title: Smad3 plays an inhibitory role in phosphate-induced vascular smooth muscle cell calcification.
abstract: 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.
authors.affiliation: First Department of Pathology, Wakayama Medical University School of Medicine, 811-1 Kimiidera, Wakayama 641-0012, Japan; Department of Cardiovascular Medicine, Wakayama Medical University School of Medicine, 811-1 Kimiidera, Wakayama 641-0012, Japan.
authors.forename: Aiko
authors.initials: A
authors.lastname: Shimokado
authors.affiliation: First Department of Pathology, Wakayama Medical University School of Medicine, 811-1 Kimiidera, Wakayama 641-0012, Japan.
authors.forename: Yujing
authors.initials: Y
authors.lastname: Sun
authors.affiliation: First Department of Pathology, Wakayama Medical University School of Medicine, 811-1 Kimiidera, Wakayama 641-0012, Japan.
authors.forename: Masako
authors.initials: M
authors.lastname: Nakanishi
authors.affiliation: First Department of Pathology, Wakayama Medical University School of Medicine, 811-1 Kimiidera, Wakayama 641-0012, Japan.
authors.forename: Fuyuki
authors.initials: F
authors.lastname: Sato
authors.affiliation: First Department of Pathology, Wakayama Medical University School of Medicine, 811-1 Kimiidera, Wakayama 641-0012, Japan.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Oikawa
authors.affiliation: Department of Cardiovascular Medicine, Wakayama Medical University School of Medicine, 811-1 Kimiidera, Wakayama 641-0012, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Akasaka
authors.affiliation: First Department of Pathology, Wakayama Medical University School of Medicine, 811-1 Kimiidera, Wakayama 641-0012, Japan. Electronic address: ymuragak@wakayama-med.ac.jp.
authors.forename: Yasuteru
authors.initials: Y
authors.lastname: Muragaki
mesh_terms: D000818:Animals; D015153:Blotting, Western; D047369:Chromatin Immunoprecipitation; D011756:Diphosphates / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000378:metabolism / Q000473:pathology*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011755:Pyrophosphatases / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D051900:Smad3 Protein / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism; D061205:Vascular Calcification / Q000378:metabolism* / Q000473:pathology
chemical_list: D011756:Diphosphates; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D053773:Transforming Growth Factor beta1; C107241:diphosphoric acid; D010727:Phosphoric Diester Hydrolases; C065962:ectonucleotide pyrophosphatase phosphodiesterase 1; D011755:Pyrophosphatases
keywords: Enpp1; Pyrophosphate; Smad3; TGF-β; Vascular calcification