Abstract
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.
Highlights
Mammary glands, the lactation organs of the mammals, are regulated by prolactin (PRL) to produce milk
Pten regulates Dairy cow mammary epithelial cells (DCMECs) functions For cultured DCMECs, cytokeratin-18 was used as a marker to ensure that we obtained pure mammary epithelial cells [27] (Figure S2)
Our results showed a correlation between mitogen-activated protein kinases (MAPK) and PTEN in DCMECs, as MAPK expression declined with Pten overexpression
Summary
The lactation organs of the mammals, are regulated by prolactin (PRL) to produce milk. Milk produced during lactation for newborns is generally considered the best nutritional source because it contains optimal ingredients for healthy growth and development [1]. An understanding of how the mammary gland is regulated to produce milk is of biomedical and agricultural importance. PRL-induced activation of Janus kinase 2 (JAK2) and Signal transducer and activator of transcription 5 (STAT5) is required to induce expression of most, possibly all, milk protein genes [2,3]. The roles of sterol regulatory element binding protein 1 (SREBP1) and peroxisome proliferatoractivated receptor gamma (PPARc) are to regulate the expression of a number of key lipid metabolism genes [5]. Glucose transporter 1 (GLUT1) is the major glucose transporter in the basal membrane and its expression is regulated by PRL when the demand for glucose during lactose synthesis is amplified [6]
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