Vascular calcification is implicated in many diseases including atherosclerosis and diabetes. Tumor necrosis factor-alpha (TNF-alpha) has been shown to promote vascular calcification both in vitro and in vivo. However, the molecular mechanism of TNF-alpha-mediated vascular calcification has not yet been fully defined. Therefore, in this study, we aimed to investigate whether MSX2 acts as a crucial regulator in TNF-alpha-induced vascular calcification and to define the regulatory mechanism of MSX2 induction in human vascular smooth muscle cells (VSMCs). TNF-alpha increased the expression of osteogenic marker genes including RUNX2, osterix, alkaline phosphatase (ALP), and bone sialoprotein, and it also promoted matrix mineralization in VSMCs. In addition, TNF-alpha enhanced MSX2 expression in a dose- and time-dependent manner. MSX2 over-expression alone induced ALP expression, whereas knockdown of MSX2 with small interfering RNA completely blocked TNF-alpha-induced ALP expression. New protein synthesis was dispensable for MSX2 induction by TNF-alpha, and the inhibition of NF-kappa B by BAY-11-7082 or by dominant negative I kappa B alpha abolished the TNF-alpha-directed induction of MSX2 expression. However, inhibition of NADPH oxidase did not affect MSX2 expression. In conclusion, our study suggests that TNF-a directly induces MSX2 expression through the NF-kappa B pathway, which in turn induces expression of ALP, a key molecule in mineralization, in VSMCs. (C) 2009 Elsevier Inc. All rights reserved.