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 ex amined in the mouse fracture model by immunohistochemistry and Northern blo t analysis and compared with the expression of type II collagen. We also st udied the expression of the transcription factor Sox9, reported to enhance type II collagen and CD-RAP gene expression in vitro. CD-RAP was first dete cted in immature chondrocytes on day 5. Intense signals for CD-RAP were fou nd 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 molec ular marker of cartilage formation during fracture healing. Northern blot a nalysis revealed similar changes in CD-RAP and type II collagen mRNA levels . However, with respect to protein levels, CD-RAP decreased faster than typ e II collagen implying the stability is lower than type II collagen. Increa sed levels of Sox9 mRNA and protein were detected on day 5 and coincided wi th the initial increase of CD-RAP and type II collagen mRNAs, Sox9 mRNA lev els declined with the progress of chondrocyte hypertrophy, followed by a co ncomitant decrease in CD-RAP and type II collagen mRNA levels. These change s in Sox9 expression compared with the cartilage-specific genes (CD-RAP and type II collagen) suggest that cell differentiation during fracture healin g may be controlled by specific transcriptional factors which regulate phen otypic changes of the cells.