Dataset Information

14-3-3? Regulates Lipopolysaccharide-Induced Inflammatory Responses and Lactation in Dairy Cow Mammary Epithelial Cells by Inhibiting NF-?B and MAPKs and Up-Regulating mTOR Signaling.

ABSTRACT: As a protective factor for lipopolysaccharide (LPS)-induced injury, 14-3-3? has been the subject of recent research. Nevertheless, whether 14-3-3? can regulate lactation in dairy cow mammary epithelial cells (DCMECs) induced by LPS remains unknown. Here, the anti-inflammatory effect and lactation regulating ability of 14-3-3? in LPS-induced DCMECs are investigated for the first time, and the molecular mechanisms responsible for their effects are explored. The results of qRT-PCR showed that 14-3-3? overexpression significantly inhibited the mRNA expression of tumor necrosis factor-? (TNF-?), interleukin-6 (IL-6), interleukin-1? (IL-1?) and inducible nitric oxide synthase (iNOS). Enzyme-linked immunosorbent assay (ELISA) analysis revealed that 14-3-3? overexpression also suppressed the production of TNF-? and IL-6 in cell culture supernatants. Meanwhile, CASY-TT Analyser System showed that 14-3-3? overexpression clearly increased the viability and proliferation of cells. The results of kit methods and western blot analysis showed that 14-3-3? overexpression promoted the secretion of triglycerides and lactose and the synthesis of ?-casein. Furthermore, the expression of genes relevant to nuclear factor-?B (NF-?B) and mitogen-activated protein kinase (MAPKs) and lactation-associated proteins were assessed by western blot, and the results suggested that 14-3-3? overexpression inactivated the NF-?B and MAPK signaling pathways by down-regulating extracellular signal regulated protein kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK) and inhibitor of NF-?B (I?B) phosphorylation levels, as well as by inhibiting NF-?B translocation. Meanwhile, 14-3-3? overexpression enhanced the expression levels of ?-casein, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), serine/threonine protein kinase Akt 1 (AKT1), sterol regulatory element binding protein 1 (SREBP1) and peroxisome proliferator-activated receptor gamma (PPAR?). These results suggest that 14-3-3? was able to attenuate the LPS-induced inflammatory responses and promote proliferation and lactation in LPS-induced DCMECs by inhibiting the activation of the NF-?B and MAPK signaling pathways and up-regulating mTOR signaling pathways to protect against LPS-induced injury.

SUBMITTER: Liu L

PROVIDER: S-EPMC4519969 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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14-3-3γ Regulates Lipopolysaccharide-Induced Inflammatory Responses and Lactation in Dairy Cow Mammary Epithelial Cells by Inhibiting NF-κB and MAPKs and Up-Regulating mTOR Signaling.

Liu Lixin L Lin Ye Y Liu Lili L Bian Yanjie Y Zhang Li L Gao Xuejun X Li Qingzhang Q

International journal of molecular sciences 20150722 7

As a protective factor for lipopolysaccharide (LPS)-induced injury, 14-3-3γ has been the subject of recent research. Nevertheless, whether 14-3-3γ can regulate lactation in dairy cow mammary epithelial cells (DCMECs) induced by LPS remains unknown. Here, the anti-inflammatory effect and lactation regulating ability of 14-3-3γ in LPS-induced DCMECs are investigated for the first time, and the molecular mechanisms responsible for their effects are explored. The results of qRT-PCR showed that 14-3- ...[more]

PMID: 26204835

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Project description:Pten is a tumor suppressor gene regulating many cellular processes, including growth, adhesion, and apoptosis. In the aim of investigating the role of Pten during mammary gland development and lactation of dairy cows, we analyzed Pten expression levels in the mammary glands of dairy cows by using western blotting, immunohistochemistry, and quantitative polymerase chain reaction (qPCR) assays. Dairy cow mammary epithelial cells (DCMECs) were used to study the function of Pten in vitro. We determined concentrations of β-casein, triglyceride, and lactose in the culture medium following Pten overexpression and siRNA inhibition. To determine whether Pten affected DCMEC viability and proliferation, cells were analyzed by CASY-TT and flow cytometry. Genes involved in lactation-related signaling pathways were detected. Pten expression was also assessed by adding prolactin and glucose to cell cultures. When Pten was overexpressed, proliferation of DCMECs and concentrations for β-casein, triglyceride, and lactose were significantly decreased. Overexpression of Pten down-regulated expression of MAPK, CYCLIN D1, AKT, MTOR, S6K1, STAT5, SREBP1, PPARγ, PRLR, and GLUT1, but up-regulated 4EBP1 in DCMECs. The Pten siRNA inhibition experiments revealed results that opposed those from the gene overexpression experiments. Introduction of prolactin (PRL) increased secretion of β-casein, triglyceride, and lactose, but decreased Pten expression levels. Introduction of glucose also increased β-casein and triglyceride concentrations, but did not significantly alter Pten expression levels. The Pten mRNA and protein expression levels were decreased 0.3- and 0.4-fold in mammary glands of lactating cows producing high quality milk (milk protein >3.0%, milk fat >3.5%), compared with those cows producing low quality milk (milk protein <3.0%, milk fat <3.5%). In conclusion, Pten functions as an inhibitor during mammary gland development and lactation in dairy cows. It can down-regulate DCMECs secretion of β-casein, triglyceride, and lactose, and plays a critical role in lactation related signaling pathways.

Dataset Information

14-3-3? Regulates Lipopolysaccharide-Induced Inflammatory Responses and Lactation in Dairy Cow Mammary Epithelial Cells by Inhibiting NF-?B and MAPKs and Up-Regulating mTOR Signaling.

Publications

14-3-3γ Regulates Lipopolysaccharide-Induced Inflammatory Responses and Lactation in Dairy Cow Mammary Epithelial Cells by Inhibiting NF-κB and MAPKs and Up-Regulating mTOR Signaling.

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