Runx1/AML1/Cbfa2 mediates onset of mesenchymal cell differentiation toward chondrogenesis.
Runx proteins mediate skeletal development. We studied the regulation of Runx1 during chondrocyte differentiation by real-time RT-PCR and its function during chondrogenesis using overexpression and RNA interference. Runx1 induces mesenchymal stem cell commitment to the early stages of chondrogenesis.


BACKGROUND
Runx1 and Runx2 are co-expressed in limb bud cell condensations that undergo both cartilage and bone differentiation during murine development. However, the cooperative and/or compensatory effects these factors exert on skeletal formation have yet to be elucidated.


METHODS
Runx1/Cbfa2 and Runx2/Cbfa1 were examined at different stages of embryonic development by immunohistochemistry. In vitro studies used mouse embryonic limb bud cells and assessed Runx expressions by immunohistochemistry and real-time RT-PCR in the presence and absence of TGFbeta and BMP2. Runx1 was overexpressed in mesenchymal cell progenitors using retroviral infection.


RESULTS
Immunohistochemistry showed that Runx1 and Runx2 are co-expressed in undifferentiated mesenchyme, had similar levels in chondrocytes undergoing transition from proliferation to hypertrophy, and that there was primarily Runx2 expression in hypertrophic chondrocytes. Overall, the expression of Runx1 remained significantly higher than Runx2 mRNA levels during early limb bud cell maturation. Treatment of limb bud micromass cultures with BMP2 resulted in early induction of both Runx1 and Runx2. However, upregulation of Runx2 by BMP2 was sustained, whereas Runx1 decreased in later time-points when type X collagen was induced. Although TGFbeta potently inhibits Runx2 and type X collagen, it induces type II collagen mRNA and mildly but significantly inhibits Runx1 isoforms in the early stages of chondrogenesis. Virus-mediated overexpression of Runx1 in mouse embryonic mesenchymal cells resulted in a potent induction of the early chondrocyte differentiation markers but not the hypertrophy marker, type X collagen. Knockdown or Runx1 potently inhibits type II collagen, alkaline phosphatase, and Runx2 and has a late inhibitory effect on type X collagen.


CONCLUSIONS
These findings show a distinct and sustained role for Runx proteins in chondrogenesis and subsequent chondrocyte maturation. Runx1 is highly expressed during chondrogenesis in comparison with Runx2, and Runx1 gain of functions stimulated this process. Thus, the Runx genes are uniquely expressed and have distinct roles during skeletal development.
Wang
YongJun
Y
Center for Musculoskeletal Research, Department of Orthopeadics, University of Rochester, Rochester, New York 14642, USA.
Belflower
Ruth M
RM
Dong
Yu-Feng
YF
Schwarz
Edward M
EM
O'Keefe
Regis J
RJ
Drissi
Hicham
H
D000423:Alcian Blue / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D001842:Bone and Bones; D002356:Cartilage / Q000166:cytology / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D019902:Chondrocytes / Q000378:metabolism*; D024043:Collagen Type II / Q000378:metabolism; D024121:Collagen Type X / Q000378:metabolism; D004396:Coloring Agents / Q000494:pharmacology; D050676:Core Binding Factor Alpha 2 Subunit / Q000502:physiology*; D017931:DNA Primers / Q000737:chemistry; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D007763:Lac Operon; D018878:Limb Buds / Q000378:metabolism; D008648:Mesoderm / Q000378:metabolism*; D051379:Mice; D008809:Mice, Inbred C3H; D008813:Mice, Inbred ICR; D010641:Phenotype; D020033:Protein Isoforms; D012313:RNA / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000378:metabolism; D015854:Up-Regulation
C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D024043:Collagen Type II; D024121:Collagen Type X; D004396:Coloring Agents; D050676:Core Binding Factor Alpha 2 Subunit; D017931:DNA Primers; D020033:Protein Isoforms; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016212:Transforming Growth Factor beta; D012313:RNA; D000423:Alcian Blue