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

TGFB超家族的配体涉及广泛的生理过程，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相关。特别是，TGFB在组织纤维化中的作用已经被强调-据报道每年导致全球约40%的死亡-其中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受体活化的下游，典型信号传导经由SMAD转录因子发生，SMAD转录因子在受体介导的磷酸化后易位至细胞核以发挥其细胞功能。此外，许多非经典的SMAD非依赖性途径对TGFB介导的功能反应至关重要，包括ERK、PI 3 K/AKT/mTOR、JNK/p38途径和Rho样GTP酶。深入了解这些途径之间的相互作用对于理解TGFB介导的纤维化和衰老中的细胞功能至关重要。PI 3 K/AKT/mTOR轴代表重要的致癌信号传导节点，整合来自各种输入的信号，包括酪氨酸激酶受体、G蛋白偶联受体和活化的Ras。我们实验室最近的研究表明，TGF β通过一种新的途径诱导肌成纤维细胞活化，该途径利用独立于PI 3 K和Akt的mTOR活化。mTOR的上游PI 3 K/Akt非依赖性激活剂尚不清楚。转化生长因子激活激酶（TAK 1）在多种体外和体内模型中参与了非经典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切割特发性肺纤维化患者肺部的人类活检材料进行的初步转录研究强调了TAK 1信号通路的公认组分，包括钙依赖性磷酸酶钙调磷酸酶及其调节伴侣钙调磷酸酶1（RCAN 1）和下游转录因子活化T细胞核因子（NFAT）。都与I型和III型前胶原基因表达高度相关。此外，在经历ER和氧化应激的细胞中，Ca 2+的细胞质水平显著升高，这是肺和其他器官中纤维化组织中普遍存在的状况。将这些流结合在一起，我们假设钙依赖性NFAT激活下游的TAK 1和mTOR伊萨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，本研究的目标是：1）扩大我们的初步转录研究结果，从激光捕获显微切割使用额外的活检组织从IPF肺，以及调查是否RCAN 1/钙调神经磷酸酶/ NFAT轴与促纤维化基因表达在其他器官设置。2)使用从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，定义钙调磷酸酶信号传导和TAK 1复合物组分在我们的体外模型系统中的表达。学生还将研究细胞外基质相关基因中NFAT共识结合位点的存在，并探索TGFB刺激后NFAT磷酸化和易位到细胞核3）访问GSK内可用的通路特异性抑制剂工具箱，以确定分离的原代成纤维细胞培养物中TAK 1信号传导和TGFB介导的mTOR激活之间的关系。此外，学生将采用GSK内部开发的器官型肺切片模型，在复杂的组织环境中探索这一途径。

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.