Mechanisms for Latent TGF-beta1 Activation In Vivo

描述(由申请人提供)：转化生长因子？从细胞中释放出来的主要形式是由转化生长因子-？同源二聚体，转化生长因子-？前肽二聚体和单分子潜伏的转化生长因子？结合蛋白(LTBP)，它与每个前肽链上的二硫键相连。与转化生长因子相关的前肽？即使前肽与转化生长因子-β？已经被切割，使转化生长因子-？潜伏期。转化生长因子-？必须从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形式。潜伏的转化生长因子的一小部分？在没有LTBP的情况下，与其前肽形成复合体。这种形式被称为小潜伏复合体(SLC)。潜伏性转化生长因子-β的LLC和SLC之间的功能差异仅在于LTBP的存在或不存在，目前尚不清楚。为了研究SLC的功能，我们建立了突变小鼠(Tgfb1C33S/C33s和Tgfb1-/C33s)，其中与LTBP结合的转化生长因子-1前肽中的半胱氨酸被丝氨酸取代。突变的转化生长因子-β1前体肽不能与LTBP结合。因此，分泌的潜伏的转化生长因子-β1不与LTBP结合，但与其前肽(SLC)络合的LTBP和转化生长因子-β1都能正常分泌。Tgfb1C33S/C33S小鼠具有多器官炎症，可发展为胃、结肠、直肠和肛门肿瘤，3-4个月后死亡。这种表型与Tgfb1-/-小鼠相似，只是Tgfb1C33S/C33s的炎症表型较轻，而肿瘤表型比Tgfb1-/-表型强。有趣的是，与Tgfb1C33S/C33s小鼠相比，100%的Tgfb1-/C33s小鼠在12周龄时会患上胃腺癌，并且具有更强的炎症反应。这些结果表明：1)LTBP与转化生长因子-？前肽对合适的转化生长因子-β？功能，2)存在激活潜伏的转化生长因子？3)Tgfb1C33S/C33s和Tgfb1-/C33s小鼠为研究Tgfb1C33S/C33S和Tgfb1-/C33S小鼠的作用提供了一个新的模型体系。在胃肠道肿瘤诱导中的作用。使用Tgfb1C33S/C33S和Tgfb1-/C33S小鼠进行实验，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，胃肠道上皮细胞增殖和凋亡的变化，以及胃肿瘤发展过程中上皮和间质之间的生化相互作用。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。拟议实验的成功完成将为理解转化生长因子-β的控制提供关键信息。行动。这些信息可能为治疗干预或预防病理情况提供见解，如纤维化、癌症和自身免疫性疾病，这些疾病是由于转化生长因子-β的不适当激活和活性引起的。公共卫生相关性：了解潜伏的转化生长因子-1如何激活？是否受控可能会对治疗条件产生影响，如纤维化，在这些情况下，过量的潜在转化生长因子？激活就会发生。我们早期观察到整合素亚单位？6参与了潜伏性的转化生长因子？几家制药公司已经启动了激活工作，以制造？V？6介导的转化生长因子？队形。此外，我们培育的突变小鼠为研究胃腺癌的快速形成提供了一种新的系统。

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.