Abstract
The prolactin/STAT5 and AKT1/mTOR pathways play a key role in milk protein transcription and translation, respectively. However, the correlation between them in bovine mammary epithelial cells remains unclear. Here, mRNA and protein expression levels of AKT1, STAT5, and mTOR and the phosphorylation of these proteins were determined. Cell proliferation and viability were examined using the CASY-TT assay. Purified bovine mammary epithelial cells were cultured in differentiation media for different periods. The basic differentiation medium contained a lactogenic hormone cocktail of insulin (5 μg/mL), hydrocortisone (1 μg/mL), and prolactin (5 μg/mL). The cells cultured in this medium grew slowly and expressed higher levels of p-STAT5, p-AKT1, and p-mTOR (activated form) than those of control cells. Although the phosphorylation ratio was not increased, transcription and translation of these proteins were upregulated by the addition of insulin-like growth factor-1 or growth hormone, which further increased β-casein mRNA expression. Furthermore, the three proteins were upregulated or downregulated synchronously, even after RNA interference silencing of either Stat5 or Akt1. These findings indicate that a few hormones and other factors play lactogenic and galactogenic roles by promoting two key lactogenic signaling associated with milk protein expression. We provide evidence of prolactin/STAT5 and AKT1/mTOR synchronization, establishing a direct correlation between transcription regulation and translation regulation of milk protein in bovine mammary epithelial cells.
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References
Abell K, Watson CJ (2005) The Jak/Stat pathway: a novel way to regulate PI3K activity. Cell Cycle 4:897–900
Akers RM (2017) A 100-year review: mammary development and lactation. J Dairy Sci 100:10332–10352
Burgos SA, Cant JP (2010) IGF-1 stimulates protein synthesis by enhanced signaling through mTORC1 in bovine mammary epithelial cells. Domest Anim Endocrinol 38:211–221
Burgos SA, Dai M, Cant JP (2010) Nutrient availability and lactogenic hormones regulate mammary protein synthesis through the mammalian target of rapamycin signaling pathway. J Dairy Sci 93:153–161
Chen CC, Boxer RB, Stairs DB, Portocarrero CP, Horton RH, Alvarez JV, Birnbaum MJ, Chodosh LA (2010) Akt is required for Stat5 activation and mammary differentiation. Breast Cancer Res 12:R72
Choi KM, Barash I, Rhoads RE (2004) Insulin and prolactin synergistically stimulate beta-casein messenger ribonucleic acid translation by cytoplasmic polyadenylation. Mol Endocrinol 18:1670–1686
Creamer BA, Sakamoto K, Schmidt JW, Triplett AA, Moriggl R, Wagner KU (2010) Stat5 promotes survival of mammary epithelial cells through transcriptional activation of a distinct promoter in Akt1. Mol Cell Biol 30:2957–2970
Dinerstein-Cali H, Ferrag F, Kayser C, Kelly PA, Postel-Vinay M (2000) Growth hormone (GH) induces the formation of protein complexes involving Stat5, Erk2, Shc and serine phosphorylated proteins. Mol Cell Endocrinol 166:89–99
Faraci-Orf E, McFadden C, Vogel WF (2006) DDR1 signaling is essential to sustain Stat5 function during lactogenesis. J Cell Biochem 97:109–121
Finucane KA, McFadden TB, Bond JP, Kennelly JJ, Zhao FQ (2008) Onset of lactation in the bovine mammary gland: gene expression profiling indicates a strong inhibition of gene expression in cell proliferation. Funct Integr Genomics 8:251–264
Gouilleux F, Wakao H, Mundt M, Groner B (1994) Prolactin induces phosphorylation of Tyr694 of Stat5 (MGF), a prerequisite for DNA binding and induction of transcription. EMBO J 13:4361–4369
Hovey RC, Trott JF, Vonderhaar BK (2002) Establishing a framework for the functional mammary gland: from endocrinology to morphology. J Mammary Gland Biol Neoplasia 7:17–38
Huynh HT, Robitaille G, Turner JD (1991) Establishment of bovine mammary epithelial cells (MAC-T): an in vitro model for bovine lactation. Exp Cell Res 197:191–199
Johnson TL, Fujimoto BA, Jimenez-Flores R, Peterson DG (2010) Growth hormone alters lipid composition and increases the abundance of casein and lactalbumin mRNA in the MAC-T cell line. J Dairy Res 77:199–204
Kfir SH, Barash I (2019) Calorie restriction and rapamycin administration induce stem cell self-renewal and consequent development and production in the mammary gland. Exp Cell Res 382:111477
Kim JJ, Yu J, Bag J, Bakovic M, Cant JP (2015) Translation attenuation via 3′ terminal codon usage in bovine csn1s2 is responsible for the difference in alphas2- and beta-casein profile in milk. RNA Biol 12:354–367
Lee HY, Heo YT, Lee SE, Hwang KC, Lee HG, Choi SH, Kim NH (2013) Short communication: retinoic acid plus prolactin to synergistically increase specific casein gene expression in MAC-T cells. J Dairy Sci 96:3835–3839
Li SS, Loor JJ, Liu HY, Liu L, Hosseini A, Zhao WS, Liu JX (2017) Optimal ratios of essential amino acids stimulate beta-casein synthesis via activation of the mammalian target of rapamycin signaling pathway in MAC-T cells and bovine mammary tissue explants. J Dairy Sci 100:6676–6688
Morrison B, Cutler ML (2009) Mouse mammary epithelial cells form mammospheres during lactogenic differentiation. J Vis Exp 32:1265
Qian X, Zhao FQ (2014) Current major advances in the regulation of milk protein gene expression. Crit Rev Eukaryot Gene Expr 24:357–378
Radler PD, Wehde BL, Wagner KU (2017) Crosstalk between STAT5 activation and PI3K/AKT functions in normal and transformed mammary epithelial cells. Mol Cell Endocrinol 451:31–39
Riley LG, Gardiner-Garden M, Thomson PC, Wynn PC, Williamson P, Raadsma HW, Sheehy PA (2010) The influence of extracellular matrix and prolactin on global gene expression profiles of primary bovine mammary epithelial cells in vitro. Anim Genet 41:55–63
Sakamoto K, Creamer BA, Triplett AA, Wagner KU (2007) The Janus kinase 2 is required for expression and nuclear accumulation of cyclin D1 in proliferating mammary epithelial cells. Mol Endocrinol 21:1877–1892
Sakamoto K, Komatsu T, Kobayashi T, Rose MT, Aso H, Hagino A, Obara Y (2005) Growth hormone acts on the synthesis and secretion of alpha-casein in bovine mammary epithelial cells. J Dairy Res 72:264–270
Schmidt JW, Wehde BL, Sakamoto K, Triplett AA, Anderson SM, Tsichlis PN, Leone G, Wagner KU (2014) Stat5 regulates the phosphatidylinositol 3-kinase/Akt1 pathway during mammary gland development and tumorigenesis. Mol Cell Biol 34:1363–1377
Sciascia Q, Pacheco D, McCoard SA (2013) Increased milk protein synthesis in response to exogenous growth hormone is associated with changes in mechanistic (mammalian) target of rapamycin (mTOR)C1-dependent and independent cell signaling. J Dairy Sci 96:2327–2338
Shao Y, Wall EH, McFadden TB, Misra Y, Qian X, Blauwiekel R, Kerr D, Zhao FQ (2013) Lactogenic hormones stimulate expression of lipogenic genes but not glucose transporters in bovine mammary gland. Domest Anim Endocrinol 44:57–69
Singh K, Vetharaniam I, Dobson JM, Prewitz M, Oden K, Murney R, Swanson KM, McDonald R, Henderson HV, Stelwagen K (2016) Cell survival signaling in the bovine mammary gland during the transition from lactation to involution. J Dairy Sci 99:7523–7543
Sornapudi TR, Nayak R, Guthikonda PK, Pasupulati AK, Kethavath S, Uppada V, Mondal S, Yellaboina S, Kurukuti S (2018) Comprehensive profiling of transcriptional networks specific for lactogenic differentiation of HC11 mammary epithelial stem-like cells. Sci Rep 8:11777
Tucker HA (2000) Hormones, mammary growth, and lactation: a 41-year perspective. J Dairy Sci 83:874–884
Wartmann M, Cella N, Hofer P, Groner B, Liu X, Hennighausen L, Hynes NE (1996) Lactogenic hormone activation of Stat5 and transcription of the beta-casein gene in mammary epithelial cells is independent of p42 ERK2 mitogen-activated protein kinase activity. J Biol Chem 271:31863–31868
Woodward TL, Sia MA, Blaschuk OW, Turner JD, Laird DW (1998) Deficient epithelial fibroblast heterocellular gap junction communication can be overcome by co-culture with an intermediate cell type but not by E-cadherin transgene expression. J Cell Sci 111(Pt 23):3529–3539
Zhao F, Liu C, Hao YM, Qu B, Cui YJ, Zhang N, Gao XJ, Li QZ (2015) Up-regulation of integrin α6β4 expression by mitogens involved in dairy cow mammary development. In Vitro Cell Dev Biol Anim 51(3): 287–299
Zhou Y, Akers RM, Jiang H (2008) Growth hormone can induce expression of four major milk protein genes in transfected MAC-T cells. J Dairy Sci 91:100–108
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We would like to thank Editage (www.editage.cn) for English language editing.
Funding
This research was funded by the National Natural Science Foundation of China, grant numbers 31571338 (to F.Z.) and 31671285 (to X.H.).
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Conceptualization, F.Z.; methodology, F.Z., B.W. and L.S.; validation, B.W. and J.M.; formal analysis, L.S. and B.W.; data curation, J.M. and X.H; writing-original draft preparation, F.Z. and L.S.; writing-review and editing, Q.L. and C.W.; supervision, X.H. and Q.L.; project administration, F.Z. and Q.L.; funding acquisition, F.Z. and X.H.
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Editor: Tetsuji Okamoto
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Wang, B., Shi, L., Men, J. et al. Controlled synchronization of prolactin/STAT5 and AKT1/mTOR in bovine mammary epithelial cells. In Vitro Cell.Dev.Biol.-Animal 56, 243–252 (2020). https://doi.org/10.1007/s11626-020-00432-x
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DOI: https://doi.org/10.1007/s11626-020-00432-x