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（TGF 2）信号传导控制许多细胞过程，包括细胞增殖和凋亡，并且对于维持组织稳态至关重要。TGF 2信号传导水平异常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TGF 2信号传导活性。本申请的目标是开发一种综合数学模型，该模型可用于预测Smad信号传导动力学和行为，并确定SnoN介导的Smad信号传导负反馈调节的功能。我们将使用野生型和SnoN-null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确认模型。我们选择与肝脏合作，因为已知TGF 2是肝细胞增殖的有效抑制剂，也是肝再生的关键负调节剂。在原代肝细胞中，TGF 2通过Smad 2和Smad 4蛋白进行信号传导。在被活性TGF β受体激酶磷酸化后，Smad 2与Smad 4同源寡聚化和异源寡聚化，导致其在细胞核中积累。然后，异聚Smad 2/Smad 4复合物与其他转录因子相互作用以调节TGF 2应答基因的表达。Smad蛋白的活性受正性和负性细胞辅因子的调节。通过系统生物学研究，我们最近确定SnoN是原代肝细胞中Smad信号转导的最重要的负调节因子。SnoN与Smad 2和Smad 4结合并抑制它们激活TGF 2靶基因的能力。有趣的是，SnoN本身是由TGF 2诱导的，这表明它以负反馈方式调节TGF 2信号传导。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Smad信号传导的初始时间，而是在高TGF 2浓度下降低靶基因表达水平。我们假设SnoN介导的负反馈功能抑制Smad靶基因表达的细胞间变异性，并使TGF 2信号传导的剂量反应行为线性化，从而保持该信号传导途径的稳健性。在这个建议中，我们将结合联合收割机数学建模与定量测量，以测试这一假设在细胞水平上，使用原代肝细胞，并在器官水平上，使用野生型和snoN突变小鼠的肝组织。提出了三个具体目标：1）分析SnoN对原代肝细胞中TGF 2信号传导的影响。2)分析SnoN对Smad依赖性基因表达的影响。3)模拟肝损伤和再生过程中Smad信号的SnoN调节。这项研究将显着推进我们对这一重要信号通路的机制和调控以及这种信号活性的失调如何导致致癌的认识。 公共卫生相关性：转化生长因子2（TGF 2）信号传导控制许多细胞过程，并且对于维持组织稳态至关重要。TGF 2信号传导水平异常导致组织再生缺陷和各种人类疾病，如癌症和糖尿病。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TGF 2信号传导活性。

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. PUBLIC HEALTH RELEVANCE: The transforming growth factor 2 (TGF2) signaling controls many cellular processes and is critical for maintaining tissue homeostasis. Aberrant levels of TGF2 signaling lead to defective tissue regeneration and various human diseases such as cancer and diabetes. 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.