Roles of Smad1 Dephosphorylation in Osteoblast Differentiation

描述（由申请人提供）：对骨形态发生蛋白（BMP）和TGF-β超家族其他成员的细胞应答的消除与人类骨骼疾病如骨质疏松症、肺动脉高压和遗传性癌症相关。我们的长期目标是了解BMP/TGF-β如何调节细胞生长和分化的分子基础，以及BMP信号转导改变导致人类骨疾病中生长和分化控制失调的潜在机制。BMP诱导间充质细胞向成骨细胞谱系分化以促进骨形成。BMP受体对转录因子Smad 1/5/8（Smad 1、Smad 5和Smad 8的统称）的磷酸化是BMP信号转导通路激活的关键事件之一。虽然磷酸化的Smad 1/5/8直接激活细胞核中的靶基因，但蛋白磷酸酶预计会使磷酸化的Smad 1/5/8去磷酸化，从而使BMP信号传导失活。直到最近，长期寻找的磷酸酶的身份一直是BMP/TGF-β生物学中未回答的主要问题之一。我们已经发现Smad 1/5/8可以被几种磷酸酶去磷酸化。我们的短期战略，这一建议是表征磷酸酶的生化特性，并调查其在成骨细胞分化过程中的BMP信号调节的生物学功能。我们的初步研究（已发表和未发表）使我们假设，Smad磷酸酶阻断或终止BMP信号在成骨细胞分化。因此，研究Smad磷酸酶在骨形成中的生理作用具有重要意义。本论文主要研究Smad去磷酸化在成骨细胞分化过程中的作用及其分子机制。提出了三个具体目标：目标1：Smad磷酸酶的BMP特异性Smad 1/5/8的去磷酸化的生物化学表征。目的2：阐明Smad磷酸酶介导的Smad去磷酸化在BMP信号转导中的作用。目的3：研究Smad磷酸酶在骨髓间充质干细胞向成骨细胞分化中的作用。 该实验为研究Smad磷酸酶如何通过Smad去磷酸化调节BMP信号通路开辟了新的研究领域。具体而言，阐明Smad磷酸酶介导的Smad 1/5/8以及可能其他R-Smads的去磷酸化的生理作用，将提供对BMP/TGF-β信号在骨生长和发育中的重要作用的见解。公共卫生相关性：本研究计划的主要目标是研究蛋白磷酸酶在骨形成中的功能。我们已经确定了三种磷酸酶作为骨形态发生蛋白（BMP）的负调控因子。我们的研究结果将对开发新的治疗方法，预防和治疗人类骨骼疾病。

DESCRIPTION (provided by applicant): Abrogation of cellular responses to Bone Morphogenetic Proteins (BMPs) and other members of TGF-( superfamily are associated with human bone diseases such as osteoporosis, pulmonary hypertension and heritable cancer. Our long-term objective is to understand the molecular basis of how BMP/TGF-( regulate cell growth and differentiation, and the underlying mechanisms through which alterations in BMP signaling leads to deregulation of growth and differentiation control in human bone diseases. BMPs induce the differentiation of mesenchymal cells towards the osteoblastic lineage to promote bone formation. One of the most critical events in activation of BMP signal transduction pathway is the phosphorylation of transcription factor Smad1/5/8 (collective term for Smad1, Smad5 and Smad8) by BMP receptors. While phosphorylated Smad1/5/8 directly activate the target genes in the nucleus, protein phosphatases are anticipated to dephosphorylate phospho-Smad1/5/8 and consequently inactivate BMP signaling. The identity of the long sought-after phosphatase has been one of the major unanswered questions in BMP/TGF-( biology until very recently. We have discovered that Smad1/5/8 can be dephosphorylated by several phosphatases. Our short-term strategy for this proposal is to characterize biochemical properties of the phosphatases and investigate their biological functions in the regulation of BMP signaling during osteoblast differentiation. Our preliminary studies (both published and unpublished) lead us to hypothesize that Smad phosphatases block or terminate BMP signaling during osteoblast differentiation. Thus, it is significantly important to investigate the physiological roles of Smad phosphatases in bone formation. In this application, we will focus our study on the functions and molecular mechanism of Smad dephosphorylation during osteoblast differentiation. Three specific aims are proposed: Aim 1: Biochemical characterization of Smad phosphatases for dephosphorylation of BMP-specific Smad1/5/8. Aim 2: Elucidation of the functions of Smad phosphatases-mediated Smad dephosphorylation in BMP signaling. Aim 3: Investigation on the roles of Smad phosphatases in osteoblast differentiation from mesenchymal stem cells. The proposed experiments will open a new area of research on how Smad phosphatases modulate BMP signaling pathways through Smad dephosphorylation. Specifically, the elucidation of the physiological roles of Smad phosphatases-mediated dephosphorylation of Smad1/5/8, and perhaps other R-Smads as well, will provide insights into the important role of BMP/TGF-( signaling in bone growth and development. PUBLIC HEALTH RELEVANCE: The major goal of this research proposal is to investigate the functions of protein phosphatases in bone formation. We have identified three phosphatases as negative regulators of bone morphogenetic proteins (BMP). Our results will be pertinent towards the development of novel therapeutic approaches for the prevention and treatment of human bone diseases.