Department of Cell Biology
Animals; Bone Morphogenetic Proteins; Cells, Cultured; Chick Embryo; Chondrocytes; Collagen; Core Binding Factor Alpha 1 Subunit; Core Binding Factor Alpha 2 Subunit; Core Binding Factor alpha Subunits; DNA-Binding Proteins; Hypertrophy; Luciferases; *Neoplasm Proteins; Phosphoproteins; Promoter Regions, Genetic; *Proto-Oncogene Proteins; *Signal Transduction; Smad Proteins; Smad5 Protein; Trans-Activators; Transcription Factors; Transcription, Genetic; Transfection
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. Clinical Relevance: 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.
J Bone Joint Surg Am. 2001;83-A Suppl 1(Pt 1):S15-22. Link to article on publisher's website
The Journal of bone and joint surgery. American volume
Leboy, Phoebe S.; Grasso-Knight, Giovi; D'Angelo, Mariana; Volk, Susan; Lian, Jane B.; Drissi, Hicham; Stein, Gary S.; and Adams, Sherrill L., "Smad-Runx interactions during chondrocyte maturation" (2001). Stein, Stein, Lian, vanWijnen Lab Publications. 64.