Systems analysis of TGFbeta signaling and SnoN function in liver

描述(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，对维持组织内环境平衡至关重要。TGF2信号的异常水平会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了解如何使用系统生物学方法在正常组织和原代细胞中维持和调节适当水平的TGF2信号活性。本申请的目的是开发一个全面的数学模型，可以用来预测Smad信号的动力学和行为，并确定SnoN介导的Smad信号的负反馈调节功能。我们将利用野生型和SnoN缺失小鼠的原代肝细胞进行定量测量并建立数学模型，并在肝组织切片中进一步验证该模型。我们选择与肝脏合作是因为已知TGF2是一种有效的肝细胞增殖抑制因子，也是肝脏再生的关键负调控因子。在原代肝细胞中，TGF2通过Smad2和Smad4蛋白传递信号。当Smad2被激活的TGFss受体激酶磷酸化后，Smad2与Smad4发生同源寡聚和异源寡聚，导致Smad4在细胞核内积聚。然后，异构体Smad2/Smad4复合体与其他转录因子相互作用，调节TGF2反应基因的表达。Smad蛋白的活性受正性和负性细胞辅助因子的调节。通过系统生物学的研究，我们最近确定SnoN是原代肝细胞中最重要的Smad信号负调控因子。SnoN与Smad2和Smad4结合，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信号的持续时间，也不影响Smad信号的初始时间，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，从而维持这一信号通路的稳定性。在这项建议中，我们将结合数学建模和定量测量，在细胞水平和器官水平上验证这一假设，使用原代肝细胞，并使用野生型和snoN突变小鼠的肝组织。1)分析SnoN对原代肝细胞TGF2信号转导的影响。2)分析SnoN对Smad依赖基因表达的影响。3)模拟SnoN在肝损伤和再生过程中对Smad信号的调节作用。这项研究将极大地促进我们对这一重要信号通路的机制和调控以及该信号活性的去调节如何导致癌症发生的了解。

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.