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.

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.

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.

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.

WNT1 mutations in early-onset osteoporosis and osteogenesis imperfecta.

This report identifies human skeletal diseases associated with mutations in WNT1. In 10 family members with dominantly inherited, early-onset osteoporosis, we identified a heterozygous missense mutation in WNT1, c.652T→G (p.Cys218Gly). In a separate family with 2 siblings affected by recessive osteogenesis imperfecta, we identified a homozygous nonsense mutation, c.884C→A, p.Ser295*. In vitro, aberrant forms of the WNT1 protein showed impaired capacity to induce canonical WNT signaling, their target genes, and mineralization. In mice, Wnt1 was clearly expressed in bone marrow, especially in B-cell lineage and hematopoietic progenitors; lineage tracing identified the expression of the gene in a subset of osteocytes, suggesting the presence of altered cross-talk in WNT signaling between the hematopoietic and osteoblastic lineage cells in these diseases.

Coordinated transcriptional regulation of bone homeostasis by Ebf1 and Zfp521 in both mesenchymal and hematopoietic lineages.

Bone homeostasis is maintained by the coupled actions of hematopoietic bone-resorbing osteoclasts (OCs) and mesenchymal bone-forming osteoblasts (OBs). Here we identify early B cell factor 1 (Ebf1) and the transcriptional coregulator Zfp521 as components of the machinery that regulates bone homeostasis through coordinated effects in both lineages. Deletion of Zfp521 in OBs led to impaired bone formation and increased OB-dependent osteoclastogenesis (OC-genesis), and deletion in hematopoietic cells revealed a strong cell-autonomous role for Zfp521 in OC progenitors. In adult mice, the effects of Zfp521 were largely caused by repression of Ebf1, and the bone phenotype of Zfp521(+/-) mice was rescued in Zfp521(+/-):Ebf1(+/-) mice. Zfp521 interacted with Ebf1 and repressed its transcriptional activity. Accordingly, deletion of Zfp521 led to increased Ebf1 activity in OBs and OCs. In vivo, Ebf1 overexpression in OBs resulted in suppressed bone formation, similar to the phenotype seen after OB-targeted deletion of Zfp521. Conversely, Ebf1 deletion led to cell-autonomous defects in both OB-dependent and cell-intrinsic OC-genesis, a phenotype opposite to that of the Zfp521 knockout. Thus, we have identified the interplay between Zfp521 and Ebf1 as a novel rheostat for bone homeostasis.

Inactivation of the androgen receptor in bone-forming cells leads to trabecular bone loss in adult female mice.

Removal of the androgen receptor (AR) from bone-forming cells has been shown to reduce trabecular bone volume in male mice. In female mice, the role of AR in the regulation of bone homeostasis has been poorly understood. We generated a mouse strain in which the AR is completely inactivated only in mineralizing osteoblasts and osteocytes by breeding mice carrying osteocalcin promoter-regulated Cre-recombinase with mice possessing loxP recombination sites flanking exon 2 of the AR gene (AR(ΔOB/ΔOB) mice). In female AR(ΔOB/ΔOB) mice, the trabecular bone volume was reduced owing to a smaller number of trabeculae at 6 months of age compared with the control AR(fl/fl) animals. In male AR(ΔOB/ΔOB) mice, an increase in trabecular bone separation could already be detected at 3.5 months of age, and at 6 months, the trabecular bone volume was significantly reduced compared with that of male AR(fl/fl) mice. No AR-dependent changes were observed in the cortical bone of either sex. On the basis of micro-computed tomography and histomorphometry, we conclude that in male mice, the AR is involved in the regulation of osteoclast number by osteoblasts, whereas in female mice, the lack of the AR in the bone-forming cells leads to a decreased number of trabeculae upon aging.