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Keywords:

  • Col10a1;
  • hypertrophic chondrocytes;
  • cis-enhancer;
  • Runx2;
  • transgenic studies

Abstract

We have recently shown that a 150-bp Col10a1 distal promoter (−4296 to −4147 bp) is sufficient to direct hypertrophic chondrocyte-specific reporter (LacZ) expression in vivo. More recently, through detailed sequence analysis we identified two putative tandem-repeat Runx2 binding sites within the 3′-end of this 150-bp region (TGTGGG-TGTGGC, −4187 to −4176 bp). Candidate electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation, and transfection studies demonstrate that these putative Runx2 sites bind Runx2 and mediate upregulated Col10a1/reporter activity in vitro. Transgenic studies using the 5′-sequence without Runx2 sites were not able to drive the cell-specific LacZ reporter activity, suggesting the in vivo requirement of the Runx2 sites located in the 3′-end in mediating Col10a1/reporter expression. Indeed, mutating the Runx2 sites in the context of the 150-bp promoter abolishes its capacity to drive hypertrophic chondrocyte-specific reporter expression in transgenic mice. We have also generated multiple transgenic mouse lines using only the 3′-sequence containing the Runx2 sites to drive the LacZ gene. Interestingly, no hypertrophic chondrocyte-specific blue staining was observed in these transgenic mice. Together, our data support that Runx2 directly interacts with murine Col10a1 cis-enhancer. This interaction is required but not sufficient for cell-specific Col10a1 promoter activity in vivo. Additional cooperative/repressive elements within the 5′- or 3′-sequences of this 150-bp promoter are needed to work with Runx2 together to mediate cell-specific Col10a1 expression. Further delineation of these elements/factors has the potential to identify novel therapeutic targets for multiple skeletal disorders, including osteoarthritis, that show abnormal Col10a1 expression and altered chondrocyte maturation. © 2011 American Society for Bone and Mineral Research