Akt1 Is Essential for Postnatal Mammary Gland Development, Function, and the Expression of Btn1a1 Jessica LaRocca*, Jodie Pietruska, Mary Hixon Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island, United States of America Abstract Akt1, a serine-threonine protein kinase member of the PKB/Akt gene family, plays critical roles in the regulation of multiple cellular processes, and has previously been implicated in lactation and breast cancer development. In this study, we utilized Akt1+/+ and Akt12/2 C57/Bl6 female mice to assess the role that Akt1 plays in normal mammary gland postnatal development and function. We examined postnatal morphology at multiple time points, and analyzed gene and protein expression changes that persist into adulthood. Akt1 deficiency resulted in several mammary gland developmental defects, including ductal outgrowth and defective terminal end bud formation. Adult Akt12/2 mammary gland composition remained altered, exhibiting fewer alveolar buds coupled with increased epithelial cell apoptosis. Microarray analysis revealed that Akt1 deficiency altered expression of genes involved in numerous biological processes in the mammary gland, including organismal development, cell death, and tissue morphology. Of particular importance, a significant decrease in expression of Btn1a1, a gene involved in milk lipid secretion, was observed in Akt12/2 mammary glands. Additionally, pseudopregnant Akt12/2 females failed to induce Btn1a1 expression in response to hormonal stimulation compared to their wild-type counterparts. Retroviral-mediated shRNA knockdown of Akt1 and Btn1a1 in MCF-7 human breast epithelial further illustrated the importance of Akt1 in mammary epithelial cell proliferation, as well as in the regulation of Btn1a1 and subsequent expression of ß-casein, a gene that encodes for milk protein. Overall these findings provide mechanistic insight into the role of Akt1 in mammary morphogenesis and function. Citation: LaRocca J, Pietruska J, Hixon M (2011) Akt1 Is Essential for Postnatal Mammary Gland Development, Function, and the Expression of Btn1a1. PLoS ONE 6(9): e24432. doi:10.1371/journal.pone.0024432 Editor: Lin Zhang, University of Pennsylvania School of Medicine, United States of America Received August 2, 2011; Accepted August 9, 2011; Published September 7, 2011 Copyright: 2011 LaRocca et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by ES015704 from the National Institute of Environmental Health Sciences/National Institutes of Health (NIH) to MH. JL was supported by 2T32E5007272 from the NIH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jessica_larocca@brown.edu Introduction The mammary gland is a complex structure composed of epithelium surrounded by stroma. Mammary gland postnatal development is an intricate process controlled by steroid and peptide hormones in specific stages that is highly conserved in mammals. Unlike many other organs which undergo development in utero, mammary gland development and ductal branching occurs during puberty [1]. While mice have five pairs of mammary glands compared to one pair in humans, mammary gland development remains similar between the two species, making the mouse a good model to study mammary gland development. During embryonic development in the mouse, a rudimentary ductal structure invades the mesenchyma, and remains dormant until approximately 21 days of age, when the onset of ovarian hormone secretion stimulates ductal growth [2]. During puberty, the ovarian hormones estradiol and progester- one stimulate stem cell proliferation in terminal end buds (TEBs) and induce side branching in the mammary gland, respectively [3]. The TEB is found exclusively in the developing mammary gland, and is the main driving force for mammary gland develop- ment [4,5]. The outer layer, or cap layer, of the TEB contains a special population of pluripotent stem cells [2], which ultimately differentiate to form the intermediate, luminal, and myoepithelial cells of the duct [6]. TEB formation and side branching drive mammary gland invasion into the fat pad until the fat pad is filled at approximately 10 weeks of age. Once the ducts have filled the entire mammary fat pad, TEBs are permanently replaced by terminal ducts and alveolar buds [7]. It has been proposed that the adult mammary gland retains a mammary stem cell population which gives rise to epithelial precursor cells [6]. The progeny of the epithelial precursor cells are either ductal precursor cells or alveolar precursor cells. The presence of these stem cells is thought to underlie the ability of the mammary gland to undergo alveolar renewal with subsequent pregnancies [3]. It has also been shown that the mammary epithelium is the major target for carcinogen- induced initiation during tumorigenesis, making the understanding of the mechanisms underlying mammary gland development and function imperative for knowledge of reproductive function and for designing effective anticancer therapies [8]. Protein kinase B/Akt is a serine/threonine kinase that plays a central role in regulation of cell metabolism, cell survival, motility, transcription, and cell-cycle progression [9,10]. The Akt gene family is activated in a phosphoinositide 3 kinase- dependent manner by a variety of signals including cytokines, growth factors, and hormones. The Akt family includes three isoforms, Akt1, Akt2, and Akt3, which share 80% amino acid homology but have distinct roles in regulating cellular activity [9]. Akt1 is the predominant isoform, and plays a role in placental development, organismal growth, adipogenesis, mammary gland lactation and involution, PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e24432