Distinct roles of the three Akt isoforms in lactogenic differentiation and involution

Abstract

The three Akt isoforms differ in their ability to transduce oncogenic signals initiated by the Neu and PyMT oncogenes in mammary epithelia. As a result, ablation of Akt1 inhibits and ablation of Akt2 accelerates mammary tumor development by both oncogenes, while ablation of Akt3 is phenotypically almost neutral. Since the risk of breast cancer development in humans correlates with multiple late pregnancies, we embarked on a study to determine whether individual Akt isoforms also differ in their ability to transduce hormonal and growth factor signals during pregnancy, lactation and post-lactation involution. The results showed that the ablation of Akt1 delays the differentiation of the mammary epithelia during pregnancy and lactation, and that the ablation of Akt2 has the opposite effect. Finally, ablation of Akt3 results in minor defects, but its phenotype is closer to that of the wild type mice. Whereas the phenotype of the Akt1 ablation is cell autonomous, that of Akt2 is not. The ablation of Akt1 promotes apoptosis and accelerates involution, whereas the ablation of Akt2 inhibits apoptosis and delays involution. Mammary gland differentiation during pregnancy depends on the phosphorylation of Stat5a, which is induced by prolactin, a hormone that generates signals transduced via Akt. Here we show that the ablation of Akt1, but not the ablation of Akt2 or Akt3 interferes with the phosphorylation of Stat5a during late pregnancy and lactation. We conclude that the three Akt isoforms have different roles in mammary gland differentiation during pregnancy and this may reflect differences in hormonal signaling.

Figure 1. Akt1 ablation inhibits whereas Akt2 ablation enhances the formation of lobuloalveolar structures in the mammary gland during pregnancy. Whereas the Akt1 ablation phenotype is cell autonomous, the Akt2 ablation phenotype is not

A. Akt isoforms are differentially expressed during pregnancy lactation and involution. Western blot analyses of mammary gland lysates harvested from wild type mice at the indicated stages of mammary gland development: p17.5 is pregnancy day 17.5; L1, L3 and L10 are postnatal (lactation) days 1,3 and 10; Inv1, Inv3 and Inv7 are involution days 1,3 and 7. Immunoblots were probed with the indicated Akt isoform-specific antibodies. β-actin was used as a loading control. B. Upper Panel: Whole mounts of mammary glands of wild type, Akt1^−/−, Akt2^−/− and Akt3^−/− mice harvested on day 17.5 of pregnancy. Mammary epithelia were stained with carmine red. Middle Panel: Mammary gland histology of wild type, Akt1^−/−, Akt2^−/−, and Akt3^−/− mice at day 17.5 of pregnancy. Lower Panel: Higher magnification of mammary gland histology on day P17.5. C. Transplantation of wild type, Akt1^−/− and Akt2^−/− mammary epithelia into wild type mammary fat pads shows that the phenotype of Akt1 ablation is cell autonomous, while the phenotype of Akt2 ablation is not.

Figure 2. Ablation of Akt1 enhances the proliferation and inhibits the survival of mammary epithelia during pregnancy

A. Tissue sections of mammary glands from wild type, Akt1^−/−, Akt2^−/− and Akt3^−/− mice, harvested at pregnancy day 17.5 (p17.5), were stained with an antibody against the proliferation marker, Ki-67. B. Graphical representation of the data shown in figure 2A. Left Panel The bars show the mean percentage of Ki-67-positive cells ^+/− the SE of the mean for each genotype. The number of Ki-67-positive cells was measured in sections of mammary glands derived from four mice of each genotype. Four randomly picked fields were counted in each mouse specimen. Right Panel. The bars show the total number of cells counted for the data analysis shown in the left panel. C. Apoptotic cells in mammary gland tissue sections of wild type, Akt1^−/−, Akt2^−/− and Akt3^−/− mice at pregnancy day 17.5 (p17.5) were detected with the TUNEL assay in a panel of slide-mounted methyl green-stained mammary tissue sections. The TUNEL assay utilizes horseradish peroxidase-conjugated anti-digoxigenin antibodies to detect digoxigenin-labeled DNA ends. D. Cumulative data of the experiment shown in figure 2C. Left Panel. The bars show the mean percentage of apoptotic cells +/− the SE of the mean for each genotype. The number of TUNEL-positive apoptotic cells was measured in sections of mammary glands derived from four mice of each genotype. Four randomly picked fields were counted in each mouse specimen. Right Panel. The bars show the total number of cells counted for the data analysis shown in the left panel.

Figure 3. Ablation of Akt1 delays luminal cell differentiation during pregnancy and lactation and impairs milk production

A. Histology of the mammary gland of wild type, Akt1^−/−, Akt2^−/− and Akt3^−/− mice on the first day of lactation. B. Histology of the mammary gland of wild type mice (upper panel) and Akt1^−/− mice (lower panel) on pregnancy day 17.5 (p17.5) and on lactation days 1 (L1) and 10 (L10). C. The expression of the milk protein WAP is delayed in the mammary gland of Akt1^−/− mice and it is accelerated in the mammary gland of Akt2^−/− mice. The expression of β-casein is also delayed in Akt1^−/− but not in Akt2^−/− mice. Milk protein expression was measured by western blotting of mammary gland lysates harvested at the indicated time points. P17.5 is pregnancy day 17.5. L1, L3 and L10 are lactation days 1, 3 and 10. These results are reproducible and were similar in three different experiments. D. The perinatal lethality of Akt1^−/− mice is due to a combination of maternal milk production defects and intrinsic embryonic defects. Percentage of surviving Akt1^−/− mice born to Akt1^+/− (n=24) and Akt1^−/− (n=15) mothers. Neonatal lethality was defined as death within the first 9 days of life. The rate of survival of the Akt1^−/− offspring of Akt1^+/− mothers and the rate of survival of the Akt1^−/− offspring of Akt1^−/− mothers were calculated as described in the results section. The statistical significance of the difference in rate of survival between mice born to Akt1^+/− and Akt1^−/− mothers is p<0.0547.

Figure 4. Ablation of Akt1 inhibits the differentiation of mammary epithelia during pregnancy by interfering with the phosphorylation of Stat5a

A1, A2 and A3. Kinetics of Stat5a phosphorylation on tyrosine 694 in wild type, Akt1^−/− and Akt2^−/− mice during late pregnancy and lactation. Western blots of lysates from wild type, Akt1^−/− and Akt2^−/− mice mammary gland tissue, harvested at adult, pregnancy day 17.5 (p17.5), and lactation days 1, 3, 10 and 14 (L1, L3, L10 and L14) time points were probed with antibodies against total and tyrosine-phosphorylated Stat5a and β-actin (loading control). B. Stat5a phosphorylation in the mammary glands of wild type, Akt1^−/− and Akt2^−/− mice on lactation days 1(L1) and 14 (L14).

Figure 5. Ablation of Akt1 accelerates, while ablation of Akt2 delays post-lactation involution of the mammary gland

A. Histology of the mammary gland of wild type, Akt1^−/−Akt2^−/−, and Akt3^−/− mice on post-lactation involution days 1, day 3, and 7. B. Sections of the mammary gland of wild type, Akt1^−/−, Akt2^−/−, and Akt3^−/− mice, harvested on the 3^rd day of post-lactation involution, were stained for apoptotic cells, using the TUNEL assay. C. Cumulative data of the experiment shown in figure 5B. The bars show the mean percentage of apoptotic cells +/− the SE of the mean for each genotype. The number of apoptotic cells was measured in sections derived from four mice of each genotype. Four randomly picked fields were counted in each mouse specimen.