14-3-3γ affects mTOR pathway and regulates lactogenesis in dairy cow mammary epithelial cells Nagam Khudhair & Chaochao Luo & Ahmed Khalid & Li Zhang & Shuang Zhang & Jinxia Ao & Qingzhang Li & Xuejun Gao Received: 14 December 2014 /Accepted: 10 February 2015 / Editor: T. Okamoto # The Society for In Vitro Biology 2015 Abstract 14-3-3 proteins are an acidic protein family that is highly conserved and widely distributed in eukaryotic cells. Recent studies have found that 14-3-3 proteins play critical roles in cell signal transductions, cell growth and differentia- tion, and protein synthesis. 14-3-3γ is an important member of 14-3-3 protein family. In our previous study, we found that 14-3-3γ was upregulated by estrogen in dairy cow mammary epithelial cell (DCMEC), but the function and mechanism of 14-3-3γ is not known. In this experiment, we first cultured and purified the primary DCMEC and found 14-3-3γ located both in the cytoplasm and nucleus by using immunofluores- cence assay. Methionine, lysine, estrogen, and prolactin could upregulate the expression of 14-3-3γ, stimulate the secretion of β-casein and triglyceride, and raise the cell viability of DCMEC. We constructed a stable 14-3-3γ overexpression cell line of DCMEC and found that the expressions of mTOR and p-mTOR, the secretion of triglyceride and β-casein (CSN2), and the cell viability of DCMEC were all upregulat- ed. We also observed the effects of 14-3-3γ gene silencing and gained consistent results with 14-3-3γ overexpression. These findings reveal that 14-3-3γ affects the mTOR pathway and regulates lactogenesis in DCMECs. Keywords 14-3-3γ . Stable overexpression . DCMECs . mTOR . Lactogenesis Introduction 14-3-3 proteins are a class of highly conserved acidic protein, and there are seven 14-3-3 isoforms found in mammals, re- spectively, β, γ, ε, η, ζ, δ, and τ (Aitken 2006). The current studies have found that 14-3-3 protein family interacts with nearly 700 species of proteins through a particular sequence of phosphorylated serine or threonine and broadly participates in cell signal transduction, cycle progression, proliferation, and protein synthesis (Zhao et al. 2011, 2012; Lam et al. 2013). Previous reports have shown that 14-3-3 is implicated in mod- ulating the TOR function in yeast (Bertram et al. 1998). Other studies indicate that 14-3-3 positively regulates mTOR path- way (Mori et al. 2000). The 14-3-3γ isoform is encoded by the YWHAZ gene. It has been reported that growth factors can induce 14-3-3γ in human vascular smooth muscle cells (Autieri et al. 1996). Exhaustion of 14-3-3γ results in a prometaphase/metaphase-like arrest in HeLa cells (Kasahara et al. 2013). The expression of 14-3-3γ also promoted cell survival and growth in hematopoietic progenitor cells (Ajjappala et al. 2009). The role of the mTOR pathway in facilitating protein and lipid synthesis and cell proliferation has been well investigat- ed (Porstmann et al. 2008; Bakan and Laplante 2012). It is well known that mTOR positively regulates protein translation through phosphorylation of S6K and of 4E-BP1/ eIF4E. Activated or phosphorylated S6K phosphorylates ribosomal protein S6 (RPS6) to stimulate translation. In our previous study, we found that 14-3-3γ was upregu- lated by estrogen in dairy cow mammary epithelial cell (DCMEC), but the function and mechanism of 14-3-3γ is not known. In this study, we observed the subcellular N. Khudhair : C. Luo : L. Zhang : S. Zhang : J. Ao : Q. Li : X. Gao (*) Key Laboratory of Agricultural Biological Functional Genes, Northeast Agricultural University, Harbin 150030, China e-mail: gaoxj5390@sina.com A. Khalid College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China N. Khudhair Biology Department, Education College for Women, Al-Anbar University, Ramadi 31001, Iraq In Vitro Cell.Dev.Biol.—Animal DOI 10.1007/s11626-015-9879-x