Akt1 in Mammary Development Figure 7. Akt1 is required for epithelial cell proliferation and Btn1a1 and ß-casein gene expression. Percent change in cell growth over 6 days in MCF7 cells expressing shAkt1 or shLuciferase control (A). Relative gene expression of Akt1, Btn1a1, and ß-casein in shAkt1, shBtn1a1, or shLuciferase cells (B). Data represented as mean plus standard error. Statistical analyses were conducted using two-tailed student’s t tests. #p,0.1, **p,0.01, *p,0.05 compared to shLuciferase control. n = 3 independent infections. doi:10.1371/journal.pone.0024432.g007 examine the influence of Akt1 on Bnt1a1 during pregnancy by PMSG and hCG injections in Akt1+/+ and Akt12/2 adult females since Akt12/2 females have high rates of fetal resorptions and are subfertile [20]. Following hormonal stimulation, Akt1+/+ mammary glands exhibited increased expression of Btn1a1, suggesting that Btn1a1 induction occurs normally during pregnan- cy, likely in preparation for milk production during nursing, as Btn1a1 is required for the secretion of milk lipid droplets [24]. Akt12/2 mammary glands failed to induce an increase in expression of Btn1a1 during pseudopregnancy compared to a virgin state, and had significantly decreased Btn1a1 expression compared to pseuodopregnant Akt1+/+ glands. Furthermore, knockdown of Akt1 in human breast epithelial cell decreased expression of ß-casein, Btn1a1 and Akt1, and knockdown of Btn1a1 decreased expression of ß-casein and Btn1a1, but not Akt1. These data suggest that Btn1a1 is downstream of Akt1, and ß-casein is further downstream. These data, taken together with the previously shown roles of Btn1a1 and Akt1 in lactation, suggest a regulation of Btn1a1 by Akt1 in the mammary gland, and that Akt1 may promote milk protein production through Btn1a1 and subsequent ß-casein expression. Collectively, while Akt12/2 mammary glands can form alveoli during pseudopregnancy, they are fewer in number and potentially dysfunctional in their milk production. IPA revealed that several genes involved in organismal development, cell death, tissue morphology, and cancer were altered Akt12/2 mammary glands. The roles of the PI3K/AKT pathway in cell survival and apoptosis have been extensively studied, but we found differential expression of additional genes that may be involved in this process, including Mtmr7, Pou2af, and Faim3, which play roles in either cell survival or apoptosis. We also found that Akt1 deficiency resulted in decreased alveolar bud diameter and increased epithelial cell apoptosis, which could decrease the quantity of alveolar buds and terminal ducts in adult Akt12/2 glands. However, cell growth in terminal end buds and alveolar buds not only relies on mitogenic signals for proliferation, but also on cell-cell contacts for forward growth. Cell-cell signaling and interaction was another biological process altered by Akt1 deficiency, which included several altered genes such as Dsc3, a member of the cadherin superfamily. Cadherins are calcium- PLoS ONE | www.plosone.org 10 September 2011 | Volume 6 | Issue 9 | e24432