Roles of Smad1 Dephosphorylation in Osteoblast Differentiation

描述（由申请人提供）：骨形态发生蛋白（Bone Morphogenetic Proteins, BMPs）和TGF-超家族其他成员的细胞应答与骨质疏松症、肺动脉高压和遗传性癌症等人类骨病有关。我们的长期目标是了解BMP/TGF-调节细胞生长和分化的分子基础，以及BMP信号改变导致人类骨病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分化，促进骨形成。BMP信号转导通路激活过程中最关键的事件之一是BMP受体磷酸化转录因子Smad1/5/8 （Smad1、Smad5和Smad8的统称）。磷酸化的Smad1/5/8直接激活细胞核中的靶基因，蛋白磷酸酶预计会使磷酸化的Smad1/5/8去磷酸化，从而使BMP信号失活。直到最近，人们长期追求的磷酸酶的身份一直是BMP/TGF-生物学中未解之谜之一。我们发现Smad1/5/8可以被几种磷酸酶去磷酸化。我们的短期策略是表征磷酸酶的生化特性，并研究它们在成骨细胞分化过程中调控BMP信号传导的生物学功能。我们的初步研究（包括已发表的和未发表的）使我们假设Smad磷酸酶在成骨细胞分化过程中阻断或终止BMP信号。因此，研究Smad磷酸酶在骨形成中的生理作用具有重要意义。在本次应用中，我们将重点研究Smad去磷酸化在成骨细胞分化过程中的功能和分子机制。提出了三个具体目标：目标1:Smad磷酸酶对bmp特异性Smad1/5/8去磷酸化的生化表征。目的2：阐明Smad磷酸酶介导的Smad去磷酸化在BMP信号传导中的作用。目的3：探讨Smad磷酸酶在间充质干细胞成骨细胞分化中的作用。

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 Our preliminary studies (both published and unpublished) lead us to hypothesize that Smad phosphatases block or terminate BMP signaling during osteoblast differentiation.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.