Stat3 controls cell death during mammary gland involution by regulating uptake of milk fat globules and lysosomal membrane permeabilization

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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Figure 1: Enlargement of the lysosomal compartment and digestion of triglyceride in the regressing mammary gland.
Figure 2: Ultrastructural analysis of lysosomal vacuoles and cargo delivery.
Figure 3: Vesicular biogenesis in EpH4 cells.
Figure 4: Free fatty acids are increased during involution and can cause death in vitro.
Figure 5: Free fatty acids induce LMP in vitro.
Figure 6: MFGs are endocytosed by mammary epithelial cells.
Figure 7: Uptake of MFGs occurs by macropinocytosis and phagocytosis.
Figure 8: A schematic model illustrating the role of Stat3 in regulating the transition from a secretory to a phagocytic cell phenotype at the onset of involution.

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Acknowledgements

We thank H. Skelton for assistance with histology, A. Gilmore (University of Manchester, UK) for the Bax–GFP construct, I. Mather (University of Maryland, USA) for the anti-BTN antibody and useful advice, and D. Neal, S. Felisbino and S. Hawkins (CRUK Cambridge Institute, University of Cambridge, UK) for providing mouse prostate tissue and advice. We thank also T. Reinheckel for providing the cathepsin L KO mice. In addition, we thank A. Tolkovsky and Z. Zakeri for helpful discussions. This work was supported by a grant from the Medical Research Council programme grant no. MR/J001023/1 (T.J.S. and B.L-L.) and a Cancer Research UK Cambridge Cancer Centre PhD studentship (H.K.R.).

Author information

T.J.S. and B.L-L. carried out most of the experiments, H.K.R. contributed the cathepsin L−/− tissue samples, A.R-M. provided the prostate samples, J.S. carried out the TEM and immunogold analysis and assisted in data interpretation. T.J.S., B.L-L. and C.J.W. designed the work, analysed the data and wrote the manuscript.

Correspondence to Christine J. Watson.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Cathepsin D and LAMP2 staining in the involuting mammary gland.

(a) Staining for cathepsin D (red) in the lactating and involuting gland. Three animals were assessed per condition. (b) The pro-form of cathepsin D was detected at approximately 46 kDa and higher levels were present at 24 h involution compared to lactating mammary glands. There was no difference between control (C) and Stat3 knockout (KO). Lanes represent independent biological samples. (c) LAMP2 staining (red) is detected lining large vacuolar structures in the control 24 h involuting mammary gland but not in the lactating mammary gland. This becomes more apparent at 48 h and 72 h. In the Stat3 KO mammary gland, LAMP2-positive vacuoles are only seen from 48 h onwards. One animal per condition was analysed. (d) Confocal images displaying immunostaining for cathepsin D (red) is shown in grey-scale and merged with staining for triglyceride (lipidtox, green). Arrowheads show lipid droplets inside lysosomal vesicles. Three animals were used. Nuclei are visualized by Hoechst (blue). Scale bars = 20 μm.

Supplementary Figure 2

(a) Milk induces lysosomal lipid accumulation. Confocal images show Lysotracker red staining overlapping with that for triglyceride (lipidtox, green) (colocalization shown by arrowheads). Four representative images from two independent experiments displayed. Nuclei are stained with Hoechst (blue). Scale bars first three rows = 1 μm, 4th row = 2 μm. (b) Free fatty acids induce cell death. Staining for triglyceride (lipidtox, green) in EpH4 cells treated overnight with 1 mM oleic acid (OA) and palmitic acid (PA) showing lipid accumulation in EpH4 cells. Nuclei are stained with Hoechst (blue); Scale bars = 10 μm. Brightfield images showing cytotoxicity in fatty acid treated EpH4 cells; Scale bars = 100 μm. (c) Fatty acid induced cell death was assessed by trypan blue positivity. Means ± s.e.m. from n = three independent experiments with 2–3 technical replicates performed per experiment shown (P < 0.05, one-way ANOVA, Dunnett’s Multiple Comparison post-test). For raw values, see the corresponding worksheet in Supplementary Table 3.

Supplementary Figure 3

(a) Optimisation of digitonin cytosol extraction assay. EpH4 cells were extracted with increasing concentrations of digitonin and cathepsin activity assayed over time with the synthetic substrate Z-Phe-Arg-AMC. Total activity was measured by extraction with 0.1% TritonX-100. A digitonin concentration of 25 μg ml−1 was selected for cytosol extraction assays. All data is plotted, optimisation performed on one occasion. (b) Fatty acids induce deacidification of the lysosomal compartment. A population of low Lysotracker Red staining (region R8) is induced with 1 mM OA or PA, indicative of de-acidification of lysosomes. Cells treated with 1 mM PA also display a population with higher lysotracker red staining. Quantification of n = four independent experiments as described in (b). Means ± s.e.m. are shown, associated statistics source data can be found in the corresponding worksheet in Supplementary Table 3. (c) OA and PA (500 μM) treated cells showing populations of Lysotracker Red fluorescence (R13), with low levels indicative of de-acidification of lysosomes (n = 1 (PA) and 2 (OA) independent experiments raw values can be found in the corresponding worksheet in Supplementary Table 3).

Supplementary Figure 4 Fatty acid treatment does not result in Bax translocation to lysosomes.

(a) EpH4 cells were transfected with GFP-Bax (green) and treated with ethanol, 500 μm oleic acid (OA), palmitic acid (PA) overnight prior to fixation and LAMP2 immunostaining (red). Cells were treated with 30 ng ml−1 TNFα and 10 μg ml−1 cycloheximide for 6.5 h in serum free conditions as a control. No obvious lysosomal co-localization was observed under these conditions. Two representative examples from all conditions displayed, experiment performed once. (b) GFP-Bax transfected EpH4 cells treated with TNFα and cycloheximide or 1 μM staurosporine as indicated for 6.5 h were fixed and immunostained for AIF (red) and show mitochondrial localization of Bax under these conditions. Nuclei are stained with Hoechst (blue). Scale bars = 10 μm.

Supplementary Figure 5 Uncropped blots and TLC plates showing experiments that appeared in figures as well as biological replicates that were not shown in main figures.

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Sargeant, T., Lloyd-Lewis, B., Resemann, H. et al. Stat3 controls cell death during mammary gland involution by regulating uptake of milk fat globules and lysosomal membrane permeabilization. Nat Cell Biol 16, 1057–1068 (2014). https://doi.org/10.1038/ncb3043

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