Department of Molecular Genetics, School of Dentistry, Dental Research Institute, BK21 Program, Seoul National University, Seoul, Korea.
J Cell Physiol. 2012 Jun;227(6):2287-96. doi: 10.1002/jcp.24038.
Matrix extracellular phosphoglycoprotein (MEPE) is a specific marker of mineralizing osteoblasts and osteocytes. Canonical BMP and Wnt signaling pathways are two of the strongest paracrine signals stimulating osteogenesis. Our previous results indicated that Mepe expression is stimulated by the BMP-2-signaling pathway. The specific aim of this study addressed whether Mepe expression is also controlled by Wnt signaling, and whether there is a cross-regulation between two major osteogenic signaling pathways. Treatment with Wnt3a, a canonical Wnt signaling stimulator, strongly enhanced Mepe mRNA expression. Knock-down of β-catenin with siRNA completely reversed Wnt3a-stimulated Mepe expression. The Mepe mRNA expression level was increased by overexpression of β-catenin and Lef-1, even in the absence of Wnt3a. Highly conserved Lef-1 response elements were identified in the mouse Mepe promoter. The direct binding of Lef-1 to these elements is critical for Mepe expression, indicating that Mepe is a direct target of canonical Wnt signaling. Meanwhile, we also found that Wnt3a treatment strongly stimulated Bmp-2 expression, and that the subsequent increase in Bmp-2 protein was determined in Wnt3a-treated conditioned medium (CM). Treatment of MC3T3-E1 cells with CM stimulated phosphorylation of the Smad1/5 proteins and their downstream Dlx5 mRNA expression. The CM-mediated increases of phospho-Smad and Dlx5 expression were not blocked completely by a Wnt3a antagonist, Dkk-1, but were almost completely suppressed by the addition of a Bmp-2 antagonist, Noggin. Collectively, Wnt3a stimulates Mepe transcription directly by a canonical Wnt signaling pathway through β-catenin and Lef-1 and indirectly through the activation of a Bmp-2 autocrine loop.
基质细胞外磷蛋白（MEPE）是成骨细胞和骨细胞矿化的特异性标志物。经典的 BMP 和 Wnt 信号通路是刺激成骨作用的两种最强的旁分泌信号。我们之前的研究结果表明，MEPE 的表达受 BMP-2 信号通路的刺激。本研究的具体目的是探讨 MEPE 的表达是否也受 Wnt 信号的控制，以及两种主要成骨信号通路之间是否存在交叉调控。用 Wnt3a（经典 Wnt 信号刺激剂）处理可强烈增强 MEPE mRNA 的表达。用 siRNA 敲低 β-catenin 可完全逆转 Wnt3a 刺激的 MEPE 表达。即使在没有 Wnt3a 的情况下，过表达 β-catenin 和 Lef-1 也会增加 MEPE mRNA 的表达水平。在小鼠 MEPE 启动子中鉴定出高度保守的 Lef-1 反应元件。Lef-1 直接结合这些元件对 MEPE 的表达至关重要，表明 MEPE 是经典 Wnt 信号的直接靶标。同时，我们还发现 Wnt3a 处理强烈刺激 Bmp-2 的表达，并且在 Wnt3a 处理的条件培养基（CM）中确定了 Bmp-2 蛋白的后续增加。用 CM 处理 MC3T3-E1 细胞可刺激 Smad1/5 蛋白的磷酸化及其下游 Dlx5 mRNA 的表达。CM 介导的磷酸化 Smad 和 Dlx5 表达的增加不能被 Wnt3a 拮抗剂 Dkk-1 完全阻断，但可以通过添加 Bmp-2 拮抗剂 Noggin 几乎完全抑制。总之，Wnt3a 通过β-catenin 和 Lef-1 直接通过经典的 Wnt 信号通路刺激 MEPE 转录，并通过激活 Bmp-2 自分泌环间接刺激 MEPE 转录。
