Verma Arvind K, Ali Syed A, Singh Parul, Kumar Sudarshan, Mohanty Ashok K
Proteomics and Cell Biology Lab, Animal Biotechnology Center, ICAR-National Dairy Research Institute, Karnal, India.
Front Cell Dev Biol. 2021 Apr 1;9:568660. doi: 10.3389/fcell.2021.568660. eCollection 2021.
The mammary gland is a unique apocrine gland made up of a branching network of ducts that end in alveoli. It is an ideal system to study the molecular mechanisms associated with cell proliferation, differentiation, and oncogenesis. MFG-E8, also known as Lactadherin, is a vital glycoprotein related to the milk fat globule membrane and initially identified to get secreted in bovine milk. Our previous report suggests that a high level of MFG-E8 is indicative of high milk yield in dairy animals. Here, we showed that MFG-E8 controls the cell growth and morphology of epithelial cells through a network of regulatory transcription factors. To understand the comprehensive action, we downregulated its expression in MECs by MFG-E8 specific shRNA. We generated a knockdown proteome profile of differentially expressed proteins through a quantitative iTRAQ experiment on a high-resolution mass spectrometer (Q-TOF). The downregulation of MFG-E8 resulted in reduced phagocytosis and cell migration ability, whereas it also leads to more lifespan to knockdown vis-a-vis healthy cells, which is confirmed through BrdU, MTT, and Caspase 3/7. The bioinformatics analysis revealed that MFG-E8 knockdown perturbs a large number of intracellular signaling, eventually leading to cessation in cell growth. Based on the directed network analysis, we found that MFG-E8 is activated by CX3CL1, TP63, and CSF2 and leads to the activation of SOCS3 and CCL2 for the regulation of cell proliferation. We further proved that the depletion of MFG-E8 resulted in activated cytoskeletal remodeling by MFG-E8 knockdown, which results in the activation of three independent pathways ZP4/JAK-STAT5, DOCK1/STAT3, and PIP3/AKT/mTOR. Overall, this study suggests that MFG-E8 expression in mammary epithelial cells is an indication of intracellular deterioration in cell health. To date, to the best of our knowledge, this is the first study that explores the downstream targets of MFG-E8 involved in the regulation of mammary epithelial cell health.
乳腺是一种独特的顶浆分泌腺，由末端为腺泡的分支导管网络组成。它是研究与细胞增殖、分化和肿瘤发生相关分子机制的理想系统。MFG-E8，也称为乳粘连蛋白，是一种与乳脂肪球膜相关的重要糖蛋白，最初被鉴定为在牛乳中分泌。我们之前的报告表明，高水平的MFG-E8表明奶牛的产奶量高。在这里，我们表明MFG-E8通过调控转录因子网络来控制上皮细胞的生长和形态。为了解其全面作用，我们通过MFG-E8特异性短发夹RNA下调其在乳腺上皮细胞中的表达。我们通过在高分辨率质谱仪（Q-TOF）上进行的定量iTRAQ实验生成了差异表达蛋白质的敲低蛋白质组图谱。MFG-E8的下调导致吞噬作用和细胞迁移能力降低，而与健康细胞相比，它也使敲低细胞的寿命延长，这通过BrdU、MTT和半胱天冬酶3/7得到证实。生物信息学分析表明，MFG-E8敲低扰乱了大量细胞内信号传导，最终导致细胞生长停止。基于定向网络分析，我们发现MFG-E8被CX3CL1、TP63和CSF2激活，并导致SOCS3和CCL2激活以调节细胞增殖。我们进一步证明，MFG-E8的缺失导致MFG-E8敲低激活细胞骨架重塑，从而激活三个独立的途径ZP4/JAK-STAT5、DOCK1/STAT3和PIP3/AKT/mTOR。总体而言，这项研究表明乳腺上皮细胞中MFG-E8的表达表明细胞健康状况的细胞内恶化。据我们所知，迄今为止，这是第一项探索参与调节乳腺上皮细胞健康的MFG-E8下游靶点的研究。
