Systems analysis of TGFbeta signaling and SnoN function in liver

描述（由申请人提供）：转化生长因子2 （TGF2）信号控制许多细胞过程，包括细胞增殖和凋亡，对维持组织稳态至关重要。TGF2信号的异常水平导致组织再生缺陷和各种人类疾病，如癌症。该项目的长期目标是利用系统生物学方法了解正常组织和原代细胞中TGF2信号活性的适当水平是如何维持和调节的。本应用程序的目标是建立一个综合的数学模型，可用于预测Smad信号的动态和行为，并确定snon介导的Smad信号的负反馈调节的功能。我们将采用野生型和non -null小鼠的原代肝细胞进行定量测量，建立数学模型，并在肝组织切片上进一步验证模型。我们选择肝脏作为研究对象，因为已知TGF2是肝细胞增殖的有效抑制剂，也是肝脏再生的关键负调节因子。在原代肝细胞中，TGF2通过Smad2和Smad4蛋白发出信号。在被活化的TGFss受体激酶磷酸化后，Smad2与Smad4发生同源寡聚和异聚，导致其在细胞核中积累。Smad2/Smad4复合物与其他转录因子相互作用，调节tgf2应答基因的表达。Smad蛋白的活性受阳性和阴性细胞辅助因子的调控。通过一项系统生物学研究，我们最近发现SnoN是原代肝细胞中Smad信号最重要的负调控因子。SnoN结合Smad2和Smad4并抑制它们激活TGF2靶基因的能力。有趣的是，SnoN本身是由TGF2诱导的，这表明它以负反馈的方式调节TGF2信号。我们的初步研究表明，SnoN既不影响Smad信号传导的持续时间，也不影响其初始时间，而是在高TGF2浓度下降低靶基因的表达水平。我们假设snon介导的负反馈功能可以抑制Smad靶基因表达的细胞间变异性，并使TGF2信号的剂量反应行为线性化，从而维持该信号通路的稳健性。在这个提议中，我们将结合数学建模和定量测量，在细胞水平（使用原代肝细胞）和器官水平（使用野生型和snoN突变小鼠的肝组织）上验证这一假设。我们提出了三个具体目标：1)分析SnoN对原代肝细胞中TGF2信号传导的影响。2) SnoN对smad依赖性基因表达的影响分析。3)模拟SnoN在肝损伤和再生过程中对Smad信号的调控。这项研究将大大提高我们对这一重要信号通路的机制和调控的认识，以及这一信号活性的失调如何导致癌变。

DESCRIPTION (provided by applicant): The transforming growth factor 2 (TGF2) signaling controls many cellular processes including cell proliferation and apoptosis and is critical for maintaining tissue homeostasis. Aberrant levels of TGF2 signaling lead to defective tissue regeneration and various human diseases such as cancer. The long-term goal of this project is to understand how proper levels of TGF2 signaling activity are maintained and regulated in normal tissues and primary cells using a system biology approach. The goal of this application is to develop a comprehensive mathematical model that can be used to predict Smad signaling dynamics and behavior and to determine the function of SnoN-mediated negative feedback regulation of Smad signaling. We will employ primary hepatocytes from wild type and SnoN-null mice to perform quantitative measurement and establish a mathematical model, and further confirm the model in liver tissue sections. We choose to work with liver because TGF2 is known to be a potent inhibitor of hepatocyte proliferation and is also the critical negative regulator of liver regeneration. In primary hepatocytes, TGF2 signals through Smad2 and Smad4 proteins. Upon phosphorylation by the active TGFss receptor kinases, Smad2 homo-oligomerizes and heterooligomerizes with Smad4, leading to its accumulation in the nucleus. The heteromeric Smad2/Smad4 complex then interacts with other transcription factors to regulate expression of TGF2-responsive genes. The activities of the Smad proteins are regulated by positive and negative cellular co-factors. Through a system biology study, we have recently determined that SnoN is the most important negative regulator of Smad signaling in primary hepatocytes. SnoN binds to Smad2 and Smad4 and represses their ability to activate TGF2 target genes. Interestingly, SnoN itself is induced by TGF2, suggesting that it modulates TGF2 signaling in a negative feedback manner. Our preliminary study suggests that SnoN neither affects the duration nor the initial timing of Smad signaling, but rather decreases the level of target gene expression at high TGF2 concentrations. We hypothesize that the SnoN-mediated negative feedback functions to suppress cell-to-cell variability in Smad target gene expression and linearize dose-response behavior of TGF2 signaling, thereby maintaining the robustness of this signaling pathway. In this proposal we will combine mathematical modeling with quantitative measurements to test this hypothesis at both the cellular level, using primary hepatocytes, and at the organ level, using liver tissues from wild type and snoN mutant mice. Three specific aims have been proposed: 1) Analysis of the impact of SnoN on TGF2 signaling in primary hepatocytes. 2) Analysis of the effects of SnoN on Smad-dependent gene expression. 3) Modeling SnoN regulation of Smad signaling during liver damage and regeneration. This study will significantly advance our knowledge regarding the mechanism and regulation of this important signaling pathway and how de-regulation of this signaling activity results in carcinogenesis.