SP3/SP1 transcription activity regulates specific expression of collagen type X in hypertrophic chondrocytes

Previously, we have shown that two non-canonical specificity protein (SP)-binding sites within the proximal promoter (nucleotide (nt) -139 to +5) of the chicken Col10a1 gene are involved in conferring tissue-specific expression of type X collagen to hypertrophic chondrocytes. In the present study, we examined the role of SP3/SP1 transcription factors in the regulation of the Col10a1 promoter. The SP3/SP1 ratio is higher in hypertrophic versus non-hypertrophic chondrocytes, due to the significant decrease in SP1 in hypertrophic cells detected by real-time PCR and Western blot analyses. Functional analyses by transfection-mediated overexpression of SP1 and SP3 suggest that SP1 inhibits the Col10a1 promoter. This effect is negated by an interaction with SP3 in hypertrophic chondrocytes. Additionally, mutation analysis showed that the 40-bp intervening sequence (nt -115 to -75) is required for expression of the Col10a1 gene. In this sequence, a binding site for Dlx5/6 transcription factors (nt -99 to -87) retards a protein specific for hypertrophic chondrocytes in electrophoretic mobility shift assay. Endogenous levels of Dlx5 are 3-fold higher in hypertrophic versus non-hypertrophic cells by real-time PCR analysis, and overexpression of Dlx5 in non-hypertrophic chondrocytes activates the proximal Col10a1 promoter 3-fold. These results indicate that the SP3/SP1 ratio and Dlx5 are important regulators of the proximal Col10a1 promoter in hypertrophic cartilage and suggest that interactions between SP3 and SP1 regulate expression of different types of collagen during chondrocyte differentiation.