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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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Uptake of MFGs occurs via macropinocytosis and phagocytosis. (a) MFGs are seen in the alveolar lumen (arrowheads). The cytosolic ring bound by milk fat globule membrane is inherited by the fat droplet as it is secreted through the apical plasmalemma (arrows). (b) Ruffles on the apical plasma membrane are indicative of macropinocytosis (arrowheads). (c) Lipid droplets (arrowhead) are present within milk laden macropinosomes in alveolar epithelium. (d) Phagocytosed milk fat droplets can be identified within epithelium by the presence of MFG associated cytosol and milk fat globule membrane (arrowheads). (e) Another example of a large phagocytosed MFG is shown by arrowheads. The boxed area is enlarged (f) to show the phagosomal membrane (arrowheads) in close apposition to the milk fat globule membrane (arrows). (g) The highly phagocytic nature of alveolar epithelium was demonstrated using cathepsin L KO (Ctsl KO) mice that were force involuted for 72 h. In comparison to wild type controls, cathepsin L KO mammary glands showed accumulation of dead cells within phagosomes (arrowhead). This phagosome is fusing with other vacuoles (arrow). n = nucleus. (h) Staining for the phagocytosis-bridging molecule MFG-E8 (red) and E-cadherin (green) in MFG-E8 heterozygous (control) and knockout mice. Images are representative of three independent biological repeats. (i) Cytosolic cathepsin activity in MFG-E8 control and knockout mice at 24 h involution. Box and whisker plot of n = 12 and n=11 cytoplasmic fractions (mice) for control and knockout groups respectively, with 2 technical replicates performed per fraction (p=0.0173, Student’s t-test). Statistics source data can be found in the associated worksheet in Supplementary Table 3. Scale bars: (a)-(d) = 1 μm, (e) = 4 μm and (g) = 2 μm, (f) = 500 nm, (h) = 20 μm.
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Figure 7: Uptake of MFGs occurs via macropinocytosis and phagocytosis. (a) MFGs are seen in the alveolar lumen (arrowheads). The cytosolic ring bound by milk fat globule membrane is inherited by the fat droplet as it is secreted through the apical plasmalemma (arrows). (b) Ruffles on the apical plasma membrane are indicative of macropinocytosis (arrowheads). (c) Lipid droplets (arrowhead) are present within milk laden macropinosomes in alveolar epithelium. (d) Phagocytosed milk fat droplets can be identified within epithelium by the presence of MFG associated cytosol and milk fat globule membrane (arrowheads). (e) Another example of a large phagocytosed MFG is shown by arrowheads. The boxed area is enlarged (f) to show the phagosomal membrane (arrowheads) in close apposition to the milk fat globule membrane (arrows). (g) The highly phagocytic nature of alveolar epithelium was demonstrated using cathepsin L KO (Ctsl KO) mice that were force involuted for 72 h. In comparison to wild type controls, cathepsin L KO mammary glands showed accumulation of dead cells within phagosomes (arrowhead). This phagosome is fusing with other vacuoles (arrow). n = nucleus. (h) Staining for the phagocytosis-bridging molecule MFG-E8 (red) and E-cadherin (green) in MFG-E8 heterozygous (control) and knockout mice. Images are representative of three independent biological repeats. (i) Cytosolic cathepsin activity in MFG-E8 control and knockout mice at 24 h involution. Box and whisker plot of n = 12 and n=11 cytoplasmic fractions (mice) for control and knockout groups respectively, with 2 technical replicates performed per fraction (p=0.0173, Student’s t-test). Statistics source data can be found in the associated worksheet in Supplementary Table 3. Scale bars: (a)-(d) = 1 μm, (e) = 4 μm and (g) = 2 μm, (f) = 500 nm, (h) = 20 μm.

Mentions: As demonstrated by BTN staining in Figure 6, MFGs observed in the alveolar lumen with TEM are surrounded by a thin rim of plasma membrane-encased cytosol and the MFG membrane (Fig. 7a). This latter structure is acquired by the lipid droplet during the secretion process from the apical surface of the epithelium13 and is direct evidence that intracellular lipid droplets are taken up by an endocytic pathway. Indication of macropinocytosis is seen at the plasmalemma, where membranous ruffles capture luminal fluid (Fig. 7b). In agreement with this, macropinosomes, some containing lipid droplets, are seen within the epithelium (Fig. 7c). However, large MFGs that have come from the alveolar lumen are also taken up by phagocytosis. MFGs are seen inside tightly fitting phagosomes within the epithelium (Fig. 7d-f). The luminal origin of fat droplets (that can measure 8 μm in diameter) is confirmed by the presence of the MFG membrane that is found in close proximity to the limiting phagosomal membrane (Fig. 7f). The highly phagocytic nature of involuting mammary gland epithelium is seen clearly in cathepsin L knockout mice that, in contrast to control animals, cannot efficiently degrade cell corpses within the phagolysosome, thereby allowing visualisation of phagocytosed material (Fig. 7g). Further support that mammary epithelial cells become phagocytes41 comes from expression of the recognition molecule CD14, on the luminal surface in a Stat3 dependent manner at 24 h involution42. These data unequivocally demonstrate that mammary epithelial cells become phagocytes during involution and thus deliver MFGs to lysosomes.


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)

Uptake of MFGs occurs via macropinocytosis and phagocytosis. (a) MFGs are seen in the alveolar lumen (arrowheads). The cytosolic ring bound by milk fat globule membrane is inherited by the fat droplet as it is secreted through the apical plasmalemma (arrows). (b) Ruffles on the apical plasma membrane are indicative of macropinocytosis (arrowheads). (c) Lipid droplets (arrowhead) are present within milk laden macropinosomes in alveolar epithelium. (d) Phagocytosed milk fat droplets can be identified within epithelium by the presence of MFG associated cytosol and milk fat globule membrane (arrowheads). (e) Another example of a large phagocytosed MFG is shown by arrowheads. The boxed area is enlarged (f) to show the phagosomal membrane (arrowheads) in close apposition to the milk fat globule membrane (arrows). (g) The highly phagocytic nature of alveolar epithelium was demonstrated using cathepsin L KO (Ctsl KO) mice that were force involuted for 72 h. In comparison to wild type controls, cathepsin L KO mammary glands showed accumulation of dead cells within phagosomes (arrowhead). This phagosome is fusing with other vacuoles (arrow). n = nucleus. (h) Staining for the phagocytosis-bridging molecule MFG-E8 (red) and E-cadherin (green) in MFG-E8 heterozygous (control) and knockout mice. Images are representative of three independent biological repeats. (i) Cytosolic cathepsin activity in MFG-E8 control and knockout mice at 24 h involution. Box and whisker plot of n = 12 and n=11 cytoplasmic fractions (mice) for control and knockout groups respectively, with 2 technical replicates performed per fraction (p=0.0173, Student’s t-test). Statistics source data can be found in the associated worksheet in Supplementary Table 3. Scale bars: (a)-(d) = 1 μm, (e) = 4 μm and (g) = 2 μm, (f) = 500 nm, (h) = 20 μm.
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Figure 7: Uptake of MFGs occurs via macropinocytosis and phagocytosis. (a) MFGs are seen in the alveolar lumen (arrowheads). The cytosolic ring bound by milk fat globule membrane is inherited by the fat droplet as it is secreted through the apical plasmalemma (arrows). (b) Ruffles on the apical plasma membrane are indicative of macropinocytosis (arrowheads). (c) Lipid droplets (arrowhead) are present within milk laden macropinosomes in alveolar epithelium. (d) Phagocytosed milk fat droplets can be identified within epithelium by the presence of MFG associated cytosol and milk fat globule membrane (arrowheads). (e) Another example of a large phagocytosed MFG is shown by arrowheads. The boxed area is enlarged (f) to show the phagosomal membrane (arrowheads) in close apposition to the milk fat globule membrane (arrows). (g) The highly phagocytic nature of alveolar epithelium was demonstrated using cathepsin L KO (Ctsl KO) mice that were force involuted for 72 h. In comparison to wild type controls, cathepsin L KO mammary glands showed accumulation of dead cells within phagosomes (arrowhead). This phagosome is fusing with other vacuoles (arrow). n = nucleus. (h) Staining for the phagocytosis-bridging molecule MFG-E8 (red) and E-cadherin (green) in MFG-E8 heterozygous (control) and knockout mice. Images are representative of three independent biological repeats. (i) Cytosolic cathepsin activity in MFG-E8 control and knockout mice at 24 h involution. Box and whisker plot of n = 12 and n=11 cytoplasmic fractions (mice) for control and knockout groups respectively, with 2 technical replicates performed per fraction (p=0.0173, Student’s t-test). Statistics source data can be found in the associated worksheet in Supplementary Table 3. Scale bars: (a)-(d) = 1 μm, (e) = 4 μm and (g) = 2 μm, (f) = 500 nm, (h) = 20 μm.
Mentions: As demonstrated by BTN staining in Figure 6, MFGs observed in the alveolar lumen with TEM are surrounded by a thin rim of plasma membrane-encased cytosol and the MFG membrane (Fig. 7a). This latter structure is acquired by the lipid droplet during the secretion process from the apical surface of the epithelium13 and is direct evidence that intracellular lipid droplets are taken up by an endocytic pathway. Indication of macropinocytosis is seen at the plasmalemma, where membranous ruffles capture luminal fluid (Fig. 7b). In agreement with this, macropinosomes, some containing lipid droplets, are seen within the epithelium (Fig. 7c). However, large MFGs that have come from the alveolar lumen are also taken up by phagocytosis. MFGs are seen inside tightly fitting phagosomes within the epithelium (Fig. 7d-f). The luminal origin of fat droplets (that can measure 8 μm in diameter) is confirmed by the presence of the MFG membrane that is found in close proximity to the limiting phagosomal membrane (Fig. 7f). The highly phagocytic nature of involuting mammary gland epithelium is seen clearly in cathepsin L knockout mice that, in contrast to control animals, cannot efficiently degrade cell corpses within the phagolysosome, thereby allowing visualisation of phagocytosed material (Fig. 7g). Further support that mammary epithelial cells become phagocytes41 comes from expression of the recognition molecule CD14, on the luminal surface in a Stat3 dependent manner at 24 h involution42. These data unequivocally demonstrate that mammary epithelial cells become phagocytes during involution and thus deliver MFGs to lysosomes.

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