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.     

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.     

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.     

Conditional repression of STAT5 expression during lactation reveals its exclusive roles in mammary gland morphology, milk-protein gene expression, and neonate growth

The role of Stat5 in maintaining adequate lactation was studied in Stat5a(-/-) mice expressing a conditionally suppressed transgenic STAT5 in their mammary glands. This system enables distinguishing STAT5's effects on lactation from its contribution to mammary development during gestation. Females were allowed to express STAT5 during their first pregnancy. After delivery, STAT5 levels were manipulated by doxycycline administration and withdrawal. In two lines of genetically modified mice, the absence of STAT5 expression during the first 10 days of lactation resulted in a decrease of 29% or 41% in newborn weight gain. The STAT5-dependent decrease in growth was recoverable, but not completely reversible, particularly when STAT5 expression was omitted for the first 4 days of lactation. Within the first 10 days of STAT5-omitted lactation, alveolar occupancy regressed by 50% compared to that measured at delivery. By Day 10, only 18% of the fat-pad area was involved in milk production. The alveolar regression caused by 4 days of STAT5 deficiency was reversible, but neonate growth remained delayed. STAT5 deficiency resulted in reduced estrogen receptor α and connexin 32 gene expression, accompanied by delayed induction of both anti- and pro-apoptotic Bcl-2 family members. An increase in Gata-3 expression may reflect an attempt to maintain alveolar progenitors. A decrease of 39% and 23% in WAP and α-lactalbumin expression, respectively, with no associated effects on β-casein, also resulted from lack of STAT5 expression in the first 10 days of lactation. This deficiency enhances the major effect of alveolar regression on delayed weight gain in newborns.     

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.     

Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice

The cyclooxygenase (COX)-2 gene encodes an inducible prostaglandin synthase enzyme that is overexpressed in adenocarcinomas and other tumors. Deletion of the murine Cox-2 gene in Min mice reduced the incidence of intestinal tumors, suggesting that it is required for tumorigenesis. However, it is not known if overexpression of Cox-2 is sufficient to induce tumorigenic transformation. We have derived transgenic mice that overexpress the human COX-2 gene in the mammary glands using the murine mammary tumor virus promoter. The human Cox-2 mRNA and protein are expressed in mammary glands of female transgenic mice and were strongly induced during pregnancy and lactation. Female virgin Cox-2 transgenic mice showed precocious lobuloalveolar differentiation and enhanced expression of the beta-casein gene, which was inhibited by the Cox inhibitor indomethacin. Mammary gland involution was delayed in Cox-2 transgenic mice with a decrease in apoptotic index of mammary epithelial cells. Multiparous but not virgin females exhibited a greatly exaggerated incidence of focal mammary gland hyperplasia, dysplasia, and transformation into metastatic tumors. Cox-2-induced tumor tissue expressed reduced levels of the proapoptotic proteins Bax and Bcl-x(L) and an increase in the anti-apoptotic protein Bcl-2, suggesting that decreased apoptosis of mammary epithelial cells contributes to tumorigenesis. These data indicate that enhanced Cox-2 expression is sufficient to induce mammary gland tumorigenesis. Therefore, inhibition of Cox-2 may represent a mechanism-based chemopreventive approach for carcinogenesis.     

Induction of mammary gland differentiation in transgenic mice by the fatty acid-binding protein MRG

A mammary-derived growth inhibitor-related gene (MRG) was previously identified and characterized. MRG induces differentiation of mammary epithelial cells in vitro and its expression is associated with mammary differentiation. To further define the role of MRG on mammary gland differentiation, a MRG transgenic mice model under the control of mouse mammary tumor virus promoter was established and the effect of MRG on mammary gland differentiation was investigated at histological and molecular levels. Expression of endogenous mouse MRG gene was significantly increased from the non-differentiated gland of control virgin mice to the functionally differentiated gland of pregnant control mice. Whole mount analyses demonstrated that ductal development was not affected by MRG transgene expression. While there was no lobuloalveolar structure in control virgin mice, expression of MRG transgene in the mammary gland resulted in the development of lobuloalveolar-like structure, which mimics the gland from early pregnancy. Consistent with the morphological change, expression of MRG also increased milk protein beta-casein expression in the gland. To study the mechanism of MRG-induced mammary differentiation, we investigated the Stat5 activation in the glands from the transgenic mouse versus virgin control mouse. While activated Stat5 was expressed at the minimal level in the non-differentiated control virgin gland, a significant Stat5 phosphorylation was observed in the virgin transgenic gland. Our data indicate that MRG is a mediator of the differentiating effects of pregnancy on breast epithelium, and overexpression of MRG in young nulliparous mice can induce differentiation.     

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.     

Ectopic expression of β-lactoglobulin/human serum albumin fusion genes in transgenic mice: hormonal regulation andin situ localization

We produced transgenic mice carrying the native sheep -lactoglobulin (BLG) or fusion genes composed of the BLG promoter and human serum albumin (HSA) minigenes. BLG was expressed exclusively in the mammary glands of the virgin and lactating transgenic mice evaluated. In contrast, transgenic females carrying the BLG/HSA fusion constructs also expressed the HSA RNA ectopically in skeletal muscle, kidney, brain, spleen, salivary gland and skin. Ectopic expression of HSA RNA was detected only in strains that express the transgene in the mammary gland. There was no obvious correlation between the level of the HSA RNA expressed in the mammary gland and that found ectopically. In three transgenic strains analysed, the expression of HSA RNA in kidney and skeletal muscle increased during pregnancy and lactation, whereas in the brain HSA expression decreased during lactation in one of the strains. HSA protein was synthesized in skeletal muscle and skin of strain #23 and its level was higher in lactating mice compared with virgin mice. Expression of HSA was also analysed in males and was found to be more stringently controlled than in females of the same strains.In situ hybridization analyses localized the expressed transgene in the skin, kidney, brain and salivary glands of various transgenic strains. Distinct strain-specific and cell-type specific HSA expression patterns were observed in the skin. This is in contrast to the exclusive expression of the HSA transgene in epithelial cells surrounding the alveoli of the mammary gland. Taken together, these results suggest that the absence of sufficient mammary-specific regulatory elements in the BLG promoter sequences and/or the juxtaposition of the BLG promoter with the HSA coding sequences leads to novel tissue- and cell-specific expression in ectopic tissues of transgenic mice.