Smad3 plays an inhibitory role in phosphate-induced vascular smooth muscle cell calcification

Exp Mol Pathol. 2014 Dec;97(3):458-64. doi: 10.1016/j.yexmp.2014.10.005. Epub 2014 Oct 7.


Arterial medial calcification is a major complication in patients with chronic kidney disease and diabetes. It has been hypothesized that a high concentration of inorganic phosphate (Pi) induces calcification in vascular smooth muscle cells (vSMCs). However, the role of transforming growth factor-β (TGF-β)/Smad3 signaling in Pi-induced vascular calcification remains controversial. The aim of this study was to investigate the possible involvement of Smad3 in Pi-induced vascular calcification. We compared the degree of Pi-induced vSMC calcification between vSMCs isolated from wild-type (Smad3(+/+)) and Smad3-deficient (Smad3(-/-)) mice. We found that vSMCs from Smad3(+/+) mice had less calcium (Ca) than those from Smad3(-/-) mice when they were exposed to high concentrations of Pi and Ca (Pi+Ca). The phosphorylation of Smad3 was induced in Smad3(+/+) vSMCs by exposure to Pi+Ca. The concentration of extracellular pyrophosphate (ePPi) was lower in Smad3(-/-) vSMCs than in Smad3(+/+) vSMCs and was significantly increased in Smad3(+/+) vSMCs by treatment with TGF-β1. Also, the addition of a small amount of PPi to culture medium significantly decreased the deposition of Ca in both Smad3(+/+) and Smad3(-/-) vSMCs. Ectonucleotide phosphatase/phosphodiesterase1 (Enpp1) was decreased at the mRNA, protein, and enzymatic activity levels in Smad3(-/-) vSMCs compared with Smad3(+/+) vSMCs. A ChIP assay showed that phosphorylated Smad3 directly binds to the Enpp1 gene. Furthermore, the calcification of aortic segments was attenuated by treatment with TGF-β1 only in Smad3(+/+) mice. Taken together, we conclude that Pi-induced vSMC calcification is suppressed by Smad3 via an increase in ePPi.

Keywords: Enpp1; Pyrophosphate; Smad3; TGF-β; Vascular calcification.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Western
  • Chromatin Immunoprecipitation
  • Diphosphates / metabolism
  • Male
  • Mice
  • Mice, Knockout
  • Muscle, Smooth, Vascular / metabolism
  • Muscle, Smooth, Vascular / pathology*
  • Phosphoric Diester Hydrolases / metabolism
  • Pyrophosphatases / metabolism
  • Real-Time Polymerase Chain Reaction
  • Signal Transduction / physiology*
  • Smad3 Protein / metabolism*
  • Transforming Growth Factor beta1 / metabolism
  • Vascular Calcification / metabolism*
  • Vascular Calcification / pathology


  • Diphosphates
  • Smad3 Protein
  • Smad3 protein, mouse
  • Transforming Growth Factor beta1
  • diphosphoric acid
  • Phosphoric Diester Hydrolases
  • ectonucleotide pyrophosphatase phosphodiesterase 1
  • Pyrophosphatases