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Tumor Necrosis Factor-&agr; Promotes In Vitro Calcification of Vascular Cells via the cAMP Pathway

BackgroundVascular calcification is an ectopic calcification that commonly occurs in atherosclerosis. Because tumor necrosis factor-&agr; (TNF-&agr;), a pleiotropic cytokine found in atherosclerotic lesions, is also a regulator of bone formation, we investigated the role of TNF-&agr; in in vitro vascular calcification. Methods and ResultsA cloned subpopulation of bovine aortic smooth muscle cells previously shown capable of osteoblastic differentiation was treated with TNF-&agr;, and osteoblastic differentiation and mineralization were assessed. Treatment of vascular cells with TNF-&agr; for 3 days induced an osteoblast-like morphology. It also enhanced both activity and mRNA expression of alkaline phosphatase, an early marker of osteoblastic differentiation. Continuous treatment with TNF-&agr; for 10 days enhanced matrix mineralization as measured by radiolabeled calcium incorporation in the matrix. Pretreatment of cells with a protein kinase A–specific inhibitor, KT5720, attenuated cell morphology, the alkaline phosphatase activity, and mineralization induced by TNF-&agr;. Consistent with this, the intracellular cAMP level was elevated after TNF-&agr; treatment. Electrophoretic mobility shift assay demonstrated that TNF-&agr; enhanced DNA binding of osteoblast specific factor (Osf2), AP1, and CREB, transcription factors that are important for osteoblastic differentiation. ConclusionsThese results suggest that TNF-&agr; enhances in vitro vascular calcification by promoting osteoblastic differentiation of vascular cells through the cAMP pathway.