Tumor necrosis factor-α increases alkaline phosphatase expression in vascular smooth muscle cells via MSX2 induction

Hye-Lim Leea, Kyung Mi Wooa, Hyun-Mo Ryooa and Jeong-Hwa Baek, a,

a Department of Cell and Developmental Biology, School of Dentistry and Dental Research Institute, Seoul National University, Seoul, Republic of Korea



Abstract
Vascular calcification is implicated in many diseases including atherosclerosis and diabetes. Tumor necrosis factor-α (TNF-α) has been shown to promote vascular calcification both in vitro and in vivo. However, the molecular mechanism of TNF-α-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-α-induced vascular calcification and to define the regulatory mechanism of MSX2 induction in human vascular smooth muscle cells (VSMCs).

TNF-α 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-α 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-α-induced ALP expression.

New protein synthesis was dispensable for MSX2 induction by TNF-α, and the inhibition of NF-κB by BAY-11-7082 or by dominant negative IκBα abolished the TNF-α-directed induction of MSX2 expression. However, inhibition of NADPH oxidase did not affect MSX2 expression.

In conclusion, our study suggests that TNF-α directly induces MSX2 expression through the NF-κB pathway, which in turn induces expression of ALP, a key molecule in mineralization, in VSMCs