Tumor Necrosis Factor-α Promotes In Vitro Calcification of Vascular Cells via the cAMP Pathway

• DOI: 10.1161/01.cir.102.21.2636
• Publication Date: 2012-06-12T00:42:29Z
• Authors: Yin Tintut; Jignesh Patel; Farhad Parhami; Linda L. Demer
• Keywords: Intracellular Fluid; Osteoblasts; Bone Matrix; Calcinosis; Cell Differentiation; Core Binding Factor Alpha 1 Subunit; Alkaline Phosphatase; Antigens, Differentiation; Second Messenger Systems; Muscle, Smooth, Vascular; Neoplasm Proteins; Transcription Factor AP-1; 03 medical and health sciences; 0302 clinical medicine; Cyclic AMP; Animals; Cattle; RNA, Messenger; Cyclic AMP Response Element-Binding Protein; Cell Adhesion Molecules; Cells, Cultured; Signal Transduction

Background —Vascular calcification is an ectopic calcification that commonly occurs in atherosclerosis. Because tumor necrosis factor-α (TNF-α), a pleiotropic cytokine found in atherosclerotic lesions, is also a regulator of bone formation, we investigated the role of TNF-α in in vitro vascular calcification.

Methods and Results —A cloned subpopulation of bovine aortic smooth muscle cells previously shown capable of osteoblastic differentiation was treated with TNF-α, and osteoblastic differentiation and mineralization were assessed. Treatment of vascular cells with TNF-α 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-α 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-α. Consistent with this, the intracellular cAMP level was elevated after TNF-α treatment. Electrophoretic mobility shift assay demonstrated that TNF-α enhanced DNA binding of osteoblast specific factor (Osf2), AP1, and CREB, transcription factors that are important for osteoblastic differentiation.

Conclusions —These results suggest that TNF-α enhances in vitro vascular calcification by promoting osteoblastic differentiation of vascular cells through the cAMP pathway.