Murine and chicken chondrocytes regulate osteoclastogenesis by producing RANKL in response to BMP2.
Chondrocytes express RANKL, but their role in osteoclastogenesis is not clear. We report that hypertrophic chondrocytes induce osteoclast formation through RANKL production stimulated by BMP2 and Runx2/Smad1 and thus they may regulate resorption of calcified matrix by osteoclasts at growth plates.


BACKGROUND
Bone morphogenetic protein (BMP) signaling and Runx2 regulate chondrogenesis during bone development and fracture repair and RANKL expression by osteoblast/stromal cells. Chondrocytes express RANKL, and this expression is stimulated by vitamin D3, but it is not known if chondrocytes directly support osteoclast formation or if BMPs or Runx2 is involved in this potential regulation of osteoclastogenesis.


METHODS
The chondrocyte cell line, ATDC5, primary mouse sternal chondrocytes, and chick sternal chondrocytes were used. Cells were treated with BMP2, and expression of RANKL and chondrocyte marker genes was determined by real-time RT-PCR and Western blot. Chondrocytes and spleen-derived osteoclast precursors +/- BMP2 were co-cultured to examine the effect of chondrocyte-produced RANKL on osteoclast formation. A reporter assay was used to determine whether BMP2-induced RANKL production is through transcriptional regulation of the RANKL promoter and whether it is mediated by Runx2.


RESULTS
BMP2 significantly increased expression of RANKL mRNA and protein in all three types of chondrocytes, particularly by Col X-expressing and upper sternal chondrocytes. Chondrocytes constitutively induced osteoclast formation. This effect was increased significantly by BMP2 and prevented by RANK:Fc. BMP2 significantly increased luciferase activity of the RANKL-luc reporter, and Smad1 increased this effect. Deletion or mutation of Runx2 binding sites within the RANKL promoter or overexpression of a dominant negative Runx2 abolished BMP2- and Smad1-mediated activation of RANKL promoter activity.


CONCLUSIONS
Hypertrophic chondrocytes may regulate osteoclastogenesis at growth plates to remove calcified matrix through BMP-induced RANKL expression.
Usui
Michihiko
M
Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, New York 14642, USA.
Xing
Lianping
L
Drissi
Hicham
H
Zuscik
Michael
M
O'Keefe
Regis
R
Chen
Di
D
Boyce
Brendan F
BF
D000818:Animals; D001846:Bone Development / Q000187:drug effects / Q000502:physiology; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D045744:Cell Line, Tumor; D002645:Chickens; D002762:Cholecalciferol / Q000494:pharmacology; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D024081:Collagen Type IX / Q000096:biosynthesis / Q000235:genetics; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism*; D005109:Extracellular Matrix / Q000235:genetics / Q000378:metabolism; D050723:Fractures, Bone / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006132:Growth Plate / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D051379:Mice; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D053245:RANK Ligand / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020218:Response Elements / Q000502:physiology; D051898:Smad1 Protein / Q000235:genetics / Q000378:metabolism*; D013154:Spleen / Q000166:cytology / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D014815:Vitamins / Q000494:pharmacology
C525718:BMP2 protein, human; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D024081:Collagen Type IX; D050659:Core Binding Factor Alpha 1 Subunit; D053245:RANK Ligand; D012333:RNA, Messenger; C493716:Runx2 protein, mouse; D051898:Smad1 Protein; C497401:Smad1 protein, mouse; C505689:Tnfsf11 protein, mouse; D016212:Transforming Growth Factor beta; D014815:Vitamins; D002762:Cholecalciferol