PP032 Expression of membrane-type matrix metalloproteinases (MT-MMPS) during osteoblast differentiation K.B. Paiva a,b, ⁎ , N. Bendini a,b , L.H. Silva a,b , J.M. Granjeiro c , M.C. Sogayar d Cell and Molecular Therapy Center a Biochemistry, Chemistry Institute, University of São Paulo, Brazil b Oral Pathology, Dental School, University of São Paulo, Sao Paulo, Brazil c Cell and Molecular Biology, Federal Fluminense University, Niteroi, Brazil d Biochemistry, University of Sao Paulo, Sao Paulo, Brazil Abstract: MMPs are zinc-dependent endopeptidases that, collectively, degrade all components of the extracellular matrix (ECM) and generate bioactive molecules. The membrane-anchored MMPs (MT-MMP), containing an anchoring motif that facilitates to reach key membrane and peripheral proteins as well as closely associated ECM components thereby play pivotal roles during alterations of the pericellular environment in both physiological and pathological conditions. Membrane insertion confers MT-MMPs with a unique set of regulatory mechanisms that serve to control the pool of active protease at the cell surface including endocytosis, recycling, autocatalytic processing, and ectodomain shedding. The biological mineralization research looking for discovering the genes involved in the molecular mechanisms that control the osteoblast differentiation process. MMPs and their inhibitors (TIMPs and RECK) are responsible for bone matrix remodeling and, probably, determinate the level of its turnover. Since there are no studies evaluating the influence of all 6 MT-MMPs members (MMP-14/MT1-MMP, MMP-15/MT2-MMP, MMP-16/MT3-MMP, MMP-17/ MT4-MMP, MMP-24/MT5-MMP, and MMP-25/MT6-MMP) on osteoblast differentia- tion in vitro, we carried out the present study. Our objective was to analyze gene expression of MT-MMPs during osteoblast differentiation by qRT-PCR. MC3T3-E1 (subclone 4), a pre-osteoblast cell line from calvaria of mice, was grown in α-MEM medium supplemented with 10% FBS and these cells were differentiated in the presence of osteogenic medium [β-glycerophosphate (10 mM) and ascorbate (50 μg/ml)] for 28 days. Our results demonstrated that transcript levels for both MMP-14/MT1-MMP and MMP-25/MT6-MMP were downregulated throughout osteoblast differentiation. Both MMP-15/MT2-MMP and MM-16/MT3-MMP were upregulated 2-fold change at 1st-day after differentiation induction, however they were downregulated at subse- quent periods. MMP-17/MT4-MMP was upregulated 10-fold and 2-fold change at 1st-day and 14th-days after differentiation induction, respectively, and downregulated at 28th-days. This is the first evidence that MT-MMPs are differentially expressed and MMP-17/MT3-MMP may be a new player in osteoblast differentiation in vitro. Keywords: MT-MMP and Osteoblast differentiation. Financial support: FAPESP. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.221 PP033 Profiling of membrane proteins of human bone marrow stromal stem cells during ex vivo osteoblast differentiation using SILAC-based quantitative proteomics H. Christiansen a,b , K.H. Larsen a, ⁎ , J.S. Burns a,c , I. Kratchmarova d , B.M. Abdallah a , J.S. Andersen d , M. Kassem a a Dept of Endocrinology and Metabolism/KMEB, Odense University Hospital, Denmark b Lundbeckfonden Center of Excellence NanoCAN and Molecular Oncology, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark c Laboratory of Cell Biology and Advanced Cancer Therapies, Department of Oncology, Hematology and Respiratory Disease, University Hospital of Modena and Reggio Emilia, Modena, Italy d Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark Abstract: Understanding the biology of bone formation has been hampered by the absence of prospective markers for skeletal stem cells (also known as bone marrow stromal or mesenchymal stem (MSC) cells) undergoing osteoblast differentiation. Thus, we employed Stable Isotope LAbeling in Cell culture (SILAC) to obtain a quantitative profile of membrane proteins during the course of ex vivo osteoblast (OB) differentiation of human MSC (hMSC). We identified a total of 972 proteins; 80% of which were either integral membrane proteins, membrane associated proteins, interactors with membrane proteins or GPI-anchored proteins and 41% of these proteins have a trans-membrane domain. Also, we identified 138 proteins that were quantifiable during 5 time-points covering 14 days ex vivo OB differentiation. These proteins were classified into several functional categories e.g. ribosomal proteins (19 proteins, 13%), Ras/Rab proteins (12 proteins, 9%), transporter proteins (10 protein, 7%) and bone-related proteins (14 proteins, 10%). In addition, 30 different CD antigens were found, 20 of which were quantified in all time-points of OB differentiation. Quantitative analysis revealed 317 proteins that were increased more than 2 fold during OB differentiation and included alkaline phosphatase and CD295 (maximal increase day 4), CD91 and Raftlin, CD107b and CD36L (maximal increase day 7) and integrin B5 (maximal increase day 14) or exhibited constant levels e.g. CD98 and CD90. Finally, we validated a subset of the potential biomarkers by fluorescence-activated cell sorting (FACS) and RT-qPCR. In conclusion, SILAC-based quantitative proteomics revealed novel biomarkers for hMSC that can prospectively be employed in monitoring OB differentiation and hMSC identity in ex vivo cultures and several of the markers may play an important role in OB biology. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.222 PP034 A specific subtype of osteoclasts secretes factors inducing nodule formation by osteoblasts K. Henriksen a, ⁎, K.V. Andreassen b , C.S. Thudium a , N.S. Gudmann a , I. Moscatelli c , J. Richter c , M.A. Karsdal a , A.V. Neutzsky-Wulff a a Bone Biology, Nordic Bioscience A/S, Denmark b Bone Biology, Nordic Bioscience, Herlev, Denmark c Department of Molecular Medicine and Gene Therapy, Lund Strategic Center for Stem Cell Biology, Lund, Sweden Abstract: Osteoclasts are known to be important for the coupling process between bone resorption and formation, at least partially by secretion of bone anabolic factor(s). The aims of this study were to address whether osteoclasts are the only source of anabolic factors, and how the ability to resorb, as well as how different matrices affect the release of bone anabolic factors from mature resorbing osteoclasts. Human monocytes were isolated from cord blood or peripheral blood and differentiated into mature osteoclasts by treatment with M-CSF and RANKL. Conditioned medium was collected from macrophages, pre-osteoclasts, and mature functional and non-resorbing osteopetrotic osteoclasts on either bone, plastic, decalcified bone or dentine. Osteoclasts on bone and plastic were treated with vehicle or diphyllin, E64 or GM6001. Conditioned medium (CM) and corresponding non- conditioned medium (non-CM) were collected and pooled. Osteoclast numbers were measured by TRACP activity. Bone resorption was evaluated by CTX-I and calcium release. The osteoblastic cell line 2T3 was treated with 50% of CM or non-CM for 12 days. Bone formation was assessed by Alizarin Red extraction. Only CM from mature osteoclasts induced bone formation, while CM from precursors or mature macrophages failed to do so. Non-resorbing osteoclasts generated from osteopetrosis patients showed very little resorption, yet a blunted, but still significant ability to induce bone formation by osteoblasts. Diphyllin and E64 all potently reduced resorption, while GM6001 did not. Collected CM from all the conditions induced bone formation significantly compared to their corresponding non- CM, with an 800% induction by vehicle CM. However, CM from diphyllin, cathepsin K inhibitor and E64 treated osteoclasts decreased the level of bone formation compared to CM from vehicle treated osteoclasts by approximately 50%, while CM from GM6001 treated osteoclasts equaled vehicle CM. Osteoclasts on either dentine or decalcified bone showed strongly attenuated anabolic capacities. In conclusion, we present evidence that osteoclasts, both dependent and independent of their resorptive activity, secrete activities stimulating osteoblastic bone formation. Further understanding of these processes should help in developing treatments for osteoporosis with less secondary inhibition of bone formation than presently possible. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.223 PP035 Wnt3a stimulates DSPP expression directly by activation of canonical Wnt signaling pathway S. Rahman a , J.-H. Oh a , Y.-D. Cho a , W.-J. Yoon a , F.H. Yu b , J.-H. Baek a , H.-M. Ryoo a , K.M. Woo a, ⁎ a Molecular Genetics, Republic of Korea b Neuroscience, Seoul National University School of Dentistry, Seoul, Republic of Korea Abstracts S77