The Wnt antagonist secreted frizzled-related protein-1 is a negative regulator of trabecular bone formation in adult mice.

Previous studies have associated activation of canonical Wnt signaling in osteoblasts with elevated bone formation. Here we report that deletion of the murine Wnt antagonist, secreted frizzled-related protein (sFRP)-1, prolongs and enhances trabecular bone accrual in adult animals. sFRP-1 mRNA was expressed in bones and other tissues of +/+ mice but was not observed in -/- animals. Despite its broad tissue distribution, ablation of sFRP-1 did not affect blood and urine chemistries, most nonskeletal organs, or cortical bone. However, sFRP-1-/- mice exhibited increased trabecular bone mineral density, volume, and mineral apposition rate when compared with +/+ controls. The heightened trabecular bone mass of sFRP-1-/- mice was observed in adult animals between the ages of 13-52 wk, occurred in multiple skeletal sites, and was seen in both sexes. Mechanistically, loss of sFRP-1 reduced osteoblast and osteocyte apoptosis in vivo. In addition, deletion of sFRP-1 inhibited osteoblast lineage cell apoptosis while enhancing the proliferation and differentiation of these cells in vitro. Ablation of sFRP-1 also increased osteoclastogenesis in vitro, although changes in bone resorption were not observed in intact animals in vivo. Our findings demonstrate that deletion of sFRP-1 preferentially activates Wnt signaling in osteoblasts, leading to enhanced trabecular bone formation in adults.

[1]  S. Wattler,et al.  Construction of Gene Targeting Vectors from λKOS Genomic Libraries , 1999 .

[2]  W. Birchmeier,et al.  New aspects of Wnt signaling pathways in higher vertebrates. , 2001, Current opinion in genetics & development.

[3]  Ivan Lobov,et al.  Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor , 2002, The Journal of cell biology.

[4]  T. Pramila,et al.  Interaction of Frizzled Related Protein (FRP) with Wnt Ligands and the Frizzled Receptor Suggests Alternative Mechanisms for FRP Inhibition of Wnt Signaling* , 1999, The Journal of Biological Chemistry.

[5]  Mark L. Johnson,et al.  A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant high-bone-mass trait. , 2002, American journal of human genetics.

[6]  D. Gazit,et al.  Bone loss (osteopenia) in old male mice results from diminished activity and availability of TGF‐β , 1998, Journal of cellular biochemistry.

[7]  B. Komm,et al.  Regulation of Bone Cell Function by Estrogens , 2001 .

[8]  S. Krane,et al.  Osteocyte and osteoblast apoptosis and excessive bone deposition accompany failure of collagenase cleavage of collagen. , 2000, The Journal of clinical investigation.

[9]  A. Zorn,et al.  Wnt signalling: Antagonistic Dickkopfs , 2001, Current Biology.

[10]  H. Varmus,et al.  Purification and molecular cloning of a secreted, Frizzled-related antagonist of Wnt action. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[11]  M. Gessler,et al.  Developmental expression patterns of mouse sFRP genes encoding members of the secreted frizzled related protein family , 1998, Mechanisms of Development.

[12]  S. Manolagas,et al.  Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. , 2000, Endocrine reviews.

[13]  S. Mundlos,et al.  Regulation of chondrocyte differentiation by Cbfa1 , 1999, Mechanisms of Development.

[14]  Tatiana Foroud,et al.  Genetics of osteoporosis. , 2002, Endocrine reviews.

[15]  D. Dempster,et al.  Isolation and culture of osteoclasts and osteoclast resorption assays , 1998 .

[16]  D. Gazit,et al.  Recombinant TGF‐β1 stimulates bone marrow osteoprogenitor cell activity and bone matrix synthesis in osteopenic, old male mice , 1999, Journal of cellular biochemistry.

[17]  B. Komm,et al.  Development and characterization of a conditionally transformed adult human osteoblastic cell line , 1996, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[18]  D. Goltzman,et al.  Discoveries, drugs and skeletal disorders , 2002, Nature Reviews Drug Discovery.

[19]  S. Teitelbaum,et al.  Bone resorption by osteoclasts. , 2000, Science.

[20]  C. Jomary,et al.  Secreted Frizzled-related proteins: searching for relationships and patterns. , 2002, BioEssays : news and reviews in molecular, cellular and developmental biology.

[21]  M. Mahadevappa,et al.  SARPs: a family of secreted apoptosis-related proteins. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[22]  R. Nusse,et al.  Mechanisms of Wnt signaling in development. , 1998, Annual review of cell and developmental biology.

[23]  B. Komm,et al.  Systemically administered rhBMP‐2 promotes MSC activity and reverses bone and cartilage loss in osteopenic mice , 2002, Journal of cellular biochemistry.

[24]  Miikka Vikkula,et al.  LDL Receptor-Related Protein 5 (LRP5) Affects Bone Accrual and Eye Development , 2001, Cell.

[25]  T. Craig,et al.  Secreted frizzled-related protein 4 is a potent tumor-derived phosphaturic agent. , 2003, The Journal of clinical investigation.

[26]  J. Lian.  Bone formation : osteoblast lineage cells, growth factors, matrix proteins and the mineralization process , 1999 .

[27]  L. Patthy,et al.  The NTR module: Domains of netrins, secreted frizzled related proteins, and type I procollagen C‐proteinase enhancer protein are homologous with tissue inhibitors of metalloproteases , 1999, Protein science : a publication of the Protein Society.

[28]  J. Nathans,et al.  A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[29]  J. A. Robinson,et al.  High Bone Mass in Mice Expressing a Mutant LRP5 Gene , 2003, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[30]  J. M. Chong,et al.  Disulfide Bond Assignments of Secreted Frizzled-related Protein-1 Provide Insights about Frizzled Homology and Netrin Modules* , 2002, The Journal of Biological Chemistry.

[31]  J. Papkoff,et al.  Activated β-catenin induces osteoblast differentiation of C3H10T1/2 cells and participates in BMP2 mediated signal transduction , 2003 .

[32]  Vicki L Church,et al.  Wnt signalling during limb development. , 2002, The International journal of developmental biology.

[33]  T. Spelsberg,et al.  Development and characterization of a conditionally immortalized human fetal osteoblastic cell line , 1995, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[34]  R. Francis,et al.  Staining of the calcification front in human bone using contrasting fluorochromes in vitro. , 1984, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[35]  R. Savani,et al.  Identification of a common hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein. , 1994, The EMBO journal.

[36]  G. Karsenty,et al.  Regulation of bone formation and vision by LRP5. , 2002, The New England journal of medicine.

[37]  Richard P Lifton,et al.  High bone density due to a mutation in LDL-receptor-related protein 5. , 2002, The New England journal of medicine.

[38]  A. Üren,et al.  Secreted Frizzled-related Protein-1 Binds Directly to Wingless and Is a Biphasic Modulator of Wnt Signaling* , 2000, The Journal of Biological Chemistry.

[39]  J. Rubin,et al.  Secreted Frizzled-related protein (sFRP-1) binds to RANKL to inhibit osteoclast formation. , 2001 .

[40]  J. Kim,et al.  Human secreted frizzled-related protein is down-regulated and induces apoptosis in human cervical cancer. , 2002, Experimental cell research.

[41]  Jens Bollerslev,et al.  Six novel missense mutations in the LDL receptor-related protein 5 (LRP5) gene in different conditions with an increased bone density. , 2003, American journal of human genetics.

[42]  R. Moon,et al.  The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape. , 2000, Trends in genetics : TIG.

[43]  A. M. Arias,et al.  Wnt signalling: a theme with nuclear variations , 2001, BioEssays : news and reviews in molecular, cellular and developmental biology.

[44]  B. Komm,et al.  Expression and regulation of Runx2/Cbfa1 and osteoblast phenotypic markers during the growth and differentiation of human osteoblasts * , 2001, Journal of cellular biochemistry.

[45]  Masato Nose,et al.  Low-density lipoprotein receptor-related protein 5 (LRP5) is essential for normal cholesterol metabolism and glucose-induced insulin secretion , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[46]  B. Komm,et al.  Tissue culture models for studies of hormone and vitamin action in bone cells. , 2002, Vitamins and hormones.