LRP4 is a novel osteoblast and osteocyte expressed specific facilitator of SOST-mediated inhibition of in vitro bone formation
Osteoblasts - Matrix Proteins and Function
Oral Presentations, Presentation Number: 1250
Session: Concurrent Oral Session 42: Osteocytes II: Wnt Pathway
Monday, September 14, 2009 5:45 PM - 6:00 PM, Colorado Convention Center, Room 205-207
* , SWITZERLAND, , SWITZERLAND, , SWITZERLAND, Shou-Ih Hu, Novartis Institutes for Biomedical Research, Chris Lu, Novartis Institutes for Biomedical Research, Andreas Bauer, Novartis Institutes for Biomedical Research, , AF
Sclerostin is a potent osteocyte secreted inhibitor of bone formation. Humans (Sclerosteosis and van Buchem disease) and mice lacking sclerostin develop a high bone mass phenotype due to increased bone formation, while sclerostin specific antibodies are bone anabolic. Sclerostin is thought to function predominantly as an inhibitor of canonical Wnt signaling by binding to the Wnt co-receptor low density lipoprotein-related protein (LRP) 5 / 6, though it had been originally demonstrated to bind to bone morphogenetic proteins (BMPs) inhibiting thereby BMP signaling. We performed a series of tandem affinity purification screens using HEK293 and UMR106 cells expressing TAP-tagged sclerostin as an unbiased approach to detect its interaction partners. The proteomics experiments identified LRP4 along with glypicans as novel sclerostin interaction partners, while confirming interaction with LRP6 and with lower confidence with LRP5. BMPs were not identified under any tested condition. We focused our work on LRP4 whose function is to date not fully known, since in vitro overexpression and RNAi mediated knockdown experiments demonstrated that LRP4 facilitates sclerostin inhibitory action on Wnt signaling reporter readout. Sclerostin-mediated inhibition of Wnt induced signaling was increased in presence of LRP4 in HEK293 and UMR106 cells, whereas the activity of the Wnt inhibitor Dkk1 was not modified suggesting specificity for sclerostin. Consistent with this observation, knockdown of LRP4 in HEK293 diminished sclerostin but not Dkk1 mediated suppression of Wnt signaling. LRP4 truncation mutation studies proved that the extracellular β-propeller structured domain of LRP4 is required for the sclerostin-facilitator activity. We found LRP4 RNA to be expressed in murine / human bone and to be up-regulated during osteoblastic differentiation (MC3T3, hMSCs). Immunohistochemistry confirmed that LRP4 protein is present in osteoblasts and -cytes both target cells of sclerostin action. Moreover, silencing of LRP4 by lentiviral-mediated shRNA delivery increased osteoblastic differentiation in MC3T3 cells (osteoblastic marker gene expression, ALP activity, calcium content). Finally silencing of LRP4 completely blocked sclerostin-inhibitory action on in vitro bone mineralization. Based on these findings, we hypothesize that the interaction of sclerostin with LRP4 is required to mediate sclerostin inhibitory function on bone formation, thus suggesting a novel role for LRP4 in bone.
C. Lu, Novartis: Employment (full or part-time). F. Morvan, Novartis: Employment (full or part-time). A. Bauer, Novartis: Employment (full or part-time). C. Halleux, Novartis: Employment (full or part-time). M. Kneissel, Novartis: Employment (full or part-time). S. Hu, Novartis: Employment (full or part-time). O. Leupin, Novartis: Employment (full or part-time).
* Presenting Authors(s):