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Wnt signaling in osteoblast differentiation and bone formation
S8
ABSTRACTS / Bone 38 (2006) S5 – S22
of gremlin and noggin, known to have a high affinity for BMP-2. Smad6 expression was also increased. These results show that the inhibitor could belong to a family of proteins able to antagonize BMPs by binding them with high affinity, preventing receptor binding. The balance between BMPs and BMP antagonists could play a crucial role in the decrease of bone formation with age. doi:10.1016/j.bone.2006.01.066
8 Arrestin-regulated genes in osteoblasts exposed to intermittent or continuous PTH E.N. Bianchi , D.D. Pierroz, D. Manen, R. Rizzoli, S.L. Ferrari Service of Bone Diseases, Geneva University Hospital, CH-1205 Geneva, Switzerland Intermittent PTH (iPTH) is anabolic for bone, whereas continuous PTH (cPTH) induces a negative balance. The molecular mechanisms implicated remain however obscure. We previously reported that h-arrestin2 (Arrb2) mediates desensitization/resensitization of PTH signaling in vitro and that Arrb2 knock-out (KO) mice have an altered response to iPTH. We hypothesized that Arrb2 may regulate osteoblast (OB) gene expression in response to iPTH versus cPTH. Primary OB cultures (n = 3/condition) from wild-type (WT) or Arrb2 KO mice calvariae were treated with 10nM of iPTH (i.e., 6h of 48h cycle), cPTH (i.e., 48h), or vehicle for 2 weeks. Gene expression profiles were carried out with Affymetrix microarrays (mouse chip, 22690 genes) and changes evaluated by Affymetrix GCOS analysis. iPTH induced mineralization in both WT and KO cultures, whereas cPTH did not. In WT cells, iPTH up-regulated 245 genes and down-regulated 213 genes (>1.5) compared to vehicle. cPTH up/down-regulated only 43 of these genes (incl. IGF-I, +1.7, and OPG, 2) in addition to 39 different genes (incl. Sfrp1, +2.3), indicating that sustained exposure to PTH primarily desensitized osteoblastic gene expression. 415 genes were exclusively regulated by iPTH, including RANKL (+5) and Il-6 (+5), genes implicated in cAMP signaling (Crem, +2.3, PTHR1, 1.7, and Arrb2, +2.4), genes associated with OB differentiation (Akp2, +1.5, Bmpr1a, 1.7, Pparg, 3, and Wisp1, 1.6), and survival (Casp12, 2.2). Interestingly, iPTH highly up-regulated (+13 –25) some genes whose skeletal function and/or response to PTH was unknown. In Arrb2 KO OB, the number of genes exclusively regulated by iPTH was decreased by 40% versus WT cells, whereas a similar number of genes were regulated exclusively by cPTH. Many genes which up/down-regulation by iPTH was lost in absence of Arrb2, are influencing OB differentiation and survival. Thus, expression profiling in OB from WT and Arrb2 KO mice exposed to iPTH or cPTH underscores the crucial role of arrestins in mediating expression of genes exclusively associated with iPTH effects on bone. doi:10.1016/j.bone.2006.01.067
9 Wnt signaling in osteoblast differentiation and bone formation P.V.N. Bodine Women’s Health Research Institute, Wyeth Research, Collegeville, PA 19426, USA Wnts are a large family of 19 growth factors that mediate fundamental biological processes like embryogenesis, organogenesis and tumorigenesis. These proteins bind to a membrane receptor complex comprised of 1 of 10 frizzled (FZD) G-protein-coupled receptors (GPCRs) and a lowdensity lipoprotein (LDL) receptor-related protein (LRP)-5 or -6. The formation of this ligand-receptor complex initiates a number of intracellular signaling cascades that includes the canonical/beta-catenin pathway, as well as several GPCRrelated noncanonical pathways. In recent years, canonical Wnt signaling has been shown to play a substantial role in the control of bone formation. Clinical investigations have found that mutations in LRP-5 are associated with bone mineral density and fractures. For example, loss-of-function mutations in LRP-5 cause osteoporosis pseudoglioma syndrome, while gain-of-function mutations lead to high bone mass phenotypes. Studies of knockout and transgenic mouse models for Wnt pathway components like Wnt-10b, LRP-5, secreted frizzledrelated protein-1, dickkopf-2 and beta-catenin have demonstrated that canonical signaling modulates most aspects of osteoblast physiology including proliferation, differentiation, bone matrix formation and apoptosis, as well as coupling to osteoclastogenesis and bone resorption. Future studies in this rapidly growing area of research should focus on elucidating Wnt/FZD specificity in the control of bone cell function, the role of noncanonical pathways in skeletal remodeling, direct effects of Wnts on cells of the osteoclast lineage, and the interplay between Wnt signaling and other bone metabolic pathways. doi:10.1016/j.bone.2006.01.068
10 Uptake of bone-related matrix proteins into implanted deproteinized bovine bone mineral D.D. Bosshardt , B. Wallkamm, R.K Schenk, D. Buser, N.P. Lang Departments of Periodontology and Prosthodontics and Oral Surgery and Stomatology, School of Dental Medicine, University of Berne, CH-3010 Berne, Switzerland Deproteinized bovine bone mineral (DBBM) (Bio-OssR, Geistlich-Pharma, Wohlhusen, Switzerland) is widely used as a bone substitute for the preservation or augmentation of bone volume. After implantation near native bone, new bone may form around the DBBM particles. Since DBBM is very resistant to resorption, it will hardly ever be replaced by bone and, therefore, the mechanical stability largely depends on the extent of bridging between the newly formed bone and the DBBM particles. The molecular factors responsible for the deposition of
ABSTRACTS / Bone 38 (2006) S5 – S22
of gremlin and noggin, known to have a high affinity for BMP-2. Smad6 expression was also increased. These results show that the inhibitor could belong to a family of proteins able to antagonize BMPs by binding them with high affinity, preventing receptor binding. The balance between BMPs and BMP antagonists could play a crucial role in the decrease of bone formation with age. doi:10.1016/j.bone.2006.01.066
8 Arrestin-regulated genes in osteoblasts exposed to intermittent or continuous PTH E.N. Bianchi , D.D. Pierroz, D. Manen, R. Rizzoli, S.L. Ferrari Service of Bone Diseases, Geneva University Hospital, CH-1205 Geneva, Switzerland Intermittent PTH (iPTH) is anabolic for bone, whereas continuous PTH (cPTH) induces a negative balance. The molecular mechanisms implicated remain however obscure. We previously reported that h-arrestin2 (Arrb2) mediates desensitization/resensitization of PTH signaling in vitro and that Arrb2 knock-out (KO) mice have an altered response to iPTH. We hypothesized that Arrb2 may regulate osteoblast (OB) gene expression in response to iPTH versus cPTH. Primary OB cultures (n = 3/condition) from wild-type (WT) or Arrb2 KO mice calvariae were treated with 10nM of iPTH (i.e., 6h of 48h cycle), cPTH (i.e., 48h), or vehicle for 2 weeks. Gene expression profiles were carried out with Affymetrix microarrays (mouse chip, 22690 genes) and changes evaluated by Affymetrix GCOS analysis. iPTH induced mineralization in both WT and KO cultures, whereas cPTH did not. In WT cells, iPTH up-regulated 245 genes and down-regulated 213 genes (>1.5) compared to vehicle. cPTH up/down-regulated only 43 of these genes (incl. IGF-I, +1.7, and OPG, 2) in addition to 39 different genes (incl. Sfrp1, +2.3), indicating that sustained exposure to PTH primarily desensitized osteoblastic gene expression. 415 genes were exclusively regulated by iPTH, including RANKL (+5) and Il-6 (+5), genes implicated in cAMP signaling (Crem, +2.3, PTHR1, 1.7, and Arrb2, +2.4), genes associated with OB differentiation (Akp2, +1.5, Bmpr1a, 1.7, Pparg, 3, and Wisp1, 1.6), and survival (Casp12, 2.2). Interestingly, iPTH highly up-regulated (+13 –25) some genes whose skeletal function and/or response to PTH was unknown. In Arrb2 KO OB, the number of genes exclusively regulated by iPTH was decreased by 40% versus WT cells, whereas a similar number of genes were regulated exclusively by cPTH. Many genes which up/down-regulation by iPTH was lost in absence of Arrb2, are influencing OB differentiation and survival. Thus, expression profiling in OB from WT and Arrb2 KO mice exposed to iPTH or cPTH underscores the crucial role of arrestins in mediating expression of genes exclusively associated with iPTH effects on bone. doi:10.1016/j.bone.2006.01.067
9 Wnt signaling in osteoblast differentiation and bone formation P.V.N. Bodine Women’s Health Research Institute, Wyeth Research, Collegeville, PA 19426, USA Wnts are a large family of 19 growth factors that mediate fundamental biological processes like embryogenesis, organogenesis and tumorigenesis. These proteins bind to a membrane receptor complex comprised of 1 of 10 frizzled (FZD) G-protein-coupled receptors (GPCRs) and a lowdensity lipoprotein (LDL) receptor-related protein (LRP)-5 or -6. The formation of this ligand-receptor complex initiates a number of intracellular signaling cascades that includes the canonical/beta-catenin pathway, as well as several GPCRrelated noncanonical pathways. In recent years, canonical Wnt signaling has been shown to play a substantial role in the control of bone formation. Clinical investigations have found that mutations in LRP-5 are associated with bone mineral density and fractures. For example, loss-of-function mutations in LRP-5 cause osteoporosis pseudoglioma syndrome, while gain-of-function mutations lead to high bone mass phenotypes. Studies of knockout and transgenic mouse models for Wnt pathway components like Wnt-10b, LRP-5, secreted frizzledrelated protein-1, dickkopf-2 and beta-catenin have demonstrated that canonical signaling modulates most aspects of osteoblast physiology including proliferation, differentiation, bone matrix formation and apoptosis, as well as coupling to osteoclastogenesis and bone resorption. Future studies in this rapidly growing area of research should focus on elucidating Wnt/FZD specificity in the control of bone cell function, the role of noncanonical pathways in skeletal remodeling, direct effects of Wnts on cells of the osteoclast lineage, and the interplay between Wnt signaling and other bone metabolic pathways. doi:10.1016/j.bone.2006.01.068
10 Uptake of bone-related matrix proteins into implanted deproteinized bovine bone mineral D.D. Bosshardt , B. Wallkamm, R.K Schenk, D. Buser, N.P. Lang Departments of Periodontology and Prosthodontics and Oral Surgery and Stomatology, School of Dental Medicine, University of Berne, CH-3010 Berne, Switzerland Deproteinized bovine bone mineral (DBBM) (Bio-OssR, Geistlich-Pharma, Wohlhusen, Switzerland) is widely used as a bone substitute for the preservation or augmentation of bone volume. After implantation near native bone, new bone may form around the DBBM particles. Since DBBM is very resistant to resorption, it will hardly ever be replaced by bone and, therefore, the mechanical stability largely depends on the extent of bridging between the newly formed bone and the DBBM particles. The molecular factors responsible for the deposition of