Tumor necrosis factor-alpha promotes in vitro calcification of vascular cells via the cAMP pathway.
BACKGROUND
Vascular calcification is an ectopic calcification that commonly occurs in atherosclerosis. Because tumor necrosis factor-alpha (TNF-alpha), a pleiotropic cytokine found in atherosclerotic lesions, is also a regulator of bone formation, we investigated the role of TNF-alpha in in vitro vascular calcification.


RESULTS
A cloned subpopulation of bovine aortic smooth muscle cells previously shown capable of osteoblastic differentiation was treated with TNF-alpha, and osteoblastic differentiation and mineralization were assessed. Treatment of vascular cells with TNF-alpha 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-alpha 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-alpha. Consistent with this, the intracellular cAMP level was elevated after TNF-alpha treatment. Electrophoretic mobility shift assay demonstrated that TNF-alpha 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-alpha enhances in vitro vascular calcification by promoting osteoblastic differentiation of vascular cells through the cAMP pathway.
Tintut
Y
Y
Division of Cardiology, Departments of Medicine and Physiology, UCLA School of Medicine, Los Angeles, CA 90095-1679, USA. ytintut@ucla.edu
Patel
J
J
Parhami
F
F
Demer
L L
LL
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000943:Antigens, Differentiation / Q000378:metabolism; D001857:Bone Matrix / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D002114:Calcinosis / Q000139:chemically induced* / Q000473:pathology; D002417:Cattle; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit; D000242:Cyclic AMP / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D009363:Neoplasm Proteins; D010006:Osteoblasts / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D012333:RNA, Messenger / Q000378:metabolism; D015290:Second Messenger Systems / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology
D000943:Antigens, Differentiation; D015815:Cell Adhesion Molecules; D050659:Core Binding Factor Alpha 1 Subunit; D017362:Cyclic AMP Response Element-Binding Protein; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D000242:Cyclic AMP; D000469:Alkaline Phosphatase