ZnT4 (SLC30A4)-null ("lethal milk") mice have defects in mammary gland secretion and hallmarks of precocious involution during lactation - PubMed (original) (raw)

ZnT4 (SLC30A4)-null ("lethal milk") mice have defects in mammary gland secretion and hallmarks of precocious involution during lactation

Nicholas H McCormick et al. Am J Physiol Regul Integr Comp Physiol. 2016.

Abstract

During lactation, highly specialized secretory mammary epithelial cells (MECs) produce and secrete huge quantities of nutrients and nonnutritive factors into breast milk. The zinc (Zn) transporter ZnT4 (SLC30A4) transports Zn into the trans-Golgi apparatus for lactose synthesis, and across the apical cell membrane for efflux from MECs into milk. This is consistent with observations in "lethal milk" (lm/lm) mice, which have a truncation mutation in SLC30A4, and present with not only low milk Zn concentration, but also smaller mammary glands, decreased milk volume, and lactation failure by lactation day 2. However, the molecular underpinnings of these defects are not understood. Here, we used lactating C57BL/6J(lm/lm) (ZnT4-null) mice to explore the consequences of a ZnT4-null phenotype on mammary gland function during early lactation. Lactating C57BL/6J(lm/lm) mice had significantly fewer, smaller, and collapsed alveoli comprising swollen, lipid-filled MECs during early lactation. These defects were associated with decreased Akt expression and STAT5 activation, indicative of defects in MEC secretion. In addition, increased expression of ZnT2, TNF-α, and cleaved e-cadherin concomitant with increased activation of STAT3 implicated the loss of ZnT4 in precocious activation of involution. Collectively, our study indicates that the loss of ZnT4 has profound consequences on MEC secretion and may promote tissue remodeling in the mammary gland during early lactation.

Keywords: SLC30A4; ZnT4; lactation; mammary gland; zinc.

Copyright © 2016 the American Physiological Society.

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Figures

Fig. 1.

Fig. 1.

A ZnT4-null phenotype in C57BL/6J_lm/lm_ mice leads to reduced secretory epithelium. A: representative immunoblot of ZnT4 in total membrane fractions prepared from wild-type (WT) and C57BL/6J_lm/lm_ (lm) mammary glands. β-actin was used as a loading control. B: representative whole mount images of mammary glands from WT and lm mice on lactation day 2. Magnification: ×0.5; scale bar = 0.5 mm. C: hematoxylin and eosin (H&E)-stained images of mammary glands from WT and lm mice on lactation day 2 at ×4 magnification (i, ii: scale bar: 250 μm) and ×10 magnification (iii, iv: scale bar = 100 μm). D: data represent mean alveolar diameter (μm) ± SD, n = 4 mice/genotype. *Significant effect of genotype, P < 0.05. E: representative immunoblots of the luminal epithelial cell marker, cytokeratin 8 (CK8) in cell lysates from WT and lm mammary glands. β-actin was used as a loading control. F: data represent mean CK8 protein abundance normalized to β-actin ± SD; n = 3 mice/genotype. *Significant effect of genotype, P < 0.05. G: representative immunoblots of phospho-STAT5 (p-STAT5) and total STAT5 in cell lysates from mammary glands of WT and lm mice. CK8 was used as a loading control. H: quantification of STAT5 activation. Data represent mean ratio of p-STAT5/total STAT5 normalized to CK8 ± SD; n = 3 mice/genotype. **Significant effect of genotype, P < 0.01.

Fig. 2.

Fig. 2.

Secretory defects in C57BL/6J_lm/lm_ mammary glands. A: H&E-stained images of mammary glands from WT and C57BL/6J_lm/lm_ (lm) mice on lactation day 2 at ×100 magnification; scale bar = 100 μm. Black arrowheads mark active fusion of lipid droplets. B: representative images of adipophilin (ADRP; green) in mammary glands from WT and lm mice on lactation day 2. Nuclei were counterstained with DAPI (blue). Magnification: ×63; scale bar = 100 μm. C: representative immunoblots of Akt in cell lysates from WT and lm mammary glands. Cytokeratin 8 (CK8) was used for normalization. D: data represent mean Akt protein abundance relative to CK8 ± SD; n = 3 mice/genotype. **Significant effect of genotype, P < 0.01.

Fig. 3.

Fig. 3.

ZnT2 abundance is increased in the C57BL/6J_lm/lm_ mammary gland. A: representative immunoblot of ZnT2 in total membrane fractions from mammary glands of C57BL/6J_lm/lm_ (lm) mice and their WT littermates. Cytokeratin 8 (CK8) was used for normalization. B: data represent mean ZnT2 protein abundance relative to cytokeratin 8 (CK8) ± SD; n = 3 mice/genotype from two different experiments. *Significant effect of genotype, P < 0.05. C: representative immunoblots of phospho-STAT3 (p-STAT3) and total STAT3 in cell lysates from mammary glands of WT and lm mice. CK8 was used for normalization. D: quantification of STAT3 activation. Data represent mean ratio of p-STAT3/total STAT3 normalized to CK8 ± SD, n = 3 mice/genotype. **Significant effect of genotype, P < 0.01. E: representative immunoblots of TNF-α in cell lysates from mammary glands of WT and lm mice. CK8 was used as a loading control. F: data represent mean TNF-α protein abundance relative to CK8 ± SD; n = 3 mice/genotype. *Significant effect of genotype, P < 0.05. G: representative immunoblots of mature (120 kDa) and truncated (97 kDa) e-cadherin (e-cad) in cell lysates from mammary glands of WT and lm mice. CK8 was used for normalization. H: E-cadherin truncation. Data represent mean ratio of truncated/mature e-cad normalized to CK8 ± SD; n = 3 mice/genotype. *Significant effect of genotype, P < 0.05.

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