BMP-2 controls alkaline phosphatase expression and osteoblast mineralization by a Wnt autocrine loop.
Wnt/beta-catenin signaling has recently been suggested to be involved in bone biology. The precise role of this cascade in osteoblast differentiation was examined. We show that a Wnt autocrine loop mediates the induction of alkaline phosphatase and mineralization by BMP-2 in pre-osteoblastic cells.


BACKGROUND
Loss of function of LRP5 leads to osteoporosis (OPPG syndrome), and a specific point mutation in this same receptor results in high bone mass (HBM). Because LRP5 acts as a coreceptor for Wnt proteins, these findings suggest a crucial role for Wnt signaling in bone biology.


METHODS
We have investigated the involvement of the Wnt/LRP5 cascade in osteoblast function by using the pluripotent mesenchymal cell lines C3H10T1/2, C2C12, and ST2 and the osteoblast cell line MC3T3-E1. Transfection experiments were carried out with a number of elements of the Wnt/LRP5 pathway. Measuring osteoblast and adipocyte differentiation markers addressed the effect of this cascade on osteoblast differentiation.


RESULTS
In mesenchymal cells, only Wnt's capable of stabilizing beta-catenin induced the expression of alkaline phosphatase (ALP). Wnt3a-mediated ALP induction was inhibited by overexpression of either Xddl, dickkopf 1 (dkk1), or LRP5deltaC, indicating that canonical beta-catenin signaling is responsible for this activity. The use of Noggin, a bone morphogenic protein (BMP) inhibitor, or cyclopamine, a Hedgehog inhibitor, revealed that the induction of ALP by Wnt is independent of these morphogenetic proteins and does not require de novo protein synthesis. In contrast, blocking Wnt/LRP5 signaling or protein synthesis inhibited the ability of both BMP-2 and Shh to induce ALP in mesenchymal cells. Moreover, BMP-2 enhanced Wntl and Wnt3a expression in our cells. In MC3T3-E1 cells, where endogenous ALP levels are maximal, antagonizing the Wnt/LRP5 pathway led to a decrease of ALP activity. In addition, overexpression of dkkl reduced extracellular matrix mineralization in a BMP-2-dependent assay.


CONCLUSIONS
Our data strongly suggest that the capacity of BMP-2 and Shh to induce ALP relies on Wnt expression and the Wnt/LRP5 signaling cascade. Moreover the effects of BMP-2 on extracellular matrix mineralization by osteoblasts are mediated, at least in part, by the induction of a Wnt autocrine/paracrine loop. These results may help to explain the phenotype of OPPG patients and HBM.
Proskelia Pharmaceuticals, Romainville, France. georges.rawadi@proskelia.com
Georges
G
Rawadi
Béatrice
B
Vayssière
Fred
F
Dunn
Roland
R
Baron
Sergio
S
Roman-Roman
D017667:Adipocytes / Q000166:cytology; D000469:Alkaline Phosphatase / Q000096:biosynthesis* / Q000378:metabolism; D000818:Animals; D001391:Azo Compounds / Q000494:pharmacology; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000378:metabolism*; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D002478:Cells, Cultured; D004396:Coloring Agents / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D053823:Hedgehog Proteins; D026502:LDL-Receptor Related Proteins; D060492:Low Density Lipoprotein Receptor-Related Protein-5; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008809:Mice, Inbred C3H; D008954:Models, Biological; D010006:Osteoblasts / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D010641:Phenotype; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D011973:Receptors, LDL / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D014162:Transfection; D016212:Transforming Growth Factor beta; D051153:Wnt Proteins; D029961:Zebrafish Proteins; D051176:beta Catenin
D001391:Azo Compounds; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; C495267:CTNNB1 protein, mouse; D004396:Coloring Agents; D003598:Cytoskeletal Proteins; D053823:Hedgehog Proteins; D026502:LDL-Receptor Related Proteins; D060492:Low Density Lipoprotein Receptor-Related Protein-5; C555483:Lrp5 protein, mouse; D011500:Protein Synthesis Inhibitors; D011518:Proto-Oncogene Proteins; D011973:Receptors, LDL; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D051153:Wnt Proteins; D029961:Zebrafish Proteins; D051176:beta Catenin; D012313:RNA; D003513:Cycloheximide; D008156:Luciferases; D000469:Alkaline Phosphatase; C011049:oil red O