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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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Ultrastructural analysis of lysosomal vacuoles and cargo delivery. (a) Transmission electron microscopy of wild type 10 d lactation, 24 h involution and Stat3 knockout glands at 24 h involution. (b) Immunogold staining for cathepsin D within large vacuoles at 24 h involution co-localising with degrading material (arrowheads). (c) Autophagic vesicles fusing with large vacuoles (arrowheads), (d) large macropinosomes full of milk inside epithelial cells and milk containing vesicles (e) are seen to fuse with larger vacuoles. (f-i) Membrane bound lipid fuses with vacuoles and can be observed inside lysosomal structures (arrowheads) that contain lipofuscin-like material (arrows). Three animals assessed for all conditions except for cathepsin D-immuno gold staining, where one animal was analysed. Scale bars: (a) epithelial cells = 2 μm, (a) mitochondria, (b) and (c) high magnification = 500 nm, (c) low magnification, (d-i) = 1 μm.
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Figure 2: Ultrastructural analysis of lysosomal vacuoles and cargo delivery. (a) Transmission electron microscopy of wild type 10 d lactation, 24 h involution and Stat3 knockout glands at 24 h involution. (b) Immunogold staining for cathepsin D within large vacuoles at 24 h involution co-localising with degrading material (arrowheads). (c) Autophagic vesicles fusing with large vacuoles (arrowheads), (d) large macropinosomes full of milk inside epithelial cells and milk containing vesicles (e) are seen to fuse with larger vacuoles. (f-i) Membrane bound lipid fuses with vacuoles and can be observed inside lysosomal structures (arrowheads) that contain lipofuscin-like material (arrows). Three animals assessed for all conditions except for cathepsin D-immuno gold staining, where one animal was analysed. Scale bars: (a) epithelial cells = 2 μm, (a) mitochondria, (b) and (c) high magnification = 500 nm, (c) low magnification, (d-i) = 1 μm.

Mentions: In order to analyse these structures in greater detail, we performed transmission electron microscopy (TEM) on perfusion fixed mammary tissue from day10 lactation and 24h involution. This revealed the presence of strikingly large, membrane-bound vesicles, many of which were at least the size of nuclei with some occupying most of the cell (Fig. 2a). These have been observed also by TEM at day 3 involution27. Notably, these were present only during involution and not at day10 lactation (Fig. 2a). Once again, in agreement with our observations on LAMP2-positive vacuoles, these large vacuoles were much less abundant in Stat3 knockout tissue (Fig. 2a). Additionally, we noted that mitochondria acquire an elongated morphology upon the switch from lactation to involution that occurred regardless of Stat3 status (Fig. 2a). This has previously been described as an adaptive response to a highly autophagic environment28. The presence of morphologically sound mitochondria, which appeared similar in both control and Stat3 deficient mammary tissue at 24h involution, further supports our previous data showing that cell death at this time is caspase independent17. Confirming results in Figure 1a and c, immunogold staining for cathepsin D showed this lysosomal enzyme to be focally associated with degrading material contained within vacuoles (Fig. 2b), revealing their lysosomal origin. Vacuoles containing autophagic contents (Fig. 2c), milk protein (Fid. 2d, e) and triglyceride (Fig. 2 f-i) were frequently observed to be in the process of fusion with other membrane bound structures (Fig. 2c, e and f), clearly demonstrating an active process of vesicle biogenesis. Furthermore, lipid was observed inside lysosomal structures that also contained electron-dense lipofuscin-like material (Fig. 2h, i), confirming hydrolysis of bulk triglyceride from lipid droplets in the lysosomal compartment.


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)

Ultrastructural analysis of lysosomal vacuoles and cargo delivery. (a) Transmission electron microscopy of wild type 10 d lactation, 24 h involution and Stat3 knockout glands at 24 h involution. (b) Immunogold staining for cathepsin D within large vacuoles at 24 h involution co-localising with degrading material (arrowheads). (c) Autophagic vesicles fusing with large vacuoles (arrowheads), (d) large macropinosomes full of milk inside epithelial cells and milk containing vesicles (e) are seen to fuse with larger vacuoles. (f-i) Membrane bound lipid fuses with vacuoles and can be observed inside lysosomal structures (arrowheads) that contain lipofuscin-like material (arrows). Three animals assessed for all conditions except for cathepsin D-immuno gold staining, where one animal was analysed. Scale bars: (a) epithelial cells = 2 μm, (a) mitochondria, (b) and (c) high magnification = 500 nm, (c) low magnification, (d-i) = 1 μm.
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Related In: Results  -  Collection

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Figure 2: Ultrastructural analysis of lysosomal vacuoles and cargo delivery. (a) Transmission electron microscopy of wild type 10 d lactation, 24 h involution and Stat3 knockout glands at 24 h involution. (b) Immunogold staining for cathepsin D within large vacuoles at 24 h involution co-localising with degrading material (arrowheads). (c) Autophagic vesicles fusing with large vacuoles (arrowheads), (d) large macropinosomes full of milk inside epithelial cells and milk containing vesicles (e) are seen to fuse with larger vacuoles. (f-i) Membrane bound lipid fuses with vacuoles and can be observed inside lysosomal structures (arrowheads) that contain lipofuscin-like material (arrows). Three animals assessed for all conditions except for cathepsin D-immuno gold staining, where one animal was analysed. Scale bars: (a) epithelial cells = 2 μm, (a) mitochondria, (b) and (c) high magnification = 500 nm, (c) low magnification, (d-i) = 1 μm.
Mentions: In order to analyse these structures in greater detail, we performed transmission electron microscopy (TEM) on perfusion fixed mammary tissue from day10 lactation and 24h involution. This revealed the presence of strikingly large, membrane-bound vesicles, many of which were at least the size of nuclei with some occupying most of the cell (Fig. 2a). These have been observed also by TEM at day 3 involution27. Notably, these were present only during involution and not at day10 lactation (Fig. 2a). Once again, in agreement with our observations on LAMP2-positive vacuoles, these large vacuoles were much less abundant in Stat3 knockout tissue (Fig. 2a). Additionally, we noted that mitochondria acquire an elongated morphology upon the switch from lactation to involution that occurred regardless of Stat3 status (Fig. 2a). This has previously been described as an adaptive response to a highly autophagic environment28. The presence of morphologically sound mitochondria, which appeared similar in both control and Stat3 deficient mammary tissue at 24h involution, further supports our previous data showing that cell death at this time is caspase independent17. Confirming results in Figure 1a and c, immunogold staining for cathepsin D showed this lysosomal enzyme to be focally associated with degrading material contained within vacuoles (Fig. 2b), revealing their lysosomal origin. Vacuoles containing autophagic contents (Fig. 2c), milk protein (Fid. 2d, e) and triglyceride (Fig. 2 f-i) were frequently observed to be in the process of fusion with other membrane bound structures (Fig. 2c, e and f), clearly demonstrating an active process of vesicle biogenesis. Furthermore, lipid was observed inside lysosomal structures that also contained electron-dense lipofuscin-like material (Fig. 2h, i), confirming hydrolysis of bulk triglyceride from lipid droplets in the lysosomal compartment.

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