Mechanisms for Latent TGF-beta1 Activation In Vivo

项目摘要/摘要 转化生长因子-ss主要以由转化生长因子-ss组成的大潜伏复合体(LLC)的形式从细胞中释放出来。 同源二聚体，转化生长因子-ss前肽二聚体，以及潜在转化生长因子-生长因子结合蛋白(LTBP)的单分子， 该二硫键连接到每个前肽链上。前肽，它仍然与 尽管前肽和转化生长因子-ss之间的结合已经断裂，但仍使转化生长因子-ss潜伏。 必须将转化生长因子-ss从LLC中释放出来，才能与其受体结合。LLC被认为是具有生物学意义的 细胞因子的形式。潜伏的转化生长因子-生长因子的一小部分与其前肽形成络合物。 没有LTBP。这种形式被称为小潜伏复合体(SLC)。两者之间的功能差异 潜伏性转化生长因子-SS的LLC和SLC仅因LTBP的存在或不存在而不同，尚不清楚。 为了研究SLC的功能，我们在中国建立了突变小鼠(Tgfb1C33S/C33s和Tgfb1-/C33s) 与LTBP结合的转化生长因子-SS1前肽中的半胱氨酸被丝氨酸取代。这个突变的转化生长因子- SS1前肽不能与LTBP结合。因此，分泌的潜在转化生长因子-SS1没有一个与LTBP结合，但两者都与LTBP结合 LTBP和与其前肽(SLC)络合的转化生长因子-SS1(SLC)正常分泌。Tgfb1C33S/C33S小鼠具有多个 器官发炎，发展为胃、结肠、直肠和肛门肿瘤，并在3-4个月内死亡。这 表型与Tgfb1-/-小鼠相似，只是Tgfb1C33S/C33s的炎症表型较轻 但肿瘤表型强于Tgfb1-/-表型。有趣的是，100%的Tgfb1/C33s小鼠发育 与12周龄的胃腺癌相比，胃腺癌有更强的炎症反应 Tgfb1C33S/C33S小鼠。这些结果表明：1)ltbp与转化生长因子-ss前体肽的结合是正常的。 转化生长因子-ss的功能，2)在缺乏的情况下存在激活潜在的转化生长因子-生长因子前肽复合体的机制 3)Tgfb1C33S/C33s和Tgfb1-/C33s小鼠为研究转化生长因子-s的作用提供了新的模型系统 在胃肠道肿瘤诱导中的作用。 在本申请中描述的实验中使用Tgfb1C33S/C33s和Tgfb1-/C33s小鼠，我们将 确定SLC的激活剂是什么，炎症对肿瘤产生的贡献-具体地说 胃腺癌、胃肠道上皮细胞增殖和凋亡的变化 胃肿瘤发生过程中上皮与间质之间的生化相互作用。我们将使用 结合小鼠遗传学、细胞生物学和生物化学来回答这些问题。成功者 拟议实验的完成将提供对理解控制的关键信息 转化生长因子-生长抑素作用。这些信息可能为治疗干预或预防病理性疾病提供见解。 疾病，如纤维化、癌症和自身免疫性疾病，由不适当的激活和 转化生长因子-生长抑素活性。

Project Summary/Abstract TGF-ss is released from cells primarily as a large latent complex (LLC) consisting of the TGF-ss homodimer, the TGF-ss propeptide dimer, and a single molecule of the latent TGF-ss binding protein (LTBP), which is disulfide bonded to each of the propeptide chains. The propeptide, which remains associated with TGF-ss even though the bond between propeptide and TGF-ss has been cleaved, renders the TGF-ss latent. TGF-ss must be freed from the LLC to bind to its receptors. The LLC is believed to be the biologically significant form of the cytokine. A minor fraction of the latent TGF-ss is secreted complexed to its propeptide in the absence of LTBP. This form is called the small latent complex (SLC). The functional differences between the LLC and SLC of latent TGF-ss, which differ only by the presence or absence of LTBP, are unknown. To interrogate the functions of the SLC, we generated mutant mice (Tgfb1C33S/C33S and Tgfb1-/C33S) in which the cysteines in the TGF-ss1 propeptide that bind to LTBP were replaced by serines. This mutant TGF- ss1 propeptide cannot bind to LTBP. Thus, none of the secreted latent TGF-ss1 is bound to an LTBP, but both LTBP and TGF-ss1 complexed to its propeptide (SLC) are secreted normally. Tgfb1C33S/C33S mice have multi- organ inflammation, develop tumors of the stomach, colon, rectum and anus, and die by 3-4 months. This phenotype resembles that of the Tgfb1-/- mouse except that the Tgfb1C33S/C33S inflammatory phenotype is milder but the tumor phenotype stronger than the Tgfb1-/- phenotypes. Interestingly, 100% of Tgfb1-/C33S mice develop gastric adenocarcinomas by 12 weeks of age and have a stronger inflammatory response compared to Tgfb1C33S/C33S mice. These results indicate 1) that LTBP binding to the TGF-ss propeptide is important for proper TGF-ss function, 2) that there are mechanisms to activate the latent TGF-ss propeptide complex in the absence of LTBP, and 3) that Tgfb1C33S/C33S and Tgfb1-/C33S mice provide a new model system to study the role of TGF-ss in GI tumor induction. Using Tgfb1C33S/C33S and Tgfb1-/C33S mice in the experiments described in this application, we will establish what are the activators for the SLC, the contribution of inflammation to tumor production - specifically gastric adenocarcinomas, the changes in cell proliferation and apoptosis in the GI epithelium, and the biochemical interactions between the epithelium and stroma during gastric tumor development. We will use a combination of mouse genetics, cell biology, and biochemistry to answer these questions. The successful completion of the proposed experiments will provide information critical to the understanding of the control of TGF-ss action. Such information may yield insights for therapeutic intervention or prevention of pathological conditions, such as fibrosis, cancer, and autoimmune disease that arise from inappropriate activation and activity of TGF-ss.