Serotonin supports bone resorption via an autocrin paracrine loop

Abstracts / Bone 48 (2011) S96–S99 revealed increases of 47% in female (p < 0.05) and 48% in male (p < 0.01) in trabecular MAR in HOM/HET mice compared to WT/WT. Further, in this region bone formation rate was increased 58% in female HOM/HET compared to WT/WT (p < 0.05). Bone resorption parameters were not different between genotypes. Importantly no skeletal phenotype has been detected in WT/HET control mice. Complete deletion of Dkk1 in the presence of reduced Wnt3 produces viable mice with an extremely high bone mass phenotype. Our histological analysis provides mechanistic data to confirm these increases are due to enhanced bone formation. Both the lack of postnatal skeletal expression of Wnt3 and absence of a skeletal phenotype in WT/HET mice suggest no functional role for Wnt3 in the postnatal skeleton. The results of this study confirm a pivotal role for Dkk1 as a negative regulator of bone formation, and inhibition of Dkk1 as a potential anabolic strategy in the treatment of osteoporosis and other bone diseases. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: M. Mcdonald: none declared, A. Morse: none declared, K. Mikulec: none declared, L. Peacock: none declared, P. Tam: none declared, D. Little Consulting fees from Novartis Pharma.

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investigated the in vivo and ex vivo bone phenotype in mice invalidated for tryptophan hydroxylase 1 (TPH−/− ), the rate limiting enzyme in peripheral serotonin synthesis. 1 The TPH−/− male mice displayed increased bone density at 6 and 10 weeks old. 1 Histomorphometric (bone formation rate) and biochemical (osteocalcin) parameters of bone formation were unchanged. By contrast, bone resorption markers (urinary deoxypyridinoline DPYR, plasma TRAP5b) were decreased at 6 (− 59%) and 10 weeks. Histomorphometric analysis, indicated an increase of trabecular bone volume (+ 67%) and markedly reduced osteoclast number (− 55%) and osteoclastic trabecular surfaces (−44%). Finally, the TPH−/− 1 mice were treated during 2 weeks with 5-HTP to override the TPH1 invalidation. DPYR ratio increased in mice treated with 5-HTP from the first week of treatment and returned to the WT level after 2 weeks of treatment. To further study the osteoclast phenotype in TPH−/− mice, we focused on ex-vivo spleen cells 1 cultured with RANKL and MCSF. Tph−/− cells gave rise to both reduced numbers of osteoclasts and 1 decreased resorption pits on dentin slices in comparison with WTcells. Moreover, osteoclastogenesis in TPH−/− cell cultures was rescued following the addition of 5-HT starting to 5 nM. 1 We investigated the 5-HT synthesis in WT osteoclast precursors and we showed an increase of TPH1 RNA expression level the presence of 5-HT in cell lysate. To define how 5-HT regulates osteoclast differentiation, we next focused on serotoninergic system:

doi:10.1016/j.bone.2011.03.140 -5-HT storage via vesicular monoamine transporters (VMAT), -Its transport via 5-HT transporter (SERT) -And its 5-HT receptors (5-HTRs).

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OPB04 (recipient of a 2011 ECTS/IBMS New Investigator Award) In osteoclasts, dynamic microtubules and their associated protein EB1 control podosomes and bone resorption through cortactin M. Biosse-Duplan a,⁎, S. Stephens a, F.P.L. Lai b, M. Oelkers c, K. Rottner c, W. Horne a, R. Baron a,d a Oral Medicine, Infection and Immunity, Harvard School Of Dental Medicine, Boston, USA b Institute of Medical Biology, Immunos, Singapore, Singapore c Institute of Genetics, University of Bonn, Bonn, Germany d Dept of Medicine Endocrine Unit, Harvard Medical School, Boston, USA

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Abstract: Mature osteoclasts (OCs) organize their actin cytoskeleton into a podosome belt (PB), a unique feature that is critical for bone resorption. The microtubule (MT) network promotes the formation and stability of the PB; however the molecules present on MTs and/or in podosomes and which mediate the interaction of the two systems have yet to be identified. Because the growing “plus” ends of MTs point toward the OC periphery, where podosomes accumulate, we used a genome-wide screen of genes expressed in OC to search for genes encoding proteins that accumulate at these MTends, called “plus end tracking proteins” (+TIPs). Several+TIPs were highly expressed and after RT-PCR confirmation, we found that expression of EB1, a master regulator of +TIP interaction complexes, increases significantly during osteoclastogenesis, with the strongest expression in mature OCs. Using live cell imaging, we observed that EB1 localizes around podosomes and is enriched in the PB. Agents that disturb or stabilize EB1 on MTs, have opposite effects on podosome belt stability, resulting respectively in a decrease or increase of podosome density at the cell periphery. Consistent with these observations, depleting EB1 or expressing a dominant-negative mutant of EB1 in OCs strongly antagonized PB formation. We then looked for targets of EB1 in podosomes and found that cortactin (CTTN) associates with EB1 and that this interaction was dependent on MT stability. Using CTTN knockout OCs, we found that CTTN deletion also altered PB formation and bone resorption activity and that the domains of CTTN that were required to rescue this phenotype were also necessary for the EB1/CTTN interaction. Most importantly, stabilization of MTs did not affect the podosome belt in the absence of CTTN. Furthermore, disruption or stabilization of MTs respectively increased or decreased CTTN phosphorylation, a post-translational modification known to affect podosome behavior. Finally, we show that the defective cytoskeletal organization observed in the absence of CTTN has in vivo consequences, since targeted deletion of CTTN in OCs by crossing cathepsin K-Cre mice with floxed CTTN mice resulted in a mild form of osteopetrosis due to impaired bone resorption. These data identify the MT-associated protein EB1 and its interaction with CTTN as critical regulators of podosome organization and bone resorption, uncovering a new role for CTTN in OCs. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.

doi:10.1016/j.bone.2011.03.141

OPB05 Serotonin supports bone resorption via an autocrin paracrine loop Y. Chabbi Achengli a,⁎, A. Coudert a, J. Callebert b, F. Côté c, C. Collet a,b, M.C. de Vernejoul a a INSERM U606, France b Laboratoire de Biochimie Hôpital Lariboisière, France c CNRS UMR 8147, Hôpital Necker, Paris, France Abstract: Recently, peripheral serotonin (5-HT or 5-Hydroxytryptamine), was shown to be a major player in bone formation. In order to determine the role of serotonin on bone resorption we

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Using reserpine a VMAT inhibitor and paroxetine, a SERT inhibitor, we observed a decrease in osteoclast number in WT. Finally, we observed a strong decrease in osteoclastogenesis in WT cells following the use of ketanserin, a 5HT2AR antagonist and SB224289 an inhibitor of 5HT1BR. In conclusion, we identify 5-HT as an important factor in the regulation of osteoclast maturation and function both in vivo and ex vivo. In addition, we demonstrate that 5-HT is synthesized in osteoclast precursor and is able to act on osteoclast differentiation by an autocrine/paracrine pathway. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.

doi:10.1016/j.bone.2011.03.142

OPB06 Osteoclast progenitors reside in ppar-gamma-expressing bone marrow cell population Y. Wan a,⁎, W. Wei a, D. Zeve b, X. Wang a, Y. Du a, W. Tang b, P.C. Dechow c, J.M. Graff b a Department of Pharmacology, USA b Department of Developmental Biology, University of Texas Southwestern Medical Center, USA c Department of Biomedical Sciences, Baylor College of Dentistry, Texas A & M University Health Sciences Center, Dallas, USA Abstract: Osteoclasts are bone resorbing cells essential for skeletal development, homeostasis and regeneration. They derive from hematopoietic progenitors in the monocyte/macrophage lineage and differentiate in response to RANKL. However, the precise nature of osteoclast progenitors remains a long-standing yet important question. Using inducible PPARg-tTA/TRE-GFP reporter mice, we show that osteoclast progenitors reside specifically in the PPARg-expressing bone marrow population, and identify the quiescent PPARg+ cells as osteoclast progenitors. Importantly, two PPARg-tTA/TRE-Cre controlled genetic models provide compelling functional evidence. First, Notch activation in PPARg+ cells causes osteopetrosis due to impaired osteoclast precursor proliferation. Second, selective ablation of PPARg+ cells by diphtheria toxin also causes osteopetrosis due to decreased osteoclast number. Furthermore, PPARg+ cells respond to both pathological and pharmacological resorption-enhancing stimuli. Mechanistically, PPARg promotes osteoclast progenitors by activating GATA2 transcription. These findings not only identify the longsought-after osteoclast progenitors but also establish unprecedented tools for their visualization, isolation, characterization and genetic manipulation. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.

doi:10.1016/j.bone.2011.03.143

OPB07 Effects of PTH(1–34) and estrogen status on osteocyte lacunar properties in rats D.B. Kimmel a,⁎, T. Fong b, M.P. Akhter a, J. Coats b, T. Wronski c a Osteoporosis Research Center, Creighton University, Omaha, NE, USA