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

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