Forced activation of Stat5 subjects mammary epithelial cells to DNA damage and preferential induction of the cellular response mechanism during proliferation

Parity‐dependent adenocarcinoma tumors developed in postestropausal transgenic mice expressing a constitutively active Stat5 variant (STAT5ca) in their mammary gland. These tumors maintained elevated expression levels of genes regulating the cellular DNA damage response (DDR) mechanism, compared to the intact gland. No correlation with STAT5ca expression was observed for these genes in the established tumors. However, activated Stat5a in individual cells of the rarely and earlier developed hyperplasia was associated with induced Chk2 activity. Deregulated Stat5 may already cause DNA damage during the fertile period. This hypothesis and the specific vulnerable stage were further studied in mammary epithelial cells that were stably transfected with β‐lactoglobulin (BLG)/STAT5ca and exposed to a reproduced reproductive cycle. During the pregnancy‐like proliferative state, STAT5ca expression was induced by the added lactogenic hormones. Production of reactive oxygen species, rather than proliferation, served as the primary mediator of DNA damage and cellular DDR. Differentiated cells expressed higher levels of STAT5ca and retained the DNA nicks. However, the elevated expression of the genes involved in DDR was downregulated. Higher levels of DNA damage were also detected in the mammary gland of transgenic mice expressing the BLG/STAT5ca during pregnancy and lactation. However, the relative number of damaged cells was much lower than that in the reproduced in vitro stages and the insults were generally associated with apoptosis and DDR. This study implicates pregnancy as the vulnerable stage for deregulated Stat5 activity, and demonstrates that DNA insults in viable differentiated mammary epithelial cells are ignored by the DDR mechanism. J. Cell. Physiol. 226: 616–626, 2011. © 2010 Wiley‐Liss, Inc.     

Cell turnover and gene activities in sheep mammary glands prior to lambing to involution

Mammary glands are special tissue characterized by proliferation of the epithelium, during puberty and pregnancy and by programmed cell death, during involution. In this study, apoptosis was identified by TUNEL staining and then related to cell proliferation, as determined by Ki-67 staining. The apoptotic index was at its highest at 8 days of involution, whereas the proliferation index was at its highest during lactation. Caspase-3 was immunolocalised only in mast cells and along the basal membrane in the mammary tissue at −10 days from lambing, 150 days of lactation and at 8 days of involution. This finding could indicate that caspase-3 is not involved in sheep mammary gland apoptosis, but that other proteins – such as apoptosis inducing factor (AIF) – can trigger apoptosis, through the mitochondrial pathway, in a caspase-independent manner. The expression of genes involved in the regulation of lactation and apoptosis was also investigated and determined relatively to −10 days from lambing. The relative expression level of LALBA, reached its maximum during lactation, whereas the expressions of BCL2, BCL2L1, BAX, STAT5A, STAT3, IGFBP5 and FOXO3A, increased significantly during involution in correlation with apoptotic index.This work shows for the first time the turnover of mammary cells and the interaction of their signals during the complete lactation cycle in sheep. The data on gene expression can contribute to elucidate the mechanisms controlling milk production and cell turnover in this species.     

Essential functions of p21-activated kinase 1 in morphogenesis and differentiation of mammary glands

Although growth factors have been shown to influence mammary gland development, the nature of downstream effectors remains elusive. In this study, we show that the expression of p21-activated kinase (Pak)1, a serine/threonine protein kinase, is activated in mammary glands during pregnancy and lactation. By targeting an ectopic expression of a kinase-dead Pak1 mutant under the control of ovine beta-lactoglobulin promoter, we found that the mammary glands of female mice expressing kinase-dead Pak1 transgene revealed incomplete lobuloalveolar development and impaired functional differentiation. The expression of whey acidic protein and beta-casein and the amount of activated Stat5 in the nuclei of epithelial cells in transgenic mice were drastically reduced. Further analysis of the underlying mechanisms revealed that Pak1 stimulated beta-casein promoter activity in normal mouse mammary epithelial cells and also cooperated with Stat5a. Pak1 directly interacted with and phosphorylated Stat5a at Ser 779, and both COOH-terminal deletion containing Ser 779 of Stat5a and the Ser 779 to Ala mutation completely prevented the ability of Pak1 to stimulate beta-casein promoter. Mammary glands expressing inactive Pak1 exhibited a reduction of Stat5a Ser 779 phosphorylation. These findings suggest that Pak1 is required for alveolar morphogenesis and lactation function, and thus, identify novel functions of Pak1 in the mammary gland development.     

Prolactin-mediated regulation of lipid biosynthesis genes in vivo in the lactating mammary epithelial cell

Prolactin (PRL) is known to play an essential role in mammary alveolar proliferation in the pregnant mouse, but its role in lactation has been more difficult to define. Genetic manipulations that alter expression of the PRL receptor and its downstream signaling molecules resulted in developmental defects that may directly or indirectly impact secretory activation and lactation. To examine the in vivo role of PRL specifically in lactation, bromocriptine (BrCr) was administered every 8 h to lactating mice on the second day postpartum, resulting in an ~95% decrease in serum PRL levels. Although morphological changes in secretory alveoli were slight, by 8 h of BrCr, pup growth was inhibited significantly. Phosphorylated STAT5 fell to undetectable levels within 4 h. Decreased milk protein gene expression, β-casein, and α-lactalbumin, was observed after 8 h of treatment. To assess mammary-specific effects on lipid synthesis genes, we isolated mammary epithelial cells (MECs) depleted of mammary adipocytes. Expression of genes involved in glucose uptake, glycolysis, pentose phosphate shunt, de novo synthesis of fatty acids, and biosynthesis of triacylglycerides was decreased up to 19-fold in MECs by just 8 h of BrCr treatment. Glands from BrCr-treated mice showed a twofold reduction in intracellular cytoplasmic lipid droplets and a reduction in cytosolic β-casein. These data demonstrate that PRL signaling regulates MEC-specific lipogenic gene expression and that PRL signals coordinate the milk synthesis and mammary epithelial cell survival during lactation in the mouse.     

Pattern of expression of the KGF receptor and its ligands KGF and FGF-10 during postnatal mouse mammary gland development

The expression of the KGF receptor (KGFR) and its stromal ligands, KGF and FGF-10, was compared during mouse mammary gland development. KGFR expression in mammary parenchyma is maximal in mature virgin mice, declines during pregnancy and lactation, but rises after weaning. The rise in KGFR mRNA in the virgin animal corresponds to parenchymal growth. The fall in KGFR expression in pregnancy is driven by hormone-induced alveolar differentiation since the level of KGFR mRNA is 5-fold higher in isolated ductal cells compared to alveolar cells. KGF and FGF-10 expression patterns differ during ductal development. FGF-10 is also expressed at about a 15-fold higher molar level than KGF. During pregnancy and lactation, expression of KGF and FGF-10 decreases in intact fat pads but is unchanged in parenchyma-free fat pads. Thus, the decrease in KGF and FGF-10 expression observed in intact glands during pregnancy and lactation is not a direct consequence of the changing hormonal milieu but more likely reflects an increase in the ratio of epithelium to stroma. Differences in the level and pattern of expression of mRNA for KGF, FGF-10, and the KGFR during postnatal development of the mouse mammary gland are a result of morphological development, changes in the ratio of stroma to epithelium, and hormonal regulation of cell differentiation. These changes suggest that the biological roles that these growth factors play are regulated by fluctuations in both growth factor and growth factor receptor expression and that KGF and FGF-10 may have different regulatory functions.     

Expression of constitutively activated Akt in the mammary gland leads to excess lipid synthesis during pregnancy and lactation

Expression of constitutively activated Akt in the mammary glands of transgenic mice results in a delay in post-lactational involution. We now report precocious lipid accumulation in the alveolar epithelium of mouse mammary tumor virus-myr-Akt transgenic mice accompanied by a lactation defect that results in a 50% decrease in litter weight over the first 9 days of lactation. Although ductal structures and alveolar units develop normally during pregnancy, cytoplasmic lipid droplets appeared precociously in mammary epithelial cells in early pregnancy and were accompanied by increased expression of adipophilin, which is associated with lipid droplets. By late pregnancy the lipid droplets had become significantly larger than in nontransgenic mice, and they persisted into lactation. The fat content of milk from lactating myr-Akt transgenic mice was 65-70% by volume compared to 25-30% in wild-type mice. The diminished growth of pups nursed by transgenic mothers could result from the high viscosity of the milk and the inability of the pups to remove sufficient quantities of milk by suckling. Transduction of the CIT3 mammary epithelial cell line with a recombinant human adenovirus encoding myr-Akt resulted in an increase in glucose transport and lipid biosynthesis, suggesting that Akt plays an important role in regulation of lipid metabolism.     

Concurrent pregnancy retards mammary involution: effects on apoptosis and proliferation of the mammary epithelium after forced weaning of mice

The effect of pregnancy on postweaning mammary gland involution was investigated in mice. On the third day after forced weaning at Lactation Day 10, the apoptotic index was 56% lower in mammary tissue of mice that were pregnant at the time of weaning than in nonpregnant mice. Conversely, the bromodeoxyuridine-labeling index was increased sevenfold in pregnant mice compared to nonpregnant controls (3.5% vs. 0.5%, respectively). Structure of mammary alveoli was largely maintained in postweaning pregnant mice. The effect of pregnancy on three specific mammary epithelial cell survival pathways was also examined. First, pregnancy blocked the loss of Stat5a phosphorylation during involution. Significantly, loss of Stat5a phosphorylation during involution was not correlated with loss of Stat5a nuclear localization. Second, pregnancy maintained nuclear-localized progesterone receptor during lactation. Third, pregnancy was associated with increased expression of bfl-1 during involution but had little effect on the expression of other bcl-2 family members. The data indicate that pregnancy inhibits mammary cell apoptosis after weaning while permitting proliferation of the mammary epithelium, and they support the hypothesis that Stat5a and progesterone-signaling pathways act in concert to mediate this effect.     

Peripheral Serotonin Regulates Maternal Calcium Trafficking in Mammary Epithelial Cells during Lactation in Mice

Lactation is characterized by massive transcellular flux of calcium, from the basolateral side of the mammary alveolar epithelium (blood) into the ductal lumen (milk). Regulation of calcium transport during lactation is critical for maternal and neonatal health. The monoamine serotonin (5-HT) is synthesized by the mammary gland and functions as a homeostatic regulation of lactation. Genetic ablation of tryptophan hydroxylase 1 (Tph1), which encodes the rate-limiting enzyme in non-neuronal serotonin synthesis, causes a deficiency in circulating serotonin. As a consequence maternal calcium concentrations decrease, mammary epithelial cell morphology is altered, and cell proliferation is decreased during lactation. Here we demonstrate that serotonin deficiency decreases the expression and disrupts the normal localization of calcium transporters located in the apical (PMCA2) and basolateral (CaSR, ORAI-1) membranes of the lactating mammary gland. In addition, serotonin deficiency decreases the mRNA expression of calcium transporters located in intracellular compartments (SERCA2, SPCA1 and 2). Mammary expression of serotonin receptor isoform 2b and its downstream pathways (PLCβ3, PKC and MAP-ERK_1/2) are also decreased by serotonin deficiency, which might explain the numerous phenotypic alterations described above. In most cases, addition of exogenous 5-hydroxy-L-tryptophan to the Tph1 deficient mice rescued the phenotype. Our data supports the hypothesis that serotonin is necessary for proper mammary gland structure and function, to regulate blood and mammary epithelial cell transport of calcium during lactation. These findings can be applicable to the treatment of lactation-induced hypocalcemia in dairy cows and can have profound implications in humans, given the wide-spread use of selective serotonin reuptake inhibitors as antidepressants during pregnancy and lactation.     

Heterogeneous Inducible Mammary-specific Expression of JAB/SOCS1 in Lactating Transgenic Mice is Associated with no Obvious Phenotype,even at the Cellular Level

The cytokine-inducible suppressor of cytokine signalling SOCS1, or JAB, has been shown to be implicated in vitro in the negative regulation of the prolactin-receptor-induced activation of JAK2 and STAT5. Disruption of this gene in vivo resulted in an accelerated mammary gland development. In the present experiment, we assessed the potential impact on the lactation process of the doxycycline-inducible mammary-controlled expression of this gene in transgenic mice. Three transgenic mouse lines that expressed JAB specifically in the mammary gland in a conditional manner following doxycycline treatment were successfully established. The resulting overall expression of JAB was high and ranged from half to four times that of the endogenously expressed homologous gene in the thymus. It was found to be highly heterogeneous in the mammary epithelium, with less than 5% of JAB-expressing cells detected. Phenotypic analysis of these transgenic mice exhibiting doxycycline-induced JAB expression did not reveal any obvious effect on the lactation process. Double immunostaining experiments suggested that JAB expression in vivo did not significantly affect the beta-casein gene expression and the STAT5a nuclear localisation. These results do not support a role for JAB in the disruption of the lactation process.