Regulation of TGFBeta-induced apoptosis in liver cells

描述(由申请人提供)：存在于正常肝组织中的肝细胞增殖和凋亡之间的严格调控的动态平衡机制在许多肝脏疾病和肝癌发生过程中被破坏。转化生长因子β(TGFbeta)信号通路是肝脏细胞生长和凋亡调控机制的核心组成部分。该项目的长期目标是了解TGFbetaa调节肝细胞增殖和凋亡的分子机制。无论是在体内还是体外，TGFbeta都是一种有效的肝细胞凋亡诱导剂。胰岛素及其下游信号分子的作用可拮抗转化生长因子β的促凋亡作用。本研究旨在了解转化生长因子β途径和胰岛素途径如何相互作用来调节肝细胞对转化生长因子β诱导的细胞凋亡的敏感性。 Smad蛋白是转化生长因子β信号转导的重要中介。在缺乏配体的情况下，Smad2和Smad3定位于细胞质中。当Smad2和Smad3与其受体结合并激活受体丝氨酸/苏氨酸激酶时，Smad2和Smad3被受体激酶磷酸化，然后移位到细胞核，在那里它们可以与Smad4形成异构体复合体，激活TGFbeta反应基因的转录。在这些Smad蛋白中，Smad3已被证明介导了TGFbeta诱导的细胞凋亡。在寻找与Smad3相互作用的分子的过程中，P.I.发现了Akt，这是一种在胰岛素下游的细胞生存途径中发挥作用的关键分子。AKT可以直接与Smad3相互作用，这种相互作用受转化生长因子β和胰岛素的调节。由于活化Akt的表达对TGFbeta诱导的细胞凋亡具有保护作用，我们推测Akt可能通过与Smad3相互作用，阻止其介导TGFbeta的凋亡信号，从而保护细胞免受TGFbeta诱导的细胞凋亡。以下特定目的旨在进一步描述Akt和Smad3之间的相互作用，并揭示这种相互作用的生理意义。其具体目的是：1)定位Akt和Smad3中相互作用所需的氨基酸残基。2)分析Smad3-Akt相互作用在抗TGFβ诱导的细胞凋亡中的作用。3)Akt抑制TGFβ诱导的细胞凋亡的分子机制分析。特别是，我们将研究Akt是通过影响Smad3的磷酸化还是通过物理隔离来抑制Smad3。这些研究将使我们更好地了解TGFa和胰岛素信号通路如何相互作用来调节对TGFbeta诱导的细胞凋亡的敏感性。由于这种串扰在控制肝脏大小方面起着重要作用，而破坏这一高度调控的过程可能会导致肝脏疾病和肝癌的发生，这些研究可能有助于更好地理解正常肝脏稳态的维持和肝脏疾病的原因。

DESCRIPTION (provided by applicant): The tightly regulated homeostatic mechanisms between hepatocyte proliferation and apoptosis that exist in normal liver tissue are disrupted in many liver diseases and during hepatocarcinogenesis. The transforming growth factor beta (TGFbeta) signaling pathway is a central component of the mechanisms by which cell growth and apoptosis are regulated in the liver. The long-term objective of this project is to understand the molecular mechanism by which TGFbetaa regulates hepatocyte proliferation and apoptosis. TGFbeta is a potent inducer of hepatocyte apoptosis both in vivo and in vitro. The apoptotic activity of TGFbeta can be antagonized by the action of insulin and its downstream signaling molecules. This proposal is designed to understand how TGFbeta pathway and insulin pathway cross talk to regulate the sensitivity of hepatocytes to TGFbeta-induced apoptosis. Smad proteins are critical mediators of TGFbeta signaling. In the absence of ligand, Smad2 and Smad3 are located in the cytoplasm. Upon binding of TGFbeta to its receptors and the subsequent activation of the receptor serine/threonine kinases, Smad2 and Smad3 become phosphorylated by the receptor kinases and then translocate to the nucleus, where they can form heteromeric complexes with Smad4 and activate transcription of TGFbeta responsive genes. Among these Smad proteins, Smad3 has been shown to mediate TGFbeta-induced apoptosis. In a search for molecules that interact with Smad3, the P.I. identified Akt, a critical molecule functioning in the cell survival pathway downstream of insulin. Akt can directly interact with Smad3, and this interaction is regulated by TGFbeta and insulin. Because expression of an activated Akt results in protection of cells from TGFbeta-induced apoptosis, we hypothesized that Akt may protect cells from TGFbeta-induced apoptosis by interacting with Smad3 and preventing it from mediating the apoptotic signals of TGFbeta. The following specific aims are designed to further characterize the interaction between Akt and Smad3 and uncover the physiological significance of this interaction. The specific aims are: 1) Mapping the amino acid residues in Akt and Smad3 required for their interaction. 2) Analysis of the role of the Smad3- Akt interaction in protection from TGFbeta-induced apoptosis. 3) Analysis of the molecular mechanism by which Akt inhibits TGFbeta-induced apoptosis. In particular, we will investigate whether Akt inhibits Smad3 through affecting its phosphorylation or through physical sequestration. These studies will allow us to have a better understanding of how TGFa and insulin signaling pathways cross talk to regulate the sensitivity to TGFbeta-induced apoptosis. Since this cross talk plays an important role in the control of liver size and disruption of this highly regulated process can result in liver diseases and hepatocarcinogenesis, these studies may contribute to a better understanding of the maintenance of normal liver homeostasis and the cause of liver diseases.