title: TNF-α stimulates alkaline phosphatase and mineralization through PPARγ inhibition in human osteoblasts.
abstract: 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.
authors.affiliation: Physiopathology of Inflammatory Bone Diseases, EA4490 University Lille North of France, Quai Masset, Bassin Napoléon BP120, 62327 Boulogne/Mer, France. philippe.lencel@univ-littoral.fr
authors.forename: P
authors.initials: P
authors.lastname: Lencel
authors.forename: S
authors.initials: S
authors.lastname: Delplace
authors.forename: P
authors.initials: P
authors.lastname: Hardouin
authors.forename: D
authors.initials: D
authors.lastname: Magne
mesh_terms: D000328:Adult; D000469:Alkaline Phosphatase / Q000378:metabolism*; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D008297:Male; D010006:Osteoblasts / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D047495:PPAR gamma / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D047495:PPAR gamma; D014409:Tumor Necrosis Factor-alpha; D000469:Alkaline Phosphatase