerated the formation of ISVs of zebrafish embryos. In contrast, canagliflozin was not able to stimulate ISV formation, and at high concentration, it was lethal to the embryos. Transcriptome analysis demonstrated that in empagliflozin-treated embryos compared to canagliflozin-treated embryos seven genes previously shown to contribute to angiogenesis were upregulated, and four downregulated. Canagliflozin at high concentrations, but not empagliflozin or dapagliflozin, decreased the viability of HUVECs and disrupted their capability to sprout. SGLT2 inhibitors differed in their effects on angiogenic processes in zebrafish embryos and on the viability of HUVECs suggesting that the risk of SGLT2 inhibitors for peripheral amputations likely differs.

Mesenchymal cell-derived Wnt1 signaling regulates subchondral bone remodeling but has no effects on the development of growth plate or articular cartilage in mice.

Chondrocyte differentiation is a principal progress in endochondral ossification and in the formation of secondary ossification center (SOC) during the long bone development. We have previously reported that targeted deletion of Wnt1 in mesenchymal progenitors (Wnt1Prrx-/-) leads to spontaneous fractures and severe osteopenia in mouse long bones, suggesting that Wnt1 is a key regulator of bone metabolism. However, the effect of Wnt1 on the regulation of cartilage development and chondrocyte differentiation remained unknown. In this study, WNT1 protein expression was observed in lateral superficial cartilage and growth plate pre-hypertrophic chondrocytes in mice. Wnt1 mRNA expression was detected in epiphyseal cartilage from E16.5 to 3 month-old mice. Detailed histological analyses revealed that the average thickness and chondrocyte density of proximal tibial articular cartilage and growth plate were unchanged between Wnt1Prrx-/- and control mice. However, µCT analysis of tibial epiphyses showed that the subchondral bone mass was reduced in Wnt1Prrx-/- mice compared to control mice, as demonstrated by decreased bone volume, trabecular number, trabecular thickness, and increased trabecular separation in Wnt1Prrx-/- mice. Mechanistically, histomorphometric analyses showed that the reduced subchondral bone mass in Wnt1Prrx-/- mice was due to impaired bone formation and enhanced bone resorption. In vitro, exogenous Wnt1 inhibited chondrogenesis and chondrocyte hypertrophy in both cell autonomous and juxtacrine manners, while matrix mineralization and the expression of Mmp13, Mmp9 and Opn were induced in a juxtacrine manner. Taken together, mesenchymal cell-derived Wnt1 is an important regulator of subchondral bone remodeling, although it has no effect on the regulation of growth plate or articular cartilage.

Lysine-Specific Demethylase 1 (LSD1) epigenetically controls osteoblast differentiation.

Epigenetic mechanisms regulate osteogenic lineage differentiation of mesenchymal stromal cells. Histone methylation is controlled by multiple lysine demethylases and is an important step in controlling local chromatin structure and gene expression. Here, we show that the lysine-specific histone demethylase Kdm1A/Lsd1 is abundantly expressed in osteoblasts and that its suppression impairs osteoblast differentiation and bone nodule formation in vitro. Although Lsd1 knockdown did not affect global H3K4 methylation levels, genome-wide ChIP-Seq analysis revealed high levels of Lsd1 at gene promoters and its binding was associated with di- and tri-methylation of histone 3 at lysine 4 (H3K4me2 and H3K4me3). Lsd1 binding sites in osteoblastic cells were enriched for the Runx2 consensus motif suggesting a functional link between the two proteins. Importantly, inhibition of Lsd1 activity decreased osteoblast activity in vivo. In support, mesenchymal-targeted knockdown of Lsd1 led to decreased osteoblast activity and disrupted primary spongiosa ossification and reorganization in vivo. Together, our studies demonstrate that Lsd1 occupies Runx2-binding cites at H3K4me2 and H3K4me3 and its activity is required for proper bone formation.

Age-Progressive and Gender-Dependent Bone Phenotype in Mice Lacking Both Ebf1 and Ebf2 in Prrx1-Expressing Mesenchymal Cells.

Ebfs are a family of transcription factors regulating the differentiation of multiple cell types of mesenchymal origin, including osteoblasts. Global deletion of Ebf1 results in increased bone formation and bone mass, while global loss of Ebf2 leads to enhanced bone resorption and decreased bone mass. Targeted deletion of Ebf1 in early committed osteoblasts leads to increased bone formation, whereas deletion in mature osteoblasts has no effect. To study the effects of Ebf2 specifically on long bone development, we created a limb bud mesenchyme targeted Ebf2 knockout mouse model by using paired related homeobox gene 1 (Prrx1) Cre. To investigate the possible interplay between Ebf1 and Ebf2, we deleted both Ebf1 and Ebf2 in the cells expressing Prrx1. Mice with Prrx1-targeted deletion of Ebf2 had a very mild bone phenotype. However, deletion of both Ebf1 and Ebf2 in mesenchymal lineage cells lead to significant, age progressive increase in bone volume. The phenotype was to some extent gender dependent, leading to an increase in both trabecular and cortical bone in females, while in males a mild cortical bone phenotype and a growth plate defect was observed. The phenotype was observed at both 6 and 12 weeks of age, but it was more pronounced in older female mice. Our data suggest that Ebfs modulate bone homeostasis and they are likely able to compensate for the lack of each other. The roles of Ebfs in bone formation appear to be complex and affected by multiple factors, such as age and gender.

Treatment with Soluble Activin Type IIB Receptor Ameliorates Ovariectomy-Induced Bone Loss and Fat Gain in Mice.

INTRODUCTION: In postmenopausal osteoporosis, hormonal changes lead to increased bone turnover and metabolic alterations including increased fat mass and insulin resistance. Activin type IIB receptors bind several growth factors of the TGF-ß superfamily and have been demonstrated to increase muscle and bone mass. We hypothesized that ActRIIB-Fc treatment could improve bone and muscle mass, inhibit fat accumulation, and restore metabolic alterations in an ovariectomy (OVX) model of postmenopausal osteoporosis. MATERIALS AND METHODS: Female C57Bl/6 N mice were subjected to SHAM or OVX procedures and received intraperitoneal injections of either PBS or ActRIIB-Fc (5 mg/kg) once weekly for 7 weeks. Glucose and insulin tolerance tests (GTT and ITT, respectively) were performed at 7 and 8 weeks, respectively. Bone samples were analyzed with micro-computed tomography imaging, histomorphometry, and quantitative RT-PCR. RESULTS: Bone mass decreased in OVX PBS mice compared to the SHAM PBS group but ActRIIB-Fc was able to prevent these changes as shown by µCT and histological analyses. This was due to decreased osteoclast numbers and function demonstrated by histomorphometric and qRT-PCR analyses. OVX induced adipocyte hypertrophy that was rescued by ActRIIB-Fc, which also decreased systemic adipose tissue accumulation. OVX itself did not affect glucose levels in GTT but ActRIIB-Fc treatment resulted in impaired glucose clearance in both SHAM and OVX groups. OVX induced mild insulin resistance in ITT but ActRIIB-Fc treatment did not affect this. CONCLUSION: Our results reinforce the potency of ActRIIB-Fc as a bone-enhancing agent but also bring new insight into the metabolic effects of ActRIIB-Fc in normal and OVX mice.

Early B-cell Factor1 (Ebf1) promotes early osteoblast differentiation but suppresses osteoblast function.

Early B cell factor 1 (Ebf1) is a transcription factor that regulates B cell, neuronal cell and adipocyte differentiation. We and others have shown that Ebf1 is expressed in osteoblasts and that global deletion of Ebf1 results in increased bone formation in vivo. However, as Ebf1 is expressed in multiple tissues and cell types, it has remained unclear, which of the phenotypic changes in bone are derived from bone cells. The aim of this study was to determine the cell-autonomous and differentiation stage-specific roles of Ebf1 in osteoblasts. In vitro, haploinsufficient Ebf1+/- calvarial cells showed impaired osteoblastic differentiation indicated by lower alkaline phosphatase (ALP) activity and reduced mRNA expression of osteoblastic genes, while overexpression of Ebf1 in wild type mouse calvarial cells led to enhanced osteoblast differentiation with increased expression of Osterix (Osx). We identified a putative Ebf1 binding site in the Osterix promoter by ChIP assay in MC3T3-E1 osteoblasts and showed that Ebf1 was able to activate Osx-luc reporter construct that included this Ebf1 binding site, suggesting that Ebf1 indeed regulates osteoblast differentiation by inducing Osterix expression. To reconcile our previous data and that of others with our novel findings, we hypothesized that Ebf1 could have a dual role in osteoblast differentiation promoting early but inhibiting late stages of differentiation and osteoblast function. To test this hypothesis in vivo, we generated conditional Ebf1 knockout mice, in which Ebf1 deletion was targeted to early or late osteoblasts by crossing Ebf1fl/fl mice with Osx- or Osteocalcin (hOC)-Cre mouse lines, respectively. Deletion of Ebf1 in early Ebf1Osx-/- osteoblasts resulted in significantly increased bone volume and trabecular number at 12 weeks by µCT analysis, while Ebf1hOC-/- mice did not have a bone phenotype. To conclude, our data demonstrate that Ebf1 promotes early osteoblast differentiation by regulating Osterix expression. However, Ebf1 inhibits bone accrual in the Osterix expressing osteoblasts in vivo but it is redundant in the maintenance of mature osteoblast function.

Osteoblastic Wnt1 regulates periosteal bone formation in adult mice.

Compelling clinical data together with genetically modified mouse models have demonstrated that Wnt1 is a key Wnt ligand in bone metabolism, regulating both osteoblast activity and osteoclast differentiation. We have previously shown that deletion of Wnt1 in limb mesenchymal cells leads to severe ostepenic bone phenotype and spontaneous fractures very early after birth. However, the function of Wnt1 in mature skeleton remained unknown. To investigate the role of Wnt1 specifically in adult bone metabolism, we generated an osteoblast lineage-targeted inducible Wnt1 knockout mouse model using tetracycline-controlled Osterix-Cre mouse line (Osx-Cre). In this model, the Cre recombinase expression is suppressed by administering doxycycline (Dox) in drinking water. As expected, Wnt1Osx-/- mice without Dox developed spontaneous fractures early by 3 weeks of age due to severe trabecular and cortical osteopenia. Administration of Dox to Wnt1Osx-Dox-/- and control mice until 4 weeks of age suppressed Wnt1 deletion and completely prevented the fractures. Withdrawal of Dox led to deletion in Wnt1 allele but the fracture incidence progressively decreased in Wnt1Osx-Dox-/- mice at 8 or 12 weeks of age (4 and 8 weeks after Dox withdrawal). Interestingly, deletion of Wnt1 at 4 weeks of age resulted only in a modest and transient trabecular osteopenia that was more pronounced in females and was normalized by 12 weeks of age. However, diaphyseal cortical bone mass and cortical thickness in the femurs were significantly decreased in Wnt1Osx-Dox-/- mice of both genders. Mechanisticly, this was due to impaired periosteal bone formation. Based on our data, in addition to its essential role in early skeletal growth, Wnt1 is an important regulator of modeling-based bone formation and cortical thickness in adult mice.

Mesenchymal Cell-Derived Juxtacrine Wnt1 Signaling Regulates Osteoblast Activity and Osteoclast Differentiation.

Human genetic evidence demonstrates that WNT1 mutations cause osteogenesis imperfecta (OI) and early-onset osteoporosis, implicating WNT1 as a major regulator of bone metabolism. However, its main cellular source and mechanisms of action in bone remain elusive. We generated global and limb bud mesenchymal cell-targeted deletion of Wnt1 in mice. Heterozygous deletion of Wnt1 resulted in mild trabecular osteopenia due to decreased osteoblast function. Targeted deletion of Wnt1 in mesenchymal progenitors led to spontaneous fractures due to impaired osteoblast function and increased bone resorption, mimicking the severe OI phenotype in humans with homozygous WNT1 mutations. Importantly, we showed for the first time that Wnt1 signals strictly in a juxtacrine manner to induce osteoblast differentiation and to suppress osteoclastogenesis, in part via canonical Wnt signaling. In conclusion, mesenchymal cell-derived Wnt1, acting in short range, is an essential regulator of bone homeostasis and an intriguing target for therapeutic interventions for bone diseases. © 2019 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.

Cilia-related protein SPEF2 regulates osteoblast differentiation.

Sperm flagellar protein 2 (SPEF2) is essential for motile cilia, and lack of SPEF2 function causes male infertility and primary ciliary dyskinesia. Cilia are pointing out from the cell surface and are involved in signal transduction from extracellular matrix, fluid flow and motility. It has been shown that cilia and cilia-related genes play essential role in commitment and differentiation of chondrocytes and osteoblasts during bone formation. Here we show that SPEF2 is expressed in bone and cartilage. The analysis of a Spef2 knockout (KO) mouse model revealed hydrocephalus, growth retardation and death prior to five weeks of age. To further elucidate the causes of growth retardation we analyzed the bone structure and possible effects of SPEF2 depletion on bone formation. In Spef2 KO mice, long bones (tibia and femur) were shorter compared to wild type, and X-ray analysis revealed reduced bone mineral content. Furthermore, we showed that the in vitro differentiation of osteoblasts isolated from Spef2 KO animals was compromised. In conclusion, this study reveals a novel function for SPEF2 in bone formation through regulation of osteoblast differentiation and bone growth.

Soluble activin type IIB receptor improves fracture healing in a closed tibial fracture mouse model.

Fractures still present a significant burden to patients due to pain and periods of unproductivity. Numerous growth factors have been identified to regulate bone remodeling. However, to date, only the bone morphogenetic proteins (BMPs) are used to enhance fracture healing in clinical settings. Activins are pleiotropic growth factors belonging to the TGF-ß superfamily. We and others have recently shown that treatment with recombinant fusion proteins of activin receptors greatly increases bone mass in different animal models by trapping activins and other ligands thus inhibiting their signaling pathways. However, their effects on fracture healing are less known. Twelve-week old male C57Bl mice were subjected to a standardized, closed tibial fracture model. Animals were divided into control and treatment groups and were administered either PBS control or a soluble activin type IIB receptor (ActRIIB-Fc) intraperitoneally once a week for a duration of two or four weeks. There were no significant differences between the groups at two weeks but we observed a significant increase in callus mineralization in ActRIIB-Fc-treated animals by microcomputed tomography imaging at four weeks. Bone volume per tissue volume was 60%, trabecular number 55% and bone mineral density 60% higher in the 4-week calluses of the ActRIIB-Fc-treated mice (p<0.05 in all). Biomechanical strength of 4-week calluses was also significantly improved by ActRIIB-Fc treatment as stiffness increased by 64% and maximum force by 45% (p<0.05) compared to the PBS-injected controls. These results demonstrate that ActRIIB-Fc treatment significantly improves healing of closed long bone fractures. Our findings support the previous reports of activin receptors increasing bone mass but also demonstrate a novel approach for using ActRIIB-Fc to enhance fracture healing.