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.     

Lactation defect in a widely used MMTV-Cre transgenic line of mice

Background

MMTV-Cre mouse lines have played important roles in our understanding about the functions of numerous genes in mouse mammary epithelial cells during mammary gland development and tumorigenesis. However, numerous studies have not included MMTV-Cre mice as controls, and many investigators have not indicated which of the different MMTV-Cre founder lines were used in their studies. Here, we describe a lactation defect that severely limits the use of one of the most commonly used MMTV-Cre founder lines.

Methodology/Principal Findings

To explore the role of protein tyrosine phosphatase Shp1 in mammary gland development, mice bearing the floxed Shp1 gene were crossed with MMTV-Cre mice and mammary gland development was examined by histological and biochemical techniques, while lactation competency was assessed by monitoring pup growth. Surprisingly, both the Shp1fl/+;MMTV-Cre and MMTV-Cre female mice displayed a severe lactation defect when compared to the Shp1 fl/+ control mice. Histological and biochemical analyses reveal that female mice expressing the MMTV-Cre transgene, either alone or in combination with floxed genes, exhibit defects in lobuloalveolar expansion, presence of large cytoplasmic lipid droplets in luminal alveolar epithelial cells postpartum, and precocious induction of involution. Using a PCR-based genotyping method, the three different founder lines can be distinguished, and we determined that the MMTV-Cre line A, the most widely used MMTV-Cre founder line, exhibits a profound lactation defect that limits its use in studies on mammary gland development.

Conclusions/Significance

The identification of a lactation defect in the MMTV-Cre line A mice indicates that investigators must use MMTV-Cre alone mice as control in studies that utilize Cre recombinase to excise genes of interest from mammary epithelial cells. Our results also suggest that previous results obtained in studies using the MMTV-Cre line A line should be re-evaluated if the controls did not include mice expressing only Cre recombinase.     

Forced involution of the functionally differentiated mammary gland by overexpression of the pro-apoptotic protein bax

The mammary gland is a developmentally dynamic, hormone-responsive organ that undergoes proliferation and differentiation within the secretory epithelial compartment during pregnancy. The epithelia are maintained by pro-survival signals (e.g., Stat5, Akt1) during lactation, but undergo apoptosis during involution through inactivation of cell survival pathways and upregulation of pro-apoptotic proteins. To assess if the survival signals in the functionally differentiated mammary epithelial cells can override a pro-apoptotic signal, we generated transgenic mice that express Bax under the whey acidic protein (WAP) promoter. WAP-Bax females exhibited a lactation defect and were unable to nourish their offspring. Mammary glands demonstrated: (1) a reduction in epithelial content, (2) hallmark signs of mitochondria-mediated cell death, (3) an increase in apoptotic cells by TUNEL assay, and (4) precocious Stat3 activation. This suggests that upregulation of a single pro-apoptotic factor of the Bcl-2 family is sufficient to initiate apoptosis of functionally differentiated mammary epithelial cells in vivo.     

Differential expression of claudin 1, 3, and 4 during normal mammary gland development in the mouse

The claudins are a family of tight junction proteins that display varied tissue distribution and preferential specificity. We recently identified by microarray analysis, members of this family, particularly claudin 1 (cldn1), as highly upregulated genes in the mouse mammary gland during early involution. Gene expression was confirmed by immunohistochemistry and real-time PCR. We then examined gene and protein expression throughout normal mammary gland development. The cldn3 gene showed a steady increase in expression from pregnancy to involution, while cldn1 and cldn4 gene expression increased during pregnancy, but decreased sharply by D10 of lactation, and once again was significantly increased by D1 of involution (P < 0.001 for both genes). The different patterns of gene expression observed between cldn3, and cldn1, and 4 suggest that different family members may be functionally important at different times during mouse mammary gland development. All three genes exhibited a high level of expression at day 1 (D1) of involution, followed by a dramatic decrease in gene expression to day 10 of involution. Immunostaining with the cldn3 antibody showed intense staining of epithelial cells; however, a lesser degree of staining was evident with the cldn1 antibody. In addition to the lateral staining of epithelial cells, basal staining was evident at D1 and D2 of involution and cytoplasmic staining was evident during lactation. Since claudins are known to play a role as tight junction proteins, lateral and basal staining may suggest a role in other functions such as vesicle trafficking or remodeling of tight junctions at different stages of mammary gland development. Cldn1 and 3 antibodies also stained epithelial cells in mouse mammary tumors. In summary, cldn1, 3, and 4 are differentially expressed in the mammary gland during pregnancy, lactation, and involution, suggesting different roles for these proteins at different stages of mammary gland function. In addition, cldn1 and cldn3 are detected in mammary tumors and the wide distribution of cldn3 in particular, suggest specific roles for these proteins in mammary tumorigenesis.     

c-myc as a mediator of accelerated apoptosis and involution in mammary glands lacking Socs3

Suppressor of cytokine signalling (SOCS) proteins are critical attenuators of cytokine-mediated signalling in diverse tissues. To determine the importance of Socs3 in mammary development, we generated mice in which Socs3 was deleted in mammary epithelial cells. No overt phenotype was evident during pregnancy and lactation, indicating that Socs3 is not a key physiological regulator of prolactin signalling. However, Socs3-deficient mammary glands exhibited a profound increase in epithelial apoptosis and tissue remodelling, resulting in precocious involution. This phenotype was accompanied by augmented Stat3 activation and a marked increase in the level of c-myc. Moreover, induction of c-myc before weaning using an inducible transgenic model recapitulated the Socs3 phenotype, and elevated expression of likely c-myc target genes, E2F-1, Bax and p53, was observed. Our data establish Socs3 as a critical attenuator of pro-apoptotic pathways that act in the developing mammary gland and provide evidence that c-myc regulates apoptosis during involution.     

Mammary gland remodeling depends on gp130 signaling through Stat3 and MAPK

The interleukin-6 (IL6) family of cytokines signals through the common receptor subunit gp130, and subsequently activates Stat3, MAPK, and PI3K. Stat3 controls cell death and tissue remodeling in the mouse mammary gland during involution, which is partially induced by IL6 and LIF. However, it is not clear whether Stat3 activation is mediated solely through the gp130 pathway or also through other receptors. This question was explored in mice carrying two distinct mutations in the gp130 gene; one that resulted in the complete ablation of gp130 and one that led to the loss of Stat3 binding sites (gp130Delta/Delta). Deletion of gp130 specifically from mammary epithelium resulted in a complete loss of Stat3 activity and resistance to tissue remodeling comparable to that seen in the absence of Stat3. A less profound delay of mammary tissue remodeling was observed in gp130Delta/Delta mice. Stat3 tyrosine and serine phosphorylation was still detected in these mice suggesting that Stat3 activation could be the result of gp130 interfacing with other receptors. Experiments in primary mammary epithelial cells and transfected COS-7 cells revealed a p44/42 MAPK and EGFR-dependent Stat3 activation. Moreover, the gp130-dependent EGFR activation was independent of EGF ligands, suggesting a cytoplasmic interaction and cross-talk between these two receptors. These experiments establish that two distinct Stat3 signaling pathways emanating from gp130 are utilized in mammary tissue.     

Developmental stage determines the effects of MYC in the mammary epithelium

Epidemiological findings suggest that the consequences of a given oncogenic stimulus vary depending upon the developmental state of the target tissue at the time of exposure. This is particularly evident in the mammary gland, where both age at exposure to a carcinogenic stimulus and the timing of a first full-term pregnancy can markedly alter the risk of developing breast cancer. Analogous to this, the biological consequences of activating oncogenes, such as MYC, can be influenced by cellular context both in terms of cell lineage and cellular environment. In light of this, we hypothesized that the consequences of aberrant MYC activation in the mammary gland might be determined by the developmental state of the gland at the time of MYC exposure. To test this hypothesis directly, we have used a doxycycline-inducible transgenic mouse model to overexpress MYC during different stages of mammary gland development. Using this model, we find that the ability of MYC to inhibit postpartum lactation is due entirely to its activation within a specific 72-hour window during mid-pregnancy; by contrast, MYC activation either prior to or following this 72-hour window has little or no effect on postpartum lactation. Surprisingly, we find that MYC does not block postpartum lactation by inhibiting mammary epithelial differentiation, but rather by promoting differentiation and precocious lactation during pregnancy, which in turn leads to premature involution of the gland. We further show that this developmental stage-specific ability of MYC to promote mammary epithelial differentiation is tightly linked to its ability to downregulate caveolin 1 and activate Stat5 in a developmental stage-specific manner. Our findings provide unique in vivo molecular evidence for developmental stage-specific effects of oncogene activation, as well as the first evidence linking MYC with activation of the Jak2-Stat5 signaling pathway.     

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.     

Leukemia inhibitory factor induces apoptosis of the mammary epithelial cells and participates in mouse mammary gland involution

Leukemia inhibitory factor (LIF) is a multifunctional glycoprotein that displays multiple biological activities in different cell types, but to date there has been no report on its expression in the normal mammary gland. In this study we found that LIF is expressed at low but detectable levels in postpubertal, adult virgin, and pregnant mouse mammary glands. However, LIF expression drops after parturition to become almost undetectable in lactating glands. Interestingly, LIF expression shows a steep increase shortly after weaning that is maintained for the following 3 days. During this period, known as the first stage of mammary gland involution, the lack of suckling induces local factors that cause extensive epithelial cell death. It has been shown that Stat3 is the main factor in signaling the initiation of apoptosis, but the mechanism of its activation remains unclear. Herein, we show that LIF expression in the gland is induced by milk stasis and not by the decrease of circulating lactogenic hormones after weaning. Implantation of LIF containing pellets in lactating glands results in a significant increase in epithelium apoptosis. In addition, this treatment also induces Stat3 phosphorylation. We conclude that LIF regulated expression in the mouse mammary gland may play a relevant role during the first stage of mammary gland involution. Our results also show that LIF-induced mammary epithelium apoptosis could be mediated, at least partially, by Stat3 activation.