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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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Vesicular biogenesis in EpH4 cells. (a) Vehicle treated Eph4 cells contain phagosomes/pinosomes (arrowheads) and lysosomes (arrows). (b) EpH4 cells treated with oncostatin M (OSM, 25 ng/ml) for 72 h have 53% more degradative vesicles, as quantified in panel (c) n = 4 individual wells of EpH4 cells seeded on one occasion. Phagolysosomes are marked by an asterisk. (d) In some cases, as a result of oncostatin M treatment, lysosomal vesicles occupied more than half of the cytosolic volume. Scale bars = 1 μm. Statistics source data for Figure 3c can be found in the corresponding worksheet in Supplementary Table 3.
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Figure 3: Vesicular biogenesis in EpH4 cells. (a) Vehicle treated Eph4 cells contain phagosomes/pinosomes (arrowheads) and lysosomes (arrows). (b) EpH4 cells treated with oncostatin M (OSM, 25 ng/ml) for 72 h have 53% more degradative vesicles, as quantified in panel (c) n = 4 individual wells of EpH4 cells seeded on one occasion. Phagolysosomes are marked by an asterisk. (d) In some cases, as a result of oncostatin M treatment, lysosomal vesicles occupied more than half of the cytosolic volume. Scale bars = 1 μm. Statistics source data for Figure 3c can be found in the corresponding worksheet in Supplementary Table 3.

Mentions: Intrigued by the association of large lysosomal vacuoles with the initiation of cell death, and their absence in the Stat3 knockout, we sought to determine a mechanistic involvement of these structures in the execution of cell death. To address this question, we utilised oncostatin M (OSM) stimulation of the EpH4 mammary epithelial cell line, which we have previously shown to mimic Stat3-induced LM-PCD17. We observed a number of vacuoles/vesicles in unstimulated EpH4 cells presumably generated by autophagy and by pinocytosis of the culture medium (Fig. 3a). We then determined whether activation of Stat3 by OSM resulted in a stimulation of the biogenesis of such vesicles. This was indeed the case with OSM treatment resulting in a 1.7-fold increase in the volume fraction of degradative vesicles after 72 h treatment as determined by TEM (Fig. 3 b-d).


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)

Vesicular biogenesis in EpH4 cells. (a) Vehicle treated Eph4 cells contain phagosomes/pinosomes (arrowheads) and lysosomes (arrows). (b) EpH4 cells treated with oncostatin M (OSM, 25 ng/ml) for 72 h have 53% more degradative vesicles, as quantified in panel (c) n = 4 individual wells of EpH4 cells seeded on one occasion. Phagolysosomes are marked by an asterisk. (d) In some cases, as a result of oncostatin M treatment, lysosomal vesicles occupied more than half of the cytosolic volume. Scale bars = 1 μm. Statistics source data for Figure 3c can be found in the corresponding worksheet in Supplementary Table 3.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4216597&req=5

Figure 3: Vesicular biogenesis in EpH4 cells. (a) Vehicle treated Eph4 cells contain phagosomes/pinosomes (arrowheads) and lysosomes (arrows). (b) EpH4 cells treated with oncostatin M (OSM, 25 ng/ml) for 72 h have 53% more degradative vesicles, as quantified in panel (c) n = 4 individual wells of EpH4 cells seeded on one occasion. Phagolysosomes are marked by an asterisk. (d) In some cases, as a result of oncostatin M treatment, lysosomal vesicles occupied more than half of the cytosolic volume. Scale bars = 1 μm. Statistics source data for Figure 3c can be found in the corresponding worksheet in Supplementary Table 3.
Mentions: Intrigued by the association of large lysosomal vacuoles with the initiation of cell death, and their absence in the Stat3 knockout, we sought to determine a mechanistic involvement of these structures in the execution of cell death. To address this question, we utilised oncostatin M (OSM) stimulation of the EpH4 mammary epithelial cell line, which we have previously shown to mimic Stat3-induced LM-PCD17. We observed a number of vacuoles/vesicles in unstimulated EpH4 cells presumably generated by autophagy and by pinocytosis of the culture medium (Fig. 3a). We then determined whether activation of Stat3 by OSM resulted in a stimulation of the biogenesis of such vesicles. This was indeed the case with OSM treatment resulting in a 1.7-fold increase in the volume fraction of degradative vesicles after 72 h treatment as determined by TEM (Fig. 3 b-d).

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