Cartilage-derived retinoic acid-sensitive protein and type II collagen expression during fracture healing are potential targets for Sox9 regulation

Cartilage-derived retinoic acid-sensitive protein (CD-RAP) and mRNA were examined in the mouse fracture model by immunohistochemistry and Northern blot analysis and compared with the expression of type H collagen. We also studied the expression of the transcription factor Sox9, reported to enhance type H collagen and CD-RAP gene expression in vitro. CD-RAP was first detected in immature chondrocytes on day 5. Intense signals for CD-RAP were found in fracture cartilage on days 7 and 9. CD-RAP decreased at the phase of endochondral ossification. Throughout fracture healing, CD-RAP was detected in cartilage and not in bone or fibrous tissue, thus CD-RAP may be a molecular marker of cartilage formation during fracture healing. Northern blot analysis revealed similar changes in CD-RAP and type H collagen mRNA levels. However, with respect to protein levels, CD-RAP decreased faster than type H collagen implying the stability is lower than type H collagen. Increased levels of Sox9 mRNA and protein were detected on day 5 and coincided with the initial increase of CD-RAP and type H collagen mRNAs. Sox9 mRNA levels declined with the progress of chondrocyte hypertrophy, followed by a concomitant decrease in CD-RAP and type H collagen mRNA levels. These changes in Sox9 expression compared with the cartilage-specific genes (CD-RAP and type II collagen) suggest that cell differentiation during fracture healing may be controlled by specific transcriptional factors which regulate phenotypic changes of the cells.