Methods: Whole sterna from embryonic day 14 chicks were placed in culture in either control media or with one of the following for eight days, PTH(1-34), MAPK inhibitor PD 98059 (100µM MAPK-I) or PLC inhibition U73122 (20µM PLC-I). Sterna were measured daily, then prepared for either immunohistochemical localization of Col-II, -IX, -X collagen via confocal microscopy or RT-PCR analysis and quantification via Stratagene MX4000 real time PCR with SYBR green labeling.
Results: As previously reported, PTH increased sternal growth rates and decreased Col-X mRNA levels compared to untreated controls. COL-X deposition was blocked by PTH, and rescued by the PD98059 but not by the U73122. COL-II and IX deposition was disrupted in the PD98059 and U73122 treated samples. There was little difference in COL-X mRNA levels of PTH-treated controls (1.37), PD98059 (1.29) or U73122 samples. There is a quarterfold decrease in Col-X mRNA with U73122 (1.77), and a ten fold decrease with PD98059I (.302) when compared to No-PTH controls (2.39). There was quarterfold decrease of COL-II mRNA with U73122 (1.27), and 100-fold inhibition with PD98059 (.015) compared to PTH controls (1.86). COL-II mRNA levels were decreased tenfold by inhibition (Control- 3.03, U73122-.574, PD98059 - .312) in No-PTHcontrols. Data for Col-IX was inconclusive.
Conclusion: PTH/PTHrP regulates growth, collagen II, IX and X deposition and mRNA levels in chondrocytes. These experiments demonstrate that PPR signals are MAPK and PLC dependant. NIH/DE-446951