Estrogen receptor alpha gene XbaI polymorphism in human mesenchymal stem cells influences osteoblast differentiation, activation and the effect of estrogen

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Abstracts / Bone 48 (2011) S104–S123

PP023-M 1alpha,25-dihydroxyvitamin D3 induces autophagy in osteoblasts V. Pierrefite-Carle, V. Breuil, M. Ettaiche, S. Santucci, D. Momier, G.F. Carle ⁎ Genetics and Physiopathology of Bone, UMR6235, CNRS, Université de Nice-Sophia Antipolis, Nice, France Abstract: 1α,25-Dihydroxyvitamin D3 [1α,25 VitD3], the hormonally active metabolite of vitamin D3, is essential for the production and maintenance of the adult skeleton. It is generally agreed that 1α,25 VitD3 affects bone formation mainly by indirect mechanisms on calcium homeostasis, but 1α,25 VitD3 has also direct effects on osteoblasts in vitro. This steroid hormone promotes osteoblast differentiation, survival and cell death, as well as osteogenic activities in vitro. In particular, it has been demonstrated that transient (<2 days) treatment of osteoblasts with 1α,25 VitD3 has anti-apoptotic effects while prolonged exposure (3 or more days) induces cell death. This dual effect based on treatment duration led us to hypothesize that 1α,25 VitD3 could induce autophagy in osteoblasts. Autophagy is a self-digestion pathway involved in protein and organelle degradations for recycling. Although autophagy is a cell survival mechanism, excessive autophagy can cause cell death. In the present work, we have used confocal and transmission electron microscopies to demonstrate that 1α,25 VitD3 induces autophagy in the rat osteosarcoma cell line UMR-106. These results were also confirmed in human primary osteoblasts. We are presently analyzing whether autophagy is involved in the anti-apoptotic effect of 1α,25 VitD3. As apoptosis regulation in bone cells is a crucial mechanism for the control of the osteoclasts–osteoblasts ratio, autophagy could appear as a new key player influencing this balance in bone remodeling. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared. doi:10.1016/j.bone.2011.03.181

PP024-T/NIPP06 The transcription factor PRDM5 is involved in endochondral bone formation by regulating collagen I in vivo G.G. Galli a,⁎, K.H. de Lichtenberg a, M. Wuelling b, R. Calogero c, A.H. Lund a a Biotech Research & Innovation Centre, Copenhagen, Denmark b Dept. Developmental Biology, Centre for Medical Biotechnology, University of Duisburg-Essen, Essen, Germany c Molecular Biotechnology Center, Turin, Italy

PP025-S Estrogen receptor alpha gene XbaI polymorphism in human mesenchymal stem cells influences osteoblast differentiation, activation and the effect of estrogen H.-V. Leskelä a,⁎, S. Lehtonen b, P. Lehenkari b a Department of Surgery, University of Oulu, Oulu, Finland b Department of Anatomy, University of Oulu, Oulu, Finland Abstract: Variation in response to hormone replacement therapy is frequently seen in postmenopausal osteoporosis. Interindividual variation in response to the 17b-estradiol (E2) treatments has been discovered to be dependent on the estrogen receptor alpha (ERα) gene in human bone marrow-derived mesenchymal stem cell (MSC) cultures. Recently, the effect of E2 on human MSCs has been shown to vary depending on the cell strain. There is still a lack of cell culture data from the association of polymorphisms with the candidate gene in osteoporosis on the human MSCs. The aim of this study was to investigate whether the gene polymorphisms of ERα (XbaI), vitamin D receptors (BsmI, FokI), calcitonin receptor (AluI), and collagen type 1 (Sp1) in MSC cultures are associated with osteoblast differentiation capacity and activity. MSCs were quantified in vitro by measuring alkaline phosphatase activity (ALP) and calcium deposition. Polymorphisms were investigated with PCR and the ERα protein expression levels were analyzed by Western blot. We show here that in ERα gene XbaI polymorphism higher osteoblast differentiation capacity of MSCs is associated with the absence of the restriction site (X) in females and the presence of the restriction site (non-X) in males, whereas higher response to E2 treatment is associated with the absence of the restriction site (X) in both sexes. No association of any other studied polymorphisms was observed. These results could help explain the interindividual variability of response to estrogen and osteogenic differentiation and activation in MSC-derived osteoblasts. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.

doi:10.1016/j.bone.2011.03.183

PP026-M GPM6B regulates osteoblast differentiation and induction of mineralization by controlling cytoskeleton and matrix vesicle release K. Drabek, J. van de Peppel, M. Eijken, H. van Leeuwen ⁎ Internal Medicine, Erasmus MC, Rotterdam, Netherlands

Abstract: Members of the PRDM protein family are transcriptional modulators involved in the differentiation of distinct tissue types. PRDMs are characterized by the presence of a PR/SET domain, often conferring histone methyltransferase activity to regulate local DNA packaging, and a variable number of zinc finger domains mediating protein–DNA and protein–protein interactions. Whereas PRDM5 was recently characterized as a putative tumor suppressor gene, very little is known about its role in tissue specific differentiation. In zebrafish Prdm5 knockdown induces cyclopia, mesoendodermal defects and impairment of morphogenetic movements at the gastrulation stage. This phenotype is dependent on the role of Prdm5 as a negative regulator of both the canonical and the noncanonical WNT pathways. In order to analyze the function of Prdm5 during tissue specific differentiation, we generated a Prdm5 gene-trap KO mouse model featuring the integration of a beta-geo cassette into the second intron of Prdm5, thus creating a null allele. Mice lacking Prdm5 are viable and fertile, without gross abnormalities and the KO allele segregates in normal Mendelian ratio. Publicly available data report that Prdm5 is highly expressed in mouse embryonic stem cells (mES), embryonic fibroblasts and osteoblasts. Microarray analysis of Prdm5 KO mES cells reveals modulation of genes involved in cardiac and skeletal system developments. By X-gal staining we show that Prdm5 is expressed in skeletal structures during mouse embryogenesis and in particular in osteoblasts regions during the process of endochondral bone formation. To demonstrate a role for Prdm5 in osteoblast differentiation we generated MC3T3 cell lines stably transfected with shRNA constructs against Prdm5. Importantly, we show that sustained knockdown of Prdm5 in the MC3T3 osteoblastic cell line impairs matrix mineralization. Furthermore, using expression analyses and chromatin immunoprecipitation we show that Col1a1 is a direct Prdm5 target gene thus sustaining the notion of Prdm5 as a transcription factor regulating osteoblast specific markers. We are currently investigating a possible phenotype in Prdm5 KO skeletal elements and preliminary data show that collagen I is deregulated in embryo bones without Prdm5. Collectively our data suggest a role for Prdm5 in regulating osteoblast biology in vivo by deregulating gene expression of collagen I. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.

Abstract: Neuronal membrane glycoprotein gene (GPM6B) encodes a four-transmembrane glycoprotein that belongs to the proteolipid protein family and initially described as a structural protein of the central nervous system. We identified GPM6B as a gene that is strongly up-regulated during osteoblast differentiation and increased and reduced by vitamin D and activin A, respectively. To investigate the role of GPM6B in bone formation we silenced GPM6B expression during osteogenic human mesenchymal stem cell (hMSC) differentiation. GPM6B silencing in hMSCs resulted in reduced alkaline phosphatase (ALP) activity along with reduced mineralization of extracellular matrix (ECM). Microarray analysis of GPM6B silenced osteogenic hMSC identified 65 genes significantly (p< 0.001 and 2-fold) and identically regulated by the 3 shRNAs tested. Bioinformatic analyses showed significant changes in genes involved in cytoskeleton organization and biogenesis. Immunocytochemistry confirmed changed distribution of actin filaments and shape and size changes of focal adhesions (vinculin) upon GPM6B silencing. GPM6B may function as a signaling molecule in the membrane coupling ECM to the cytoplasm and nucleus thereby linking ECM interaction to gene transcription. In this way GPM6B may also play an important role in mechanotransduction. Support for an intracellular signaling role of GPM6B in osteoblasts is derived from our observation that silencing GPM6B significantly down-regulated expression of PDLIM7 (also known as LMP1: LIM mineralization protein-1). PDLIM7 has been shown to be involved in BMP-6 stimulated osteoblast differentiation and mineralization and antisense block of PDLIM7 prevented osteoblast differentiation (Boden et al., Endocrinology. 139:5125; 1998). Based on this it is tempting to speculate that GPM6B functions as a membrane localized switch in osteoblasts that integrates ECM derived signals into an intracellular signal and eventually altered osteoblast activity. Finally, we demonstrated a role for GPM6B in production of ALP-positive matrix vesicles (MVs), which was reduced after silencing of GPM6B. In conclusion, we identified GPM6B as a novel regulator of osteoblast differentiation and bone formation and demonstrate the significance of cytoskeleton organization for osteoblast differentiation, MV production and eventual mineralization. In general, our current data strengthen a role for the cytoskeleton in the MV production and/or release from the osteoblasts. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.

doi:10.1016/j.bone.2011.03.182

doi:10.1016/j.bone.2011.03.184