Figure 8.

ECM1 is required for the normal function of PTHrP ex vivo. A) ECM1 blocking antibody completely neutralized PTHrP-mediated chondrocyte hypertrophy, mineralization, and bone length detected by safranin O–fast green staining of metatarsals. Metatarsals were explanted from 15-d-old mouse embryos and cultured in the absence [control (ctr)] or presence of PTHrP (10⁻⁷ M), with or without anti-ECM1 (1 µg/ml) antibodies. Explants were cultured for 5 d and safranin O–fast green staining was observed by using low-power and high-power microphotography. B, C) Percent increase in total (B) and mineralization (C) length of metatarsal bones. Metatarsals were cultured as described above, total or mineralization length was determined, and percent increase was calculated [percent increase = (length at d 5 − length at d 0)/length at d 0]. D) ECM1 corrects defects in chondrocyte hypertrophy in metatarsal bones of PTHrP-null embryos. Metatarsals were explanted from 14.5-d-old WT and PTHrP^−/− mouse embryos and cultured in the absence [control (ctr)] or presence of ECM1-conditioned medium. Explants were cultured for 5 d, and safranin O staining was observed. E, F) Percent increase in total (E) and mineralization (F) length of metatarsal bones. Rel. level, relative level; siECM1, pSuper-ECM1. Values are means ± sd. *P < 0.05 vs. PBS treatment control group. ^#P < 0.05 between PTHrP alone treatment group and additional use of ECM1 antibody (B, C). ^#P < 0.05 between PTHrP knockout group and additional use of ECM1(E, F).