FIG. 10.

ADAMTS-7 inactivates the chondroinductive action of GEP. (A) ADAMTS-7 inhibits GEP-induced Col X and MMP-13 expression, assayed by real-time PCR. ATDC5 cells pretreated with BMP-2 for 5 days were cultured in the absence or presence of GEP, ADAMTS-7 (TS7)-, ADAMTS-7 point mutant (TS7PM)-, or granulin B (GRN B)-conditioned medium or various combinations as indicated for an additional 3 days, and Col X and MMP-13 expression was measured by real-time PCR. The units are arbitrary, and the normalized values were calibrated against controls, here given the value of 1. Asterisks indicate a significant difference from the control (P < 0.05). (B) ADAMTS-7 abolishes GEP-stimulated endochondral ossification. (a and b) Safranin O-fast green staining of metatarsals. Metatarsals were explanted from 15-day-old mouse embryos and cultured in the absence (CTR) or presence of GEP with or without ADAMTS7-conditioned medium. After 5 days of culture, safranin O-fast green staining was observed by using low-power (a) or high-power (b) microphotography. (c) Alizarin red S and alcian blue staining of metatarsals. The explants were fixed and processed for alizarin red S and alcian blue staining; a representative photograph is presented. (d) Percent increase in total and mineralization length of metatarsal bones. Metatarsals were cultured as described above, the total or mineralization length was determined, and the percent increase was calculated (percent increase = [length at day 5 − length at day 0]/length at day 0). Asterisks indicate a significant difference from the control (P < 0.05). (C) Proposed model for explaining the role and regulation of ADAMTS-7 in chondrocyte differentiation. ADAMTS-7 metalloproteinase, a direct target of PTHrP, negatively regulates chondrocyte differentiation by associating with GEP and inactivating chondrogenic activity of GEP growth factor. Rel.level, relative level.