Dome formation in cell cultures as expression of an early stage of lactogenic differentiation of the mammary gland

The study of the development of the mammary gland at the molecular level in the animal is difficult because of the complex tissue organization of the gland. We have previously developed an in vitro system for genetic analysis of mammary cell differentiation, based on the cell line LA7 clonally derived from a rat mammary adenocarcinoma. This cell line, after induction with DMSO, differentiates forming structures called domes. This process is under strict gene regulation, and we have previously identified several of the genes involved. In the present paper, we have defined the meaning of dome formation in relation to mammary development, by showing that treatment of LA7 cells with the lactogenic hormones hydrocortisone and prolactin induces dome formation; in the animal, these hormones precede and accompany milk production. Moreover, dome formation is accompanied by expression within the cells of the milk protein genes WDMN1 and beta-casein, which are differentiation markers for the gland during pregnancy and lactation. We also show that two proteins, highly expressed in the mammary gland during lactation, HSP90-beta and annexin I, are strongly expressed in DMSO-induced LA7 cells. Both proteins are essential in the formation of domes because when their synthesis is blocked by antisense RNA oligonucleotides, dome formation is abolished. Thus our in vitro system is a model for lobulo-alveolar development, and the genes identified in the pathway of dome formation are likely to be involved in the early differentiation steps occurring in the rat mammary gland during pregnancy and lactation.     

Changes in secretory cell turnover, and mitochondrial oxidative damage in the mouse mammary gland during a single prolonged lactation cycle suggest the possibility of accelerated cellular aging

Milk synthesis by the mammary gland declines during prolonged lactation despite the continued suckling stimulus and complete removal of mammary secretions. Although this process has been hypothesized to result from cellular aging there has been no reported analysis of aging markers in the lactating mammary gland. The goal of these studies was to relate lactation performance in the mouse during a single prolonged lactation cycle to changes in mammary development and mitochondrial oxidative damage. During an artificially prolonged lactation cycle, the capacity of the dams to support litter growth decreased over time. This decrease was associated with decreased mammary epithelial content. Cell proliferation, along with the percentage of mammary progenitor cells, was high during early lactation, but low during prolonged lactation. Apoptosis increased during prolonged lactation. Oxidative damage to mitochondrial DNA increased during the early postpartum period and remained elevated through the end of the cycle. In contrast oxidative damage to mitochondrial protein was high during early lactation and decreased through mid lactation to increase again with prolonged lactation. The results suggest that a single prolonged lactation cycle may replicate on an accelerated basis some of the changes that occur with a lifetime of aging in organs possessing more stable cell populations.     

STAT signaling in mammary gland differentiation,cell survival and tumorigenesis

The mammary gland is a unique organ that undergoes extensive and profound changes during puberty, menstruation, pregnancy, lactation and involution. The changes that take place during puberty involve large-scale proliferation and invasion of the fat-pad. During pregnancy and lactation, the mammary cells are exposed to signaling pathways that inhibit apoptosis, induce proliferation and invoke terminal differentiation. Finally, during involution the mammary gland is exposed to milk stasis, programmed cell death and stromal reorganization to clear the differentiated milk-producing cells. Not surprisingly, the signaling pathways responsible for bringing about these changes in breast cells are often subverted during the process of tumorigenesis. The STAT family of proteins is involved in every stage of mammary gland development, and is also frequently implicated in breast tumorigenesis. While the roles of STAT3 and STAT5 during mammary gland development and tumorigenesis are well studied, others members, e.g. STAT1 and STAT6, have only recently been observed to play a role in mammary gland biology. Continued investigation into the STAT protein network in the mammary gland will likely yield new biomarkers and risk factors for breast cancer, and may also lead to novel prophylactic or therapeutic strategies against breast cancer.     

Paracrine regulation of mammary gland growth

The growth and differentiation of the mammary gland is a complex process involving the interactions of various steroid and polypeptide hormones. The mammary growth occurs in a discontinued way during five distinct phases, i.e. fetal, prepubertal, postpubertal, pregnancy, and early lactation periods. The gland expresses its differentiated function by producing milk during the period of lactation. Although the mammary gland has been regarded as one of the well-known target tissues for various types of hormones, evidence has been accumulating in recent years indicating the involvement of other factors and substances in the process of mammary growth and differentiation. In this chapter the importance of the mesenchymal component in mammary epithelial cell growth has been documented. This component, including embryonic mesenchyme and adipocytes in adult tissue, play an essential role by not only serving as a structural entity of the gland but also by producing extracellular matrix substances and various factors that promote the growth, morphologic development and differentiation of mammary epithelium in a paracrine fashion. Other possible paracrine peptide factors for mammary cell growth have been isolated from several other sources including mammary tumors and milk. Thus, it is possible that paracrine growth factors play a role in mammary tumorigenesis. Since most of these factors are present in minute amounts, it is difficult to obtain pure forms of these factors in sufficient amounts for detailed physicochemical characterization. Moreover, further studies are needed to assess the physiological importance of these growth factors, their mode of action, and the mechanism of regulation relating to their production. It is conceivable that some mammary paracrine agents interact with each other or with endocrine agents in promoting the normal and neoplastic growth of mammary cells. Furthermore, the possibility exists that the production and release of paracrine factors are under the endocrine control. In view of the rapid progress and great interest in this area, these questions may be answered before long, along with the discovery of some other new growth regulating agents in this system. Clearly such information is important for understanding the complex process of normal and neoplastic growth of the mammary gland.     

Glucocorticoids and progesterone prevent apoptosis in the lactating rat mammary gland.

Apoptosis, a form of noninflammatory cell death, plays a central role in mammary gland involution after weaning. Previous studies have shown that apoptosis in the postweaning mammary gland is substantially reduced by treatment with glucocorticoids or progesterone, but whether these steroids exert a similar antiapoptotic effect during normal lactation is not known. Therefore, the present study used an in vivo rat model to assess the effects of progesterone and glucocorticoids on apoptosis in the lactating mammary gland. Rats were untreated, sham operated, ovariectomized (OVX), and/or adrenalectomized (ADX) on d 10 of lactation. Additional groups of OVX/ADX rats were treated with either progesterone or corticosterone. Mammary gland apoptosis was determined 3 d later by 3'-end labeling of fragmented DNA and by in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labeling analysis (TUNEL). DNA fragmentation was relatively low in the mammary gland from untreated and sham-operated rats and was unaffected by either ADX or OVX alone. In contrast, DNA fragmentation was markedly elevated in OVX/ADX rats (P < 0.01), but this effect on mammary gland apoptosis was prevented by replacement with either corticosterone or progesterone. Consistent with these data, dying cells identified by TUNEL analysis were readily observed in the alveolar epithelium of mammary tissue from OVX/ADX rats but not in any of the other groups. These data demonstrate that during normal lactation, mammary gland apoptosis is inhibited by endogenous progesterone and glucocorticoids. Importantly, the presence of either steroid alone was sufficient to prevent apoptosis, suggesting that their antiapoptotic effects in the lactating mammary gland may be mediated via similar signaling pathways.     

Localization of activin and inhibin subunits,receptors and SMADs in the mouse mammary gland

Activin and inhibin, two closely related protein hormones, are members of the transforming growth factor beta (TGF beta) superfamily of growth factors. Activin and TGF beta have been associated with mouse mammary gland development and human breast carcinogenesis. TGF beta expression in the mammary gland has been previously described, and was found to be expressed in nonparous tissue and during pregnancy, down-regulated during lactation, and then up-regulated during involution. The expression pattern of activin subunits, receptors and cytoplasmic signaling molecules has not been thoroughly described in post-natal mammary gland development. We hypothesize that activin signaling components are dynamically regulated during mammary gland development, thereby permitting activin to have distinct temporal growth regulatory actions on this tissue. To examine the activin signal transduction system in the mammary gland, tissue from CD1 female mice was dissected from nonparous, lactating day 1, 10, and 20 and post-weaning day 4 animals. The expression of the activin receptors (ActRIIA, ActRIIB and ActRIB), the inhibin co-receptor (betaglycan), and ligand subunit (alpha, beta A and beta B), mRNA was measured by semi-quantitative RT-PCR in these tissues. In addition, the cellular compartmentalization of the activin signaling proteins, including the cytoplasmic signaling co-activators, Smads 2, 3 and 4, were examined by immunohistochemistry. Generally, mRNA abundance of activin signaling components was greatest in the nonparous tissue, and then decreased, whereas protein immunoreactivity for activin signaling components increased during lactation and decreased during involution. The alpha-subunit protein was detected in nonparous and lactating day 1 tissue only. Importantly, Smad 3, but not Smad 2, was detected in epithelial cell nuclei during all time points examined, indicating that activin signaling is mediated by Smad 3 at these times. These findings suggest that activin's growth regulatory role during lactation may be distinguished from that of TGF beta during post-natal mammary development. Future studies will focus on determining the exact role this ligand plays in mammary tissue differentiation and neoplasia.     

Reversible transdifferentiation of secretory epithelial cells into adipocytes in the mammary gland

Mammalian breast adipose tissue is replaced by a milk-secreting gland during pregnancy; the reverse process takes place upon interruption of lactation. Morphological and bromodeoxyuridine studies provide indirect evidence that mouse mammary adipocytes transform into secretory epithelial cells during pregnancy and revert to adipocytes after lactation. By using the Cre-loxP recombination system we show that the mammary gland of whey acidic protein (WAP)-Cre/R26R mice, in which secretory epithelial cells express the lacZ gene during pregnancy, contains labeled adipocytes during involution. Conversely, adipocyte P2-Cre/R26R mice, in which adipocytes are labeled before pregnancy, contain labeled secretory epithelial cells during pregnancy. We conclude that reversible adipocyte-to-epithelium and epithelium-to-adipocyte transdifferentiation occurs in the mammary gland of adult mice during pregnancy and lactation.     

Quantitative analysis of estrogen receptor proteins in rat mammary gland.

Estrogen receptor alpha and beta proteins (ERalpha and ERbeta) at various stages of development of the rat mammary gland were quantified by Western blotting. ERalpha and ERbeta recombinant proteins were used as standards, and their molar concentrations were measured by ligand binding assays. In 3-week-old pregnant, lactating, and postlactating rats the ERalpha content ranged from 0.30-1.55 fmol/microg total protein (mean values). The ERbeta content of the same samples ranged between 1.06-7.50 fmol/microg total protein. At every developmental stage, the ERbeta content of the mammary gland was higher than that of ERalpha. When receptor levels were normalized against beta-actin, it was evident that ER expression changed during development, with maximum expression of both receptors during the lactation period. With an antibody raised against the 18-amino acid insert of the ERbeta variant, originally called ERbeta2 but named ERbetains in this paper, Western blots revealed that ERbetains protein was up-regulated during the lactation period. RT-PCR showed that the levels of messenger RNA of ERbetains paralleled those of the protein. Double immunohistochemical staining with anti-ERalpha and anti-ERbetains antibodies revealed that ERbetains protein colocalized with ERalpha in 70-80% of the ERalpha-expressing epithelial cells during lactation and with 30% of these cells during pregnancy. These observations indicate that expression of ERbetains is regulated not only quantitatively, but also with regard to its cellular distribution. As ERbetains acts as the dominant repressor of ERalpha, we suggest that its coexpression with ERalpha quenches ERalpha function and may be one of the factors that contribute to the previously described insensitivity of the mammary gland to estrogens during lactation.     

Ontogeny and control of prolactin receptors in the mammary gland and liver of virgin, pregnant and lactating rats.

Prolactin receptors were identified and partially characterized in the mammary gland of the rat. The binding of 125I-labelled ovine prolactin to a subcellular particulate fraction of rat mammary gland decreased between days 30 and 100 of age. Over the same period, binding to the liver increased and there was a significant negative correlation between prolactin binding in the two tissues. Binding to the mammary gland was low during pregnancy, increased in early lactation and declined after the litters were weaned. Binding to the liver was lower during lactation than during pregnancy or the period after weaning suggesting that tissue-specific factors may operate in the control of this receptor. In virgin rats, prolactin binding by the mammary gland was increased by oestrogen. This effect was blocked by hypophysectomy and partially restored by replacement therapy with prolactin. Hypothyroidism and treatment with progesterone also reduced the response to oestrogen. The maintenance of prolactin binding by the mammary gland of lactating rats depends on the presence of the ovaries and pituitary, thyroid and adrenal glands. Examination of the ratio epithelium: stroma suggests that prolactin acts by increasing the number of epithelial cells in the mammary gland and that thyroid, adrenal and ovarian hormones modulate the number of receptors per cell.     

Acidic and basic fibroblast growth factor mRNA and protein in mouse mammary glands

Previous studies suggest that members of the fibroblast growth factor (FGF) family are mitogenic to mammary epithelium. In order to determine expression of acidic and basic FGF (aFGF and bFGF) during mammary development, mice were euthanized as virgins, early pregnant, mid-pregnant, late-pregnant, or during early lactation. Mammary expression of both aFGF and bFGF mRNA increased through pregnancy. Acidic FGF mRNA continued to increase during early lactation, but basic FGF message level decreased drastically during early lactation. Western blots probed with anti-aFGF showed four immunoreactive bands approx 30, 48, 52, and 55-kDa in size. The 30-, 48-, and 55-kDa bands for aFGF were expressed at low levels during virgin and early pregnant stages but were more prominent during the later stages. The 52-kDa band was high during the virgin and early pregnant stages and low in mid-pregnancy through early lactation. Blots probed with anti-bFGF showed two bands approx 30 and 50 kDa in size. Both bands increased through early-pregnancy, but during late-pregnancy there was a decrease in immunoreactive protein levels, which remained low during early lactation. Experiments to determine where FGF mRNAs are produced in the mammary gland suggest that both FGFs may be produced in the stroma, leading to the hypothesis that aFGF and bFGF are stromally produced growth factors and probably act on the epithelial component of the gland in a paracrine fashion.     

Expression of calcitonin receptor in rat mammary gland during lactation

Calcitonin (CT) and calcitonin receptor (CTR) have been reported to play an important role in mammary tissue during pregnancy, lactation, and involution. In the present study, the expression and distribution of CTR mRNA in rat mammary tissue during pregnancy and lactation were investigated. As measured by real-time RT-PCR, CTR mRNA levels were increased only slightly during pregnancy, but increased markedly immediately postpartum and remained elevated through lactation, with the highest levels observed 14 days postpartum. In situ hybridization analysis showed that intense CTR mRNA signals were detected in the whole mammary gland. We performed immunohistochemistry to determine distribution of CTR in the mammary epithelium. CTR has been reported to act as an amylin receptor when heterodimerized with receptor activity modifying protein-1 (RAMP1) or RAMP3. mRNA expression of RAMP1 and RAMP3 in mammary tissue decreased during pregnancy and lactation, and amylin mRNA was undetectable, suggesting that up-regulated CTR in lactating mammary tissues binds CT rather than amylin. In primary cultures of mammary cells isolated from rat dams 14 days postpartum, CT produced a statistically significant decrease in thymidine incorporation. These results suggest that up-regulation of CTR during lactation may contribute to inhibition of mammary epithelial cell proliferation.     

Enhancement of maternal lactation performance during prolonged lactation in the mouse by mouse GH and long-R3-IGF-I is linked to changes in mammary signaling and gene expression

GH, prolactin (PRL), and IGF-I stimulate lactation-related metabolic processes in mammary epithelial cells. However, the ability of these factors to stimulate milk production in animals varies depending on species and experimental variables. Previous work in our laboratory demonstrated that transgenic overexpression of des(1-3)IGF-I within the mammary glands of lactating mouse dams increased lactation capacity during prolonged lactation. This work also suggested that some of the effects of the overexpressed IGF-I may have been mediated through elevated concentrations of IGF-I or PRL in the systemic circulation. In the present study, murine GH and PRL, and a human IGF-I analog, long-R3-IGF-I (LR3), were administered as s.c. injections to compare their ability to enhance milk production, and alter mammary gland signaling and gene expression. Lactation capacity, as measured by litter gain, was increased (P<0.05) by GH, but not by PRL. LR3 increased (P<0.05) mammary phospho-Akt and suppressors of cytokines signaling 3 (SOCS3) gene expression, and had a modest ability to increase (P<0.05) lactation capacity. GH both increased (P<0.05) mammary SOCS1 expression and decreased (P<0.05) mammary expression of tryptophan hydroxylase 1, the rate-limiting enzyme in the synthesis of serotonin and a potential feedback inhibitor of lactation. These results suggest that while both GH and IGF-I stimulate milk production in the lactating mouse, the effect of GH may be additionally mediated through IGF-I-independent effects associated with repression of mammary serotonin synthesis.     

Receptor activator of nuclear factor kappa B ligand (RANKL): another link between breast and bone.

Mice rendered null for the genes encoding receptor activator of nuclear factor kappa B ligand (RANKL) or its receptor, RANK, are osteopetrotic because of failure of osteoclast development. The failure of lactation owing to the lack of development of lobulo-alveolar structures during pregnancy, despite earlier stages of mammary gland development being normal, is now added to each of these phenotypes. The breast phenotype in RANKL-/- (but not in RANK-/-) mice is rescued by treatment of pregnant mice with RANKL, indicating a key role for these tumour necrosis factor (TNF) ligand and receptor family members in a crucial terminal step in breast development and lactation. Both RANKL and RANK are synthesized by mammary epithelial cells, with both prolactin and parathyroid hormone-related protein (PTHrP) able to enhance production of mRNA for RANKL. These findings reveal a paracrine-autocrine system in lactation control, with novel signalling pathways that reflect intercellular communication processes in bone.     

The calcium-sensing receptor regulates plasma membrane calcium adenosine triphosphatase isoform 2 activity in mammary epithelial cells: a mechanism for calcium-regulated calcium transport into milk

The calcium-sensing receptor (CaR) regulates transepithelial calcium transport into milk by mammary epithelial cells. Using a genome-wide screening strategy, we identified the plasma membrane calcium ATPase isoform 2 (PMCA2) as a potential downstream target of the CaR. We show that PMCA2 expression in the mouse mammary gland increases during lactation and that PMCA2 is localized solely to the apical plasma membrane of mammary epithelial cells. In milk from deafwaddler mice, which have mutations in the gene encoding PMCA2, calcium concentrations were reduced, confirming its importance in calcium transport into milk. Furthermore, in cultured primary and EpH4 mouse mammary epithelial cells, CaR stimulation up-regulated calcium-dependent ATPase activity in plasma membrane preparations. By small interfering RNA-mediated gene knockdown of PMCA2, we show that PMCA2 accounts for the preponderance of calcium-ATPase activity. We also show that reduction of CaR expression with small interfering RNA eliminates the ability of extracellular calcium to elicit an increase in calcium-dependent ATPase activity in EpH4 cell membranes. These results demonstrate that activation of the CaR increases PMCA2 activity in mouse mammary epithelial cells, providing a mechanism for the regulation of transepithelial calcium transport by calcium in the lactating mouse mammary gland.     

Developmental changes in the protein and mRNA content of a p115/transcytosis-associated protein in the bovine mammary gland

We measured the amounts of a vesicular transport factor, p115/transcytosis-associated protein (p115/TAP) and its mRNA, in mammary glands from cows in which lactation was induced hormonally. The highest level of p115/TAP mRNA, determined by Northern blotting, was detected in the developing stage. In contrast to the mRNA level, the amount of protein, determined by immunoblot analysis using anti-p115/TAP antibodies raised against a p115/TAP-derived recombinant fusion protein, was higher during the lactating stages than at other times. Immunohistochemical study showed that p115/TAP was predominantly localized in mammary epithelial cells. The p115/TAP was also detected in tissues other than the mammary gland but, in contrast to the situation in the mammary gland, the protein and its mRNA levels in those tissues were independent of the stage of lactation. The increased level of p115/TAP mRNA during the developing stage and the maintenance of p115/TAP protein during lactation suggest that the synthesis of p115/TAP is regulated during mammary development and differentiation, and also that the protein is involved in a function related to lactation.     

Uncoupling the mechanisms that facilitate cell survival in hormone-deprived bovine mammary explants

Mammary explants can be hormonally stimulated to mimic the biochemical changes that occur during lactogenesis. Previous studies using mammary explants concluded that the addition of exogenous macromolecules were required for mammary epithelial cells to remain viable in culture. The present study examines the survival of mammary explants from the dairy cow using milk protein gene expression as a functional marker of lactation and cell viability. Mammary explants cultured from late pregnant cows mimicked lactogenesis and showed significantly elevated milk protein gene expression after 3 days of culture with lactogenic hormones. The subsequent removal of exogenous hormones from the media for 10 days resulted in the down-regulation of milk protein genes. During this time, the mammary explants remained hormone responsive, the alveolar architecture was maintained and the expression of milk protein genes was re-induced after a second challenge with lactogenic hormones. We report that a population of bovine mammary epithelial cells have an intrinsic capacity to remain viable and hormone responsive for extended periods in chemically defined media without any exogenous macromolecules. In addition, we found mammary explant viability was dependent on de novo protein and RNA synthesis. Global functional microarray analysis showed that differential expression of genes involved in energy production, immune responses, oxidative stress and apoptosis signalling might contribute to cell survival. As the decline in milk production in dairy cattle after peak lactation results in considerable economic loss, the identification of novel survival genes may be used as genetic markers for breeding programmes to improve lactational persistency in dairy cows.     

Relaxin detected by immunocytochemistry and northern analysis in the mammary gland of the guinea pig

Relaxin is a product of the endometrial gland cells in the guinea pig. Mammary tissue was collected from intact cyclic animals, on days 35 and 63 of pregnancy, days 5 and 21 of lactation, and day 28 postpartum. Tissue was collected from six cyclic hysterectomized animals. Sections of mammary gland were immunostained with the avidin-biotin immunoperoxidase method, and antisera to porcine relaxin which were raised to different preparations in different laboratories. Light uniform staining was evident in the cytoplasm of all of the cuboidal epithelial cells forming the mammary duct system in cyclic animals; mammary gland from hysterectomized animals showed similar staining. At midpregnancy light staining was seen in some epithelial cells, and by late pregnancy there was only faint staining. On day 5 of lactation there was intense and uniform staining throughout the epithelial cells of the alveoli. By day 21 of lactation the cells still immunostained for relaxin, but on day 28 postpartum there was a return to the cyclic staining pattern. Endometrium from animals at 55-60 days of pregnancy and mammary gland from day 6 of lactation were used for poly(A)+ RNA isolation and Northern analysis. Three 48-mer oligonucleotide probes were used. Poly(A)+ RNA from endometrium of late pregnant guinea pigs and from the mammary gland in lactation hybridized with the same two probes and failed to hybridize with a third under moderate stringency conditions. The results suggest that the major source of relaxin in the guinea pig is sequential, the pregnant uterus and the lactating mammary gland. The local and/or systemic significance is not known.