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

转化生长因子β通过细胞死亡/凋亡、生长停滞、存活和运动等多种细胞效应来调节上皮细胞的动态平衡。例如，转化生长因子作为生长抑制因子调节乳腺形态发生，以及哺乳期后退缩作为细胞凋亡诱导剂等作用。转化生长因子信号通过规范途径(Smad)和非规范途径(如Par6和PI3K/Akt)，这两种途径都促进了细胞对转化生长因子的反应：上皮-间充质转化(EMT)。EMT的一个关键特征是细胞-细胞连接(紧密连接和粘连连接)和尖-基极性的丧失。我们发现，在接受EMT的正常乳腺细胞中，对转化生长因子的反应，细胞凋亡的发生与紧密连接和尖-基极性的破坏平行发生，并促进细胞外小泡的释放。PAR6通过调节PI3K/Akt/FoxO信号通路以及其他信号通路来调节这些过程。我们假设，转化生长因子在正常乳腺中的功能依赖于其调节连接完整性的能力，以及一个相互连接的细胞极性-细胞生存网络。为了验证这一点，我们提出了三个目标：1)证明Par6-PI3K/Akt-FoxO信号轴的存在及其在转化生长因子诱导的细胞凋亡中的作用；2)确定Par6/TJ-细胞生存网络的其他信号介质；3)研究细胞外小泡在细胞凋亡和EMT之间的联系中的作用。 研究将在正常乳腺上皮细胞上进行，培养成单层或三维腺泡样结构，建立适当的细胞-细胞连接和尖端-基底极性。对连接中断敏感或不敏感的这些细胞的变体(例如，具有过度活跃或受阻的Par6信号的细胞)将在加或不加转化生长因子、信号抑制剂和/或沉默RNA的情况下进行治疗。结果将通过免疫沉淀、免疫印迹、免疫荧光成像、报告分析和qRT-PCR进行分析，以验证蛋白质-蛋白质相互作用、信号通路的激活状态、治疗对蛋白质水平和亚细胞定位的影响，以及感兴趣的靶基因的调节。目标2将采用基于siRNA的高通量筛查，在通过免疫荧光对TJ丢失和凋亡进行双重评估后，将识别与TJ完整性相关的生存信号中介。目标3将涉及在上述条件下处理的细胞释放的胞外囊泡的分离、纯化、蛋白质组学和功能鉴定。 这项研究将提供第一个证据，证明通过将正常组织结构与细胞生存联系起来，细胞-细胞连接在转化生长因子依赖的乳腺动态平衡中发挥积极作用。

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.