Background: Chondrocyte maturation and hypertrophy during endochondral bone formation are stimulated by both retinoids and bone morphogenetic proteins (BMPs). The type-X collagen gene, which is expressed only in hypertrophic chondrocytes, provides an excellent marker for chondrocyte maturation. We previously identified a 651-base-pair region of the type-X collagen promoter that is essential for its activation by BMP. We examined the relationship between the retinoid and BMP signaling pathways in transcriptional stimulation of the type-X collagen gene to determine whether they act independently or interact to regulate endochondral bone formation.
Methods: Prehypertrophic chondrocytes from embryonic chick sterna cultured in the presence or absence of retinoic acid or BMP-2 were transiently transfected with plasmids containing various mutations of the type-X collagen promoter directing expression of a luciferase reporter gene. In addition, real-time polymerase chain reaction was used to examine the effects of retinoic acid on expression of genes encoding BMP-2, 4, and 6.
Results: The previously identified BMP-responsive region of the type-X collagen promoter also mediated stimulation by physiological concentrations of retinoic acid in prehypertrophic chondrocytes. Systematic deletion mutagenesis of the BMP/retinoid-responsive region of the type-X collagen promoter identified distinct regions that are responsible for promoter stimulation by retinoids and BMP. Retinoic acid rapidly and dramatically stimulated accumulation of BMP-2 and BMP-6 messenger RNAs.
Conclusions: These results suggest that, while retinoic acid appears to stimulate type-X collagen gene transcription in part by stimulating the BMP signaling pathway, it also acts in part through mechanisms that are independent of BMP.
Clinical Relevance: Retinoids are essential for chondrocyte maturation during endochondral bone formation, but at high concentrations vitamin A is a potent teratogen; thus, both excessive and deficient vitamin A cause skeletal abnormalities by mechanisms that are not well understood. Since synthetic derivatives of vitamin A are widely used therapeutic agents for the treatment of several types of diseases, it is important to understand their effects on endochondral bone formation.