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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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MFGs are endocytosed by mammary epithelial cells. (a) Staining for MFG membrane protein BTN (red) in 10 d lactating and 24 h involuting mammary gland. (b) Number of butyrophilin-positive structures per nuclei quantified in control and Stat3 knockout 24 h involuting mammary glands. Bars represent means +/− s.e.m. of n = 3 mice per genotype, with 34 alveoli counted per animal (*p<0.05; Student’s t-test). Statistics source data can be found in the associated worksheet in Supplementary Table 3. E-cadherin staining is green. Images are representative of three independent biological repeats. (c) Microarray analysis of twelve different timepoints during the mammary gland cycle showing expression of BTN and XDH – “Mammary Gland Pregnancy Cycle Data (.xls)” (http://www.path.cam.ac.uk/~madgroup/microarraysummary.shtml). (d) Western blot for BTN (70 kDa) showing BTN downregulation at 24 h involution in control but not in Stat3 knockout samples. Asterisk indicates a spurious band. Lanes show samples from independent biological repeats. Scale bars: (a) low magnification and (b) = 20 μm, (a) high magnification = 10 μm. Uncropped images of blots appear in supplementary figure 5.
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Figure 6: MFGs are endocytosed by mammary epithelial cells. (a) Staining for MFG membrane protein BTN (red) in 10 d lactating and 24 h involuting mammary gland. (b) Number of butyrophilin-positive structures per nuclei quantified in control and Stat3 knockout 24 h involuting mammary glands. Bars represent means +/− s.e.m. of n = 3 mice per genotype, with 34 alveoli counted per animal (*p<0.05; Student’s t-test). Statistics source data can be found in the associated worksheet in Supplementary Table 3. E-cadherin staining is green. Images are representative of three independent biological repeats. (c) Microarray analysis of twelve different timepoints during the mammary gland cycle showing expression of BTN and XDH – “Mammary Gland Pregnancy Cycle Data (.xls)” (http://www.path.cam.ac.uk/~madgroup/microarraysummary.shtml). (d) Western blot for BTN (70 kDa) showing BTN downregulation at 24 h involution in control but not in Stat3 knockout samples. Asterisk indicates a spurious band. Lanes show samples from independent biological repeats. Scale bars: (a) low magnification and (b) = 20 μm, (a) high magnification = 10 μm. Uncropped images of blots appear in supplementary figure 5.

Mentions: In the process of being secreted, MFGs become coated with BTN, which is essential for their secretion38. BTN can be visualised as a thin ring surrounding MFGs secreted into the alveolar lumen at day 10 lactation (Fig. 6a). Notably, unsecreted lipid droplets within the mammary epithelium do not stain for BTN38. At 24 h involution, the appearance of MFGs coated with BTN becomes strikingly evident within the mammary epithelium (Fig. 6a). This is Stat3 dependent as considerably fewer MFGs coated with BTN are evident in Stat3fl/fl;BLG-Cre glands at 24 h involution (Fig. 6b) indicating reduced uptake of MFGs. Interestingly, microarray analysis indicates that expression of BTN and XDH is suppressed at the onset of involution (Fig. 6c) and 39 (www.path.cam.ac.uk/~madgroup). Quantitative PCR analysis shows these genes are upregulated in Stat3 KO mammary gland at 24 h involution, suggesting Stat3 represses XDH and BTN transcription (Supplementary Table 2). Immunoblot of Stat3 knockout and control mammary tissue corroborates the role of Stat3 in suppressing BTN expression (Fig. 6d).


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)

MFGs are endocytosed by mammary epithelial cells. (a) Staining for MFG membrane protein BTN (red) in 10 d lactating and 24 h involuting mammary gland. (b) Number of butyrophilin-positive structures per nuclei quantified in control and Stat3 knockout 24 h involuting mammary glands. Bars represent means +/− s.e.m. of n = 3 mice per genotype, with 34 alveoli counted per animal (*p<0.05; Student’s t-test). Statistics source data can be found in the associated worksheet in Supplementary Table 3. E-cadherin staining is green. Images are representative of three independent biological repeats. (c) Microarray analysis of twelve different timepoints during the mammary gland cycle showing expression of BTN and XDH – “Mammary Gland Pregnancy Cycle Data (.xls)” (http://www.path.cam.ac.uk/~madgroup/microarraysummary.shtml). (d) Western blot for BTN (70 kDa) showing BTN downregulation at 24 h involution in control but not in Stat3 knockout samples. Asterisk indicates a spurious band. Lanes show samples from independent biological repeats. Scale bars: (a) low magnification and (b) = 20 μm, (a) high magnification = 10 μm. Uncropped images of blots appear in supplementary figure 5.
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Figure 6: MFGs are endocytosed by mammary epithelial cells. (a) Staining for MFG membrane protein BTN (red) in 10 d lactating and 24 h involuting mammary gland. (b) Number of butyrophilin-positive structures per nuclei quantified in control and Stat3 knockout 24 h involuting mammary glands. Bars represent means +/− s.e.m. of n = 3 mice per genotype, with 34 alveoli counted per animal (*p<0.05; Student’s t-test). Statistics source data can be found in the associated worksheet in Supplementary Table 3. E-cadherin staining is green. Images are representative of three independent biological repeats. (c) Microarray analysis of twelve different timepoints during the mammary gland cycle showing expression of BTN and XDH – “Mammary Gland Pregnancy Cycle Data (.xls)” (http://www.path.cam.ac.uk/~madgroup/microarraysummary.shtml). (d) Western blot for BTN (70 kDa) showing BTN downregulation at 24 h involution in control but not in Stat3 knockout samples. Asterisk indicates a spurious band. Lanes show samples from independent biological repeats. Scale bars: (a) low magnification and (b) = 20 μm, (a) high magnification = 10 μm. Uncropped images of blots appear in supplementary figure 5.
Mentions: In the process of being secreted, MFGs become coated with BTN, which is essential for their secretion38. BTN can be visualised as a thin ring surrounding MFGs secreted into the alveolar lumen at day 10 lactation (Fig. 6a). Notably, unsecreted lipid droplets within the mammary epithelium do not stain for BTN38. At 24 h involution, the appearance of MFGs coated with BTN becomes strikingly evident within the mammary epithelium (Fig. 6a). This is Stat3 dependent as considerably fewer MFGs coated with BTN are evident in Stat3fl/fl;BLG-Cre glands at 24 h involution (Fig. 6b) indicating reduced uptake of MFGs. Interestingly, microarray analysis indicates that expression of BTN and XDH is suppressed at the onset of involution (Fig. 6c) and 39 (www.path.cam.ac.uk/~madgroup). Quantitative PCR analysis shows these genes are upregulated in Stat3 KO mammary gland at 24 h involution, suggesting Stat3 represses XDH and BTN transcription (Supplementary Table 2). Immunoblot of Stat3 knockout and control mammary tissue corroborates the role of Stat3 in suppressing BTN expression (Fig. 6d).

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