Increased gene expression of endothelin-1 and vasoactive intestinal contractor/endothelin-2 in the mammary gland of lactating mice

In an attempt to understand the roles of endothelin-1 (ET-1) and vasoactive intestinal contractor/endothelin-2 (VIC/ET-2), we have studied the genes for both peptides to be expressed in the mammary gland of lactating mice. We observed through real-time PCR analysis that ET-1 and VIC/ET-2 gene expression gradually increase after parturition and that ET-1 gene expression is significantly higher than that of VIC/ET-2. The distribution of ET-1 peptide was found to be localized mainly in the epithelial cells of the mammary gland at 14th day of lactation. ET-1 gene expression increases significantly, parallel to the increase in beta-casein gene expression, in epithelial cell lines (HC11) of mouse mammary gland after hormonal stimulation by addition of dexamethazone and prolactin. The observed increase in ET-1 expression in differentiated epithelial cells suggests physiological roles for ET-1, including milk production and secretion in the mammary gland of lactating mice.     

Mammary gland copper transport is stimulated by prolactin through alterations in Ctr1 and Atp7A localization

Milk copper (Cu) concentration declines and directly reflects the stage of lactation. Three Cu-specific transporters (Ctr1, Atp7A, Atp7B) have been identified in the mammary gland; however, the integrated role they play in milk Cu secretion is not understood. Whereas the regulation of milk composition by the lactogenic hormone prolactin (PRL) has been documented, the specific contribution of PRL to this process is largely unknown. Using the lactating rat as a model, we determined that the normal decline in milk Cu concentration parallels declining Cu availability to the mammary gland and is associated with decreased Atp7B protein levels. Mammary gland Cu transport was highest during early lactation and was stimulated by suckling and hyperprolactinemia, which was associated with Ctr1 and Atp7A localization at the plasma membrane. Using cultured mammary epithelial cells (HC11), we demonstrated that Ctr1 stains in association with intracellular vesicles that partially colocalize with transferrin receptor (recycling endosome marker). Atp7A was primarily colocalized with mannose 6-phosphate receptor (M6PR; late endosome marker), whereas Atp7B was partially colocalized with protein disulfide isomerase (endoplasmic reticulum marker), TGN38 (trans-Golgi network marker) and M6PR. Prolactin stimulated Cu transport as a result of increased Ctr1 and Atp7A abundance at the plasma membrane. Although the molecular mechanisms responsible for these posttranslational changes are not understood, transient changes in prolactin signaling play a role in the regulation of mammary gland Cu secretion during lactation.     

Characterisation of the potential SNARE proteins relevant to milk product release by mouse mammary epithelial cells

Casein micelles and fat globules are essential components of milk and are both secreted at the apical side of mammary epithelial cells during lactation. Milk fat globules are excreted by budding, being enwrapped by the apical plasma membrane, while caseins contained in transport vesicles are released by exocytosis. Nevertheless, the molecular mechanisms governing casein exocytosis are, to date, not fully deciphered. SNARE proteins are known to take part in cellular membrane trafficking and in exocytosis events in many cell types and we therefore attempted to identify those relevant to casein secretion. With this aim, we performed a detailed analysis of their expression by RT-PCR in both whole mouse mammary gland and in purified mammary acini at various physiological stages, as well as in the HC11 cell line. The expression of some regulatory proteins involved in SNARE complex formation such as Munc-13, Munc-18 and complexins was also explored. The amount of certain SNAREs appeared to be regulated depending on the physiological stage of the mammary gland. Co-immunoprecipitation experiments indicated that SNAP-23 interacted with syntaxin-6, -7 and -12, as well as with VAMP-3, -4 and -8 in mammary epithelial cells during lactation. Finally, the subcellular localisation of candidate SNAREs in these cells was determined both by indirect immunofluorescence and immunogold labelling. The present work provides important new data concerning SNARE proteins in mammary epithelial cells and points to SNAP-23 as a potential central player for the coupling of casein and milk fat globule secretion during lactation.     

Expression, activity, and localization of hormone-sensitive lipase in rat mammary gland during pregnancy and lactation

We examined the presence of hormone-sensitive lipase (HSL) in mammary glands of virgin, pregnant (12, 20, and 21 days), and lactating (1 and 4 days postpartum) rats. Immunohistochemistry with antibody against rat HSL revealed positive HSL in the cytoplasm of both alveolar epithelial cells and adipocytes. In virgin rats, immunoreactive HSL was observed in mammary adipocytes, whereas diffuse staining was found in the epithelial cells. Positive staining for HSL was seen in the two types of cells in pregnant and lactating rats. However, as pregnancy advanced, the staining intensity of immunoreactive HSL increased in the epithelial cells parallel to their proliferation, attaining the maximum during lactation. An immunoreactive protein of 84 kDa and a HSL mRNA of 3.3. kb were found in the rat mammary gland as in white adipose tissue. Both HSL protein and activity were lower in mammary glands from 20 and 21 day pregnant rats than from those of virgin rats, although they returned to virgin values on days 1 and 4 of lactation. Mammary gland HSL activity correlated negatively to plasma insulin levels. Immunoreactive HSL and HSL activity were found in lactating rats' milk. The observed changes indicate an active role of HSL in mammary gland lipid metabolism.     

Glucocorticoid and progesterone inhibit involution and programmed cell death in the mouse mammary gland

Milk production during lactation is a consequence of the suckling stimulus and the presence of glucocorticoids, prolactin, and insulin. After weaning the glucocorticoid hormone level drops, secretory mammary epithelial cells die by programmed cell death and the gland is prepared for a new pregnancy. We studied the role of steroid hormones and prolactin on the mammary gland structure, milk protein synthesis, and on programmed cell death. Slow-release plastic pellets containing individual hormones were implanted into a single mammary gland at lactation. At the same time the pups were removed and the consequences of the release of hormones were investigated histologically and biochemically. We found a local inhibition of involution in the vicinity of deoxycorticosterone- and progesterone-release pellets while prolactin-release pellets were ineffective. Dexamethasone, a very stable and potent glucocorticoid hormone analogue, inhibited involution and programmed cell death in all the mammary glands. It led to an accumulation of milk in the glands and was accompanied by an induction of protein kinase A, AP-1 DNA binding activity and elevated c-fos, junB, and junD mRNA levels. Several potential target genes of AP-1 such as stromelysin-1, c-jun, and SGP-2 that are induced during normal involution were strongly inhibited in dexamethasone-treated animals. Our results suggest that the cross-talk between steroid hormone receptors and AP-1 previously described in cells in culture leads to an impairment of AP-1 activity and to an inhibition of involution in the mammary gland implying that programmed cell death in the postlactational mammary gland depends on functional AP-1.     

Overexpression of des(1-3) insulin-like growth factor 1 in the mammary glands of transgenic mice delays the loss of milk production with prolonged lactation

During prolonged lactation, the mammary gland gradually loses the capacity to produce milk. In agricultural species, this decline can be slowed by administration of exogenous growth hormone (GH), which is believed to act through insulin-like growth factor 1 (IGF1). Our previous work demonstrated delayed natural mammary gland involution in des(1-3)IGF1-overexpressing transgenic mice (Tg[Wap-des{1-3}IGF1]8266 Jmr), hereafter referred to as WAP-DES mice. The present study tested the hypothesis that overexpressed des(1-3)IGF1 would delay the loss of milk production during prolonged lactation. Accordingly, we examined lactational performance in WAP-DES mice by artificially prolonging lactation with continual litter cross-fostering. Over time, lactational capacity and mammary development declined in both WAP-DES and control mice. However, the rate of decline was 40% slower in WAP-DES mice. Mammary cell apoptosis increased by 3-fold in both groups during prolonged lactation but was not different between genotypes. Plasma concentrations of murine IGF1 were decreased in WAP-DES mice, while those of the transgenic human IGF1 were elevated during prolonged lactation. Phosphorylation of the mammary IGF1 receptor was increased in the WAP-DES mice, but only during prolonged lactation. Plasma prolactin decreased with prolonged lactation in nontransgenic mice but remained high in WAP-DES mice. The WAP-DES mice maintained a higher body mass and a greater lean body mass during prolonged lactation. These data support the conclusion that overexpressed des(1-3)IGF1 enhanced milk synthesis and mammary development during prolonged lactation through localized and direct activation of the mammary gland IGF1 receptor and through systemic effects on prolactin secretion and possibly nutrient balance.     

Pregnancy-Induced Noncoding RNA (PINC) Associates with Polycomb Repressive Complex 2 and Regulates Mammary Epithelial Differentiation

Pregnancy-induced noncoding RNA (PINC) and retinoblastoma-associated protein 46 (RbAp46) are upregulated in alveolar cells of the mammary gland during pregnancy and persist in alveolar cells that remain in the regressed lobules following involution. The cells that survive involution are thought to function as alveolar progenitor cells that rapidly differentiate into milk-producing cells in subsequent pregnancies, but it is unknown whether PINC and RbAp46 are involved in maintaining this progenitor population. Here, we show that, in the post-pubertal mouse mammary gland, mPINC is enriched in luminal and alveolar progenitors. mPINC levels increase throughout pregnancy and then decline in early lactation, when alveolar cells undergo terminal differentiation. Accordingly, mPINC expression is significantly decreased when HC11 mammary epithelial cells are induced to differentiate and produce milk proteins. This reduction in mPINC levels may be necessary for lactation, as overexpression of mPINC in HC11 cells blocks lactogenic differentiation, while knockdown of mPINC enhances differentiation. Finally, we demonstrate that mPINC interacts with RbAp46, as well as other members of the polycomb repressive complex 2 (PRC2), and identify potential targets of mPINC that are differentially expressed following modulation of mPINC expression levels. Taken together, our data suggest that mPINC inhibits terminal differentiation of alveolar cells during pregnancy to prevent abundant milk production and secretion until parturition. Additionally, a PRC2 complex that includes mPINC and RbAp46 may confer epigenetic modifications that maintain a population of mammary epithelial cells committed to the alveolar fate in the involuted gland.     

ABC- and SLC-Transporters in Murine and Bovine Mammary Epithelium - Effects of Prochloraz

Some chemicals are ligands to efflux transporters which may result in high concentrations in milk. Limited knowledge is available on the influence of maternal exposure to chemicals on the expression and function of transporters in the lactating mammary gland. We determined gene expression of ABC and SLC transporters in murine mammary tissue of different gestation and lactation stages, in murine mammary cells (HC11) featuring resting and secreting phenotypes and in bovine mammary tissue and cells (BME-UV). Effects on transporter expression and function of the imidazole fungicide prochloraz, previously reported to influence BCRP in mammary cells, was investigated on transporter expression and function in the two cell lines. Transporters studied were BCRP, MDR1, MRP1, OATP1A5/OATP1A2, OCTN1 and OCT1. Gene expressions of BCRP and OCT1 in murine mammary glands were increased during gestation and lactation, whereas MDR1, MRP1, OATP1A5 and OCTN1 were decreased, compared to expressions in virgins. All transporters measured in mammary glands of mice were detected in bovine mammary tissue and in HC11 cells, while only MDR1 and MRP1 were detected in BME-UV cells. Prochloraz treatment induced MDR1 gene and protein expression in both differentiated HC11 and BME-UV cells and increased protein function in HC11 cells, resulting in decreased accumulation of the MDR1 substrate digoxin. In conclusion, our results demonstrate that murine (HC11) and bovine (BME-UV) mammary epithelial cells can be applied to characterize expression and function of transporters as well as effects of contaminants on the mammary transporters. An altered expression, induced by a drug or toxic chemical, on any of the transporters expressed in the mammary epithelial cells during lactation may modulate the well-balanced composition of nutrients and/or secretion of contaminants in milk with potential adverse effects on breast-fed infants and dairy consumers.     

Lactogenic hormones regulate xanthine oxidoreductase and beta-casein levels in mammary epithelial cells by distinct mechanisms

Xanthine oxidoreductase (XOR) is a prominent component of the milk lipid globule, whose concentration is selectively increased in mammary epithelial cells during the transition from pregnancy to lactation. To understand how XOR expression is controlled in the mammary gland, we investigated its properties and regulation by lactogenic hormones in cultured HC11 mammary epithelial cells. XOR was purified as the NAD(+)-dependent dehydrogenase by benzamidine-Sepharose chromatography and was shown to be intact and to have biochemical properties similar to those of enzyme from other sources. Treating confluent HC11 cells with prolactin and cortisol produced a progressive, four- to fivefold, increase in XOR activity, while XOR activity in control cells remained constant. Elevated cellular XOR activity was correlated with increased XOR protein and was due to both increased synthesis and decreased degradation of XOR. Prolactin and cortisol increased XOR protein and mRNA in the presence of epidermal growth factor, which blocked the stimulation of beta-casein synthesis by these hormones. Further, hormonal stimulation of XOR was inhibited by genistein (a protein tyrosine kinase inhibitor) and by PD 98059 (a specific inhibitor of the MAP kinase cascade). These findings indicate that lactogenic hormones stimulate XOR and beta-casein expression via distinct pathways and suggest that a MAP kinase pathway mediates their effects on XOR. Our results provide evidence that lactogenic hormones regulate milk protein synthesis by multiple signaling pathways.     

Expression and localisation of GLUT1 and GLUT12 glucose transporters in the pregnant and lactating rat mammary gland

Glucose plays a major role in mammary gland function during lactation as it is used both as a fuel and as a precursor of milk components. In rats, previous studies have shown that the facilitative glucose transporter GLUT1 is expressed in mammary epithelial cells. We have used confocal immunofluorescence to localise GLUT1 and GLUT12, a recently identified member of the sugar transporter family, in pregnant and lactating rat mammary gland. GLUT12 staining was observed in the cytoplasm of mammary epithelial cells at day 20 of pregnancy, and at 1 and 6 days postpartum. Furthermore, GLUT12 staining was present at the apical plasma membrane of epithelial cells during lactation. In contrast, GLUT1 protein localised to the cytoplasm and basolateral surface of mammary epithelial cells. Forced weaning resulted in decreased cytoplasmic GLUT1 staining intensity, but no change in GLUT12 staining. The results suggest a possible role for GLUT12 in the metabolism of mammary epithelial cells during pregnancy and lactation.     

Milk protein expression and ductal morphogenesis in the mammary gland in vitro: hormone-dependent and -independent phases of adipocyte-mammary epithelial cell interaction

Epithelial cell differentiation frequently occurs in situ in conjunction with supporting mesenchyme or connective tissue. In embryonic development the importance of the supporting mesenchyme for cytodifferentiation and morphogenesis has been demonstrated in several epithelial tissues, but the importance of epithelial-connective tissue interactions is less well studied in adult epithelial organs. We have investigated the interaction of adult mammary epithelial cells with adipocytes, which compose the normal supporting connective tissue in the mammary gland. Mammary epithelial cells from mice in various physiological states were cultured on cellular substrates of adipocytes formed from cells of the 3T3-L1 preadipocyte cell line. We found that there were two distinct phases to the interaction of epithelial cells with adipocytes. Cytodifferentiation of the epithelial cells and milk protein production were dependent on lactogenic hormones (insulin, hydrocortisone, and prolactin), whereas ductal morphogenesis was lactogenic hormone independent. When cultured on preadipocytes or adipocytes, mammary epithelial cells from never pregnant, pregnant, lactating, and involuting mice responded to lactogenic hormones rapidly by producing and secreting large amounts of alpha-, beta-, and gamma-casein and alpha-lactalbumin. This response was seen in individual as well as in clusters of epithelial cells, but was not seen if the same cells were cultured on tissue culture dishes without adipocytes, on fibroblasts (human newborn foreskin fibroblasts) or in the presence of adipocytes but in the absence of lactogenic hormones. Continued incubation of mammary epithelial cells on adipocytes in the presence or absence of lactogenic hormones resulted in the formation of a branching ductal system. Mammary epithelial cells in ducts that formed in the absence of lactogenic hormones produced no casein, but rapidly synthesized casein when subsequently exposed to these hormones. Ultrastructural studies revealed that the formation of a basement membrane occurs only in co-cultures of mammary epithelium with adipocytes or preadipocytes. Ultrastructural changes associated with secretion occurred only in the presence of lactogenic hormones. We propose that growth and formation of a ductal system in vitro can occur in the absence of lactogenic hormones, but that certain environment-associated events must occur if the epithelium is to become responsive to lactogenic hormones and undergo the cytodifferentiation associated with lactation.     

The mineralocorticoid receptor may compensate for the loss of the glucocorticoid receptor at specific stages of mammary gland development

To study the role of glucocorticoid receptor (GR) at different stages of mammary gland development, mammary anlage were rescued from GR-/- mice by transplantation into the cleared fat pad of wild-type mice. In virgin mice, GR-/- outgrowths displayed abnormal ductal morphogenesis characterized by distended lumena, multiple layers of luminal epithelial cells in some regions along the ducts, and increased periductal stroma. In contrast, the loss of GR did not result in overt phenotypic changes in mammary gland development during pregnancy, lactation, and involution. Surprisingly, despite the known synergism between glucocorticoids and prolactin in the regulation of milk protein gene expression, whey acidic protein and beta-casein mRNA levels were unaffected in GR-/- transplants as compared with wild-type transplants. That mineralocorticoid receptor (MR) might compensate for the loss of GR was suggested by the detection of MR in the mammary gland at d 1 of lactation. This hypothesis was tested using explant cultures derived from the GR-/- transplants in which the mineralocorticoid fludrocortisone was able to synergistically induce beta-casein gene expression in the presence of prolactin and insulin. These studies suggest that MR may compensate for the absence of GR at some, but not at all stages of mammary gland development.     

转基因小鼠乳腺上皮细胞的体外培养及其对催乳素反应的研究

实现转基因生物乳腺反应器对外源蛋白的高效表达是目前生物制药亟待解决的难题。催乳素对泌乳期乳蛋白的合成与分泌具有重要的调控功能。通过转基因小鼠乳腺上皮细胞模型的建立,研究催乳素如何调控乳蛋白的表达,为提高乳腺反应器高效表达外源蛋白提供技术及理论支撑。应用机械破碎及胶原酶消化法,经差速贴壁纯化,成功培养含人转铁蛋白基因的小鼠乳腺上皮细胞,细胞上清液中检测到人转铁蛋白表达。细胞经牛催乳素诱导后人转铁蛋白的表达水平明显升高。利用转基因小鼠乳腺上皮细胞模型,可以进行催乳素和环境因素等对乳腺上皮细胞合成及分泌蛋白能力影响的研究。     

Pten对奶牛乳腺上皮细胞活力及乳蛋白合成的影响

Pten作为抑癌基因,参与调控细胞生长、粘附、凋亡以及其它细胞活动.目前,国内外关于Pten在奶牛乳腺发育过程中表达及调节的研究鲜有报道.为了揭示Pten的表达与奶牛乳腺发育与泌乳之间的关系,本研究应用qRT-PCR技术检测Pten在不同泌乳时期和不同乳品质的奶牛乳腺组织中的表达差异,进而应用脂质体转染方法,通过siRNA介导的RNA干扰技术改变Pten基因在奶牛乳腺上皮细胞中的表达量,CASY法检测细胞活力,用ELISA试剂盒检测细胞分泌β-酪蛋白的含量,采用qRT-PCR、Western 印迹等技术检测Pten对奶牛乳腺上皮细胞中乳蛋白相关信号通路基因表达的影响.结果显示,泌乳期高乳品质奶牛乳腺组织中Pten表达水平显著低于泌乳期低乳品质及干乳期奶牛;Pten基因沉寂后,细胞活力提高,β-酪蛋白质量浓度增加,CSN2、AKT、MTOR、STAT5表达量增加.研究表明,Pten可通过抑制细胞活力和乳蛋白分泌而影响泌乳.     

Regulation and functional relevance of milk fat globules and their components in the mammary gland

Mammary gland and epithelial cells are unique to mammals and are under the control of lactogenic hormones such as prolactin. Recent findings indicated that major components of milk fat globule membrane (MFGM) are under the control of lactogenic hormones, and that the major components butyrophilin and xanthine oxidoreductase are indispensable for milk fat secretion. Further, prolactin signaling is negatively controlled by two highly related protein tyrosine phosphatases, PTP1B and TC-PTP. Milk fat globule EGF factor 8 (MFG-E8) is one of the major components of MFGM and is upregulated during lactation. MFG-E8 is further upregulated in the involuting mammary gland. MFG-E8 on exosome-like membrane vesicles in the milk recovered from post-weaning but not lactating mammary glands exhibits higher binding activity to phosphatidylserine and apoptotic mammary epithelial cells, and serves as a link between apoptotic mammary epithelial cells and phagocytes. Recent reports using MFG-E8 deficient mice support the view that MFG-E8 is indispensable for eliminating apoptotic mammary epithelial cells during involution.     

miR-27a suppresses triglyceride accumulation and affects gene mRNA expression associated with fat metabolism in dairy goat mammary gland epithelial cells

MicroRNAs (miRNAs), a well-defined group of small RNAs containing about 22 nucleotides, participate in various biological metabolic processes. miR-27a is a miRNA that is known to regulate fat synthesis and differentiation in preadipocyte cells. However, little is known regarding the role that miR-27a plays in regulating goat milk fat synthesis. In this study, we determined the miR-27a expression profile in goat mammary gland and found that miR-27a expression was correlated with the lactation cycle. Additionally, prolactin promoted miR-27a expression in goat mammary gland epithelial cells. Further functional analysis showed that over-expression of miR-27a down-regulated triglyceride accumulation and decreased the ratio of unsaturated/saturated fatty acid in mammary gland epithelial cells. miR-27a also significantly affected mRNA expression related to milk fat metabolism. Specifically, over-expression of miR-27a reduced gene mRNA expression associated with triglyceride synthesis by suppressing PPARγ protein levels. This study provides the first experimental evidence that miR-27a regulates triglyceride synthesis in goat mammary gland epithelial cells and improves our understanding about the importance of miRNAs in milk fat synthesis.