Smad-Runx interactions during chondrocyte maturation.
BACKGROUND
Intracellular signaling triggered by bone morphogenetic proteins (BMPs) results in activated Smad complexes that regulate transcription of BMP-responsive genes. However, the low specificity of Smad binding to regulatory sequences implies that additional tissue-specific transcription factors are also needed. Runx2 (Cbfal) is a transcription factor required for bone formation. We have examined the role of Smads and Runx2 in BMP induction of type X collagen, which is a marker of chondrocyte hypertrophy leading to endochondral bone formation.


METHODS
Pre-hypertrophic chondrocytes from the cephalic portion of the chick embryo sternum were placed in culture in the presence or absence of rhBMP-2. Cultures were transiently transfected with DNA containing the BMP-responsive type X collagen promoter upstream of the luciferase gene. The cultures were also transfected with plasmids, causing over-expression of Smads or Runx2, or both. After 24-48 hours, cell extracts were examined for levels of luciferase expression.


RESULTS
In the presence of BMP-2, chondrocytes over-expressing BMP-activated Smadl or Smad5 showed significant enhancement of luciferase production compared with that seen with BMP alone. This enhancement was not observed with over-expression of Smad2, a transforming growth factor beta (TGF-beta)-activated Smad. Overexpression of Runx2 in BMP-treated cultures increased transcriptional activity to levels similar to those seen with Smads 1 or 5. When chondrocytes were simultaneously transfected with both Runx2 and Smad 1 or 5, promoter activity was further increased, indicating that BMP-stimulated Smad activity can be augmented by increasing the levels of Runx2.


CONCLUSIONS
These results implicate the skeletal tissue transcription factor Runx2 in regulation of chondrocyte hypertrophy and suggest that maximal transcription of the type X collagen gene in pre-hypertrophic chondrocytes involves interaction of BMP-stimulated Smads with Runx2.


CONCLUSIONS
Many skeletal abnormalities are associated with impaired regulation of chondrocyte hypertrophy in growth plates. These studies demonstrate that both BMP-activated Smads and Runx2 levels can modulate chondrocyte transition to hypertrophy.
Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia 19104-6003, USA. phoebe@biochem.dental.upenn.edu
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Leboy
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Grasso-Knight
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D'Angelo
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Volk
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Lian
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Drissi
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Stein
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Adams
D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000502:physiology*; D002478:Cells, Cultured; D002642:Chick Embryo; D019902:Chondrocytes / Q000473:pathology / Q000502:physiology*; D003094:Collagen / Q000502:physiology; D050659:Core Binding Factor Alpha 1 Subunit; D050676:Core Binding Factor Alpha 2 Subunit; D050657:Core Binding Factor alpha Subunits; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D006984:Hypertrophy; D008156:Luciferases / Q000502:physiology; D009363:Neoplasm Proteins; D010750:Phosphoproteins / Q000502:physiology; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins; D015398:Signal Transduction; D051785:Smad Proteins; D051902:Smad5 Protein; D015534:Trans-Activators / Q000235:genetics / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection
D019485:Bone Morphogenetic Proteins; D050659:Core Binding Factor Alpha 1 Subunit; D050676:Core Binding Factor Alpha 2 Subunit; D050657:Core Binding Factor alpha Subunits; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D051785:Smad Proteins; D051902:Smad5 Protein; D015534:Trans-Activators; D014157:Transcription Factors; D003094:Collagen; D008156:Luciferases