Abstract
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.
Highlights
Milk fat globule-EGF-factor is a 72-kDa secreted glycoprotein initially recognized in milk fat globules membrane (MFGM) released into milk by mammary epithelial cells (MECs)
The multiple sequence alignment (MSA) of milk fat globuleEGF factor 8 protein (MFG-E8) from 35 organisms showed the absolute conservation of the RGD motif that is involved in cellular interactions with cell-surface integrins (Taylor et al, 1997), but we argued that its presence may be fortuitous and may imply for another cellular process
We revealed that MFG-E8 mediated DOCK1/Rac1 signaling is essential for non-professional epithelial phagocytes to endorse the engulfment of apoptotic cells
Summary
Milk fat globule-EGF-factor is a 72-kDa secreted glycoprotein initially recognized in MFGM released into milk by MECs. The most critical described function of MFG-E8 is to regulate immune homeostasis through the phagocytosis of apoptotic cells by signaling through αvβ− integrins linking phosphatidylserine at the surface of membrane vesicles (Oshima et al, 2002) of apoptotic cells (Hanayama et al, 2002; Lotfan et al, 2018; Peterman et al, 2019). In addition to the immunological process, it involves multiple regulatory functions, including in humans, SNP in MFGE8 is associated with SLE (Hu et al, 2009). This protein shares structural domain homology with Del-1 (developmental endothelial locus 1), constituting a two-gene family of αvβ integrin ligands (Hidai et al, 1998). It drives us to study the impact of MFG-E8 for other functions, including the role in the crucial cellular proliferation and homeostasis through phagocytosis (Li et al, 2019)
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