Reciprocal interferences of TNF-α and Wnt1/β-catenin signaling axes shift bone marrow-derived stem cells towards osteoblast lineage after ethanol exposure

Abstract

Background/Aims: We have reported in a separate study that alcohol exposure triggers activation of the TNF-α signaling pathway leading to an adverse shift of multipotential mesenchymal stem cells in bone marrow (BMSCs) away from osteogenesis towards adipogenesis. However, inhibition of TNF-α signaling only yielded moderate inhibition of adipogenesis. Here we showed that in addition to promoting the TNF-α signaling, alcohol also suppressed the Wnt1/β-catenin signaling pathway. Methods: We treated primary BMSCs from human subjects with alcohol for 24 days. We measured changes of genes related to endoplasmic reticulum (ER) stress, adipogenic markers and osteogenic markers using quantitative real-time RT-PCR and Western blot analysis. We performed Alizarin red staining for osteogenesis. We also conducted assays for osteogenic biomarkers alkaline phosphatase, collagen-I and osteocalcin. Results: Wnt/β-catenin signaling was markedly activated in BMSCs treated with osteogenic inducers relative to the control cells, as indicated by the increased levels of nuclear β-catenin along with reduced levels of cytosolic β-catenin, as well as increased protein levels of Wnt1. Activation of Wnt/β-catenin signaling was significantly suppressed in BMSCs exposed to alcohol, which was reflected by downregulated expression of osteogenic marker genes Osf2/Cbfa1, osteopontin and osteocalcin, upregulated adipogenic marker PPARγ2 and aP2, and reduced number of calcifcation nodules. In contrast, activation of Wnt/β-catenin signaling by BIO favored osteogenesis even in the presence of alcohol. Simultaneous activation of Wnt1 by BIO and inhibition of TNF-α by 3,6'-dithiothalidomide produced synergetic suppression of ethanol-induced adipogenic lineage compared to interference with either of them alone. Conclusion: This remarkable shift of BMSCs towards osteoblast lineage suggests the superiority of concordant and reciprocal interferences of the TNF-α and Wnt/β-catenin pathways for promoting osteogenesis. © 2013 S. Karger AG, Basel.