Role of EGFR signaling in bone formation and the anabolic actions of PTH

描述（由申请人提供）：间歇性甲状旁腺激素（PTH）注射是骨质疏松症最有效的合成代谢治疗方法之一，因为它对骨形成有显着作用。成骨细胞来源于间充质祖细胞，包括间充质干细胞（MSC）和定向骨祖细胞。已经提出了多种机制来解释PTH如何发挥其有益作用，但这些作用是否包括或介导的骨髓间充质祖细胞活性的刺激尚不清楚。我们的新数据支持这一机制，证明存在一个功能不同的人口间充质祖细胞的小梁骨，是非常敏感的PTH。PTH强烈刺激成骨细胞和骨细胞中双调蛋白（表皮生长因子受体（EGFR）配体）的表达。EGFR在间充质祖细胞中高度表达，其活化刺激这些细胞的增殖、存活和迁移。我们的初步数据显示，阻断小鼠中EGFR活性导致骨形成缺陷和骨质减少表型，这伴随着间充质祖细胞数量的减少。有趣的是，条件培养基从PTH处理的成骨细胞chemoattracts间充质祖细胞的双调蛋白-EGFR依赖的方式。此外，成骨细胞系细胞中EGFR活性缺陷的小鼠对PTH注射的合成代谢反应较差。这些和其他数据导致我们的中心假设，EGFR信号传导是骨髓间充质祖细胞的重要调节因子，并介导至少部分间歇性PTH给药的合成代谢作用。我们将测试我们的 1）通过评估有条件EGFR敲除小鼠的骨骼表型，研究MSC中EGFR失活是否抑制骨形成和对PTH的合成代谢反应，所述小鼠具有巢蛋白启动子驱动的诱导型Cre，有或没有PTH治疗; 2）阐明EGFR是否在正常和PTH-1条件下维持骨髓间充质祖细胞群中起关键作用。诱导骨形成。我们将使用在不同谱系定型阶段的间充质祖细胞中EGFR活性缺陷的动物模型来研究EGFR活性与居住在长骨不同区域的间充质祖细胞群体之间的关系。我们还将使用细胞培养方法来确定一组转录因子Egr是否介导和调节EGFR刺激的间充质祖细胞的增殖和存活; 3）通过使用体内移植方法来确定由PTH注射激活的EGFR信号传导是否向骨表面募集间充质祖细胞，其中GFP标记的间充质祖细胞可以可视化。这三个目标的完成将使我们能够首次确定EGFR在骨形成中的作用以及骨对骨质疏松症治疗PTH的合成代谢反应。长期来看，我们寻求确定我们是否可以靶向EGFR信号传导作为一种新的合成代谢策略来治疗骨质疏松症和其他骨相关疾病。

DESCRIPTION (provided by applicant): Intermittent parathyroid hormone (PTH) injection is one of the most effective anabolic treatments for osteoporosis because of its remarkable actions on bone formation. Osteoblasts are derived from mesenchymal progenitors, including mesenchymal stem cells (MSCs) and committed osteoprogenitors. Multiple mechanisms have been proposed to explain how PTH exerts its beneficial effects, but whether these effects include, or are mediated by, a stimulation of bone marrow mesenchymal progenitor activities, is not clear. Our new data support this mechanism by demonstrating the presence of a functionally distinct population of mesenchymal progenitors within the trabecular bone that are very responsive to PTH. PTH strongly stimulates the expression of amphiregulin, an epidermal growth factor receptor (EGFR) ligand, in osteoblasts and osteocytes. EGFR is highly expressed in mesenchymal progenitors and its activation stimulates proliferation, survival, and migration of these cells. Our preliminary data show that blocking EGFR activity in mice leads to defective bone formation and an osteopenic phenotype which are accompanied by a reduction in the number of mesenchymal progenitors. Interestingly, conditioned media from PTH-treated osteoblastic cells chemoattract mesenchymal progenitors in an amphiregulin-EGFR-dependent manner. Moreover, mice with deficient EGFR activity in osteoblast lineage cells have a poor anabolic response to PTH injection. These and other data lead to our central hypothesis that EGFR signaling is an essential regulator of bone marrow mesenchymal progenitors and mediates at least in part the anabolic effects of intermittent PTH administration. We will test our central hypothesis by pursuing the following aims: 1) investigate whether EGFR inactivation in MSCs inhibits bone formation and the anabolic response to PTH by assessing skeletal phenotypes of conditional EGFR knockout mice with a nestin promoter-driven inducible Cre with or without PTH treatment; 2) elucidate whether EGFR plays a critical role in maintaining the bone marrow mesenchymal progenitor population under conditions of normal and PTH-induced bone formation. We will use animal models with deficient EGFR activity in mesenchymal progenitors at different stages of lineage commitment to investigate the relationship between EGFR activity and mesenchymal progenitor populations residing in different regions of the long bone. We will also use cell culture approaches to determine whether a group of transcription factors, Egrs, mediates and regulates EGFR-stimulated proliferation and survival of mesenchymal progenitors; 3) determine whether EGFR signaling activated by PTH injection recruits mesenchymal progenitors toward the bone surface by using an in vivo transplantation approach, in which GFP-labeled mesenchymal progenitors can be visualized. Completion of these three aims will enable us to determine for the first time the role of EGFR in bone formation and the anabolic response of bone to the osteoporosis therapy PTH. Long-term, we seek to determine whether we can target EGFR signaling as a novel anabolic strategy to treat osteoporosis and other bone-related diseases.