Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, Brazil.
J Cell Biochem. 2024 Jul;125(7):e30606. doi: 10.1002/jcb.30606. Epub 2024 May 23.
The Hippo pathway, a signaling cascade involved in the regulation of organ size and several other processes, acts as a conduit between extracellular matrix (ECM) cues and cellular responses. We asked whether the basement membrane (BM), a specialized ECM component known to induce quiescence and differentiation in mammary epithelial cells, would regulate the localization, activity, and interactome of YAP, a Hippo pathway effector. To address this question, we used a broad range of experimental approaches, including 2D and 3D cultures of both mouse and human mammary epithelial cells, as well as the developing mouse mammary gland. In contrast to malignant cells, nontumoral cells cultured with a reconstituted BM (rBM) displayed higher concentrations of YAP in the cytoplasm. Incidentally, when in the nucleus of rBM-treated cells, YAP resided preferentially at the nuclear periphery. In agreement with our cell culture experiments, YAP exhibited cytoplasmic predominance in ductal cells of developing mammary epithelia, where a denser BM is found. Conversely, terminal end bud (TEB) cells with a thinner BM displayed higher nucleus-to-cytoplasm ratios of YAP. Bioinformatic analysis revealed that genes regulated by YAP were overrepresented in the transcriptomes of microdissected TEBs. Consistently, mouse epithelial cells exposed to the rBM expressed lower levels of YAP-regulated genes, although the protein level of YAP and Hippo components were slightly altered by the treatment. Mass spectrometry analysis identified a differential set of proteins interacting with YAP in cytoplasmic fractions of mouse epithelial cells in the absence or presence of rBM. In untreated cells, YAP interactants were enriched in processes related to ubiquitin-mediated proteolysis, whereas in cells exposed to rBM YAP interactants were mainly key proteins related to amino acid, amino sugar, and carbohydrate metabolism. Collectively, we unraveled that the BM induces YAP translocation or retention in the cytoplasm of nontumoral epithelial cells and that in the cytoplasm YAP seems to undertake novel functions in metabolic pathways.
Hippo 通路是一个参与调节器官大小和其他几个过程的信号级联，它作为细胞外基质 (ECM) 线索和细胞反应之间的通道。我们想知道基底膜 (BM) 是否会调节 Hippo 通路效应物 YAP 的定位、活性和相互作用组，基底膜是一种已知能诱导乳腺上皮细胞静止和分化的特殊 ECM 成分。为了解决这个问题，我们使用了广泛的实验方法，包括培养来自小鼠和人类的乳腺上皮细胞的 2D 和 3D 培养物，以及正在发育的小鼠乳腺。与恶性细胞不同，用重建基底膜 (rBM) 培养的非肿瘤细胞在细胞质中显示出更高浓度的 YAP。偶然的是，当 rBM 处理的细胞的核内时，YAP 优先位于核周。与我们的细胞培养实验一致，YAP 在发育中的乳腺上皮的导管细胞中表现出细胞质优势，在那里发现了更密集的 BM。相反，具有较薄 BM 的终末芽 (TEB) 细胞显示出更高的 YAP 核质比。生物信息学分析表明，YAP 调节的基因在微分离的 TEB 转录组中过度表达。一致地，暴露于 rBM 的小鼠上皮细胞表达较低水平的 YAP 调节基因，尽管该处理略微改变了 YAP 和 Hippo 成分的蛋白水平。质谱分析鉴定了在缺乏或存在 rBM 的情况下，在小鼠上皮细胞的细胞质部分与 YAP 相互作用的不同蛋白质组。在未处理的细胞中，YAP 相互作用物富含与泛素介导的蛋白水解相关的过程，而在暴露于 rBM 的细胞中，YAP 相互作用物主要是与氨基酸、氨基糖和碳水化合物代谢相关的关键蛋白。总的来说，我们揭示了 BM 诱导非肿瘤上皮细胞中的 YAP 易位或保留在细胞质中，并且在细胞质中 YAP 似乎在代谢途径中承担新的功能。
