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 II collagen. We also studied the expression of the transcription factor Sox9, reported to enhance type II 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 II collagen mRNA levels. However, with respect to protein levels, CD-RAP decreased faster than type II collagen implying the stability is lower than type II collagen. Increased levels of Sox9 mRNA and protein were detected on day 5 and coincided with the initial increase of CD-RAP and type II collagen mRNAs. Sox9 mRNA levels declined with the progress of chondrocyte hypertrophy, followed by a concomitant decrease in CD-RAP and type II 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.