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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 induce LMP in vitro. (a) Cathepsin activity assay on EpH4 cytosolic extracts treated with 500 μM fatty acids for 16 h demonstrating release of cathepsins to the cytosol by OA. Means +/−s.e.m of n = 4 independent experiments (*p<0.05; Kruskal-Wallis test, Dunn’s multiple comparison post-test). (b) Representative sample from (a) was analysed by western blot for Cathepsin L in total and cytosolic EpH4 extracts, showing release of cathepsin L to the cytosol with 500 μM OA. M denotes marker lane. (c) Schematic of iron nanoparticle lysosomal purification protocol. (d) Optimisation of iron nanoparticle mediated purification of lysosomes from EpH4 cells. Cells were labelled and chased as indicated followed by extraction and immunoblotting. LAMP2, COXIV, RAB5 and RAB7 are shown as markers for lysosomes, mitochondria, early endosomes and late endosomes respectively. Time-course optimisation performed on one occasion. pns; post nuclear supernatant, sn; post magnetic supernatant, mp; magnetic pellet. (e) Cathepsin L immunoblot showing extent of leakiness from the lysosomal pellet (P) into the supernatant (S) at 30 min with 70 μM OA or PA. Representative blot from three independent experiments. LAMP2 was used as a lysosomal marker and COXIV used to show undetectable mitochondrial contamination. (f) Structures of free fatty acids used in this study. Chemical structures for palmitic acid, stearic acid and oleic acid are depicted. Statistics source data can be found in the corresponding worksheet in Supplementary Table 3. Uncropped images of blots appear in supplementary figure 5.
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Figure 5: Free fatty acids induce LMP in vitro. (a) Cathepsin activity assay on EpH4 cytosolic extracts treated with 500 μM fatty acids for 16 h demonstrating release of cathepsins to the cytosol by OA. Means +/−s.e.m of n = 4 independent experiments (*p<0.05; Kruskal-Wallis test, Dunn’s multiple comparison post-test). (b) Representative sample from (a) was analysed by western blot for Cathepsin L in total and cytosolic EpH4 extracts, showing release of cathepsin L to the cytosol with 500 μM OA. M denotes marker lane. (c) Schematic of iron nanoparticle lysosomal purification protocol. (d) Optimisation of iron nanoparticle mediated purification of lysosomes from EpH4 cells. Cells were labelled and chased as indicated followed by extraction and immunoblotting. LAMP2, COXIV, RAB5 and RAB7 are shown as markers for lysosomes, mitochondria, early endosomes and late endosomes respectively. Time-course optimisation performed on one occasion. pns; post nuclear supernatant, sn; post magnetic supernatant, mp; magnetic pellet. (e) Cathepsin L immunoblot showing extent of leakiness from the lysosomal pellet (P) into the supernatant (S) at 30 min with 70 μM OA or PA. Representative blot from three independent experiments. LAMP2 was used as a lysosomal marker and COXIV used to show undetectable mitochondrial contamination. (f) Structures of free fatty acids used in this study. Chemical structures for palmitic acid, stearic acid and oleic acid are depicted. Statistics source data can be found in the corresponding worksheet in Supplementary Table 3. Uncropped images of blots appear in supplementary figure 5.

Mentions: Cathepsin activity assays and immunoblot analysis on digitonin-extracted cytosolic fractions (optimised for digitonin concentration to avoid damage to lysosomes; Supplementary Fig. 3a) of fatty acid treated EpH4 cells revealed that oleic acid caused a significant release of cathepsins to the cytosol (Fig. 5a). Immunoblot analysis of these samples further demonstrated that only oleic acid induces significant release of cathepsin L to the cytosol (Fig. 5b). In contrast, palmitic acid treatment resulted in limited release of cathepsins to the cytosol, which was not significant. Importantly stearic acid, the saturated carbon chain counterpart of oleic acid, also had no effect on cathepsin release as measured by cytosolic cathepsin activity assays and immunoblot (Fig. 5a,b). Staining with the lysosomotropic dye LysoTracker® Red showed that both oleic and palmitic acid treatment resulted in an appearance of a population of cells with low Lysotracker® intensities, indicative of lysosomal de-acidification (Supplementary Fig. 3b,c). Of note, fatty acid treatment did not appear to induce the translocation of the pore-forming protein Bax to lysosomes in EpH4 cells (Supplementary Fig. 4). Therefore, these data clearly demonstrate that lysosomal membranes can be permeabilised efficiently by oleic acid. However, an effect of palmitic acid cannot be completely excluded.


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 induce LMP in vitro. (a) Cathepsin activity assay on EpH4 cytosolic extracts treated with 500 μM fatty acids for 16 h demonstrating release of cathepsins to the cytosol by OA. Means +/−s.e.m of n = 4 independent experiments (*p<0.05; Kruskal-Wallis test, Dunn’s multiple comparison post-test). (b) Representative sample from (a) was analysed by western blot for Cathepsin L in total and cytosolic EpH4 extracts, showing release of cathepsin L to the cytosol with 500 μM OA. M denotes marker lane. (c) Schematic of iron nanoparticle lysosomal purification protocol. (d) Optimisation of iron nanoparticle mediated purification of lysosomes from EpH4 cells. Cells were labelled and chased as indicated followed by extraction and immunoblotting. LAMP2, COXIV, RAB5 and RAB7 are shown as markers for lysosomes, mitochondria, early endosomes and late endosomes respectively. Time-course optimisation performed on one occasion. pns; post nuclear supernatant, sn; post magnetic supernatant, mp; magnetic pellet. (e) Cathepsin L immunoblot showing extent of leakiness from the lysosomal pellet (P) into the supernatant (S) at 30 min with 70 μM OA or PA. Representative blot from three independent experiments. LAMP2 was used as a lysosomal marker and COXIV used to show undetectable mitochondrial contamination. (f) Structures of free fatty acids used in this study. Chemical structures for palmitic acid, stearic acid and oleic acid are depicted. Statistics source data can be found in the corresponding worksheet in Supplementary Table 3. Uncropped images of blots appear in supplementary figure 5.
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Related In: Results  -  Collection

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Figure 5: Free fatty acids induce LMP in vitro. (a) Cathepsin activity assay on EpH4 cytosolic extracts treated with 500 μM fatty acids for 16 h demonstrating release of cathepsins to the cytosol by OA. Means +/−s.e.m of n = 4 independent experiments (*p<0.05; Kruskal-Wallis test, Dunn’s multiple comparison post-test). (b) Representative sample from (a) was analysed by western blot for Cathepsin L in total and cytosolic EpH4 extracts, showing release of cathepsin L to the cytosol with 500 μM OA. M denotes marker lane. (c) Schematic of iron nanoparticle lysosomal purification protocol. (d) Optimisation of iron nanoparticle mediated purification of lysosomes from EpH4 cells. Cells were labelled and chased as indicated followed by extraction and immunoblotting. LAMP2, COXIV, RAB5 and RAB7 are shown as markers for lysosomes, mitochondria, early endosomes and late endosomes respectively. Time-course optimisation performed on one occasion. pns; post nuclear supernatant, sn; post magnetic supernatant, mp; magnetic pellet. (e) Cathepsin L immunoblot showing extent of leakiness from the lysosomal pellet (P) into the supernatant (S) at 30 min with 70 μM OA or PA. Representative blot from three independent experiments. LAMP2 was used as a lysosomal marker and COXIV used to show undetectable mitochondrial contamination. (f) Structures of free fatty acids used in this study. Chemical structures for palmitic acid, stearic acid and oleic acid are depicted. Statistics source data can be found in the corresponding worksheet in Supplementary Table 3. Uncropped images of blots appear in supplementary figure 5.
Mentions: Cathepsin activity assays and immunoblot analysis on digitonin-extracted cytosolic fractions (optimised for digitonin concentration to avoid damage to lysosomes; Supplementary Fig. 3a) of fatty acid treated EpH4 cells revealed that oleic acid caused a significant release of cathepsins to the cytosol (Fig. 5a). Immunoblot analysis of these samples further demonstrated that only oleic acid induces significant release of cathepsin L to the cytosol (Fig. 5b). In contrast, palmitic acid treatment resulted in limited release of cathepsins to the cytosol, which was not significant. Importantly stearic acid, the saturated carbon chain counterpart of oleic acid, also had no effect on cathepsin release as measured by cytosolic cathepsin activity assays and immunoblot (Fig. 5a,b). Staining with the lysosomotropic dye LysoTracker® Red showed that both oleic and palmitic acid treatment resulted in an appearance of a population of cells with low Lysotracker® intensities, indicative of lysosomal de-acidification (Supplementary Fig. 3b,c). Of note, fatty acid treatment did not appear to induce the translocation of the pore-forming protein Bax to lysosomes in EpH4 cells (Supplementary Fig. 4). Therefore, these data clearly demonstrate that lysosomal membranes can be permeabilised efficiently by oleic acid. However, an effect of palmitic acid cannot be completely excluded.

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