TNF-α stimulates alkaline phosphatase and mineralization through PPARγ inhibition in human osteoblasts

The aims of the present study were to determine whether prostaglandins (PGs) and PPARγ are involved in the stimulation of tissue-non-specific alkaline phosphatase (TNAP) activity and mineralization by TNF-α in human osteoblasts. We used osteoblasts differentiated from MSCs from three different donors and MG-63 osteoblast-like cells. Inhibition of prostaglandin synthesis with the cyclooxygenase (COX) inhibitor indomethacin or the specific COX-2 blocker NS-398 abolished mineralization in the absence and presence of 1 ng/ml of TNF-α, suggesting that PGs were involved. The TNAP inhibitor levamisole abolished TNF-α effects on mineralization, suggesting that PGs were involved in TNAP expression and mineralization. TNF-α stimulated expression of COX-2 and PG E synthase before that of TNAP, but expression of PG D synthase later suggesting that PGE₂ and PGF₂α but not 15d-PGJ₂ were involved in TNF-α effects. However, both PGE₂ and PGF₂α dose-dependently inhibited mineralization indicating that endogenous PG are required for mineralization but that TNF-α does not increase mineralization by increasing PG synthesis. Interestingly, TNF-α inhibited PPARγ expression and binding activity to PPRE consensus sequences independently of 15d-PGJ₂. Inhibition of PPARγ activity with GW-9662 mimicked TNF-α effects in MG-63 cells, indicating that TNF-α stimulates mineralization by inhibiting PPARγ in osteoblasts. In MSC-derived osteoblast cultures, inhibition of PPARγ dropped TNAP expression and mineralization. Treatment of MG-63 cells with conditioned media from MSC-derived osteoblasts or MSC-derived adipocytes treated or not with GW-9662 revealed that TNF-α inhibition of PPARγ in undifferentiated MSCs and/or adipocytes was responsible for the decreased expression of TNAP in osteoblasts. In conclusion, TNF-α increases TNAP expression and stimulates mineralization by inhibiting PPARγ in osteoblasts, but PPARγ in adipocytes or undifferentiated MSCs controls the secretion of a factor leading to TNAP stimulation in osteoblasts.