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Stat3 controls cell death during mammary gland involution by regulating uptake of milk fat globules and lysosomal membrane permeabilization.

Sargeant TJ, Lloyd-Lewis B, Resemann HK, Ramos-Montoya A, Skepper J, Watson CJ - Nat. Cell Biol. (2014)

Bottom Line: We show here that Stat3 regulates the formation of large lysosomal vacuoles that contain triglyceride.Furthermore, we demonstrate that milk fat globules (MFGs) are toxic to epithelial cells and that, when applied to purified lysosomes, the MFG hydrolysate oleic acid potently induces lysosomal leakiness.Additionally, uptake of secreted MFGs coated in butyrophilin 1A1 is diminished in Stat3-ablated mammary glands and loss of the phagocytosis bridging molecule MFG-E8 results in reduced leakage of cathepsins in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cambridge, Tennis Court Road Cambridge CB2 1QP, UK.

ABSTRACT
We have previously demonstrated that Stat3 regulates lysosomal-mediated programmed cell death (LM-PCD) during mouse mammary gland involution in vivo. However, the mechanism that controls the release of lysosomal cathepsins to initiate cell death in this context has not been elucidated. We show here that Stat3 regulates the formation of large lysosomal vacuoles that contain triglyceride. Furthermore, we demonstrate that milk fat globules (MFGs) are toxic to epithelial cells and that, when applied to purified lysosomes, the MFG hydrolysate oleic acid potently induces lysosomal leakiness. Additionally, uptake of secreted MFGs coated in butyrophilin 1A1 is diminished in Stat3-ablated mammary glands and loss of the phagocytosis bridging molecule MFG-E8 results in reduced leakage of cathepsins in vivo. We propose that Stat3 regulates LM-PCD in mouse mammary gland by switching cellular function from secretion to uptake of MFGs. Thereafter, perturbation of lysosomal vesicle membranes by high levels of free fatty acids results in controlled leakage of cathepsins culminating in cell death.

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Free fatty acids are increased during involution and can cause death in vitro. (a) Fluorescence and brightfield microscopy shows milk derived lipid accumulation (lipidtox, green) and cell death of EpH4 cells incubated in milk overnight. Scale bars: Lipidtox staining panels =10 μm, phase images = 100 μm. Nuclei are visualised by Hoechst (blue). (b) Milk induced cell death assessed by trypan blue positivity. Means +/− s.e.m. from n=5 independent experiments with 3 technical replicates performed per experiment (*p<0.05; Mann Whitney U test). (c) Bafilomycin A1 treatment partially rescues milk induced cell death as assessed by brightfield microscopy and trypan blue positivity. (d) Milk induced cell death assessed by trypan blue positivity. Means +/− s.e.m. from n = 4 independent experiments with 3-4 technical replicates performed per experiment (**p<0.01; Kruskal-Wallis test, Dunn’s multiple comparison post-test). Scale bars: 250 μm. Cells were pre-treated with Bafilomycin A1 for 1 h prior to milk addition for further 8 h. (e) Thin layer chromatography was used to demonstrate the presence of free fatty acids in mammary gland extracts. Free fatty acids were seen in force involuted mammary gland extracts but not in lactating mammary glands (arrowhead). TG = triglyceride, PA = palmitic acid. (f) A concentration curve of oleic acid is quantified using densitometry. (g) Quantification of the free fatty acid band showed a significant difference between lactating and involuting mammary gland (p<0.05, Student’s t-test, n = 4 animals). (h) Fluorescence and brightfield microscopy showing lipid accumulation (lipidtox, green) and cell death of EpH4 cells treated with 500 μM oleic acid (OA), palmitic acid (PA) and stearic acid (SA) overnight. Scale bars: Lipidtox staining panels bars =10 μm, phase images = 100 μm. (i) Fatty acid induced cell death assessed by trypan blue positivity. Means +/− s.e.m. from n = 4 independent experiments with 2-3 technical replicates performed per experiment (**p<0.01 vs ETOH control; one-way ANOVA, Dunnett’s Multiple Comparison post-test). Statistics source data can be found in the corresponding worksheets in Supplementary Table 3.
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Figure 4: Free fatty acids are increased during involution and can cause death in vitro. (a) Fluorescence and brightfield microscopy shows milk derived lipid accumulation (lipidtox, green) and cell death of EpH4 cells incubated in milk overnight. Scale bars: Lipidtox staining panels =10 μm, phase images = 100 μm. Nuclei are visualised by Hoechst (blue). (b) Milk induced cell death assessed by trypan blue positivity. Means +/− s.e.m. from n=5 independent experiments with 3 technical replicates performed per experiment (*p<0.05; Mann Whitney U test). (c) Bafilomycin A1 treatment partially rescues milk induced cell death as assessed by brightfield microscopy and trypan blue positivity. (d) Milk induced cell death assessed by trypan blue positivity. Means +/− s.e.m. from n = 4 independent experiments with 3-4 technical replicates performed per experiment (**p<0.01; Kruskal-Wallis test, Dunn’s multiple comparison post-test). Scale bars: 250 μm. Cells were pre-treated with Bafilomycin A1 for 1 h prior to milk addition for further 8 h. (e) Thin layer chromatography was used to demonstrate the presence of free fatty acids in mammary gland extracts. Free fatty acids were seen in force involuted mammary gland extracts but not in lactating mammary glands (arrowhead). TG = triglyceride, PA = palmitic acid. (f) A concentration curve of oleic acid is quantified using densitometry. (g) Quantification of the free fatty acid band showed a significant difference between lactating and involuting mammary gland (p<0.05, Student’s t-test, n = 4 animals). (h) Fluorescence and brightfield microscopy showing lipid accumulation (lipidtox, green) and cell death of EpH4 cells treated with 500 μM oleic acid (OA), palmitic acid (PA) and stearic acid (SA) overnight. Scale bars: Lipidtox staining panels bars =10 μm, phase images = 100 μm. (i) Fatty acid induced cell death assessed by trypan blue positivity. Means +/− s.e.m. from n = 4 independent experiments with 2-3 technical replicates performed per experiment (**p<0.01 vs ETOH control; one-way ANOVA, Dunnett’s Multiple Comparison post-test). Statistics source data can be found in the corresponding worksheets in Supplementary Table 3.

Mentions: Having confirmed that EpH4 cells can take up substances from the culture medium we incubated cells with freshly collected mouse milk which resulted in lipid accumulation (Fig. 4a) and induction of cell death, as measured by phase contrast microscopy (Fig. 4a) and cell viability analysis (Fig. 4b).


Stat3 controls cell death during mammary gland involution by regulating uptake of milk fat globules and lysosomal membrane permeabilization.

Sargeant TJ, Lloyd-Lewis B, Resemann HK, Ramos-Montoya A, Skepper J, Watson CJ - Nat. Cell Biol. (2014)

Free fatty acids are increased during involution and can cause death in vitro. (a) Fluorescence and brightfield microscopy shows milk derived lipid accumulation (lipidtox, green) and cell death of EpH4 cells incubated in milk overnight. Scale bars: Lipidtox staining panels =10 μm, phase images = 100 μm. Nuclei are visualised by Hoechst (blue). (b) Milk induced cell death assessed by trypan blue positivity. Means +/− s.e.m. from n=5 independent experiments with 3 technical replicates performed per experiment (*p<0.05; Mann Whitney U test). (c) Bafilomycin A1 treatment partially rescues milk induced cell death as assessed by brightfield microscopy and trypan blue positivity. (d) Milk induced cell death assessed by trypan blue positivity. Means +/− s.e.m. from n = 4 independent experiments with 3-4 technical replicates performed per experiment (**p<0.01; Kruskal-Wallis test, Dunn’s multiple comparison post-test). Scale bars: 250 μm. Cells were pre-treated with Bafilomycin A1 for 1 h prior to milk addition for further 8 h. (e) Thin layer chromatography was used to demonstrate the presence of free fatty acids in mammary gland extracts. Free fatty acids were seen in force involuted mammary gland extracts but not in lactating mammary glands (arrowhead). TG = triglyceride, PA = palmitic acid. (f) A concentration curve of oleic acid is quantified using densitometry. (g) Quantification of the free fatty acid band showed a significant difference between lactating and involuting mammary gland (p<0.05, Student’s t-test, n = 4 animals). (h) Fluorescence and brightfield microscopy showing lipid accumulation (lipidtox, green) and cell death of EpH4 cells treated with 500 μM oleic acid (OA), palmitic acid (PA) and stearic acid (SA) overnight. Scale bars: Lipidtox staining panels bars =10 μm, phase images = 100 μm. (i) Fatty acid induced cell death assessed by trypan blue positivity. Means +/− s.e.m. from n = 4 independent experiments with 2-3 technical replicates performed per experiment (**p<0.01 vs ETOH control; one-way ANOVA, Dunnett’s Multiple Comparison post-test). Statistics source data can be found in the corresponding worksheets in Supplementary Table 3.
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Related In: Results  -  Collection

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Figure 4: Free fatty acids are increased during involution and can cause death in vitro. (a) Fluorescence and brightfield microscopy shows milk derived lipid accumulation (lipidtox, green) and cell death of EpH4 cells incubated in milk overnight. Scale bars: Lipidtox staining panels =10 μm, phase images = 100 μm. Nuclei are visualised by Hoechst (blue). (b) Milk induced cell death assessed by trypan blue positivity. Means +/− s.e.m. from n=5 independent experiments with 3 technical replicates performed per experiment (*p<0.05; Mann Whitney U test). (c) Bafilomycin A1 treatment partially rescues milk induced cell death as assessed by brightfield microscopy and trypan blue positivity. (d) Milk induced cell death assessed by trypan blue positivity. Means +/− s.e.m. from n = 4 independent experiments with 3-4 technical replicates performed per experiment (**p<0.01; Kruskal-Wallis test, Dunn’s multiple comparison post-test). Scale bars: 250 μm. Cells were pre-treated with Bafilomycin A1 for 1 h prior to milk addition for further 8 h. (e) Thin layer chromatography was used to demonstrate the presence of free fatty acids in mammary gland extracts. Free fatty acids were seen in force involuted mammary gland extracts but not in lactating mammary glands (arrowhead). TG = triglyceride, PA = palmitic acid. (f) A concentration curve of oleic acid is quantified using densitometry. (g) Quantification of the free fatty acid band showed a significant difference between lactating and involuting mammary gland (p<0.05, Student’s t-test, n = 4 animals). (h) Fluorescence and brightfield microscopy showing lipid accumulation (lipidtox, green) and cell death of EpH4 cells treated with 500 μM oleic acid (OA), palmitic acid (PA) and stearic acid (SA) overnight. Scale bars: Lipidtox staining panels bars =10 μm, phase images = 100 μm. (i) Fatty acid induced cell death assessed by trypan blue positivity. Means +/− s.e.m. from n = 4 independent experiments with 2-3 technical replicates performed per experiment (**p<0.01 vs ETOH control; one-way ANOVA, Dunnett’s Multiple Comparison post-test). Statistics source data can be found in the corresponding worksheets in Supplementary Table 3.
Mentions: Having confirmed that EpH4 cells can take up substances from the culture medium we incubated cells with freshly collected mouse milk which resulted in lipid accumulation (Fig. 4a) and induction of cell death, as measured by phase contrast microscopy (Fig. 4a) and cell viability analysis (Fig. 4b).

Bottom Line: We show here that Stat3 regulates the formation of large lysosomal vacuoles that contain triglyceride.Furthermore, we demonstrate that milk fat globules (MFGs) are toxic to epithelial cells and that, when applied to purified lysosomes, the MFG hydrolysate oleic acid potently induces lysosomal leakiness.Additionally, uptake of secreted MFGs coated in butyrophilin 1A1 is diminished in Stat3-ablated mammary glands and loss of the phagocytosis bridging molecule MFG-E8 results in reduced leakage of cathepsins in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cambridge, Tennis Court Road Cambridge CB2 1QP, UK.

ABSTRACT
We have previously demonstrated that Stat3 regulates lysosomal-mediated programmed cell death (LM-PCD) during mouse mammary gland involution in vivo. However, the mechanism that controls the release of lysosomal cathepsins to initiate cell death in this context has not been elucidated. We show here that Stat3 regulates the formation of large lysosomal vacuoles that contain triglyceride. Furthermore, we demonstrate that milk fat globules (MFGs) are toxic to epithelial cells and that, when applied to purified lysosomes, the MFG hydrolysate oleic acid potently induces lysosomal leakiness. Additionally, uptake of secreted MFGs coated in butyrophilin 1A1 is diminished in Stat3-ablated mammary glands and loss of the phagocytosis bridging molecule MFG-E8 results in reduced leakage of cathepsins in vivo. We propose that Stat3 regulates LM-PCD in mouse mammary gland by switching cellular function from secretion to uptake of MFGs. Thereafter, perturbation of lysosomal vesicle membranes by high levels of free fatty acids results in controlled leakage of cathepsins culminating in cell death.

Show MeSH
Related in: MedlinePlus