Milk Fat globule-EGF Research Articles

MFGE8 promotes gastric cancer progression by activating the IL-6/JAK/STAT3 signaling

ObjectiveGastric cancer is malignant cancer with high morbidity and mortality worldwide. Milk fat globule EGF and factor V/VIII domain containing (MFGE8) was involved in many cancers. Nevertheless, the role of MFGE8 in gastric cancer remained indistinct. To probe the role of MFGE8 in gastric cancer and further explore the regulating mechanism. MethodsGEPIA was employed for analysis of MFGE8 expression and survival of gastric cancer patients. MFGE8 expression in gastric cancer was determined by immunohistochemistry, PCR, and western blot. The effect of MFGE8 on gastric cancer cells were evaluated by a series of cell function experiments. The mechanism of MFGE8 on gastric cancer was analyzed by GSEA and verified by in vitro and in vivo experiments. ResultsMFGE8 was over-expressed in gastric cancer. Silence of MFGE8 suppressed cell viability, proliferated ability, migrated and invasive ability, and EMT, but accelerated cell apoptosis. The opposite results were obtained in MFGE8-overexpressed gastric cancer cells. Zinc finger and BTB domain containing 7 A (ZBTB7A) was a transcription factor of MFGE8. ZBTB7A overexpression eliminated the effect of MFGE8 on gastric cancer cells. MFGE8 activated the IL-6/JAK/STAT3 signaling. Inhibition of IL-6/JAK/STAT3 signaling by Stattic (pathway inhibitor) could eliminate the promoting effect of MFGE8 on IL-6/JAK/STAT3 signaling. In addition, MFGE8 shRNA inhibited tumor growth. ConclusionMFGE8 promoted cell proliferation, EMT progress, and tumor growth of gastric cancer by activating the IL-6/JAK/STAT3 signaling.

Extracellular vesicle-derived silk fibroin nanoparticles loaded with MFGE8 accelerate skin ulcer healing by targeting the vascular endothelial cells

BackgroundReduced supplies of oxygen and nutrients caused by vascular injury lead to difficult-to-heal pressure ulcers (PU) in clinical practice. Rapid vascular repair in the skin wound is the key to the resolution of this challenge, but clinical measures are still limited. We described the beneficial effects of extracellular vesicle-derived silk fibroin nanoparticles (NPs) loaded with milk fat globule EGF factor 8 (MFGE8) on accelerating skin blood vessel and PU healing by targeting CD13 in the vascular endothelial cells (VECs).MethodsCD13, the specific targeting protein of NGR, and MFGE8, an inhibitor of ferroptosis, were detected in VECs and PU tissues. Then, NPs were synthesized via silk fibroin, and MFGE8-coated NPs (NPs@MFGE8) were assembled via loading purified protein MFGE8 produced by Chinese hamster ovary cells. Lentivirus was used to over-express MFGE8 in VECs and obtained MFGE8-engineered extracellular vesicles (EVs-MFGE8) secreted by these VECs. The inhibitory effect of EVs-MFGE8 or NPs@MFGE8 on ferroptosis was detected in vitro. The NGR peptide cross-linked with NPs@MFGE8 was assembled into NGR-NPs@MFGE8. Collagen and silk fibroin were used to synthesize the silk fibroin/collagen hydrogel. After being loaded with NGR-NPs@MFGE8, silk fibroin/collagen hydrogel sustained-release carrier was synthesized to investigate the repair effect on PU in vivo.ResultsMFGE8 was decreased, and CD13 was increased in PU tissues. Similar to the effect of EVs-MFGE8 on inhibiting ferroptosis, NPs@MFGE8 could inhibit the mitochondrial autophagy-induced ferroptosis of VECs. Compared with the hydrogels loaded with NPs or NPs@MFGE8, the hydrogels loaded with NGR-NPs@MFGE8 consistently released NGR-NPs@MFGE8 targeting CD13 in VECs, thereby inhibiting mitochondrial autophagy and ferroptosis caused by hypoxia and accelerating wound healing effectively in rats.ConclusionsThe silk fibroin/collagen hydrogel sustained-release carrier loaded with NGR-NPs@MFGE8 was of great significance to use as a wound dressing to inhibit the ferroptosis of VECs by targeting CD13 in PU tissues, preventing PU formation and promoting wound healing.Graphical

Does the RGD region of certain proteins affect metabolic activity?

A better understanding of the role of mineralized tissues and their associated factors in governing whole-body metabolism should be of value toward informing clinical strategies to treat mineralized tissue and metabolic disorders, such as diabetes and obesity. This perspective provides evidence suggesting a role for the arginine-glycine-aspartic acid (RGD) region, a sequence identified in several proteins secreted by bone cells, as well as other cells, in modulating systemic metabolic activity. We focus on (a) two of the SIBLING (small integrin-binding ligand, N-linked glycoprotein) family genes/proteins, bone sialoprotein (BSP) and osteopontin (OPN), (b) insulin-like growth factor-binding protein-1 & 2 (IGFBP-1, IGFBP-2) and (c) developmental endothelial locus 1 (DEL1) and milk fat globule–EGF factor-8 (MFG-E8). In addition, for our readers to appreciate the mounting evidence that a multitude of bone secreted factors affect the activity of other tissues, we provide a brief overview of other proteins, to include fibroblast growth factor 23 (FGF23), phosphatase orphan 1 (PHOSPHO1), osteocalcin (OCN/BGLAP), tissue non-specific alkaline phosphatase (TNAP) and acidic serine aspartic-rich MEPE-associated motif (ASARM), along with known/suggested functions of these factors in influencing energy metabolism.

MFG-E8 Maintains Cellular Homeostasis by Suppressing Endoplasmic Reticulum Stress in Pancreatic Exocrine Acinar Cells.

Excessive endoplasmic reticulum (ER) stress contributes significantly to the pathogenesis of exocrine acinar damage in acute pancreatitis. Our previous study found that milk fat globule EGF factor 8 (MFG-E8), a lipophilic glycoprotein, alleviates acinar cell damage during AP via binding to αvβ3/5 integrins. Ligand-dependent integrin-FAK activation of STAT3 was reported to be of great importance for maintaining cellular homeostasis. However, MFG-E8’s role in ER stress in pancreatic exocrine acinar cells has not been evaluated. To study this, thapsigargin, brefeldin A, tunicamycin and cerulein + LPS were used to induce ER stress in rat pancreatic acinar cells in vitro. L-arginine- and cerulein + LPS-induced acute pancreatitis in mice were used to study ER stress in vivo. The results showed that MFG-E8 dose-dependently inhibited ER stress under both in vitro and in vivo conditions. MFG-E8 knockout mice suffered more severe ER stress and greater inflammatory response after L-arginine administration. Mechanistically, MFG-E8 increased phosphorylation of FAK and STAT3 in cerulein + LPS-treated pancreatic acinar cells. The presence of specific inhibitors of αvβ3/5 integrin, FAK or STAT3 abolished MFG-E8’s effect on cerulein + LPS-induced ER stress in pancreatic acinar cells. In conclusion, MFG-E8 maintains cellular homeostasis by alleviating ER stress in pancreatic exocrine acinar cells.

Maternal Variants in the MFGE8 Gene are Associated with Perceived Breast Milk Supply.

Background: The World Health Organization recommends exclusive breastfeeding for ≥6 months, but many mothers are unable to meet this goal. A major reason why mothers undergo early, unplanned breastfeeding cessation is perceived inadequate of milk supply (PIMS). We hypothesized that defining genetic polymorphisms associated with PIMS could aid early identification of at-risk mothers, providing an opportunity for targeted lactation support. Materials and Methods: This prospective observational cohort study followed 221 breastfeeding mothers for 12 months, collecting medical, demographic, and breastfeeding characteristics. Eighteen mammary secretory genes were assessed for single-nucleotide polymorphisms in 88 women (45 with PIMS and 43 with perceived adequate milk supply [PAMS]), matched by age/race/parity. Hierarchical regressions were used to assess the ability of genotype to aid PIMS prediction. Results: Mothers with PIMS exclusively breastfed for a shorter period (7 ± 12 weeks; p = 0.001) and reported lower milk production (17.6 ± 13.3 oz/day; p = 0.001), and their infants displayed reduced weight-for-length Z-score gains (0.74 ± 1.4; p = 0.038) relative to mothers with PAMS (22 ± 19 weeks; 27.03 ± 12.2 oz/day; 1.4 ± 1.5). Maternal genotype for the rs2271714 variant within milk fat globule EGF and factor V/VIII domain containing gene (MFGE8) was associated with PIMS status (p = 0.009, adjusted p = 0.09, likelihood ratio = 9.33) and duration of exclusive breastfeeding (p = 0.009, adjusted p = 0.09, χ2 = 9.39). Addition of MFGE8 genotype to a model employing maternal characteristics (age, parity, previous breast-feeding duration, body mass index, education, and depression status) significantly increased predictive accuracy for PIMS status (p = 0.001; χ2 = 13.5; area under the curve = 0.813 versus 0.725). Conclusions: Genotyping one lactogenic gene aided identification of mothers at risk for PIMS. If validated in a larger cohort, such an approach could be used to identify mothers who may benefit from increased lactation support.

Abstract 11596: Amyloidogenic Medin Induces Endothelial Cell Prothrombotic Activation

Background: Vascular aging is characterized by acquisition of a pro-inflammatory and prothrombotic vascular phenotype independent of cardiovascular metabolic risk factors through still unknown mechanisms. Medin is an amyloidogenic peptide formed as a cleavage product of milk fat globule EGF factor 8 protein. It accumulates in the vasculature with aging and is one of the most common yet poorly understood human amyloid proteins. Cerebrovascular medin was shown to be strongly associated with vascular dementia and Alzheimer’s disease. It was shown to induce endothelial pro-inflammatory activation and endothelial dysfunction. We aim to test the hypothesis that medin induces prothrombotic activation in endothelial cells. Methods: Recombinant medin was expressed in Lemo 21 (DE3 cells) and purified. Human umbilical vein endothelial cells (HUVECs) were exposed to either vehicle or medin (5 μM, a dose found to be physiologically relevant in human tissue) for 20 hours. Cell lysates were measured for protein content of tissue factor (TF, receptor and cofactor for factor VII and initiates coagulation), plasminogen activator inhibitor-1 (PAI-1, inhibits plasminogen activators that promote fibrinolysis) and thrombomodulin (cell surface glycoprotein that inhibits procoagulant function of thrombin) using standard Western blot and compared. Results: See Figure. Physiologic dose of medin induced increased endothelial cell protein expression of TF, PAI-1 and reduced expression of thrombomodulin. Conclusions: Medin induces prothrombotic activation in human endothelial cells and may be a key novel mediator in the development of vascular aging phenotype.

Camostat mesilate inhibits pro-inflammatory cytokine secretion and improves cell viability by regulating MFGE8 and HMGN1 in lipopolysaccharide-stimulated DF-1 chicken embryo fibroblasts.

Camostat mesilate (CM) possesses potential anti-viral and anti-inflammatory activities. However, it remains unknown whether CM is involved in lipopolysaccharide (LPS)-mediated inflammatory responses and cell injury. In this project, differentially expressed proteins (DEPs, fold change ≥ 1.2 or ≤ 0.83 and Q value ≤ 0.05) in response to LPS stimulation alone or in combination with CM were identified through tandem mass tags (TMT)/mass spectrometry (MS)-based proteomics analysis in DF-1 chicken embryo fibroblasts. The mRNA expression levels of filtered genes were determined by RT-qPCR assay. The results showed that CM alleviated the detrimental effect of LPS on cell viability and inhibited LPS-induced TNF-α and IL-6 secretions in DF-1 chicken embryo fibroblasts. A total of 141 DEPs that might be involved in mediating functions of both LPS and CM were identified by proteomics analysis in DF-1 chicken embryo fibroblasts. LPS inhibited milk fat globule EGF and factor V/VIII domain containing (MFGE8) expression and induced high mobility group nucleosome binding domain 1 (HMGN1) expression, while these effects were abrogated by CM in DF-1 chicken embryo fibroblasts. MFGE8 knockdown facilitated TNF-α and IL-6 secretions , reduced cell viability, stimulated cell apoptosis in DF-1 chicken embryo fibroblasts co-treated with LPS and CM. HMGN1 loss did not influence TNF-α and IL-6 secretions, cell viability, and cell apoptosis in DF-1 chicken embryo fibroblasts co-treated with LPS and CM. In conclusion, CM exerted anti-inflammatory and pro-survival activities by regulating MFGE8 in LPS-stimulated DF-1 chicken embryo fibroblasts, deepening our understanding of the roles and molecular basis of CM in protecting against Gram-negative bacteria.

Inhibition of Efferocytosis by Extracellular CIRP-Induced Neutrophil Extracellular Traps.

Phagocytic clearance of apoptotic cells by the macrophages (efferocytosis) is impaired in sepsis, but its mechanism is poorly understood. Extracellular cold-inducible RNA-binding protein (eCIRP) is a novel damage-associated molecular pattern that fuels inflammation. We identify that eCIRP-induced neutrophil extracellular traps (NETs) impair efferocytosis through a novel mechanism. Coculture of macrophages and apoptotic thymocytes in the presence of recombinant murine CIRP (rmCIRP)-induced NETs significantly inhibited efferocytosis. Efferocytosis was significantly inhibited in the presence of rmCIRP-treated wild-type (WT), but not PAD4-/- neutrophils. Efferocytosis in the peritoneal cavity of rmCIRP-injected PAD4-/- mice was higher than WT mice. Milk fat globule-EGF-factor VIII (MFG-E8), an opsonin, increased macrophage efferocytosis, whereas the inhibition of efferocytosis by NETs was not rescued upon addition of MFG-E8, indicating disruption of MFG-E8's receptor(s) αvβ3 or αvβ5 integrin by the NETs. We identified neutrophil elastase in the NETs significantly inhibited efferocytosis by cleaving macrophage surface integrins αvβ3 and αvβ5 Using a preclinical model of sepsis, we found that CIRP-/- mice exhibited significantly increased rate of efferocytosis in the peritoneal cavity compared with WT mice. We discovered a novel role of eCIRP-induced NETs to inhibit efferocytosis by the neutrophil elastase-dependent decrease of αvβ3/αvβ5 integrins in macrophages. Targeting eCIRP ameliorates sepsis by enhancing efferocytosis.

Milk fat globule EGF factor 8 restores mitochondrial function via integrin-medicated activation of the FAK-STAT3 signaling pathway in acute pancreatitis.

Acute pancreatitis (AP) remains a significant clinical challenge. Mitochondrial dysfunction contributes significantly to the pathogenesis of AP. Milk fat globule EGF factor 8 (MFG‐E8) is an opsonizing protein, which has many biological functions via binding to αvβ3/5 integrins. Ligand‐dependent integrin‐FAK activation of STAT3 was reported to be of great importance for maintaining a normal mitochondrial function. However, MFG‐E8's role in AP has not been evaluated.MethodsBlood samples were acquired from 69 healthy controls and 134 AP patients. Serum MFG‐E8 levels were measured by ELISA. The relationship between serum concentrations of MFG‐E8 and disease severity were analyzed. The role of MFG‐E8 was evaluated in experimental models of AP.ResultsSerum concentrations of MFG‐E8 were lower in AP patients than healthy controls. And serum MFG‐E8 concentrations were negatively correlated with disease severity in AP patients. In mice, MFG‐E8 administration decreased L‐arginine‐induced pancreatic injury and mortality. MFG‐E8's protective effects in experimental AP were associated with improvement in mitochondrial function and reduction in oxidative stress. MFG‐E8 knockout mice suffered more severe pancreatic injury and greater mitochondrial damage after l‐arginine administration. Mechanistically, MFG‐E8 activated the FAK‐STAT3 pathway in AP mice. Cilengitide, a specific αvβ3/5 integrin inhibitor, abolished MFG‐E8's beneficial effects in AP. PF00562271, a specific FAK inhibitor, blocked MFG‐E8‐induced STAT3 phosphorylation. APTSTAT3‐9R, a specific STAT3 antagonist, also eliminated MFG‐E8's beneficial effects under such a condition.ConclusionsMFG‐E8 acts as an endogenous protective mediator in the pathogenesis of AP. MFG‐E8 administration protects against AP possibly by restoring mitochondrial function via activation of the integrin‐FAK‐STAT3 signaling pathway. Targeting the action of MFG‐E8 may present a potential therapeutic option for AP.

Knockdown of milk-fat globule EGF factor-8 suppresses glioma progression in GL261 glioma cells by repressing microglial M2 polarization.

Tumor-associated microglial cells promote glioma growth, invasion, and chemoresistance by releasing inflammatory factors. Milk fat globule EGF factor 8 protein (MFG-E8), a secreted glycoprotein, is closely related to tissue homeostasis and anti-inflammation. In the present study, we investigated the role of MFG-E8 in microglial polarization and glioma progression in vitro and in vivo. We found that glioma cells secrete comparable amounts of MFG-E8 in culture media to astrocytes. Recombinant MFG-E8 triggered microglia to express the M2 polarization markers, such as arginase-1 (ARG-1), macrophage galactose-type C-type lectin-2 (MGL-2), and macrophage mannose receptor (CD206). Forced expression of MFG-E8 in BV-2 microglia cells not only promoted IL-4-induced M2 polarization but also inhibited lipopolysaccharide (LPS)-induced M1 microglial polarization. Mechanistic studies demonstrated that recombinant MFG-E8 markedly induced signal transducer and activator of transcription 3 (STAT3) phosphorylation, and the STAT3 inhibitor stattic significantly blocked MFG-E8-induced ARG-1 expression. Administration of antibody against MFG-E8 and knockdown of its receptor, integrin β3, significantly attenuated MFG-E8-induced ARG-1 expression. Similarly, knockdown of MFG-E8 also markedly reduced IL-4-induced M2 marker expression and increased LPS-induced M1 marker expression in microglia cells. Moreover, the knockdown of MFG-E8 in GL261 glioma cells inhibited cell proliferation and enhanced chemosensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), which was likely associated with the downregulation of FAK/AKT activation and STAT3/cyclin D1 signaling. The murine GL261 glioma experimental model demonstrated that knockdown of MFG-E8 significantly reduced tumor size and extended survival times. Additionally, attenuated CD11b+ cell infiltration and reduced CD206+ expression in CD11b+ cells were also observed in an MFG-E8 knockdown GL261 murine glioma model. These results suggested that inhibition of MFG-E8 might hamper the immunosuppressive microenvironment in gliomas and therefore ameliorate tumor progression.

Electrostatic and Lipid Packing Effects on the Binding of Milk Fat Globule EGF Factor 8 to Phospholipid Membranes

Electrostatic and Lipid Packing Effects on the Binding of Milk Fat Globule EGF Factor 8 to Phospholipid Membranes

The expression levels and clinical significance of MFG-E8 and CD133 in epithelial ovarian cancer

The aim of our study was to test whether there is an association between high expression of milk fat globule EGF factor 8 (MFG-E8) and CD133 presence or clinical outcomes of patients with epithelial ovarian cancer (EOC). MFG-E8 and CD133 expression levels were analyzed by immunohistochemistry in 88 EOC tumor specimens. High expression of MFG-E8 directly and significantly correlated with the presence of CD133 immunostaining (R = 0.353, p=.001), whereas immunostaining of MFG-E8 and CD133 significantly correlated with FIGO stage, tumor grade, debulking status, the dualistic model, ascites status, and nonresponse to chemotherapy (p<.05). It was also found that high expression of MFG-E8 and CD133 presence is a potent predictor of poor clinical outcomes among patients with EOC. Our study is the first to show that high expression of MFG-E8 in EOCs positively correlates with CD133 presence. Further research on MFG-E8 in EOC is needed to determine whether MFG-E8 is a new tumor marker of ovarian cancer and a new target for anticancer therapy as well as whether it can interact with cancer stem cell inhibitors for the treatment of refractory tumors.

Abstract 247: Phagocytosis-mediated Activation of Smad3 in Macrophages Mediates Anti-inflammatory Transition and Protects From Adverse Post-infarction Remodeling

Macrophages are essential regulators in tissue injury, repair and fibrosis. Although activation of TGF-β signaling cascades critically modulates macrophage phenotype in vitro, the in vivo role of macrophage TGF-β signaling in homeostasis and disease is unknown. Our study examined the role of macrophage-specific TGF-β/Smad3 signaling in a mouse model of myocardial infarction (MI). TGF-βs markedly activated Smad3 in isolated macrophages, without affecting Smad-independent pathways. Infarct macrophages, but not neutrophils exhibited Smad3 activation. Phagocytosis rapidly and directly activated macrophage Smad3, in the absence of active TGF-β1 release. Myeloid cell-specific Smad3 knockout mice (MyS3KO) had no baseline defects, but exhibited increased late mortality and accentuated remodeling following MI. Adverse outcome in MyS3KO mice after MI was associated with perturbations in phagocytic activity, with defective transition of macrophages to an anti-inflammatory phenotype and with scar expansion. Milk-fat globule EGF factor-8 (Mfge8) partly rescued the phagocytic defect of Smad3 null macrophages, without affecting inflammatory activity. Perturbed anti-inflammatory transition of Smad3 null macrophages was associated with marked attenuation of phagocytosis-mediated PPAR induction. We demonstrate that Smad3 is activated in phagocytic macrophages, critically regulates their function and mediates anti-inflammatory transition, and protects the infarcted heart from adverse remodeling.

Variation of proteomic profile during lactation in Girgentana goat milk: a preliminary study

The knowledge of milk proteome has been greatly enhanced by technological advances in the proteomics field as the use of the two-dimensional differential in-gel electrophoresis, a gel-based approach which allowed the analysis of proteins from complex mixtures and the comparing of several protein samples in the same experiment. The aim of this study was to characterise the whole milk proteomic profile in Girgentana dairy goat breed by two-dimensional differential in-gel elecrophoresis. The obtained representative 2D whole milk proteomic map showed a general picture of the protein distributions over the pH 3–10 NL including about 100 spots, most of them organised like a spot train. Among differentially abundant spots in the three experimental conditions, milk fat globule EGF factor 8 protein, β-lactoglobulin, β-casein and serum albumin were successfully identified. The three-dyes system employed in two-dimensional differential in-gel electrophoresis (2D-DIGE) analysis allowed us to obtain a global representation of the Girgentana whole milk proteome. These preliminary results could be used to generate a milk reference proteomics map for the Girgentana goat breed.

Mfge8 Binding of the avß5 Integrin Promotes Insulin Resistance in Mice

Milk fat globule EGF factor 8 (Mfge8) promotes obesity by inducing fatty acid uptake through ligation of the αvβ5 integrin. Global deletion of Mfge8 in mice increases insulin sensitivity through effects on lean body mass. The primary objective of the present study was to assess whether acute disruption of the Mfge8 signaling pathway in wild type mice modulates insulin sensitivity independent of body composition. We therefore evaluated insulin sensitivity in age and sex-matched wild type mice with insulin (ITT) and glucose tolerance tests (GTT) in the presence of a blocking antibody (ALULA) targeting the β5 integrin. Intraperitoneal administration of 5mg/kg ALULA 1h prior to ITT resulted in significantly lower blood glucose levels compared to control antibody treated mice. ALULA treated mice had a significantly lower blood glucose levels in GTT. Serum insulin level measured during GTT in ALULA treated mice was lower compared to control group, suggesting enhanced sensitivity to insulin. We validated our findings in vitro using differentiated 3T3L1 adipocytes and hepG2 liver cells. Both cell types had significantly higher uptake of the glucose analog 2NBDG after insulin treatment in presence of ALULA as compared with insulin treatment with control antibody. Insulin is known to induce glucose uptake in adipocytes through a PI3K/Akt signaling pathway. β5 integrin blockade followed by insulin administration augmented insulin mediated phosphorylation of Akt and As160 in white adipose tissue (WAT) and skeletal muscle (SM) in mice indicating that the 㬥 integrin normally dampens insulin signaling. In preliminary data we have found that after insulin treatment, the β5 integrin associates with the mature insulin receptor 㬥 (IR㬥) in WAT and SM as shown in coimmunoprecipitation studies. Further mechanistic insight to this novel regulation of IR㬥 by 㬥5 integrin will direct us towards testing the therapeutic potential of blocking 㬥5 integrin producing insulin resistance in diabetes. Disclosure R. Datta: None. A. Khalifeh-Soltani: None. A. Ha: None. K. Atabai: None.

Microglia Endocytose Amyloid β Through the Binding of Transglutaminase 2 and Milk Fat Globule EGF Factor 8 Protein.

Activation of glial cells has been observed in neurodegenerative diseases including Alzheimer's disease (AD). Aggregation of amyloid β (Aβ) is profusely observed as characteristic pathology in AD brain. In our previous study using microglial cell line BV-2, tissue-type transglutaminase (TG2) was found to be involved in phagocytosis (Kawabe et al., in Neuroimmunomodulation 22(4):243-249, 2015; Kawabe et al., Neurochem Res 2017). In the present study, we examined whether TG2 and milk fat globule EGF factor 8 protein (MFG-E8), an adaptor protein promotes macrophage to engulf apoptotic cells, were involved in Aβ endocytosis. When the neuronal/glial mixed culture was stimulated freshly prepared Aβ1-42 for 3 days, the incorporation of Aβ was observed by immunofluorescence staining technique in Iba-1-positive microglia. Cystamine, a broad competitive inhibitor of TGs, suppressed it. When aggregated Aβ was added to the mixed culture, the immunoreactivity of MFG-E8 surrounding Aβ was observed, and then followed by microglial endocytosis. Using western blotting technique, MFG-E8 was detected in cell lysate of astrocyte culture, and was also detected in the medium. When microglia culture was incubated with astrocyte conditioned medium, MFG-E8 levels in microglia tended to increase. It is likely that microglia might utilize MFG-E8 released from astrocytes as well as that expressed in themselves in order to endocytose Aβ aggregation. Furthermore, we confirmed that MFG-E8 could bind with TG2 in microglia culture by immunoprecipitate technique. These results suggest that microglia might uptake Aβ as a complex of aggregated Aβ/MFG-E8/TG2.

Recombinant milk fat globule-EGF factor-8 reduces oxidative stress via integrin β3/nuclear factor erythroid 2-related factor 2/heme oxygenase pathway in subarachnoid hemorrhage rats.

Milk fat globule-EGF factor-8 (MFGE8) has been reported to be neuroprotective in ischemic stroke. However, the effects of MFGE8 in early brain injury after subarachnoid hemorrhage (SAH) have not been investigated. We investigated the role of MFGE8 in early brain injury and the potential mechanisms in antioxidation after SAH. Two dosages (1 μg and 3.3 μg) of recombinant human MFGE8 were injected intracerebroventricularly at 1.5 hours after SAH. SAH grades, neurological scores, and brain water content were measured at 24 and 72 hours. For mechanistic study, MFGE8 siRNA, integrin β3 siRNA, and heme oxygenase (HO) inhibitor SnPP IX were used for intervention. The oxidative stress and expression of MFGE8, integrin β3, HO-1, extracellular signal-regulated kinase, and nuclear factor erythroid 2-related factor 2 were measured by Western blots 24 hours after SAH. The expression of MFGE8 and HO-1 increased and peaked 24 hours after SAH. Administration of recombinant human MFGE8 decreased brain water content and improved neurological functions both at 24 hours and at 72 hours after SAH. Recombinant human MFGE8 reduced oxidative stress and enhanced the expression of extracellular signal-regulated kinase, nuclear factor erythroid 2-related factor 2, and HO-1; and the effects were abolished by integrin β3 siRNA and HO inhibitor SnPP IX. Recombinant MFGE8 attenuated oxidative stress that may be mediated by integrin β3/nuclear factor erythroid 2-related factor 2/HO pathway after SAH. Recombinant MFGE8 may serve as an alternative treatment to ameliorate early brain injury for SAH patients.

Signaling thru milk fat globule EGF factor 8 on human endometrial endothelial cells promotes angiogenesis

Endometrial milk fat globule EGF factor 8 (MFG-E8) has been discovered and studied by our group with predominant localization in the epithelium during the window of implantation. Our recent findings have shown that dysregulated MFG-E8 impairs endometrial receptivity and embryo attachment. This protein is traditionally thought to play a role in promoting tumor cell invasion, metastasis and angiogenesis. However, it is not known whether MFG-E8 regulates angiogenesis in the endometrium. Thus, we sought to determine the function of MFG-E8 on human endometrial endothelial cells (HEEC), hypothesizing that activation of the MFG-E8 production would stimulate angiogenesis.

Milk fat globule EGF factor 8 (MFG-E8) mediates transforming growth factor beta (TGF- β) induced epithelial mesenchymal transition (EMT) in human endometrial epithelium contributing to implantation

MFG-E8 is a protein recently discovered by us in the human endometrium, with predominant localization in the epithelium during the window of implantation. Our recent findings have shown that MFG-E8 promotes endometrial epithelial cell migration. Studies on non-uterine cancer cells have implicated that MFG-E8 promotes cell invasion and metastasis, in which epithelial cells acquire molecular features leading to EMT. It is not known whether MFG-E8 regulates EMT in the endometrium. TGF-β is a cytokine synthesized by trophoblast and a known EMT inducer in many tissues including endometrium.

MFGE8 inhibits inflammasome-induced IL-1β production and limits postischemic cerebral injury

Milk fat globule-EGF 8 (MFGE8) plays important, nonredundant roles in several biological processes, including apoptotic cell clearance, angiogenesis, and adaptive immunity. Several recent studies have reported a potential role for MFGE8 in regulation of the innate immune response; however, the precise mechanisms underlying this role are poorly understood. Here, we show that MFGE8 is an endogenous inhibitor of inflammasome-induced IL-1β production. MFGE8 inhibited necrotic cell-induced and ATP-dependent IL-1β production by macrophages through mediation of integrin β(3) and P2X7 receptor interactions in primed cells. Itgb3 deficiency in macrophages abrogated the inhibitory effect of MFGE8 on ATP-induced IL-1β production. In a setting of postischemic cerebral injury in mice, MFGE8 deficiency was associated with enhanced IL-1β production and larger infarct size; the latter was abolished after treatment with IL-1 receptor antagonist. MFGE8 supplementation significantly dampened caspase-1 activation and IL-1β production and reduced infarct size in wild-type mice, but did not limit cerebral necrosis in Il1b-, Itgb3-, or P2rx7-deficient animals. In conclusion, we demonstrated that MFGE8 regulates innate immunity through inhibition of inflammasome-induced IL-1β production.