The influence of intracellular calcium-release in non-canonical TGFB signalling and myofibroblast differentiation

细胞内钙释放对非经典 TGFB 信号传导和肌成纤维细胞分化的影响

• 批准号: 1792388
• 金额: --
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1792388
• 关键词: influence; intracellular; calcium; release; non

Ligands of the TGFB superfamily have been implicated in a wide range of physiological processes involving cell growth, migration, survival, differentiation and extracellular matrix production. Consistent with its role in these critical functions, dysregulated TGFB signalling has been associated with diverse disease processes and in aging. In particular the role of TGFB has been highlighted in tissue fibrosis - reported to contribute to around 40% of deaths worldwide annually- where it is recognised as a critical mediator of fibroblast to myofibroblast differentiation and aberrant matrix deposition. Downstream of TGFB receptor activation, canonical signalling occurs via SMAD transcription factors which translocate to the nucleus following receptor mediated phosphorylation to exert their cellular functions. In addition, a number of non-canonical, SMAD-independent pathways have emerged to be critical for TGFB mediated functional responses , including the ERK, PI3K/AKT/mTOR, JNK/p38 pathways and the Rho-like GTPases. Insight into the cross talk between these pathways will be critical in understanding TGFB mediated cellular function in fibrosis and aging.The PI3K/AKT/mTOR axis represents an important oncogenic signalling node, integrating signals from a variety of inputs including tyrosine kinase receptors, G-protein coupled receptors and activated Ras. Recent studies from our laboratory suggest that TGFb induces myofibroblast activation via a novel pathway which utilises mTOR activation independent of PI3K and Akt. The upstream PI3K/Akt independent activator of mTOR is as yet unknown. Transforming growth factor activated kinase (TAK1) has been and implicated in non-canonical TGFB mediated profibrotic gene expression in a variety of in vitro and in vivo models. Importantly recent preliminary transcriptional studies in our laboratory using laser capture microdissection of human biopsy material from the lungs of patients with idiopathic pulmonary fibrosis have highlighted that recognised components of the TAK1 signalling pathway including the calcium dependent phosphatase calcineurin its regulatory partner regulator of calcineurin 1 (RCAN1) and downstream transcription factor nuclear factor of activated T-cells (NFAT) are all highly associated with procollagen type I and III gene expression. Moreover, cytoplasmic levels of Ca2+ are significantly elevated in cells undergoing ER and oxidative stress, conditions prevalent in fibrotic tissue in the lung and other organs. Bringing these streams together, we hypothesise that calcium dependent NFAT activation downstream of TAK1 and mTOR isa critical pathway for myofibroblast differentiation and pro-fibrotic gene expression.In order to test this hypothesis, this studentship will aim to:1) Expand on our preliminary transcriptional findings from laser capture microdissection using additional biopsy tissue from IPF lungs as well as investigating whether the RCAN1/ calcineurin/ NFAT axis correlates with profibrotic gene expression in other organ settings. 2) Define the expression of components of calcineurin signalling and TAK1 complex in our in vitro model systems, using primary fibroblasts isolated from IPF and non-IPF lung tissue as well as primary fibroblasts isolated from other organs. The student will also investigate the presence of NFAT consensus binding sites in extracellular matrix related genes and explore NFAT phosphorylation and translocation to the nucleus following TGFB stimulation3) Access a toolbox of pathway specific inhibitors available within GSK to define the relationship between TAK1 signalling and TGFB mediated mTOR activation in isolated primary fibroblast cultures. Moreover, the student will employ an organotypic lung slice model developed within GSK to pharmacologically explore this pathway in a complex tissue environment.

TGFB超家族的配体参与了广泛的生理过程，包括细胞生长、迁移、存活、分化和细胞外基质的产生。与其在这些关键功能中的作用一致，失调的TGFB信号传导与多种疾病过程和衰老有关。特别是TGFB在组织纤维化中的作用已被强调，据报道，每年全球约40%的死亡是由TGFB造成的，它被认为是成纤维细胞向肌成纤维细胞分化和异常基质沉积的关键介质。在TGFB受体激活的下游，典型信号通过SMAD转录因子发生，SMAD转录因子在受体介导的磷酸化后转运到细胞核以发挥其细胞功能。此外，一些非规范的、不依赖smad的通路对TGFB介导的功能反应至关重要，包括ERK、PI3K/AKT/mTOR、JNK/p38通路和rho样GTPases。深入了解这些通路之间的串扰对于理解TGFB介导的纤维化和衰老中的细胞功能至关重要。PI3K/AKT/mTOR轴是一个重要的致癌信号传导节点，整合来自酪氨酸激酶受体、g蛋白偶联受体和活化Ras等多种输入的信号。我们实验室最近的研究表明，TGFb通过一种新的途径诱导肌成纤维细胞激活，该途径利用独立于PI3K和Akt的mTOR激活。上游PI3K/Akt独立的mTOR激活因子尚不清楚。在多种体外和体内模型中，转化生长因子活化激酶（TAK1）已经参与了非典型TGFB介导的促纤维化基因表达。重要的是，我们实验室最近使用激光捕获显微解剖特发性肺纤维化患者肺部活检材料的初步转录研究强调了TAK1信号通路的公认成分，包括钙依赖性磷酸酶钙调磷酸酶及其调节伙伴钙调磷酸酶1 （RCAN1）和下游转录因子活化t细胞核因子（NFAT）都与TAK1高度相关前胶原I型和III型基因表达。此外，在经历内质网和氧化应激的细胞中，细胞质Ca2+水平显著升高，这种情况在肺和其他器官的纤维化组织中普遍存在。综上所述，我们假设TAK1和mTOR下游的钙依赖性NFAT激活是肌成纤维细胞分化和促纤维化基因表达的关键途径。为了验证这一假设，该学生的目标是：1)扩展我们从IPF肺的额外活检组织中激光捕获显微解剖的初步转录发现，以及研究RCAN1/钙调磷酸酶/ NFAT轴是否与其他器官中的纤维化基因表达相关。2)利用从IPF和非IPF肺组织分离的原代成纤维细胞以及从其他器官分离的原代成纤维细胞，确定我们体外模型系统中钙调磷酸酶信号传导成分和TAK1复合物的表达。该学生还将研究细胞外基质相关基因中NFAT的一致结合位点的存在，并探索TGFB刺激后NFAT的磷酸化和向细胞核的易位。3)在分离的原代成纤维细胞培养中，使用GSK提供的途径特异性抑制剂工具箱来定义TAK1信号传导与TGFB介导的mTOR激活之间的关系。此外，学生将采用葛兰素史克公司开发的器官型肺切片模型，在复杂的组织环境中对这一途径进行药理学探索。