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

转化生长因子β (TGF)通过多种环境依赖的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复归等作用。TGF信号通过典型（Smad）和非典型途径（如Par6和PI3K/Akt）传递，这两种途径都促进了对TGF的最佳细胞反应之一：上皮-间质转化（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）和顶基极性的丧失。我们发现，在正常乳腺细胞响应TGF进行EMT时，细胞凋亡的发生与紧密连接和顶基极性的破坏并行，细胞外囊泡的释放增强。Par6通过调节PI3K/Akt/FoxO信号通路以及其他信号通路介导这些过程。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，以及相互连接的细胞极性-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Par6- pi3k /Akt-FoxO信号轴的存在及其在tgf诱导的细胞凋亡中的作用，2)确定Par6/ tj细胞存活网络的其他信号介质，3)解决细胞外囊泡在细胞凋亡和EMT之间的联系中的作用。

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.