TGF-b Prevents Phosphate-Induced Osteogenesis through Inhibition of BMP and Wnt / b-Catenin Pathways

Background: Transforming growth factor-b (TGF-b) is a key cytokine during differentiation of mesenchymal stem cells (MSC) into vascular smooth muscle cells (VSMC). High phosphate induces a phenotypic transformation of vascular smooth muscle cells (VSMC) into osteogenic-like cells. This study was aimed to evaluate signaling pathways involved during VSMC differentiation of MSC in presence or not of high phosphate. Results: Our results showed that TGF-b induced nuclear translocation of Smad3 as well as the expression of vascular smooth muscle markers, such as smooth muscle alpha actin, SM22a, myocardin, and smooth muscle-myosin heavy chain. The addition of high phosphate to MSC promoted nuclear translocation of Smad1/5/8 and the activation of canonical Wnt/bcatenin in addition to an increase in BMP-2 expression, calcium deposition and alkaline phosphatase activity. The administration of TGF-b to MSC treated with high phosphate abolished all these effects by inhibiting canonical Wnt, BMP and TGF-b pathways. A similar outcome was observed in high phosphate-treated cells after the inhibition of canonical Wnt signaling with Dkk-1. Conversely, addition of both Wnt/b-catenin activators CHIR98014 and lithium chloride enhanced the effect of high phosphate on BMP-2, calcium deposition and alkaline phosphatase activity. Conclusions: Full VSMC differentiation induced by TGF-b may not be achieved when extracellular phosphate levels are high. Moreover, TGF-b prevents high phosphate-induced osteogenesis by decreasing the nuclear translocation of Smad 1/5/8 and avoiding the activation of Wnt/b-catenin pathway. Citation: Guerrero F, Herencia C, Almadén Y, Martı́nez-Moreno JM, Montes de Oca A, et al. (2014) TGF-b Prevents Phosphate-Induced Osteogenesis through Inhibition of BMP and Wnt/b-Catenin Pathways. PLoS ONE 9(2): e89179. doi:10.1371/journal.pone. 0089179 Editor: Christos Chadjichristos, National Institute of Health and Medical Research, France Received September 24, 2013; Accepted January 16, 2014; Published February 27, 2014 Copyright: 2014 Guerrero et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Sources of Funding: This study has been carried out with the funding of ‘‘Proyecto de Excelencia P09-CTS-5205’’ from Consejerı́a de Economı́a, Ciencia y Empresa from Junta de Andalucı́a, FIS PI11/02055 from Instituto de Salud Carlos III and PI-0127 from Consejerı́a de Salud, Junta de Andalucia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have read the journal’s policy and have the following conflicts: WGR and MF are an employees and stockholder of Amgen, Inc". This does not alter the authors’ adherence to all the Plos One policies on sharing data and materials. * E-mail: juanr.munoz. exts@juntadeandalucia.es . These authors contributed equally to this work.