The chondrogenic transcription factor Sox9 is a target of signaling by theparathyroid hormone-related peptide in the growth plate of endochondral bones

In the growth plate of endochondral bones, parathyroid hormone (PTH)-relate d peptide (PTHrP) regulates the rate of chondrocyte maturation from prehype rtrophic chondrocytes to hypertrophic chondrocytes. Using an antibody speci fic for Sox9 phosphorylated at serine 181 (S-181), one of the two consensus protein kinase A phosphorylation sites of Sox9, we showed that the additio n of PTHrP strongly increased the phosphorylation of SOX9 in COS7 cells tra nsfected with both SOX9- and PTH/PTHrP receptor-expressing vectors. PTHrP a lso increased the SOX9-dependent activity of chondrocyte-specific enhancers in the gene for type II collagen (Col2a1) in transient transfection experi ments. This increased enhancer activity did not occur with a Sox9 mutant ha rboring serine-to-alanine substitutions in its two consensus protein kinase A phosphorylation sites. Consistent with these results, PTHrP also increas ed Col2a1 mRNA levels in rat chondrosarcoma cells as well as 10T1/2 mesench ymal cells transfected with a PTH/PTHrP receptor expressing plasmid. No pho sphorylation of Sox9 at S-181 was detected in prehypertrophic chondrocytes of the growth plate or any chondrocytes of PTH/PTHrP receptor null mutants. In contrast in wild-type mouse embryos, previous immunohistochemistry expe riments indicated that Sox9 phosphorylated at S-181 was detected almost exc lusively in chondrocytes of the prehypertrophic zone. Sox9, regardless of t he phosphorylation state, was present in all chondrocytes of both genotypes except hypertrophic chondrocytes. Our results indicated that Sox9 is a tar get of PTHrP signaling in prehypertrophic chondrocytes in the growth plate. We hypothesize that Sox9 mediates at least some effects of PTHrP in the gr owth plate and that the PTHrP-dependent increased transcriptional activity of Sox9 helps maintain the chondrocyte phenotype of cells in the prehypertr ophic zone and inhibits their maturation to hypertrophic chondrocytes.