Mechanisms for Latent TGF-beta1 Activation In Vivo

性状（申请人提供）：TGF-？是从细胞释放主要是作为一个大的潜伏复合物（LLC）组成的TGF-？同源二聚体，TGF-？前肽二聚体，和一个潜在的TGF-？结合蛋白（LTBP），其与每个前肽链二硫键结合。前肽，这仍然与TGF-？尽管前肽和TGF-？已被切割，使TGF-？潜伏的TGF-β？必须从LLC中释放出来才能与其受体结合。LLC被认为是细胞因子的生物学重要形式。一小部分潜在的TGF-？在不存在LTBP的情况下分泌与其前肽复合。这种形式被称为小潜在复合物（SLC）。LLC和SLC之间潜伏性TGF-β的功能差异，其区别仅在于LTBP的存在或不存在，是未知的。为了询问SLC的功能，我们产生了突变小鼠（Tgfb1C33S/C33S和Tgfb1-/C33S），其中TGF-？结合LTBP的1个前肽被丝氨酸取代。这个突变的TGF-？1前肽不能与LTBP结合。因此，没有分泌的潜伏TGF-？1结合LTBP，但LTBP和TGF-？1与其前肽（SLC）复合的蛋白质正常分泌。Tgfb1C33S/C33S小鼠具有多器官炎症，发生胃、结肠、直肠和肛门的肿瘤，并在3 - 4个月内死亡。该表型类似于Tgfb1-/-小鼠的表型，除了Tgfb1C33S/C33S炎性表型比Tgfb1-/-表型更温和，但肿瘤表型比Tgfb1-/-表型更强。有趣的是，100%的Tgfb1-/C33S小鼠在12周龄时发生胃腺癌，并且与Tgfb1C33S/C33S小鼠相比具有更强的炎症反应。这些结果表明：1）LTBP结合TGF-？前肽是重要的适当的TGF-？功能，2）有机制激活潜在的TGF-？Tgfb1-C33S/C33S和Tgfb1-/C33S小鼠为研究TGF-？胃肠道肿瘤诱导。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-特别是胃腺癌，GI上皮中细胞增殖和凋亡的变化，以及胃肿瘤发展过程中上皮和基质之间的生物化学相互作用。我们将结合小鼠遗传学、细胞生物学和生物化学来回答这些问题。成功完成拟议的实验将提供关键信息的控制TGF-？行动上这些信息可能有助于治疗干预或预防病理状况，如由TGF-β不适当激活和活性引起的纤维化、癌症和自身免疫性疾病。公共卫生相关性：了解如何激活潜在的TGF-？是控制可能有影响到治疗条件，如纤维化，其中过量的潜在TGF-？激活发生。我们先前的观察，整合素亚基？6参与潜伏的TGF-？激活已经开始在几家制药公司的工作，使抑制剂？v？6介导的TGF-？阵此外，我们已经产生的突变小鼠提供了一个新的系统的研究胃腺癌的快速形成。

DESCRIPTION (provided by applicant): TGF-? is released from cells primarily as a large latent complex (LLC) consisting of the TGF-? homodimer, the TGF-? propeptide dimer, and a single molecule of the latent TGF-? binding protein (LTBP), which is disulfide bonded to each of the propeptide chains. The propeptide, which remains associated with TGF-? even though the bond between propeptide and TGF-? has been cleaved, renders the TGF-? latent. TGF-? 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-? 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-?, 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-?1 propeptide that bind to LTBP were replaced by serines. This mutant TGF-?1 propeptide cannot bind to LTBP. Thus, none of the secreted latent TGF-?1 is bound to an LTBP, but both LTBP and TGF-?1 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-? propeptide is important for proper TGF-? function, 2) that there are mechanisms to activate the latent TGF-? 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-? 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-? 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-?. PUBLIC HEALTH RELEVANCE: Understanding how the activation of latent TGF-? is controlled may have impact into treating conditions, such as fibrosis, in which excess latent TGF-? activation occurs. Our earlier observation that the integrin subunit ?6 is involved in latent TGF-? activation has initiated work in several pharmaceutical companies to make inhibitors of ?v?6 mediated TGF-? formation. In addition, the mutant mice we have produced provide a novel system for the study of the rapid formation of gastric adenocarcinomas.