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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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Enlargement of the lysosomal compartment and digestion of triglyceride in the regressing mammary gland. (a) Large secondary lysosomes are shown by cathepsin D staining (red, arrowheads) in different involuting tissues. Mouse uterus at 24 h post-partum and during late pregnancy, prostate gland at 7 d post castration and uncastrated and mammary gland at 24 h involution and 10 d lactation. Tissue was stained for cathepsin D (red) and E-cadherin (green). (b) Hoescht staining showing dead cells (arrowheads) in the involuting mouse uterus, prostate and mammary gland. Three independent biological repeats per condition for prostate and mammary glands, one biological repeat per condition for uterus. (c) Dual staining for LAMP2 and cathepsin D shows cathepsin D (red) localising to the inside of a LAMP2-positive (green) vacuole. Three independent biological repeats were assessed. (d) LAMP2 (red) and E-cadherin (green) staining in 10 d lactating and 24 h involuting mammary glands. Six independent biological repeats per condition analysed. (e) LAMP2-positive vacuoles (red) are found in the control but not the Stat3 KO mammary gland at 24 h involution. (f) Staining for LAMP2 at 24 h involution in control and Stat3 knockout animals was quantified. Bars represent means +/− s.e.m. of n = 3 mice per genotype with 12-13 fields counted per mouse (*p<0.05; Student’s t-test). Statistics source data can be found in the associated worksheet in Supplementary Table 3 (g) Confocal images showing that cathepsin D-positive vacuoles (arrowhead) contain lipid droplets (lipidtox staining, green) in the 24 h involuting mammary gland. Three independent biological repeats per condition analysed. Nuclei are visualised by Hoescht stain (blue). Scale bars: (a), (d), (e) and (g) = 20 μm, (b) and (c) = 10 μm.
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Figure 1: Enlargement of the lysosomal compartment and digestion of triglyceride in the regressing mammary gland. (a) Large secondary lysosomes are shown by cathepsin D staining (red, arrowheads) in different involuting tissues. Mouse uterus at 24 h post-partum and during late pregnancy, prostate gland at 7 d post castration and uncastrated and mammary gland at 24 h involution and 10 d lactation. Tissue was stained for cathepsin D (red) and E-cadherin (green). (b) Hoescht staining showing dead cells (arrowheads) in the involuting mouse uterus, prostate and mammary gland. Three independent biological repeats per condition for prostate and mammary glands, one biological repeat per condition for uterus. (c) Dual staining for LAMP2 and cathepsin D shows cathepsin D (red) localising to the inside of a LAMP2-positive (green) vacuole. Three independent biological repeats were assessed. (d) LAMP2 (red) and E-cadherin (green) staining in 10 d lactating and 24 h involuting mammary glands. Six independent biological repeats per condition analysed. (e) LAMP2-positive vacuoles (red) are found in the control but not the Stat3 KO mammary gland at 24 h involution. (f) Staining for LAMP2 at 24 h involution in control and Stat3 knockout animals was quantified. Bars represent means +/− s.e.m. of n = 3 mice per genotype with 12-13 fields counted per mouse (*p<0.05; Student’s t-test). Statistics source data can be found in the associated worksheet in Supplementary Table 3 (g) Confocal images showing that cathepsin D-positive vacuoles (arrowhead) contain lipid droplets (lipidtox staining, green) in the 24 h involuting mammary gland. Three independent biological repeats per condition analysed. Nuclei are visualised by Hoescht stain (blue). Scale bars: (a), (d), (e) and (g) = 20 μm, (b) and (c) = 10 μm.

Mentions: Our initial approach to investigate the hypothesis that Stat3 both enhances, and exploits, the lysosomal system to mediate cell death was to examine the lysosomal compartment during mammary gland regression in more detail. We noted the appearance of large cathepsin D-positive vacuoles upon initiation of involution (Fig. 1a). Significantly, similar vacuoles also appear upon induction of involution in the uterus and prostate (Fig. 1a), and were often larger than nuclei (> 5 μm). Importantly, such a dramatic increase in size is a factor that could, of itself, sensitise lysosomes to become leaky24. Notably, the morphology of dead cells in all three tissues was similar, being atypical for either classical apoptosis or necrosis (Fig. 1b), and characterised by hypercondensed nuclei and a complete absence of membrane blebbing18. This suggests a common mechanism of cell death during regression of hormone-dependent tissues.


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)

Enlargement of the lysosomal compartment and digestion of triglyceride in the regressing mammary gland. (a) Large secondary lysosomes are shown by cathepsin D staining (red, arrowheads) in different involuting tissues. Mouse uterus at 24 h post-partum and during late pregnancy, prostate gland at 7 d post castration and uncastrated and mammary gland at 24 h involution and 10 d lactation. Tissue was stained for cathepsin D (red) and E-cadherin (green). (b) Hoescht staining showing dead cells (arrowheads) in the involuting mouse uterus, prostate and mammary gland. Three independent biological repeats per condition for prostate and mammary glands, one biological repeat per condition for uterus. (c) Dual staining for LAMP2 and cathepsin D shows cathepsin D (red) localising to the inside of a LAMP2-positive (green) vacuole. Three independent biological repeats were assessed. (d) LAMP2 (red) and E-cadherin (green) staining in 10 d lactating and 24 h involuting mammary glands. Six independent biological repeats per condition analysed. (e) LAMP2-positive vacuoles (red) are found in the control but not the Stat3 KO mammary gland at 24 h involution. (f) Staining for LAMP2 at 24 h involution in control and Stat3 knockout animals was quantified. Bars represent means +/− s.e.m. of n = 3 mice per genotype with 12-13 fields counted per mouse (*p<0.05; Student’s t-test). Statistics source data can be found in the associated worksheet in Supplementary Table 3 (g) Confocal images showing that cathepsin D-positive vacuoles (arrowhead) contain lipid droplets (lipidtox staining, green) in the 24 h involuting mammary gland. Three independent biological repeats per condition analysed. Nuclei are visualised by Hoescht stain (blue). Scale bars: (a), (d), (e) and (g) = 20 μm, (b) and (c) = 10 μm.
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Figure 1: Enlargement of the lysosomal compartment and digestion of triglyceride in the regressing mammary gland. (a) Large secondary lysosomes are shown by cathepsin D staining (red, arrowheads) in different involuting tissues. Mouse uterus at 24 h post-partum and during late pregnancy, prostate gland at 7 d post castration and uncastrated and mammary gland at 24 h involution and 10 d lactation. Tissue was stained for cathepsin D (red) and E-cadherin (green). (b) Hoescht staining showing dead cells (arrowheads) in the involuting mouse uterus, prostate and mammary gland. Three independent biological repeats per condition for prostate and mammary glands, one biological repeat per condition for uterus. (c) Dual staining for LAMP2 and cathepsin D shows cathepsin D (red) localising to the inside of a LAMP2-positive (green) vacuole. Three independent biological repeats were assessed. (d) LAMP2 (red) and E-cadherin (green) staining in 10 d lactating and 24 h involuting mammary glands. Six independent biological repeats per condition analysed. (e) LAMP2-positive vacuoles (red) are found in the control but not the Stat3 KO mammary gland at 24 h involution. (f) Staining for LAMP2 at 24 h involution in control and Stat3 knockout animals was quantified. Bars represent means +/− s.e.m. of n = 3 mice per genotype with 12-13 fields counted per mouse (*p<0.05; Student’s t-test). Statistics source data can be found in the associated worksheet in Supplementary Table 3 (g) Confocal images showing that cathepsin D-positive vacuoles (arrowhead) contain lipid droplets (lipidtox staining, green) in the 24 h involuting mammary gland. Three independent biological repeats per condition analysed. Nuclei are visualised by Hoescht stain (blue). Scale bars: (a), (d), (e) and (g) = 20 μm, (b) and (c) = 10 μm.
Mentions: Our initial approach to investigate the hypothesis that Stat3 both enhances, and exploits, the lysosomal system to mediate cell death was to examine the lysosomal compartment during mammary gland regression in more detail. We noted the appearance of large cathepsin D-positive vacuoles upon initiation of involution (Fig. 1a). Significantly, similar vacuoles also appear upon induction of involution in the uterus and prostate (Fig. 1a), and were often larger than nuclei (> 5 μm). Importantly, such a dramatic increase in size is a factor that could, of itself, sensitise lysosomes to become leaky24. Notably, the morphology of dead cells in all three tissues was similar, being atypical for either classical apoptosis or necrosis (Fig. 1b), and characterised by hypercondensed nuclei and a complete absence of membrane blebbing18. This suggests a common mechanism of cell death during regression of hormone-dependent tissues.

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