While cyclooxygenases are important in endochondral bone formation during fracture healing, mechanisms involved in prostaglandin E2 (PGE2) regulation of chondrocyte maturation are incompletely understood. The present study was undertaken to determine if PGE2 effects on chondrocyte differentiation are related to modulation of the bone morphogenetic protein (BMP) signaling pathway. In primary murine sternal chondrocytes, PGE2 differentially regulated genes involved in differentiation. PGE2 induced type II collagen and MMP-13, had minimal effects on alkaline phosphatase, and inhibited the expression of the maturational marker, type X collagen. In BMP-2-treated cultures, PGE2 blocked the induction of type X collagen. All four EP receptors were expressed in chondrocytes and tended to be inhibited by BMP-2 treatment. RCJ3.1C5.18 chondrocytes transfected with the protein kinase A (PKA) responsive reporter, CRE-luciferase, showed luciferase induction following exposure to PGE2, consistent with activation of PKA signaling and the presence of the EP2 and EP4 receptors. Both PGE2 and the PKA agonist, dibutyryl cAMP, blocked the induction of the BMP-responsive reporter, 12XSBE, by BMP-2 in RCJ3.1C5.18 chondrocytes. In contrast, PGE2 increased the ability of TGF-b to activate the TGF-b-responsive reporter, 4XSBE. Keywords: prostaglandin E2; bone morphogenetic protein; chondrocyte; type X collagen; Smad Endochondral ossification occurs during long bone development and in response to injury during the process of bone repair.1,2 During this process, mesenchymal stem cells proliferate, undergo condensation, and begin producing cartilage-specific type II collagen.1 Immature chondrocytes form a template for bone formation. A key event is the transition from a proliferating to a hypertrophic chondrocyte. Pre-hypertrophic chondrocytes exit the cell cycle and express the genes alkaline phosphatase and type X collagen (colX), and calcify the matrix.
1,3Subsequently, these cells terminally differentiate, express matrix metalloproteinase 13 (MMP-13) and vascular endothelial growth factor (VEGF) and undergo apoptosis, 3,4 leaving behind a cartilage matrix that provides a scaffold for vascular invasion and primary bone formation by osteoblasts. 1,2,5,6 The progression of chondrocytes through the process of endochondral ossification is controlled by growth factors and signaling molecules. One signaling molecule implicated in this process, particularly in bone reparative events, is PGE2. PGE2 is a major metabolite in the cyclooxygenase pathway. PGE2 is synthesized and secreted by growth plate chondrocytes and chondrocytes express all four receptor isoforms, EP1, EP2, EP3, and EP4.7-10 Addition of PGE2 to cell cultures increases expression of Sox9, type II collagen, aggrecan, and proteoglycan and induces proliferation. [7][8][9]11,12 The expression of later maturational markers seen in hypertrophic and terminally differentiated chondrocytes is delayed with continued PGE2 treatment.9 PGE2 dosedependently inhibits the expression of t...