Synthesis and secretion of the mouse whey acidic protein in transgenic sheep

The synthesis of foreign proteins can be targeted to the mammary gland of transgenic animals, thus permitting commercial purification of otherwise unavailable proteins from milk. Genetic regulatory elements from the mouse whey acidic protein (WAP) gene have been used successfully to direct expression of transgenes to the mammary gland of mice, goats and pigs. To extend the practical usefulness of WAP promoter-driven fusion genes and further characterize WAP expression in heterologous species, we introduced a 6.8 kb DNA fragment containing the genomic form of the mouse WAP gene into sheep zygotes. Two lines of transgenic sheep were produced. The transgene was expressed in mammary tissue of both lines and intact WAP was secreted into milk at concentrations estimated to range from 100 to 500 mg/litre. Ectopic WAP gene expression was found in salivary gland, spleen, liver, lung, heart muscle, kidney and bone marrow of one founder ewe. WAP RNA was not detected in skeletal muscle and intestine. These data suggest that unlike pigs, sheep may possess nuclear factors in a variety of tissues that interact with WAP regulatory sequences. Though the data presented are based on only two lines, these findings suggest WAP regulatory sequences may not be suitable as control elements for transgenes in sheep bioreactors.     

Expression and function of CCAAT/enhancer binding proteinbeta (C/EBPbeta) LAP and LIP isoforms in mouse mammary gland, tumors and cultured mammary epithelial cells

CCAAT/Enhancer binding proteins (C/EBPs) play important roles in the regulation of cell growth and differentiation. This study investigated the expression and function of C/EBPbeta isoforms in the mouse mammary gland, mammary tumors, and a nontransformed mouse mammary epithelial cell line (HC11). C/EBPbeta mRNA levels are 2-5-fold higher in mouse mammary tumors derived from MMTV/c-neu transgenic mice compared with lactating and involuting mouse mammary gland. The "full-length" 38 kd C/EBPbeta LAP ("Liver-enriched Activator Protein") isoform is the predominant C/EBPbeta protein isoform in mammary tumor whole cell lysates, however, the truncated 20 kd C/EBPbeta LIP ("Liver-enriched Inhibitory Protein") isoform is also present at detectable levels (mean LAP:LIP ratio 5.3:1). The mammary tumor C/EBPbeta LAP:LIP ratio decreases 70% (from 5.3:1 to 1.6:1) when lysate preparation is switched from a rapid whole cell lysis protocol to a multistep nuclear/cytoplasmic fractionation protocol. In contrast to mammary tumors, only the C/EBPbeta LAP isoform is detectable in the mammary gland whole cell and nuclear lysates; the truncated "LIP" isoform is undetectable regardless of isolation protocol. Ectopic over expression of C/EBPbeta LIP or C/EBPbeta LAP did not alter HC11 growth rates. However, C/EBPbeta LIP over expressing HC11 cells (LAP:LIP ratio of approximately 1:1) exhibited a consistent 2-4 h delay in G(0)/S phase transition. C/EBPbeta LIP overexpressing HC11 cells did not express beta-casein mRNA (mammary epithelial cell differentiation marker) in response to lactogenic hormones. This defect in beta-casein expression was not corrected by carrying out the differentiation protocol in the presence of an artificial extracellular matrix. These results demonstrate that the "full-length" C/EBPbeta LAP isoform is the predominant C/EBPbeta protein isoform expressed in mouse mammary gland in vivo and mouse mammary epithelial cell cultures in vitro. C/EBPbeta LIP detected in mammary tumor lysates may result from in vivo production or ex vivo isolation-induced proteolysis of C/EBPbeta LAP. Ectopic overexpression of C/EBPbeta LIP (LAP:LIP ratio of approximately 1:1) inhibits mammary epithelial cell differentiation (beta-casein expression).     

Inappropriate P-cadherin expression in the mouse mammary epithelium is compatible with normal mammary gland function

Cadherins comprise a family of cell-cell adhesion proteins critical to the architecture and function of tissues. Expression of family members E-, N-, and P-cadherin is regulated in a spatial and temporal fashion in the developing and adult organism. Using in vivo and in vitro experimental systems, perturbation of cadherin expression by genetic deletion, overexpression, mutant dominant-negative constructs, and, to a lesser degree, expression of an inappropriate cadherin have all been shown to alter embryogenesis, tissue architecture, and cell behavior. Here we studied how expression of an inappropriate cadherin affects the adult mouse mammary gland. Human P-cadherin was expressed in mammary epithelial cells under control of the mouse mammary tumor virus (MMTV) promoter, and the effect on mammary gland behavior was studied. Typically, E-cadherin is expressed by mammary epithelial cells, whereas P-cadherin is found in myoepithelial cells and cap cells of the ductal terminal end bud. However, breast cancers frequently express P-cadherin, even though they are thought to arise from epithelial cells, and it is a marker of poor prognosis. We developed two independent transgenic mouse lines that exhibited high levels of P-cadherin protein expression in the mammary epithelium. P-cadherin was detected in most, but not all, luminal epithelial cells, and was appropriately localized to cell-cell borders. It was detected in the mammary glands of virgin, pregnant, lactating, post-lactation, and aged parous female mice. Despite the robust and widespread expression of an inappropriate cadherin, no effect was observed on mammary gland morphogenesis, architecture, lactation, or involution in transgenic mice compared to wild-type mice. No mammary tumors formed spontaneously in either wild-type or transgenic mice. Moreover, mammary tumors induced by the neu oncogene, which was introduced by a breeding strategy, showed no differences between mice with or without hP-cadherin. Surprisingly, however, none of the tumors expressed hP-cadherin protein. Together, our studies show no apparent effect on adult mammary gland or tumor behavior by inappropriate expression of P-cadherin in normal mammary epithelial cells.     

乳腺脂肪组织调节乳腺发育的研究进展

乳腺是哺乳动物哺育子代的重要器官,其通过分泌乳汁给子代提供充足的营养物质,乳腺的健康发育对泌乳以及提高子代的存活率具有重要意义.脂肪组织是乳腺重要的组成部分,在乳腺发育和循环重构过程中,乳腺脂肪组织随之呈现规律性的形态和功能变化,乳腺脂肪组织的动态变化是乳腺循环性发育重构的重要特征.脂肪组织能够分泌特殊的"脂肪因子"调节上皮细胞的功能和乳腺的发育,并且存在与上皮细胞相互转换的潜能.本综述综合近年来乳腺脂肪组织的相关研究进展,为后续研究脂肪组织调节乳腺发育的机制提供基础数据.     

Real-time imaging of beta-lactoglobulin-targeted luciferase activity in the mammary glands of transgenic mice.

This study was aimed at establishing a new platform for real-time monitoring of milk-protein gene expression in the mammary glands. A transgenic reporter composed of the beta-lactoglobulin (BLG)/luciferase hybrid gene was targeted to the mammary glands of pregnant and lactating mice and luciferase activity was imaged in vivo with a low-light imaging system. The mammary glands of a 17-day pregnant mouse occupied an area comparable to that of a 6-day lactating mouse. Nevertheless, the intensity of the luciferase signal was much weaker and confined to regions in the inguinal and thoracic glands. A few small and defined locations of higher expression were also detected, indicating diversity in the initiation of this transgenic milk protein expression. In the lactating mice, high inter- and intra-heterogeneity among regions in a particular gland and among glands was demonstrated, and confirmed by ex vivo analysis of luciferase activity in mammary biopsies. The lack of correlation between luciferase activities and levels of beta-casein accumulation in these biopsies resulted, most probably, from the longer half-life of the native milk protein, compared to the activity of the transgenic marker in the tissue. Unilateral sealing of mammary glands for 4 hr resulted in complete abrogation of luciferase activity, establishing the BLG/luciferase transgene as a reliable tool to follow short-term stimuli. Dispersed mammary epithelial cells preserved luciferase activity in culture, and thus could be used for following mammary gland development after re-implantation. The bioluminescence-based methodology presented here eliminates averaging of heterogeneity in gene expression among glands, and misinterpretations resulting from sampling biopsies taken from inactive regions. Imaging luciferase expression in the mammary glands may enable an accurate monitoring of milk-protein gene expression during cyclic periods of development and apoptosis in a limited number of animals, and could be applied for reporting the consequences of selected drugs on milk-protein gene expression.     

Elf5 is essential for early embryogenesis and mammary gland development during pregnancy and lactation

Elf5 is an epithelial-specific ETS factor. Embryos with a null mutation in the Elf5 gene died before embryonic day 7.5, indicating that Elf5 is essential during mouse embryogenesis. Elf5 is also required for proliferation and differentiation of mouse mammary alveolar epithelial cells during pregnancy and lactation. The loss of one functional allele led to complete developmental arrest of the mammary gland in pregnant Elf5 heterozygous mice. A quantitative mRNA expression study and Western blot analysis revealed that decreased expression of Elf5 correlated with the downregulation of milk proteins in Elf5(+/-) mammary glands. Mammary gland transplants into Rag(-/-) mice demonstrated that Elf5(+/-) mammary alveolar buds failed to develop in an Elf5(+/+) mammary fat pad during pregnancy, demonstrating an epithelial cell autonomous defect. Elf5 expression was reduced in Prolactin receptor (Prlr) heterozygous mammary glands, which phenocopy Elf5(+/-) glands, suggesting that Elf5 and Prlr are in the same pathway. Our data demonstrate that Elf5 is essential for developmental processes in the embryo and in the mammary gland during pregnancy.     

Long-term organ culture of mouse mammary gland

Summary A method for maintaining mouse mammary gland in organ culture for periods of at least 30 days is described. Strips of the number four mammary glands were cultured in individual tubes while fully submerged in Medium 199 supplemented with insulin, aldosterone, ovine prolactin and bovine growth hormone. Exchange processes were aided by slowly rotating the tubes during culture. Mammary tissue from midpregnant BALB/c and virgin GR/A mice was induced to undergo lobulo-alveolar development, secrete and remain differentiated and metabolically active for the period of culture. Cells of both the ductal and alveolar epithelium continued to synthesize DNA and divide. The submerged roller-tube culture allows the use of larger pieces of tissue than can be accommodated in static culture, and the technique may prove applicable to the culture of a variety of tissues.     

EGF Modulates Expression of STAT5 in Mammary Epithelial Cells

HC11 mouse mammary epithelial cells are capable of differentiatingin vitro.By growing cells in EGF-containing medium, and upon confluence withdrawing EGF, these cells become competent at responding to lactogenic hormone treatment and expressing milk proteins. We found that during proliferation and at confluence STAT5A and STAT5B proteins were expressed at equal levels or with STAT5B being predominant. In competent cells, expression levels of STAT5A and STAT5B increased markedly with STAT5A now being the predominant form, an expression pattern resembling the expression patterns of STAT5 proteins seen during mammary gland differentiationin vivo.This suggests that EGF has a suppressive effect on STAT5 expression, in particular, STAT5A, which we conclude to be mediated through ras/raf/MEK/MAPK pathway and to a lesser extent through a PI3-kinase-mediated pathway. Furthermore, we also found that EGF regulated a nuclear phosphatase capable of dephosphorylating tyrosine-phosphorylated STAT5. Our data show that HC11 cells have retained the expression patterns of STAT5 proteins seenin vivo.This makes HC11 cells useful for studying molecular mechanisms regulating expression of STAT factors and their participation in differentiation processes of mammary gland.     

Establishment of La-tPA/G-CSF dual transgenic mice and expression in their mammary gland

Expression vectors of human granulocyte colony stimulating factor (G-CSG) and long acting tissue plasminogen activator (La-tPA) in mammary gland were constructed using promoters of mouse whey acid protein gene (WAP) and sheep β-lactoglobulin gene (BLG) with sizes of 2.6 and 5 kb respectively. Two kinds of transgenic mice of G-CSF and La-tPA were produced with microinjection. The expression of G-CSF and La-tPA was achieved in mammary glands of transgenic mice, respectively. In order to establish dual transgenic mice of La-tPA/G-CSF, transgenic mice carrying G-CSF and La-tPA gene characterized with specific expression in mammary gland were mated. La-tPA/G-CSF dual transgenic mice were screened out from the hybrid offspring by Once-PCR. The co-expression of La-tPA and G-CSF in mammary gland of the dual transgenic mice was confirmed by the milk assayed and Northern blot analysis. Some parameters about the dual transgenic mice indicated that there were fewer litters than that of normal mice. The ratio of du     

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。     

Stem Cell Research: A Novel Boulevard towards Improved Bovine Mastitis Management

The dairy industry is a multi-billion dollar industry catering the nutritional needs of all age groups globally through the supply of milk. Clinical mastitis has a severe impact on udder tissue and is also an animal welfare issue. Moreover, it significantly reduces animal value and milk production. Mammary tissue damage reduces the number and activity of epithelial cells and consequently contributes to decreased milk production. The high incidence, low cure rate of this highly economic and sometimes deadly disease is an alarming for dairy sector as well as policy makers. Bovine mammary epithelial cells (MECs) and their stem cells are very important in milk production and bioengineering. The adult mammary epithelium consists of two main cell types; an inner layer of luminal epithelial cells, which produce the milk during lactation, and an outer layer of myoepithelial cells resting on a basement membrane, which are responsible for pushing the milk through the ductal network to the teat cistern. Inner layer of columner/luminal cells of bovine MECs, is characterized by cytokeratin18, 19 (CK18, CK19) and outer layer such as myoepithelial cells which are characterized by CK14, α-smooth muscle actin (α-SMA) and p63. Much work has been done in mouse and human, on mammary gland stem cell research, particularly in cancer therapy, but stem cell research in bovine is still in its infancy. Such stem/progenitor cell discoveries in human and mouse mammary gland bring some hope for application in bovines. These progenitors may be therapeutically adopted to correct the structural/cytological defects in the bovine udder due to mastitis. In the present review we focused on various kinds of stem/progenitor cells which can have therapeutic utility and their possibilities to use as a potential stem cell therapy in the management of bovine post-mastitis damage in orders to restore milk production. The possibilities of bovine mammary stem cell therapy offers significant potential for regeneration of tissues that can potentially replace/repair diseased and damaged tissue through differentiation into epithelial, myoepithelial and/or cuboidal/columnar cells in the udder with minimal risk of rejection and side effects.     

Specific neutralizing antiserum against a polypeptide growth inhibitor for mammary cells purified from bovine mammary gland

A recently published method for purification of a new inhibitor of growth of mammary cells in vitro from bovine mammary gland has been modified to yield several hundred μg of inhibitor per kg of glandular tissue. The inhibitory effect exerted by this preparation to Ehrlich ascites mammary carcinoma cells fulfilled all biological criteria of specificity established earlier for preparations obtained by other means, the most important being that the inhibitory effect is abolished by the epidermal growth factor and insulin. The preparation is shown to consist mainly of a protein of 13 kDa which appears to be not glycosylated. An antiserum raised in mice against the inhibitor is demonstrated to be specific for the 13 kDa bovine mammary gland protein. Neutralization of the inhibitory activity by the specific antiserum strongly supports the view that the 13 kDa protein is indeed the carrier of inhibitory activity. First data on tissue distribution obtained with an enzyme-linked immunosorbent assay revealed a high concentration of the anti-inhibitor-antiserum-reactive antigen in bovine lactating but not in non-lactating mammary gland tissue and in milk fat globule membranes. Some reactivity was also found in bovine lung. These data are interpreted with respect to a possible physiological significance of the growth inhibitor.     

Proteomic analysis of endogenous conjugated linoleic acid biosynthesis in lactating rats and mouse mammary gland epithelia cells (HC11)

This study was conducted to investigate the amount of CLA synthesized endogenously by rat mammary tissues in response to TVA (a precursor for cis-9, trans-11 CLA endogenous synthesis) treatment as well as the differences in the protein expression of genes encoding the biosynthesis of CLA in rat mammary tissue and mouse mammary gland epithelia cells (HC11). Treatment with TVA resulted in improved CLA productivity. Furthermore, 2-DE revealed two spots in samples of mammary tissues and one spot in samples of mammary gland epithelia cells (HC11) that were consistently altered in the TVA treatment groups when compared with the control group (non-fatty acid). The mRNA expression patterns of three of the proteins (PDI, PRDX2, LAMR1), as measured by real-time PCR, were similar to the pattern of protein abundance. In addition, the expression of SCD mRNA in the mammary tissue of rats and HC11 cell treated with TVA was higher than in the control group. Our results suggest that the identified proteins may be related to CLA biosynthesis in mammary tissue.     

Calpains mediate epithelial-cell death during mammary gland involution: mitochondria and lysosomal destabilization

Our aim was to elucidate the physiological role of calpains (CAPN) in mammary gland involution. Both CAPN-1 and -2 were induced after weaning and its activity increased in isolated mitochondria and lysosomes. CAPN activation within the mitochondria could trigger the release of cytochrome c and other pro-apoptotic factors, whereas in lysosomes it might be essential for tissue remodeling by releasing cathepsins into the cytosol. Immunohistochemical analysis localized CAPNs mainly at the luminal side of alveoli. During weaning, CAPNs translocate to the lysosomes processing membrane proteins. To identify these substrates, lysosomal fractions were treated with recombinant CAPN and cleaved products were identified by 2D-DIGE. The subunit b(2) of the v-type H(+) ATPase is proteolyzed and so is the lysosomal-associated membrane protein 2a (LAMP2a). Both proteins are also cleaved in vivo. Furthermore, LAMP2a cleavage was confirmed in vitro by addition of CAPNs to isolated lysosomes and several CAPN inhibitors prevented it. Finally, in vivo inhibition of CAPN1 in 72-h-weaned mice decreased LAMP2a cleavage. Indeed, calpeptin-treated mice showed a substantial delay in tissue remodeling and involution of the mammary gland. These results suggest that CAPNs are responsible for mitochondrial and lysosomal membrane permeabilization, supporting the idea that lysosomal-mediated cell death is a new hallmark of mammary gland involution.     

ATP7B expression in human breast epithelial cells is mediated by lactational hormones.

A role for the copper transporter, ATP7B, in secretion of copper from the human breast into milk has previously not been reported, although it is known that the murine ortholog of ATP7B facilitates copper secretion in the mouse mammary gland. We show here that ATP7B is expressed in luminal epithelial cells in both the resting and lactating human breast, where it has a perinuclear localization in resting epithelial cells and a diffuse location in lactating tissue. ATP7B protein was present in a different subset of vesicles from those containing milk proteins and did not overlap with Menkes ATPase, ATP-7A, except in the perinuclear region of cells. In the cultured human mammary line, PMC42-LA, treatment with lactational hormones induced a redistribution of ATP7B from a perinuclear region to a region adjacent, but not coincident with, the apical plasma membrane. Trafficking of ATP7B was copper dependent, suggesting that the hormone-induced redistribution of ATP7A was mediated through an increase in intracellular copper. Radioactive copper ((64)Cu) studies using polarized PMC42-LA cells that overexpressed mAtp7B protein showed that this transporter facilitates copper efflux from the apical surface of the cells. In summary, our results are consistent with an important function of ATP7B in the secretion of copper from the human mammary gland.     

An iterative calibration method with prediction of post-translational modifications for the construction of a two-dimensional electrophoresis database of mouse mammary gland proteins

Protein databases serve as general reference resources providing an orientation on two-dimensional electrophoresis (2-DE) patterns of interest. The intention behind constructing a 2-DE database of the water soluble proteins from wild-type mouse mammary gland tissue was to create a reference before going on to investigate cancer-associated protein variations. This database shall be deemed to be a model system for mouse tissue, which is open for transgenic or knockout experiments. Proteins were separated and characterized in terms of their molecular weight (M(r)) and isoelectric point (pI) by high resolution 2-DE. The proteins were identified using prevalent proteomics methods. One method was peptide mass fingerprinting by matrix-assisted laser desorption/ionization-mass spectrometry. Another method was N-terminal sequencing by Edman degradation. By N-terminal sequencing M(r) and pI values were specified more accurately and so the calibration of the master gel was obtained more systematically and exactly. This permits the prediction of possible post-translational modifications of some proteins. The mouse mammary gland 2-DE protein database created presently contains 66 identified protein spots, which are clickable on the gel pattern. This relational database is accessible on the WWW under the URL: http://www.mpiib-berlin.mpg.de/2D-PAGE.     

Role of ovarian secretions in mammary gland development and function in ruminants

The mammary gland is a dynamic organ that undergoes cyclic developmental and regressive changes during the lifetime of a female mammal. Mammogenesis begins during embryonic life with the development of the first mammary gland rudiments and ductal system. After birth, during the pre-pubertal period, the ductal growth of the mammary parenchyma occurs through the fat pad. In most of the ruminant species allometric mammary parenchyma development begins with the onset of cyclic ovarian secretions activity. The two main hormones secreted during an ovarian cycle are estradiol and progesterone. These steroid hormones are derived from cholesterol and are synthesized by theca and granulosa cells in ovaries. During puberty, the mammary parenchyma develops in a compact, highly arborescent parenchymal mass surrounded by a dense connective matrix. Ductal elongation and lobulo-alveolar development are accomplished during growth and pregnancy to prepare for future milk production. At the end of lactation, the mammary gland undergoes involution, which corresponds to a regression of the secretory tissue, a reduction in the alveolar size and a loss of mammary epithelial cells (MECs). Ovarian steroids (estradiol and progesterone) appear to be key regulators of the different stages of mammogenesis and mammary function. Through this review, the role and the importance of ovarian steroids on mammary gland and on MECs is described.     

Soluble fragment of P-cadherin adhesion protein found in human milk

Classical cadherins such as E- and P-cadherin are transmembrane proteins that mediate specific cell-to-cell adhesion and are important to tissue development and function. Cadherin function can be modulated by various means, including proteolytic cleavage of the extracellular adhesion domain from the cells' surface, yielding large soluble fragments termed (soluble) sE- or sP-cadherin. In people with certain carcinomas, sE-cadherin can be detected at elevated levels in the serum and sometimes can serve as a prognostic marker. Soluble E-cadherin also is found in urine of patients with bladder cancer. In addition to being present in bodily fluids of cancer patients, sE- and sP-cadherin are present in the serum of healthy people, suggesting that shedding of cadherins is a normal event. Here, we report high levels of 80 kDa sP-cadherin in human milk. In the lactating mammary gland tissue, P-cadherin appears to be a protein secreted by epithelial cells, rather than an adhesion protein. This is in contrast to the non-lactating mammary gland where P-cadherin is restricted to myoepithelial cells, and is present at sites of cell-cell contact.     

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.