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Adverse effects of LPS on membrane proteins in lactating bovine mammary epithelial cells

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Abstract

Mastitis causes a decrease in milk yield and abnormalities in milk components from dairy cows. Escherichia coli and the E. coli lipopolysaccharide (LPS) cell wall component directly downregulate milk production in bovine mammary epithelial cells (BMECs). However, the detailed mechanism by which this occurs in BMECs remains unclear. Various membrane proteins, such as immune sensors (Toll-like receptors, TLR), nutrient transporters (glucose transporter and aquaporin), and tight junction proteins (claudin and occludin) are involved in the onset of mastitis or milk production in BMECs. In this study, we investigated the influence of LPS on membrane proteins using an in vitro culture model. This mastitis model demonstrated a loss of glucose transporter-1 and aquaporin-3 at lateral membranes and a decrease in milk production in response to LPS treatment. LPS disrupted the tight junction barrier and caused compositional changes in localization of claudin-3 and claudin-4, although tight junctions were maintained to separate the apical membrane domains and the basolateral membrane domains. LPS did not significantly affect the expression level and subcellular localization of epidermal growth factor receptor in lactating BMECs with no detectable changes in MEK1/2-ERK1/2 signaling. In contrast, NFκB was concurrently activated with temporal translocation of TLR-4 in the apical membranes, whereas TLR-2 was not significantly influenced by LPS treatment. These findings indicate the importance of investigating the subcellular localization of membrane proteins to understand the molecular mechanism of LPS in milk production in mastitis.

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Abbreviations

AQP:

Aquaporin

BMEC:

Bovine mammary epithelial cell

BSA:

Bovine serum albumin

EGFR:

Epidermal growth factor receptor

FBS:

Fetal bovine serum

FITC:

Fluorescein isothiocyanate

GLUT1:

Glucose transporter-1

LPS:

Lipopolysaccharide

PBS-T:

PBS containing 0.05% Tween 20

STAT:

Signal transducer and activator of transcription

SDS:

Sodium dodecyl sulfate

TJ:

Tight junction

TLR:

Toll-like receptor

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Acknowledgments

We are grateful to Kota Matsunaga, Naoki Omatsu, and Futa Shinagawa, Laboratory of Dairy Food Science, Research, Faculty of Agriculture, Hokkaido University, for their helpful research advice.

Funding

This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS Kakenhi Grant Number 18H0232009 and JP19J10189).

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Authors and Affiliations

  1. Laboratory of Cell and Tissue Biology, Research Faculty of Agriculture, Hokkaido University, North 9, West 9, Sapporo, Hokkaido, 060-8589, Japan

    Yusaku Tsugami, Haruka Wakasa, Takahiro Suzuki, Takanori Nishimura & Ken Kobayashi

  2. Laboratory of Animal Genetics and Reproduction, Research Faculty of Agriculture, Hokkaido University, North 9, West 9, Sapporo, Hokkaido, 060-8589, Japan

    Manabu Kawahara

  3. Hokkaido Research Station, National Institute of Animal Health, National Agriculture and Food Research Organization, 4 Hitsujigaoka, Toyohira, Sapporo, Hokkaido, 062-0045, Japan

    Atsushi Watanabe

Authors
  1. Yusaku Tsugami
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  2. Haruka Wakasa
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  3. Manabu Kawahara
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  4. Atsushi Watanabe
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  5. Takahiro Suzuki
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  6. Takanori Nishimura
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  7. Ken Kobayashi
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Correspondence to Ken Kobayashi.

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The authors declare that they have no conflict of interest.

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All experiments were approved by the Animal Resource Committee of Hokkaido University (permission number: 15–0085).

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Tsugami, Y., Wakasa, H., Kawahara, M. et al. Adverse effects of LPS on membrane proteins in lactating bovine mammary epithelial cells. Cell Tissue Res 384, 435–448 (2021). https://doi.org/10.1007/s00441-020-03344-0

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  • DOI: https://doi.org/10.1007/s00441-020-03344-0

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