Regulation of TGFBeta-induced apoptosis in liver cells

描述（由申请人提供）：在许多肝脏疾病和肝癌发生过程中，存在于正常肝组织中的肝细胞增殖和凋亡之间的严格调节的稳态机制被破坏。转化生长因子β（TGF β）信号传导途径是肝脏中调节细胞生长和凋亡的机制的核心组成部分。本项目的长期目标是了解TGF β a调节肝细胞增殖和凋亡的分子机制。TGF β是体内和体外肝细胞凋亡的有效诱导剂。TGF β的凋亡活性可以通过胰岛素及其下游信号分子的作用来拮抗。本研究旨在了解TGF β通路和胰岛素通路如何相互作用调节肝细胞对TGF β诱导的细胞凋亡的敏感性。 Smad蛋白是TGF β信号传导的关键介质。在没有配体的情况下，Smad 2和Smad 3位于细胞质中。在TGF β与其受体结合并随后激活受体丝氨酸/苏氨酸激酶后，Smad 2和Smad 3被受体激酶磷酸化，然后易位到细胞核，在那里它们可以与Smad 4形成异聚复合物并激活TGF β应答基因的转录。在这些Smad蛋白中，已显示Smad 3介导TGF β诱导的细胞凋亡。在寻找与Smad 3相互作用的分子时，P.I.确定了Akt，一种在胰岛素下游细胞存活途径中起作用的关键分子。Akt可以直接与Smad 3相互作用，并且这种相互作用受到TGF β和胰岛素的调节。由于激活的Akt的表达导致保护细胞免受TGF β诱导的凋亡，我们假设Akt可能通过与Smad 3相互作用并阻止其介导TGF β的凋亡信号来保护细胞免受TGF β诱导的凋亡。以下具体目的旨在进一步表征Akt和Smad 3之间的相互作用，并揭示这种相互作用的生理意义。具体目的是：1）定位Akt和Smad 3相互作用所需的氨基酸残基。2)分析Smad 3- Akt相互作用在保护TGF β诱导的细胞凋亡中的作用。3)Akt抑制TGF β诱导的细胞凋亡的分子机制分析。特别是，我们将研究Akt是否通过影响其磷酸化或通过物理隔离来抑制Smad 3。这些研究将使我们能够更好地了解TGF α和胰岛素信号通路如何相互作用以调节对TGF β诱导的细胞凋亡的敏感性。由于这种串扰在肝脏大小的控制中起着重要的作用，并且这种高度调节的过程的破坏可能导致肝脏疾病和肝癌发生，因此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.