Chen Yueping, Chen Liang, Yin Qingshui, Gao Hui, Dong Panfeng, Zhang Xiaoyun, Kang Jie
Southern Medical University, 2010 MD TongHe, Guangzhou, China.
Cell Physiol Biochem. 2013;32(3):755-65. doi: 10.1159/000354477. Epub 2013 Sep 13.
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.
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.
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.
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.
背景/目的：我们在另一项研究中报告，酒精暴露会触发肿瘤坏死因子-α（TNF-α）信号通路的激活，导致骨髓间充质干细胞（BMSCs）的多能性发生不利转变，从成骨方向转向脂肪生成方向。然而，抑制TNF-α信号通路仅对脂肪生成产生适度抑制作用。在此我们表明，除了促进TNF-α信号通路外，酒精还会抑制Wnt1/β-连环蛋白信号通路。
我们用酒精处理来自人类受试者的原代BMSCs 24天。我们使用定量实时逆转录-聚合酶链反应（RT-PCR）和蛋白质印迹分析来测量与内质网（ER）应激、脂肪生成标志物和成骨标志物相关的基因变化。我们进行茜素红染色以检测成骨情况。我们还对成骨生物标志物碱性磷酸酶、I型胶原蛋白和骨钙素进行了检测。
与对照细胞相比，用成骨诱导剂处理的BMSCs中Wnt/β-连环蛋白信号通路明显激活，表现为核β-连环蛋白水平升高，胞质β-连环蛋白水平降低，以及Wnt1蛋白水平增加。暴露于酒精的BMSCs中Wnt/β-连环蛋白信号通路的激活受到显著抑制，这表现为成骨标志物基因Osf2/Cbfa1、骨桥蛋白和骨钙素的表达下调，脂肪生成标志物PPARγ2和aP2的表达上调，以及钙化结节数量减少。相比之下，即使在有酒精存在的情况下，BIO激活Wnt/β-连环蛋白信号通路也有利于成骨。与单独干扰其中任何一个相比，BIO同时激活Wnt1和3,6'-二硫代硫代酰胺抑制TNF-α对乙醇诱导的脂肪生成谱系产生协同抑制作用。
BMSCs向成骨细胞谱系的这种显著转变表明，TNF-α和Wnt/β-连环蛋白通路的协同和相互干扰在促进成骨方面具有优越性。
