Insulin-like growth factor binding protein-5 (IGFBP-5) induces premature cell death in the mammary glands of transgenic mice

We have previously demonstrated that IGFBP-5 production by mammary epithelial cells increases dramatically during involution of the mammary gland. To demonstrate a causal relationship between IGFBP-5 and cell death we created transgenic mice expressing IGFBP-5 in the mammary gland using a mammary-specific promoter, beta-lactoglobulin. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Histological analysis indicated reduced numbers of alveolar end buds, with decreased ductal branching. Transgenic dams produced IGFBP-5 in their milk at concentrations similar to those achieved at the end of normal lactation. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. BrdU labelling was decreased, whereas DNA ladders were increased in transgenic animals on day 1 of lactation. On day 2 postpartum, the epithelial invasion of the mammary fat pad was clearly impaired in transgenic animals. The concentrations of the pro-apoptotic molecule caspase-3 and of plasmin were both increased in transgenic animals whilst the concentrations of 2 prosurvival molecules Bcl-2 and Bcl-x(L)were both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I we examined IGF receptor phosphorylation and Akt phosphorylation and showed that both were inhibited. We attempted to "rescue" the transgenic phenotype by using growth hormone to increase endogenous IGF-I concentrations or by implanting minipumps delivering an IGF-1 analogue, R(3)-IGF-1, which binds weakly to IGFBP-5. Growth hormone treatment failed to affect mammary development suggesting that increased concentrations of endogenous IGF-1 are insufficient to overcome the high concentrations of IGFBP-5 produced by these transgenic animals. In contrast mammary development (gland weight and DNA content) was normalised by R3-IGF-I although milk production was only partially restored. This is the first demonstration that over-expression of IGFBP-5 can lead to; impaired mammary development, increased expression of the pro-apoptotic molecule caspase-3, increased plasmin generation and decreased expression of pro-survival molecules of the Bcl-2 family. It clearly demonstrates that IGF-I is an important developmental/survival factor for the mammary gland and, furthermore, this cell death programme may be utilised in a wide variety of tissues.     

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。     

Increased milk yield in transgenic mice expressing insulin-like growth factor 1

Insulin-like growth factor 1 (IGF-1) mediates many of the actions of growth hormone. Overexpression of IGF-1 was reported to have endocrine and paracrine/autocrine effects on somatic growth in transgenic mice. To study the paracrine/autocrine effects of IGF-1 in mammary gland, transgenic mice were produced by pronuclear microinjection of a construct containing a bovine alpha-lactalbumin (alpha-LA) promoter linked to an ovine IGF-1 cNDA. This alpha-LA promoter has previously been shown to direct expression of a human factor VIII gene specifically to the mammary gland of transgenic mice. Three transgenic mouse lines were established as a result of microinjection of 398 embryos. Transgene expression was found in mammary gland at day 1 of lactation from these three lines. Progeny test were carried out by mating two transgenic males/one transgenic female to two nontransgenic females/one nontransgenic male. Mice from one line (line 1225) were all nonexpressors and the other (line 1372) failed to produce offspring. Milk yield was analyzed in the line 1137 that produced 10 mice, of which three were transgenic females and three nontransgenic females. All of the three transgenic females showed integration of the transgene and expressed transgene IGF-1 mRNA in the mammary gland. Milk yields from days 5, 10, and 15 of lactation were significant greater in transgenic expressors than in their nontransgenic littermates. Specifically, there is 17.9% increase in total milk yield from these three days for transgenics compared with nontransgenics. These results demonstrate that local overexpression of IGF-1 in transgenic mice is capable to stimulating milk yield during the first lactation.     

Suppression of lactation by pregnancy-dependent mammary tumors in GR/A mice

Pregnancy-dependent mammary tumors (PDMT) in GR/A mice appear during pregnancy, disappear soon after parturition, and appear again during subsequent pregnancies. The retardation of pup growth, an indication of the level of milk production, was also observed with the advance of lactation numbers in this strain. This study was performed to elucidate the relationship between PDMT and lactational performance. At the end of the second pregnancy, mice were divided into two groups according to the presence of PDMT [PDMT(-) and PDMT(+) groups]. Although all PDMT disappeared within a day after parturition, the weight and growth of pups on Day 12 of lactation were significantly less in the PDMT(+) group than in the PDMT(-) group. Associated with this, the DNA and RNA contents of the mammary glands were apparently lower in the former than in the latter, although the differences were not statistically significant. There was little difference in mammary RNA/DNA ratio between groups. No difference was also observed between groups in endocrine organ weights, mother body weights, morphology of the mammary glands, adrenals and ovaries and plasma prolactin and progesterone levels. These results suggest that PDMT suppression of lactation is principally due to the retardation of mammary gland growth. Furthermore, no significant correlations were obtained between the size of PDMT and the parameters for mammary gland function. The data suggest that the development of PDMT per se is important for the retarded mammary gland growth.     

Increased Milk Yield in Transgenic Mice Expressing Insulin-like Growth Factor 1

Abstract

Insulin-like growth factor 1 (IGF-1) mediates many of the actions of growth hormone. Overexpression of IGF-1 was reported to have endocrine and paracrine/autocrine effects on somatic growth in transgenic mice. To study the paracrine/autocrine effects of IGF-1 in mammary gland, transgenic mice were produced by pronuclear microinjection of a construct containing a bovine α-lactalbumin (α-LA) promoter linked to an ovine IGF-1 cNDA. This α-LA promoter has previously been shown to direct expression of a human factor VIII gene specifically to the mammary gland of transgenic mice. Three transgenic mouse lines were established as a result of microinjection of 398 embryos. Transgene expression was found in mammary gland at day 1 of lactation from these three lines. Progeny test were carried out by mating two transgenic males/one transgenic female to two nontransgenic females/one nontransgenic male. Mice from one line (line 1225) were all nonexpressors and the other (line 1372) failed to produce offspring. Milk yield was analyzed in the line 1137 that produced 10 mice, of which three were transgenic females and three nontransgenic females. All of the three transgenic females showed integration of the transgene and expressed transgene IGF-1 mRNA in the mammary gland. Milk yields from days 5, 10, and 15 of lactation were significant greater in transgenic expressors than in their nontransgenic littermates. Specifically, there is 17.9% increase in total milk yield from these three days for transgenics compared with nontransgenics. These results demonstrate that local overexpression of IGF-1 in transgenic mice is capable to stimulating milk yield during the first 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.     

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.     

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.     

Caveolin-1-deficient mice show accelerated mammary gland development during pregnancy,premature lactation,and hyperactivation of the Jak-2/STAT5a signaling cascade

It is well established that mammary gland development and lactation are tightly controlled by prolactin signaling. Binding of prolactin to its cognate receptor (Prl-R) leads to activation of the Jak-2 tyrosine kinase and the recruitment/tyrosine phosphorylation of STAT5a. However, the mechanisms for attenuating the Prl-R/Jak-2/STAT5a signaling cascade are just now being elucidated. Here, we present evidence that caveolin-1 functions as a novel suppressor of cytokine signaling in the mammary gland, akin to the SOCS family of proteins. Specifically, we show that caveolin-1 expression blocks prolactin-induced activation of a STAT5a-responsive luciferase reporter in mammary epithelial cells. Furthermore, caveolin-1 expression inhibited prolactin-induced STAT5a tyrosine phosphorylation and DNA binding activity, suggesting that caveolin-1 may negatively regulate the Jak-2 tyrosine kinase. Because the caveolin-scaffolding domain bears a striking resemblance to the SOCS pseudosubstrate domain, we examined whether Jak-2 associates with caveolin-1. In accordance with this homology, we demonstrate that Jak-2 cofractionates and coimmunoprecipitates with caveolin-1. We next tested the in vivo relevance of these findings using female Cav-1 (-/-) null mice. If caveolin-1 normally functions as a suppressor of cytokine signaling in the mammary gland, then Cav-1 null mice should show premature development of the lobuloalveolar compartment because of hyperactivation of the prolactin signaling cascade via disinhibition of Jak-2. In accordance with this prediction, Cav-1 null mice show accelerated development of the lobuloalveolar compartment, premature milk production, and hyperphosphorylation of STAT5a (pY694) at its Jak-2 phosphorylation site. In addition, the Ras-p42/44 MAPK cascade is hyper-activated. Because a similar premature lactation phenotype is observed in SOCS1 (-/-) null mice, we conclude that caveolin-1 is a novel suppressor of cytokine signaling.     

A short form of the prolactin (PRL) receptor is able to rescue mammopoiesis in heterozygous PRL receptor mice

The heterozygous prolactin (PRL) receptor (PRLR +/-) mouse fails to develop a fully functional mammary gland at the end of the first pregnancy and shows markedly impaired lobuloalveolar development and milk secretion in young females. The PRLR is expressed ubiquitously, with various proportions of long and short isoforms in different tissues. Conflicting data have appeared on the putative role of the receptor short forms, with both agonist and antagonistic actions proposed. To assess whether the mouse PR-1 short isoform of the PRLR is potentially able to transduce a signal, we overexpressed it in heterozygous mice and investigated its effect on the rescue of mammary development. PRLR+/- mice were not able to develop a functional mammary gland, but restoration of mammary alveolar development and an increase in the expressions of casein and whey acidic protein genes were observed in transgenic PRLR+/- mice expressing the short form of the PRLR, leading to a complete rescue of mammary gland development and function in young females. These results demonstrate that PR-1, the short form of the PRLR, can improve mammary development in PRLR+/- mice, which compensates for the haploinsufficiency of the receptor long form; this effect is probably caused by accelerated proliferation and an activation of the PRLR signaling cascade, resulting in activation of target genes involved in mammary development and milk synthesis.     

Cold-inducible RNA binding protein in mouse mammary gland development

RNA binding proteins (RBPs) regulate gene expression by controlling mRNA export, translation, and stability. When altered, some RBPs allow cancer cells to grow, survive, and metastasize. Cold-inducible RNA binding protein (CIRP) is overexpressed in a subset of breast cancers, induces proliferation in breast cancer cell lines, and inhibits apoptosis. Although studies have begun to examine the role of CIRP in breast and other cancers, its role in normal breast development has not been assessed. We generated a transgenic mouse model overexpressing human CIRP in the mammary epithelium to ask if it plays a role in mammary gland development. Effects of CIRP overexpression on mammary gland morphology, cell proliferation, and apoptosis were studied from puberty through pregnancy, lactation and weaning. There were no gross effects on mammary gland morphology as shown by whole mounts. Immunohistochemistry for the proliferation marker Ki67 showed decreased proliferation during the lactational switch (the transition from pregnancy to lactation) in mammary glands from CIRP transgenic mice. Two markers of apoptosis showed that the transgene did not affect apoptosis during mammary gland involution. These results suggest a potential in vivo function in suppressing proliferation during a specific developmental transition.     

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.     

Production of low-lactose milk by ectopic expression of intestinal lactase in the mouse mammary gland

We have investigated, in mice, an in vivo method for producing low-lactose milk, based on the creation of transgenic animals carrying a hybrid gene in which the intestinal lactase-phlorizin hydrolase cDNA was placed under the control of the mammary-specific alpha-lactalbumin promoter. Transgenic females expressed lactase protein and activity during lactation at the apical side of mammary alveolar cells. Active lactase was also secreted into milk, anchored in the outer membrane of fat globules. Lactase synthesis in the mammary gland caused a significant decrease in milk lactose (50-85%) without obvious changes in fat and protein concentrations. Sucklings nourished with low-lactose milk developed normally. Hence, these data validate the use of transgenic animals expressing lactase in the mammary gland to produce low-lactose milk in vivo, and they demonstrate that the secretion of an intestinal digestive enzyme into milk can selectively modify its composition.     

Over-expression of human lipoprotein lipase in mouse mammary glands leads to reduction of milk triglyceride and delayed growth of suckling pups

Background

The mammary gland is a conserved site of lipoprotein lipase expression across species and lipoprotein lipase attachment to the luminal surface of mammary gland vascular endothelial cells has been implicated in the direction of circulating triglycerides into milk synthesis during lactation.

Principal Findings

Here we report generation of transgenic mice harboring a human lipoprotein lipase gene driven by a mammary gland-specific promoter. Lipoprotein lipase levels in transgenic milk was raised to 0.16 mg/ml, corresponding to an activity of 8772.95 mU/ml. High lipoprotein lipase activity led to a significant reduction of triglyceride concentration in milk, but other components were largely unchanged. Normal pups fed with transgenic milk showed inferior growth performances compared to those fed with normal milk.

Conclusion

Our study suggests a possibility to reduce the triglyceride content of cow milk using transgenic technology.     

Prolactin evokes lactational transmission of larvae in mice infected with Toxocara canis

We investigated the trans-lactational maternal–neonatal transmission of Toxocara canis larvae in mice, with particular interest in the role of prolactin in their migration to the mammary gland. Two female mice were infected with 300 T. canis eggs soon after delivery of 27 offspring. After 1 week of breast-feeding, seven larvae were recovered from 4 of 13 offspring. After 2 weeks of lactation, 101 larvae were recovered from all the remaining offspring. Daily prolactin administration (5 μg) was performed 2 weeks before T. canis infection and continued until 2 weeks after infection in six non-pregnant female mice, which resulted in larval accumulation in the mammary gland. Furthermore, prolactin administration in female mice that had been infected with T. canis 4 weeks prior to prolactin treatment induced migration of larvae into the mammary gland. These findings suggest that prolactin is a promoting factor contributing to lactational transmission of T. canis larvae in mice.     

Lactogenic hormones increase epidermal growth factor messenger RNA content of mouse mammary glands.

Epidermal growth factor (EGF) is known to stimulate mammary epithelial proliferation, has been identified in milk and is expressed in lactating mammary epithelia. This study examined hormonal control of EGF mRNA in mammary glands of mice. Prepro-EGF mRNA (4.7 kb) was detected during lactation (and increased significantly during this period), whereas a smaller EGF-like RNA (.5 kb) was at highest levels in mammary glands of virgin and pregnant mice. The 4.7 kb RNA was polyadenylated, whereas .5 kb RNA was not. In mammary gland organ cultures from steroid-primed mice, the combinations of insulin + hydrocortisone and insulin + prolactin + hydrocortisone increased both prepro-EGF and beta-casein mRNA expression. When hydrocortisone was present there was a decrease in mammary gland content of EGF-like RNA (.5 kb band). We conclude that prepro-EGF mRNA expression in mouse mammary tissue is under the control of the lactogenic hormones prolactin and hydrocortisone.