Linking junctional integrity and cell polarity to TGF-beta function in the normal mammary gland

转化生长因子β（TGF）通过许多环境依赖性细胞效应介导上皮稳态，包括细胞死亡/凋亡、生长停滞、存活和运动。例如，TGF作为生长抑制剂介导乳腺形态发生，作为细胞凋亡诱导剂介导泌乳后退化，以及其他作用。TGF信号通过经典（Smad）和非经典途径（例如Par 6和PI 3 K/Akt），这两种途径都促进了对TGF的最佳记录的细胞反应之一：上皮-间充质转化（EMT）。EMT的一个关键特征是细胞-细胞连接（紧密连接和粘附连接）和顶端-基底极性的丧失。我们发现，在正常的乳腺细胞经历EMT响应TGF，细胞凋亡发生在平行的紧密连接和顶端-基底极性的破坏，并增强细胞外囊泡的释放。Par 6介导这些过程与PI 3 K/Akt/FoxO信号传导以及其他信号传导途径的调节相关。我们假设TGF在正常乳腺中的功能依赖于其调节连接完整性的能力，以及相互关联的细胞极性-细胞存活网络。为了验证这一点，我们提出了3个目标：1）证明Par 6-PI 3 K/Akt-FoxO信号传导轴的存在及其在TGF诱导的细胞凋亡中的作用，2）鉴定Par 6/TJ细胞存活网络的另外的信号传导介质，3）解决细胞外囊泡在细胞凋亡和EMT之间的联系中的作用。作为单层或作为三维腺泡样结构培养，其建立适当的细胞-细胞连接和顶端-基底极性。这些细胞的变体，其对连接破坏敏感或不敏感（例如，具有过度活性或阻断的Par 6信号传导的细胞）将被加或减TGF、信号传导抑制剂和/或沉默RNA处理。将通过免疫沉淀、免疫印迹、免疫荧光成像、报告基因测定和qRT-PCR分析结局，以验证蛋白质-蛋白质相互作用、信号传导途径的激活状态、治疗对蛋白质水平和亚细胞定位的影响以及目标靶基因的调节。目标2将采用基于siRNA的高通量筛选，其在通过免疫荧光对TJ损失和凋亡进行双重评估后，将鉴定与TJ完整性相关的存活信号传导介质。目的三是对上述条件下细胞释放的细胞外囊泡进行分离、纯化、蛋白质组学研究和功能鉴定，首次证明细胞-细胞连接在TGF依赖的乳腺稳态中起着积极作用，并将正常组织结构与细胞存活联系起来。

Transforming growth factor beta (TGF) mediates epithelial homeostasis via a number of context-dependent cellular effects, including cell death/apoptosis, growth arrest, survival, and motility. For instance, TGF mediates mammary gland morphogenesis as a growth inhibitor, and post-lactation involution as an apoptosis inducer, among other effects. TGF signals via canonical (Smad) and non-canonical pathways (e.g. Par6 and PI3K/Akt), both of which facilitate one of the best-documented cellular responses to TGF: the epithelial-mesenchymal transition (EMT). A key feature of EMT is the loss of cell-cell junctions (tight and adherens junctions) and apical-basal polarity. We found that in normal mammary cells undergoing EMT in response to TGF, apoptosis occurs in parallel to the disruption of tight junctions and apical-basal polarity, and an enhanced release of extracellular vesicles. Par6 mediates these processes in association with modulation of PI3K/Akt/FoxO signalling, as well as other signalling pathways. We hypothesize that TGF's function in the normal mammary gland relies on its capacity to modulate junctional integrity, and an interconnected cell polarity-cell survival network. To validate this, we propose 3 objectives: 1) demonstrate the existence of a Par6-PI3K/Akt-FoxO signalling axis and its role in TGF-induced apoptosis, 2) identify additional signalling mediators of the Par6/TJ-cell survival network, 3) address the role of extracellular vesicles in the link between apoptosis and EMT.Research will be conducted on normal mammary epithelial cells, cultured as monolayers or as three-dimensional acini-like structures, which establish proper cell-cell junctions and apical-basal polarity. Variants of these cells, which are susceptible or not to junctional disruption (e.g., cells with overactive or blocked Par6 signalling), will be treated plus or minus TGF, signalling inhibitors and/or silencing RNA. Outcomes will be analyzed by immunoprecipitation, immunoblotting, immunofluorescence imaging, reporter assays and qRT-PCR, to verify protein-protein interactions, activation status of signalling pathways, the effect of treatments on protein levels and sub-cellular localization, and modulation of target genes of interest. Objective 2 will employ a siRNA-based high-throughput screen which, upon dual assessment of TJ loss and apoptosis via immunofluorescence, will identify signalling mediators of survival in association with TJ integrity. Objective 3 will involve isolation, purification, proteomics, and functions characterization of extracellular vesicles released by cells treated under the above-described conditions.This research will provide the first evidence that, by linking normal tissue architecture to cellular survival, cell-cell junctions plays an active role in TGF-dependent mammary gland homeostasis.