Abstract
Cyclooxygenase-2 (COX-2) activity is necessary for fracture healing to proceed normally. In most cell types, COX-2 is inductively expressed and acts in a coordinated pathway to produce prostaglandins, which affect many physiological processes including inflammation. In the fracture callus, however, COX-2 expression and the molecular and cellular processes affected by COX-2 activity remain poorly understood. Using LC-MS/MS and xMAP, we measured fracture callus prostaglandin and inflammatory cytokine levels. We found that inflammatory cytokines rapidly peaked after fracture before declining to normal levels by day 4 after fracture. However, callus prostaglandin levels did not peak until 4 days after fracture before returning to normal levels by day 10. We used immunohistochemistry to detected COX-2 expression in callus cells and found that COX-2 was expressed in callus chondrocytes and osteoclasts during endochondral ossification, including those osteoclasts at the callus chondro-osseous junction. Targeted deletion of the COX-2 gene (Ptgs2) in osteoclasts or in chondrocytes was found to delay fracture healing. Using cell-based experiments, we found that COX-2 expression could be induced in osteoclasts by osteopontin treatment, suggesting an integrin-dependent induction of COX-2 expression in osteoclasts. This was confirmed in vivo using mice lacking osteopontin or integrin ß3. Immunohistochemistry also showed abundant osteopontin expression at the callus chondro-osseous junction. The results indicate that COX-2 expression in osteoclasts is controlled by integrin-dependent signalling, that COX-2 expression in osteoclasts and chondrocytes is necessary for fracture healing to proceed normally, and that COX-2 expression in chondro-osseous junction osteoclasts may be induced by osteopontin-dependent signalling by chondrocytes.
