Transcription factors Ebf1 and Ebf2 in bone metabolism

Bone formation and bone metabolism are controlled by several different factors and signaling pathways. Transcription factors participate in this cascade by regulating the differentiation of osteoblasts, osteoclasts and chondrocytes. One of the additions to the group of bone-related transcription factors are early B-cell factors. Early Bcell factor 1 (Ebf1) was first identified as a key transcription factor regulating B-cell differentiation. Further work has found that it also regulates neuronal and adipocyte differentiation, as does its family member Ebf2. We and others have shown that Ebf1 is expressed in osteoblasts and that genomewide deletion of Ebf1 results in increased bone formation in vivo. In contrast, global Ebf2 knock out mice have osteopenia in cancellous and cortical bone due to enhanced bone resorption and increased number of osteoclasts. However, interpreting the effects of global Ebf1 and Ebf2 deletion on bone is challenging, as both factors are expressed in multiple tissues. This results in failure to thrive and could therefore affect the skeletal development. Therefore, the functions and molecular mechanisms of action at specific stages of osteoblasts differentiation remained to be elucidated in detail. This thesis investigates the role of Ebf1 and Ebf2 in osteoblast differentiation and function, at different stages of bone development. To achieve this, we analysed several conditional, osteoblast targeted Ebf1 and Ebf2 knockout mouse models. Results of our work show that Ebf1 promotes early osteoblast differentiation by regulating Osterix expression. We also concluded that Ebf1 inhibits bone accrual in the Osterix-expressing osteoblasts in vivo, but it is redundant in the function of mature, osteocalcin-expressing osteoblasts. Deletion of both Ebf1 and Ebf2 in mesenchymal lineage cells led to significant, age progressive increase in bone volume. The phenotype was to some extent gender dependent. Results presented in this thesis have established that Ebf1 and Ebf2 have prominent effect on bone formation. By understanding the specific roles of Ebf transcription factors in osteoblast differentiation, we are one step closer to guiding MSC differentiation to facilitate bone repair and developing future therapies.Transkriptiotekijät Ebf1 ja Ebf2 luun aineenvaihdunnassa Luunmuodostusta ja luun aineenvaihduntaa säätelevät useat eri tekijät sekä signalointireitit. Tähän prosessiin osallistuvat transkriptiotekijät säätelevät osteoblastien, osteoklastien sekä kondrosyyttien erilaistumista. Ebf-ryhmän (engl. Early B-cell factor) transkriptiotekijät kuuluvat luunmuodostuksen säätelijöihin. Ebf1 (Early B-cell factor 1) tunnistettiin ensimmäistä kertaa B-solujen erilaistumisen säätelyssä. Myöhemmin sen on todettu osallistuvan myös adiposyyttien sekä hermosolujen erilaistumisen säätelyyn, yhdessä saman säätelyperheen toisen tekijän, Ebf2:n kanssa. Aiemmat tutkimukset osoittavat Ebf1:n ilmenevän osteoblasteissa. Globaaleissa poistogeenisissä hiirimalleissa Ebf1:n puuttuminen johti kasvaneeseen luumassaan. Ebf2:n globaali poistogeenisyys taas johtaa hiirimalleissa osteopeniaan lisääntyneen luun resorption ja kasvaneen osteoklastimäärän myötä. Luustovaikutusten tulkinta globaalisti poistogeenisissä hiirimalleissa on kuitenkin haastavaa, sillä Ebf1 ja Ebf2 ilmenevät useissa eri kudostyypeissä. Tämä voi johtaa sekundäärisiin muutoksiin, jotka osaltaan vaikuttavat luunmuodostukseen. Näin ollen spesifit vaikutukset luusolujen erilaistumiseen ja molekyylitason mekanismeihin ovat yhä epäselviä. Tutkimuksemme selvitti Ebf1- ja Ebf2- transkriptiotekijöiden merkitystä osteoblastien erilaistumisessa ja toiminnassa luunmuodostuksen eri vaiheissa. Analysoimme useita Ebf1- ja Ebf2- hiirimalleja, joissa poistogeenisyys oli kohdennettu osteoblasteihin. Tutkimuksen tulokset osoittavat, että Ebf1 edistää varhaista osteoblastien erilaistumista. Totesimme kuinka osteoblastien erilaistumisen edetessä Ebf1 heikentää luun kertymistä in vivo, mutta ei enää osallistu kypsien osteoblastien toimintaan. Ebf1:n ja Ebf2:n samanaikainen puute mesenkymaalisissa esiastesoluissa johti iän myötä etenevään luumassan lisääntymiseen. Ilmiasu oli joiltain osin riippuvainen sukupuolesta. Tämän tutkimuksen tulokset vahvistavat Ebf-transkriptiotekijöiden merkitystä luunmuodostuksessa. Ymmärtämällä paremmin Ebf-transkriptiotekijöiden spesifejä vaikutuksia osteoblastien erilaistumisessa, olemme askeleen lähempänä mesenkymaalisten stroomasolujen hyödyntämistä luuvaurioiden korjaamisessa sekä tulevaisuuden lääkekehityskohteena

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-beta 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 mu CT 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.Peer reviewe

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

IntroductionIn 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-beta 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 MethodsFemale 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.ResultsBone mass decreased in OVX PBS mice compared to the SHAM PBS group but ActRIIB-Fc was able to prevent these changes as shown by mu CT 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.ConclusionOur 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.</p

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.</p

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.</p

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