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ZnT2 is a critical mediator of lysosomal-mediated cell death during early mammary gland involution.

Hennigar SR, Seo YA, Sharma S, Soybel DI, Kelleher SL - Sci Rep (2015)

Bottom Line: Zinc (Zn) activates both LCD and apoptosis in vitro.Following weaning, ZnT2 abundance increased in lysosomes and mitochondria, which paralleled Zn accumulation in each of these organelles.Our data implicate ZnT2 as a critical mediator of cell death during involution and importantly, that as an initial involution signal, TNFα redistributes ZnT2 to lysosomes to activate LCD.

View Article: PubMed Central - PubMed

Affiliation: Department of Nutritional Sciences, The Pennsylvania State University, University Park, PA, USA.

ABSTRACT
Mammary gland involution is the most dramatic example of physiological cell death. It occurs through an initial phase of lysosomal-mediated cell death (LCD) followed by mitochondrial-mediated apoptosis. Zinc (Zn) activates both LCD and apoptosis in vitro. The Zn transporter ZnT2 imports Zn into vesicles and mitochondria and ZnT2-overexpression activates cell death in mammary epithelial cells (MECs). We tested the hypothesis that ZnT2-mediated Zn transport is critical for mammary gland involution in mice. Following weaning, ZnT2 abundance increased in lysosomes and mitochondria, which paralleled Zn accumulation in each of these organelles. Adenoviral expression of ZnT2 in lactating mouse mammary glands in vivo increased Zn in lysosomes and mitochondria and activated LCD and apoptosis, promoting a profound reduction in MECs and alveoli. Injection of TNFα, a potent activator of early involution, into the mammary gland fat pads of lactating mice increased ZnT2 and Zn in lysosomes and activated premature involution. Exposure of cultured MECs to TNFα redistributed ZnT2 to lysosomes and increased lysosomal Zn, which activated lysosomal swelling, cathepsin B release, and LCD. Our data implicate ZnT2 as a critical mediator of cell death during involution and importantly, that as an initial involution signal, TNFα redistributes ZnT2 to lysosomes to activate LCD.

No MeSH data available.


Related in: MedlinePlus

ZnT2 accumulates Zn in lysosomes and mitochondria in mouse mammary glands during involution.(a) Mammary glands (~0.1–0.2 g) from virgin, lactating, and 24- and 48 h involuting mice were digested in nitric acid and Zn concentration was measured by atomic absorption spectroscopy. Data represent mean μg Zn/g tissue ± SD; n = 5/group, *P < 0.05, n.s., not significant. (b)–(e) Lysosome- and mitochondria-enriched fractions were isolated from lactating and 24- and 48 h involuting mammary glands by differential centrifugation. (b) Lysosomal Zn concentration from lactating and involuting mammary glands. Fractions were digested in nitric acid and concentration was measured by atomic absorption spectroscopy. Data represent mean μg Zn/mg protein ± SD; n = 4/group, *P < 0.05. (c) Representative immunoblot of ZnT2 and Lamp1 (lysosomal marker) in lysosome-enriched fractions isolated from lactating and involuting mammary glands. β-actin was used as a loading control and ratios of signal intensities are reported under the blot. (d) Isolated mitochondria-enriched fractions from lactating and involuting mammary glands were digested in nitric acid and Zn concentration was measured by atomic absorption spectroscopy. Data represent mean μg Zn/mg protein ± SD; n = 3/group, *P < 0.05. (e) Protein abundance of ZnT2 in mitochondria-enriched fractions isolated from lactating and involuting mammary glands was determined by immunoblot and expressed relative to the mitochondrial marker succinate dehydrogenase (SDH). Ratios of signal intensities are reported under the blot.
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f1: ZnT2 accumulates Zn in lysosomes and mitochondria in mouse mammary glands during involution.(a) Mammary glands (~0.1–0.2 g) from virgin, lactating, and 24- and 48 h involuting mice were digested in nitric acid and Zn concentration was measured by atomic absorption spectroscopy. Data represent mean μg Zn/g tissue ± SD; n = 5/group, *P < 0.05, n.s., not significant. (b)–(e) Lysosome- and mitochondria-enriched fractions were isolated from lactating and 24- and 48 h involuting mammary glands by differential centrifugation. (b) Lysosomal Zn concentration from lactating and involuting mammary glands. Fractions were digested in nitric acid and concentration was measured by atomic absorption spectroscopy. Data represent mean μg Zn/mg protein ± SD; n = 4/group, *P < 0.05. (c) Representative immunoblot of ZnT2 and Lamp1 (lysosomal marker) in lysosome-enriched fractions isolated from lactating and involuting mammary glands. β-actin was used as a loading control and ratios of signal intensities are reported under the blot. (d) Isolated mitochondria-enriched fractions from lactating and involuting mammary glands were digested in nitric acid and Zn concentration was measured by atomic absorption spectroscopy. Data represent mean μg Zn/mg protein ± SD; n = 3/group, *P < 0.05. (e) Protein abundance of ZnT2 in mitochondria-enriched fractions isolated from lactating and involuting mammary glands was determined by immunoblot and expressed relative to the mitochondrial marker succinate dehydrogenase (SDH). Ratios of signal intensities are reported under the blot.

Mentions: We previously reported that the mammary gland accumulates Zn during lactation21. Herein, we found that similar to calcium22, Zn accumulation in the mammary gland was further augmented during involution (Fig. 1a). To determine where Zn accumulated, we isolated subcellular fractions enriched in specific organelles using density fractionation. Organelle enrichment was confirmed by immunoblotting for specific organelle markers for mitochondria, lysosomes, and the endoplasmic reticulum/Golgi apparatus (Supplementary Fig. S1a). We found that the Zn concentration in lysosome-enriched fractions isolated from involuting mammary glands was significantly higher than identical fractions isolated from lactating mammary glands (Fig. 1b). Moreover, we noted that the abundance of ZnT2 in lysosome-enriched fractions was higher during the initial phase of involution (24 h post weaning) (Fig. 1c). Acid phosphatase (EC 3.1.3.2) is a lysosomal enzyme that requires Zn to hydrolyze the substrate, nitrophenyl phosphate23. Consistent with previous reports noting that acid phosphatase activity is highest during early involution and declines 48 h post-weaning24, we found that peak ZnT2 abundance in lysosomes corresponded with peak acid phosphatase activity (Supplementary Fig. S1b). Concurrently, Figure 1d shows that the Zn concentration of mitochondria increased as involution progressed which paralleled an increase in mitochondrial ZnT2 abundance (Fig. 1e). Although the abundance of ZnT2 in specific fractions was altered, this was not a consequence of changes in the total abundance of ZnT2 (Supplementary Fig. S2a). Taken together, these data suggest that ZnT2 expression is associated with Zn accumulation in lysosomes and mitochondria during the early stages of mammary gland involution.


ZnT2 is a critical mediator of lysosomal-mediated cell death during early mammary gland involution.

Hennigar SR, Seo YA, Sharma S, Soybel DI, Kelleher SL - Sci Rep (2015)

ZnT2 accumulates Zn in lysosomes and mitochondria in mouse mammary glands during involution.(a) Mammary glands (~0.1–0.2 g) from virgin, lactating, and 24- and 48 h involuting mice were digested in nitric acid and Zn concentration was measured by atomic absorption spectroscopy. Data represent mean μg Zn/g tissue ± SD; n = 5/group, *P < 0.05, n.s., not significant. (b)–(e) Lysosome- and mitochondria-enriched fractions were isolated from lactating and 24- and 48 h involuting mammary glands by differential centrifugation. (b) Lysosomal Zn concentration from lactating and involuting mammary glands. Fractions were digested in nitric acid and concentration was measured by atomic absorption spectroscopy. Data represent mean μg Zn/mg protein ± SD; n = 4/group, *P < 0.05. (c) Representative immunoblot of ZnT2 and Lamp1 (lysosomal marker) in lysosome-enriched fractions isolated from lactating and involuting mammary glands. β-actin was used as a loading control and ratios of signal intensities are reported under the blot. (d) Isolated mitochondria-enriched fractions from lactating and involuting mammary glands were digested in nitric acid and Zn concentration was measured by atomic absorption spectroscopy. Data represent mean μg Zn/mg protein ± SD; n = 3/group, *P < 0.05. (e) Protein abundance of ZnT2 in mitochondria-enriched fractions isolated from lactating and involuting mammary glands was determined by immunoblot and expressed relative to the mitochondrial marker succinate dehydrogenase (SDH). Ratios of signal intensities are reported under the blot.
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Related In: Results  -  Collection

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Show All Figures
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f1: ZnT2 accumulates Zn in lysosomes and mitochondria in mouse mammary glands during involution.(a) Mammary glands (~0.1–0.2 g) from virgin, lactating, and 24- and 48 h involuting mice were digested in nitric acid and Zn concentration was measured by atomic absorption spectroscopy. Data represent mean μg Zn/g tissue ± SD; n = 5/group, *P < 0.05, n.s., not significant. (b)–(e) Lysosome- and mitochondria-enriched fractions were isolated from lactating and 24- and 48 h involuting mammary glands by differential centrifugation. (b) Lysosomal Zn concentration from lactating and involuting mammary glands. Fractions were digested in nitric acid and concentration was measured by atomic absorption spectroscopy. Data represent mean μg Zn/mg protein ± SD; n = 4/group, *P < 0.05. (c) Representative immunoblot of ZnT2 and Lamp1 (lysosomal marker) in lysosome-enriched fractions isolated from lactating and involuting mammary glands. β-actin was used as a loading control and ratios of signal intensities are reported under the blot. (d) Isolated mitochondria-enriched fractions from lactating and involuting mammary glands were digested in nitric acid and Zn concentration was measured by atomic absorption spectroscopy. Data represent mean μg Zn/mg protein ± SD; n = 3/group, *P < 0.05. (e) Protein abundance of ZnT2 in mitochondria-enriched fractions isolated from lactating and involuting mammary glands was determined by immunoblot and expressed relative to the mitochondrial marker succinate dehydrogenase (SDH). Ratios of signal intensities are reported under the blot.
Mentions: We previously reported that the mammary gland accumulates Zn during lactation21. Herein, we found that similar to calcium22, Zn accumulation in the mammary gland was further augmented during involution (Fig. 1a). To determine where Zn accumulated, we isolated subcellular fractions enriched in specific organelles using density fractionation. Organelle enrichment was confirmed by immunoblotting for specific organelle markers for mitochondria, lysosomes, and the endoplasmic reticulum/Golgi apparatus (Supplementary Fig. S1a). We found that the Zn concentration in lysosome-enriched fractions isolated from involuting mammary glands was significantly higher than identical fractions isolated from lactating mammary glands (Fig. 1b). Moreover, we noted that the abundance of ZnT2 in lysosome-enriched fractions was higher during the initial phase of involution (24 h post weaning) (Fig. 1c). Acid phosphatase (EC 3.1.3.2) is a lysosomal enzyme that requires Zn to hydrolyze the substrate, nitrophenyl phosphate23. Consistent with previous reports noting that acid phosphatase activity is highest during early involution and declines 48 h post-weaning24, we found that peak ZnT2 abundance in lysosomes corresponded with peak acid phosphatase activity (Supplementary Fig. S1b). Concurrently, Figure 1d shows that the Zn concentration of mitochondria increased as involution progressed which paralleled an increase in mitochondrial ZnT2 abundance (Fig. 1e). Although the abundance of ZnT2 in specific fractions was altered, this was not a consequence of changes in the total abundance of ZnT2 (Supplementary Fig. S2a). Taken together, these data suggest that ZnT2 expression is associated with Zn accumulation in lysosomes and mitochondria during the early stages of mammary gland involution.

Bottom Line: Zinc (Zn) activates both LCD and apoptosis in vitro.Following weaning, ZnT2 abundance increased in lysosomes and mitochondria, which paralleled Zn accumulation in each of these organelles.Our data implicate ZnT2 as a critical mediator of cell death during involution and importantly, that as an initial involution signal, TNFα redistributes ZnT2 to lysosomes to activate LCD.

View Article: PubMed Central - PubMed

Affiliation: Department of Nutritional Sciences, The Pennsylvania State University, University Park, PA, USA.

ABSTRACT
Mammary gland involution is the most dramatic example of physiological cell death. It occurs through an initial phase of lysosomal-mediated cell death (LCD) followed by mitochondrial-mediated apoptosis. Zinc (Zn) activates both LCD and apoptosis in vitro. The Zn transporter ZnT2 imports Zn into vesicles and mitochondria and ZnT2-overexpression activates cell death in mammary epithelial cells (MECs). We tested the hypothesis that ZnT2-mediated Zn transport is critical for mammary gland involution in mice. Following weaning, ZnT2 abundance increased in lysosomes and mitochondria, which paralleled Zn accumulation in each of these organelles. Adenoviral expression of ZnT2 in lactating mouse mammary glands in vivo increased Zn in lysosomes and mitochondria and activated LCD and apoptosis, promoting a profound reduction in MECs and alveoli. Injection of TNFα, a potent activator of early involution, into the mammary gland fat pads of lactating mice increased ZnT2 and Zn in lysosomes and activated premature involution. Exposure of cultured MECs to TNFα redistributed ZnT2 to lysosomes and increased lysosomal Zn, which activated lysosomal swelling, cathepsin B release, and LCD. Our data implicate ZnT2 as a critical mediator of cell death during involution and importantly, that as an initial involution signal, TNFα redistributes ZnT2 to lysosomes to activate LCD.

No MeSH data available.


Related in: MedlinePlus