The effect of co-integration of the entire beta-lactoglobulin (BLG) gene or matrix attachment region (MAR) sequences on the expression of various BLG/ human serum albumin (HSA) gene constructs was tested in transgenic mice. These former... more
The effect of co-integration of the entire beta-lactoglobulin (BLG) gene or matrix attachment region (MAR) sequences on the expression of various BLG/ human serum albumin (HSA) gene constructs was tested in transgenic mice. These former sequences were chosen because of their reported ability to insulate transgenes from the neighboring host genomic DNA sequences and/or to provide a more permissive transcriptional environment. When introduced alone, a cDNA-based BLG/HSA construct was expressed in 60% of transgenic strains and HSA was secreted at levels up to 0.3 mg/ml into the milk. Upon co-integration with either the entire BLG gene or MAR element, HSA RNA and protein expression were completely abrogated. While the co-integrated BLG gene suppressed the proportion of expresser strains carrying cDNA as well as genomic BLG/HSA constructs, the MAR element only exerted its negative effect on the cDNA-based BLG/HSA construct. In transgenics expressing both HSA and BLG, the tissue specificity and developmental patterns of BLG expression were altered and resembled the less stringent pattern of the BLG/HSA expression. These results demonstrate that rescue of transgene expression through co-integration with BLG or MAR sequences do not apply universally.
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Mammary epithelial stem cells differentiate to create the basal and luminal layers of the gland. Inducing the number of differentiating bovine mammary stem cells may provide compensating populations for the milk-producing cells that die... more
Mammary epithelial stem cells differentiate to create the basal and luminal layers of the gland. Inducing the number of differentiating bovine mammary stem cells may provide compensating populations for the milk-producing cells that die during lactation. Inhibition of mTOR activity by rapamycin signals self-renewal of intestinal stem cells, with similar consequences in the mouse mammary gland and in bovine mammary implants maintained in mice. The implementation of these results in farm animals for better mammary development and production was studied in 3-month-old calves. mTOR activity decreased by ~50% in mammary epithelial cells subjected to 3-week rapamycin administration, with no negative consequences on mammary morphology or β-casein expression. Subsequently, stem cell self-renewal was induced, reflected by a higher propagation rate of cultures from rapamycin-treated glands compared to respective controls and higher expression of selected markers. Followed by 4-day estrogen an...
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Original objectives: The long term objective of the project is to achieve higher content of protein in the milk of ruminants by modulating the translational machinery in the mammary gland. The first specific aim of the BARD proposal was... more
Original objectives: The long term objective of the project is to achieve higher content of protein in the milk of ruminants by modulating the translational machinery in the mammary gland. The first specific aim of the BARD proposal was to characterize responsiveness of various experimental systems to combination of lactogenic hormones and amino acids with particular emphasis on discrimination between the control of total protein synthesis and milk protein synthesis. Based on the results, we planned to proceed by characterizing the stage of protein synthesis in which the stimulation by lactogenic hormones and amino acid occur and finally we proposed to identify which components of the translation machinery are modified. Background to the topic: Milk protein is the most valuable component in milk, both for direct human consumption and for manufacturing cheese and other protein-based products. Attempts to augment protein content by the traditional methods of genetic selection and impr...
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Altering milk composition via transgenesis depends on three main factors. (1) The availability of an efficient regulatory sequences for targeting transgene(s) to the mammary gland; (2) a reliable in vitro model to test the expression of... more
Altering milk composition via transgenesis depends on three main factors. (1) The availability of an efficient regulatory sequences for targeting transgene(s) to the mammary gland; (2) a reliable in vitro model to test the expression of transgenes prior to their introduction to the animal genome; and (3) better understanding of the major factors which determine the rate of gene expression and protein synthesis. The current studies provide the necessary means and knowledge to alter milk protein composition via transgenesis. The following specific goals were achieved: a: Identifying regulatory regions in the b-lactoglobulin (BLG) gene and the cross-talk between elements which enabled us to construct an efficient vector for the expression of desirable cDNA's in the mammary gland. b: The establishment of a sheep mammary cell line that serves as a model for the analysis of endogenous and exogenous milk protein synthesis in the mammary gland of livestock. c: An accurate comparison of ...
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Publication View. 55582701. BMC Cancer BioMed Central (2008). Laure Voisin,; Catherine Julien,; StÃĐphanie Duhamel,; Kailesh Gopalbhai,; Isabelle Claveau,; Marc K Saba-el-leil,; Ian GaÃŦl Rodrigue-gervais,; Louis Gaboury,; Daniel... more
Publication View. 55582701. BMC Cancer BioMed Central (2008). Laure Voisin,; Catherine Julien,; StÃĐphanie Duhamel,; Kailesh Gopalbhai,; Isabelle Claveau,; Marc K Saba-el-leil,; Ian GaÃŦl Rodrigue-gervais,; Louis Gaboury,; Daniel Lamarre,; Mark Basik,; Sylvain Meloche. ...
Calorie restriction enhances stem cell self-renewal in various tissues, including the mammary gland. We hypothesized that similar to their intestinal counterparts, mammary epithelial stem cells are insulated from sensing changes in energy... more
Calorie restriction enhances stem cell self-renewal in various tissues, including the mammary gland. We hypothesized that similar to their intestinal counterparts, mammary epithelial stem cells are insulated from sensing changes in energy supply, depending instead on niche signaling. The latter was investigated by subjecting cultures of mammary epithelial stem cells for 8 days to in vivo paracrine calorie-restriction signals collected from a 4-day-conditioned medium of individual mammary cell populations. Conditioned medium from calorie-restricted non-epithelial cells induced latent cell propagation and mammosphere formation—established markers of stem cell self-renewal. Combined RNA-Seq, immunohistochemistry and immunofluorescence analyses of the non-epithelial population identified macrophages and secreted CSF1 as the energy sensor and paracrine signal, respectively. Calorie restriction-induced pStat6 expression in macrophages suggested that skewing to the M2 phenotype contributes...
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Lactating females of eight independent strains of transgenic mice carrying a human serum albumin (HSA) gene or minigene under the control of the B‐lactoglobulin (BLG) promoter secrete strain‐specific amounts of HSA into their milk,... more
Lactating females of eight independent strains of transgenic mice carrying a human serum albumin (HSA) gene or minigene under the control of the B‐lactoglobulin (BLG) promoter secrete strain‐specific amounts of HSA into their milk, ranging from 0‐4.5 mg per ml. The HSA produced in the milk of all transgenic strains appeared to be identical. The ability of mammary explants from
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Limited outgrowth development of bovine mammary epithelial stem cells transplanted into de-epithelialized mouse fat pads restricts advanced studies on this productive organ's development and renewal. We challenged the mouse-bovine... more
Limited outgrowth development of bovine mammary epithelial stem cells transplanted into de-epithelialized mouse fat pads restricts advanced studies on this productive organ's development and renewal. We challenged the mouse-bovine incompatibility by implanting parenchymal adjacent or distant bovine stromal layers (close and far stroma, respectively) into the mouse fat pad to serve as an endogenous niche for transplanted stem cells. The close stroma better supported stem cell take rate and outgrowth development. The diameter of these open duct-like structures represented and occasionally exceeded that of the endogenous ducts and appeared 8.3-fold wider than the capsule-like structures developed in the mouse fat pad after similar cell transplantation. RNA-Seq revealed lower complement activity in this layer, associated with secretion of specific laminins and WNT proteins favoring epithelial outgrowth development. The close stroma appeared genetically more similar to the parenchyma than to the far stroma due to epithelial characteristics, mainly of fibroblasts, including expression of epithelial markers, milk protein genes, and functional mammary claudins. Gene markers and activators of the mesenchymal-to-epithelial transition were highly enriched in the epithelial gene cluster and may contribute to the acquired epithelial properties of this stromal layer.
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Expansion of the mammary epithelial stem cell pool holds promise for consequent mammary gland development and production. Complementary analyses of bovine mammary implants maintained in de-epithelialized mouse mammary fat pad and... more
Expansion of the mammary epithelial stem cell pool holds promise for consequent mammary gland development and production. Complementary analyses of bovine mammary implants maintained in de-epithelialized mouse mammary fat pad and endogenous mouse mammary gland were performed to elucidate the effect of calorie restriction (CR) on stem cell self-renewal. CR elevated propagation rate and non-adherent mammosphere generation in cultured bovine mammary cells. A corresponding decrease in progenitor-induced colony formation and differentiation marker expression was noted. In the mouse gland, CR enhanced the take rate of transplanted cells and outgrowths' fat pad occupancy. Downregulating mTOR activity by rapamycin administration reproduced CR's effects on stem cell self-renewal within a shorter period. Flow cytometry demonstrated a significant 1.5-fold increase in stem cell number and a corresponding decrease in luminal progenitor and differentiated cells. Consequent effects of rapamycin administration included enhanced ductlet generation in bovine implants and higher milk-protein gene expression in cultured mouse mammary cells. The stimulatory effect of CR on BST-1 expression in both bovine implants and mouse glands resembled that noted in the intestinal Paneth stem cell niche (Yilmaz et al., 2012). A putative niche may also exist in the mammary gland, conveying energy-status information to the insulated stem cells.
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Elucidating cell hierarchy in the mammary gland is fundamental for understanding the mechanisms governing its normal development and malignant transformation. There is relatively little information on cell hierarchy in the bovine mammary... more
Elucidating cell hierarchy in the mammary gland is fundamental for understanding the mechanisms governing its normal development and malignant transformation. There is relatively little information on cell hierarchy in the bovine mammary gland, despite its agricultural potential and relevance to breast cancer research. Challenges in bovine-to-mouse xenotransplantation and difficulties obtaining bovine-compatible antibodies hinder the study of mammary stem-cell dynamics in this species. In-vitro indications of distinct bovine mammary epithelial cell populations, sorted according to CD24 and CD49f expression, have been provided. Here, we successfully transplanted these bovine populations into the cleared fat pads of immunocompromised mice, providing in-vivo evidence for the multipotency and self-renewal capabilities of cells that are at the top of the cell hierarchy (termed mammary repopulating units). Additional outgrowths from transplantation, composed exclusively of myoepithelial c...
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ABSTRACT
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Signal transducer and activator of transcription 5 (Stat5) transduces extracellular cytokine and growth factor signals to the nucleus of mammary epithelial cells and thereby regulates gene transcription during pregnancy, lactation, and... more
Signal transducer and activator of transcription 5 (Stat5) transduces extracellular cytokine and growth factor signals to the nucleus of mammary epithelial cells and thereby regulates gene transcription during pregnancy, lactation, and weaning. Gene constructs were prepared which subject the wild-type Stat5 or a constitutively active variant of Stat5 to the control of the beta-lactoglobulin (BLG) regulatory sequences and direct it to the mammary epithelium. The integrity and functionality of these constructs were confirmed through introduction into cultured mammary epithelial cells and hormone induction experiments. Expression levels and states of activity of Stat5 in mammary gland tissue were manipulated by introducing Stat5 variants as transgenes into the pronuclei of transgenic mice. The consequences of enhanced Stat5 expression and activation on the development of alveoli, their differentiated functions, and on postlactational involution were investigated. As expected, the trans...
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Human skin fibroblasts synthesize and secrete complement Factor B, a component of the complement alternative pathway, when stimulated by mediators of inflammation such as lipopolysaccharide and various cytokines. Recombinant IL-6/IFN-beta... more
Human skin fibroblasts synthesize and secrete complement Factor B, a component of the complement alternative pathway, when stimulated by mediators of inflammation such as lipopolysaccharide and various cytokines. Recombinant IL-6/IFN-beta 2 (E. coli) stimulates Factor B synthesis in fibroblasts but the effect is strongly potentiated by the addition of IFN-gamma. When both cytokines are added, the skin fibroblasts secrete significant amounts of biologically active Factor B detectable in a hemolysis test. This cooperative effect of IL-6, which is made by most tissue cells and monocytes and of IFN-gamma which is made by T-lymphocytes may play a role in local inflammatory processes. IL-6 and IFN-gamma also cooperate in the induction of (2'-5') A synthetase, a mediator of IFN action.
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We have tested the feasibility of producing large quantities of human serum albumin (HSA) in the milk of transgenic livestock by generating transgenic mice as a model system. The sheep beta-lactoglobulin (BLG)... more
We have tested the feasibility of producing large quantities of human serum albumin (HSA) in the milk of transgenic livestock by generating transgenic mice as a model system. The sheep beta-lactoglobulin (BLG) 5'-regulatory promoter sequences were used to support expression of BLG or HSA in transgenic mice. Transgenic animals generated from the entire BLG gene including 3, 5.5 or 10.8 kb of 5'-sequences demonstrated that 3 kb of 5'-sequences were sufficient to support high levels of expression of BLG, and that the longer 5'-sequences did not improve upon the levels of expression. As such, the 3 kb 5'-sequences were used to drive expression of HSA in BLG-HSA constructs. HSA was not detectably expressed in eight transgenic lines generated from a BLG-HSA construct containing the HSA cDNA. Two transgenic lines of 26 generated, using five different constructs, with an HSA minigene possessing the first intron expressed HSA in their milk. One of these expressed HSA at high levels (2.5 mg ml-1) and has stably transmitted this ability to its progeny. A high percentage of transgenic mouse lines (four of six) generated from a vector containing an HSA minigene possessing introns 1 and 2 expressed HSA in their milk at levels which ranged from 1 to 35 micrograms ml-1. In a similar trend, levels of expression of HSA by transfected tissue culture cells from BLG-HSA vectors containing an introduced SV40 enhancer were low with the HSA cDNA, increased with the HSA minigene with intron 1 and increased further with the minigene containing introns 1 and 2. This study demonstrates that high levels of HSA can be expressed in the milk of transgenic animals, that introns of the HSA gene play a role in its expression and that transfected cell lines may be used to quickly evaluate the relative expression efficiencies of various vector constructs intended for future transgenic evaluation.
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Limited information on the production and secretion of the individual milk proteins at the cellular level in the mammary gland is available. It is generally assumed that all active epithelial cells within the lactating mammary gland of... more
Limited information on the production and secretion of the individual milk proteins at the cellular level in the mammary gland is available. It is generally assumed that all active epithelial cells within the lactating mammary gland of mice share a comparable ability to produce and secrete all the components of milk. This is based on ultrastructural studies and observations on cell membrane turnover related to fat globule membrane loss and vesicular protein exocytosis.
The challenge in manipulating the proportion of somatic stem cells lies in having to override tissue homeostasis. Xanthosine infusion via the teat canal has been reported to augment the number of label-retaining cells in the mammary gland... more
The challenge in manipulating the proportion of somatic stem cells lies in having to override tissue homeostasis. Xanthosine infusion via the teat canal has been reported to augment the number of label-retaining cells in the mammary gland of 3-month-old bovine calves. To further delineate xanthosine׳s effect on defined stem cells in the mammary gland of heifers-which are candidates for increased prospective milk production following such manipulation-bovine mammary parenchymal tissue was transplanted and integrated into the cleared mammary fat pad of immunodeficient mice. Xanthosine administration for 14 days did not affect the number of label-retaining cells after 10- and 11-week chases. No change in stem cell proportion, analyzed according to CD49f and CD24 expression, was noted. Clone formation and propagation rate of cultured cells, as well as expression of stem cell markers, were also unaffected. In contrast, a latent 50% decrease in bovine mammary cell proliferation rate was o...
Research Interests: Stem Cells, Flow Cytometry, Biology, Stem Cell, Apoptosis, and 12 moreMedicine, Western blotting, Mice, Female, Animals, Clinical Sciences, Cattle, Epithelial cells, Cell Proliferation, Biochemistry and cell biology, real time polymerase chain reaction, and reverse transcriptase polymerase chain reaction
A ß-lactoglobulin (BLG)/luciferase gene vector (p907), composed of a luciferase intronless gene inserted between the second and sixth BLG exons was constructed. Stable transfections of CID-9 cells with this vector, as well as with a... more
A ß-lactoglobulin (BLG)/luciferase gene vector (p907), composed of a luciferase intronless gene inserted between the second and sixth BLG exons was constructed. Stable transfections of CID-9 cells with this vector, as well as with a series of additional vectors, were performed to define regulatory regions within the BLG sequence, and the contribution of the SV40 polyadenylation (PA) site to luciferase
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A dramatic uncoupling of the expression of chimaeric ß-lactoglobulin (BLG)/human serum albumin (HSA) gene constructs at the RNA and protein levels was observed in cultured mammary explants of virgin transgenic mice. Upon explantation,... more
A dramatic uncoupling of the expression of chimaeric ß-lactoglobulin (BLG)/human serum albumin (HSA) gene constructs at the RNA and protein levels was observed in cultured mammary explants of virgin transgenic mice. Upon explantation, both HSA RNA and protein were expressed at high levels. However, when the explants were grown in hormone-free medium, HSA RNA continued to accumulate, whereas the synthesis
Research Interests: RNA, Biology, Immunohistochemistry, Medicine, Gene expression, and 15 moreIn Situ Hybridization, Protein synthesis, Biological Sciences, Humans, Prolactin, Insulin, Mice, Female, Animals, Polymerase Chain Reaction, Mammary gland, Milk Protein, Protein Biosynthesis, Drug synergism, and Human serum albumin
Transgenic mice were produced, carrying hybrid genes comprised of the ovine beta-lactoglobulin (BLG) milk protein gene promoter and human serum albumin (HSA) coding sequences. In situ hybridization revealed high levels of BLG/HSA hybrid... more
Transgenic mice were produced, carrying hybrid genes comprised of the ovine beta-lactoglobulin (BLG) milk protein gene promoter and human serum albumin (HSA) coding sequences. In situ hybridization revealed high levels of BLG/HSA hybrid mRNA, confined to the epithelial cells of the lactating mammary gland with a several hundred fold lower concentration in virgin mammary glands. During the first 24 h in culture, exceptionally high levels of HSA were secreted from explants of virgin mice, independent of hormonal control. HSA secretion was reduced considerably during subsequent days in culture and became dependent on the presence of insulin, hydrocortisone and prolactin. This temporal and hormonal pattern of regulation of HSA was different than that found for the secretion of caseins. In contrast to the vast difference in the mRNA content, the amount of HSA secreted from explants derived from lactating mice during the first 24 h in culture was only 2- to 5-fold higher than that found with explants from virgin transgenic mice, suggesting post-transcriptional control of HSA synthesis. The high-level synthesis and secretion of HSA in mammary explants of lactating mice was also dependent on the presence of insulin, hydrocortisone and prolactin. This study confirms previous suggestion that mammary explants from virgin transgenics may serve as a powerful tool for screening the potential of transgenic animals to secrete foreign proteins in their milk.
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We produced transgenic mice carrying the native sheep ~-lactoglobulin (BLG) or fusion genes composed of the BLG promoter and human serum albumin (HSA) minigenes. BLG was expressed exclusively in the mammary glands of the virgin and... more
We produced transgenic mice carrying the native sheep ~-lactoglobulin (BLG) or fusion genes composed of the BLG promoter and human serum albumin (HSA) minigenes. BLG was expressed exclusively in the mammary glands of the virgin and lactating transgenic mice evaluated. ...
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Research Interests: Engineering, Chemistry, Molecular Biology, Biology, Medicine, and 15 moreHumans, Female, Animals, Milk products, Cattle, Fluorescent Antibody Technique, Epithelial cells, Mammary gland, Biological markers, Cell Surface Markers, Aldehyde Dehydrogenase, Differential Gene Expression, Gene Expression Regulation, Cell Aggregation, and Cell count
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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... more
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.
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The expression pattern of human serum albumin (HSA) in transgenic mice carrying various HSA genomic sequences driven either by the mouse whey acidic protein (WAP) or the sheep beta-lactoglobulin (BLG) promoters, was compared. The pattern... more
The expression pattern of human serum albumin (HSA) in transgenic mice carrying various HSA genomic sequences driven either by the mouse whey acidic protein (WAP) or the sheep beta-lactoglobulin (BLG) promoters, was compared. The pattern of HSA expression in both WAP/HSA and BLG/HSA transgenic lines was copy number independent, and the major site of ectopic expression was the skeletal muscle. Although an equal proportion of expressors was determined in both sets of mice (approximately 25% secreting >0.1 mg/ml), the highest level of HSA secreted into the milk in the WAP/HSA transgenic lines was one order of magnitude lower than in the BLG/HSA lines. Despite this difference, the HSA expression patterns in the mammary gland were similar and consisted of two levels of variegated expression. Studies using mammary explant cultures revealed a comparable responsiveness to the lactogenic hormones insulin, hydrocortisone, and prolactin, although the WAP/HSA gene constructs were more sensitive to the hydrocortisone effect than were the BLG/HSA vectors. When HSA vectors were stably transfected into the mouse mammary cell line CID-9, they displayed a hierarchy of expression, dependent upon the specific complement of HSA introns included. Nevertheless, the expression of HSA in four out of five WAP/HSA constructs was similar to their BLG/HSA counterparts. This construct-dependent, and promoter-independent, hierarchy was also found following transfection into the newly established Golda-1 ovine mammary epithelial cell line.
Research Interests: Molecular Biology, Biology, Lactation, Medicine, Gene expression, and 15 moreCell line, Humans, Mice, Sheep, Female, Animals, Gene Dosage, Milk, Epithelial cells, Genome sequence, Biochemistry and cell biology, Milk proteins, Serum albumin, Human serum albumin, and Paediatrics and reproductive medicine
Signal transducer and activator of transcription (Stat5) is a transcription factor, which transduces extracellular cytokine and growth-factor signals to the nuclei of mammalian cells. As a major mediator of prolactin action, it is... more
Signal transducer and activator of transcription (Stat5) is a transcription factor, which transduces extracellular cytokine and growth-factor signals to the nuclei of mammalian cells. As a major mediator of prolactin action, it is involved in the regulation of the development, function, and survival of mammary epithelial cells. The carboxyl terminal of Stat5 encodes a transactivation domain (TAD), which interacts with coactivators and is crucial for the transcriptional induction of Stat5 target genes. To study the role of the Stat5 TAD in mediating Stat5 functions, a carboxy terminally truncated Stat5 variant (Stat5Delta750) was directed for expression in the mammary glands of transgenic mice by regulatory sequences of the beta-lactoglobulin (BLG) gene. Expression of Stat5Delta750 in mammary tissue reduced the rates of cell proliferation at mid and late pregnancy. Subsequently, morphological signs of milk secretion upon parturition were delayed. In double-transgenic mice, expression of Stat5Delta750 drastically decreased BLG/luciferase activity during lactation, but did not affect the expression and secretion of the endogenous beta-casein or alpha-lactalbumin into the milk. Expression of Stat5Delta750 also caused an increase in the number of apoptotic cells during mammary involution by a factor of 3 relative to control glands. Our data established a role for the Stat5 TAD in mediating the effects of Stat5 on mammary development, regulation of milk protein gene activity, and cell survival. The full effects of Stat5Delta750 may be partially buffered by the expression of endogenous wild-type Stat5 and the formation of truncated and wild-type heterodimers.
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Research Interests: Biology, Polyadenylation, Medicine, Molecular endocrinology, Protein synthesis, and 15 moreSignal Transduction, Biological Sciences, Prolactin, Insulin, Mice, Female, Animals, Epithelial cells, Cytoplasm, Milk Protein, Protein Biosynthesis, Milk proteins, Polyribosomes, Drug synergism, and Medical and Health Sciences
Research Interests: Kinetics, Biology, Medicine, Biological Sciences, Cell Differentiation, and 15 moreInsulin, Mice, Female, Animals, Kinase inhibitor, Mitogen Activated Protein Kinase, Epithelial cells, Mammary gland, DNA binding, MAP Kinase, DNA binding proteins, Milk proteins, janus kinase, Drug synergism, and Medical and Health Sciences
Short-term regulation of prolactin (PRL) receptors was studied in ketamine-anaesthetized 18-day pregnant or 7-day lactating female rats, by infusing them with various doses of oPRL or human growth hormone (hGH) for 0-3 h and measuring the... more
Short-term regulation of prolactin (PRL) receptors was studied in ketamine-anaesthetized 18-day pregnant or 7-day lactating female rats, by infusing them with various doses of oPRL or human growth hormone (hGH) for 0-3 h and measuring the binding of [125I]oPRL of [125I]hGH to the microsomal fractions prepared from the liver, mammary gland and kidneys of animals sacrificed at various states of infusion. Our main findings are: In pregnant rats, only 30% of liver receptors are unoccupied and infusion with 25 micrograms/h for 3 h of either oPRL of hGH decreased both free and total receptors by 22-30% while infusion with 250 micrograms/ml caused an additional decrease only in the free receptors. In the mammary gland and the kidney of pregnant rats, all receptors seem to be unoccupied; infusion with 25 micrograms/ml had none or a slight elevating effect on the number of both free and total receptors in the mammary gland but caused a significant 3-fold increase in the kidney; infusion with 250 micrograms/ml, however, resulted in a slight decrease in the mammary gland and a significant decrease in the kidney in both total and free receptors. In the liver of the lactating rats, there was no significant difference between the number of free and total receptors, but in mammary gland, specific binding to the total receptor was higher than to the free ones indicating partial occupancy; infusion with 25 micrograms/ml caused a significant decrease in free and total liver receptors without a remarkable change in the mammary gland and some decrease (by infusion with hGH only) in the kidney. In all cases, the changes in the specific binding resulted from the increase or decrease in receptor number and not from the change in receptor-hormone affinity. In almost all cases, infusion with oPRL or hGH yielded similar results. Infusion with both hormones did not affect the level of the endogenous rat prolactin. In conclusion, our results indicate the short-term regulation of PRL receptors by exogenous hormones is a complicated process which is affected by the level of the infused hormone, physiological state of the animal and may yield, simultaneously, different or even opposite changes in receptor number in various organs.
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Research Interests: Pharmaceutical Technology, Signal Transduction, RNA interference, Humans, Prolactin, and 14 moreCancer Therapy, Mice, Female, Animals, Transcription Factor, Clinical Sciences, Pharmaceutical Formulation Technology, Mammary gland, Chromatin Immunoprecipitation, Growth Factor, Cell Surface Markers, Hematopoietic Stem Cell, Tyrosine Phosphorylation, and Breast Neoplasms
Parity-dependent adenocarcinoma tumors developed in postestropausal transgenic mice expressing a constitutively active Stat5 variant (STAT5ca) in their mammary gland. These tumors maintained elevated expression levels of genes regulating... more
Parity-dependent adenocarcinoma tumors developed in postestropausal transgenic mice expressing a constitutively active Stat5 variant (STAT5ca) in their mammary gland. These tumors maintained elevated expression levels of genes regulating the cellular DNA damage response (DDR) mechanism, compared to the intact gland. No correlation with STAT5ca expression was observed for these genes in the established tumors. However, activated Stat5a in individual cells of the rarely and earlier developed hyperplasia was associated with induced Chk2 activity. Deregulated Stat5 may already cause DNA damage during the fertile period. This hypothesis and the specific vulnerable stage were further studied in mammary epithelial cells that were stably transfected with β-lactoglobulin (BLG)/STAT5ca and exposed to a reproduced reproductive cycle. During the pregnancy-like proliferative state, STAT5ca expression was induced by the added lactogenic hormones. Production of reactive oxygen species, rather than proliferation, served as the primary mediator of DNA damage and cellular DDR. Differentiated cells expressed higher levels of STAT5ca and retained the DNA nicks. However, the elevated expression of the genes involved in DDR was downregulated. Higher levels of DNA damage were also detected in the mammary gland of transgenic mice expressing the BLG/STAT5ca during pregnancy and lactation. However, the relative number of damaged cells was much lower than that in the reproduced in vitro stages and the insults were generally associated with apoptosis and DDR. This study implicates pregnancy as the vulnerable stage for deregulated Stat5 activity, and demonstrates that DNA insults in viable differentiated mammary epithelial cells are ignored by the DDR mechanism.
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The signal transducer and activator of transcription (Stat5) funnels extracellular signals of cytokines, hormones, and growth factors into transcriptional activity in the mammary gland. Postnatal development and functionality of this... more
The signal transducer and activator of transcription (Stat5) funnels extracellular signals of cytokines, hormones, and growth factors into transcriptional activity in the mammary gland. Postnatal development and functionality of this tissue is synchronized with the reproductive cycle. Consequently, Stat5 involvement in lobuloalveolar development, milk-protein synthesis, or tissue remodeling is dictated by the particular reproductive stage. Latent deregulation of Stat5 activity during the reproductive cycle predisposes the tissue to tumorigenesis at a later stage, when the female is no longer fertile. Accumulating data from studies with mouse models and breast-cancer specimens demonstrate a dual role for Stat5 in this context. It causes tumorigenesis, but delays metastasis progression. Consequently, Stat5 activity in breast-cancer specimens marks a better prognosis for survival.
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The role of amino acids (AA) on translational regulation in mammary epithelial cells cultured under lactogenic conditions was studied. The rates of total protein synthesis and beta-lactoglobulin (BLG) synthesis in mouse CID-9 cells were... more
The role of amino acids (AA) on translational regulation in mammary epithelial cells cultured under lactogenic conditions was studied. The rates of total protein synthesis and beta-lactoglobulin (BLG) synthesis in mouse CID-9 cells were 2.1- or 3.1-fold higher, respectively, than in their bovine L-1 counterparts. Total AA deprivation or selective deprivation of Leu had a negative protein-specific effect on BLG synthesis that was more pronounced in bovine cells than in murine cells. Dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and S6 kinase (S6K1) on Thr(389) but not on Ser(411) was also more prominent in bovine cells. Noteably, deprivation of Leu had a less marked effect on BLG synthesis and 4E-BP1 or S6K1 phosphorylation than deprivation of all AA. In AA-deprived CID-9 cells, Leu specifically restored BLG synthesis from pre-existing mRNA whereas AA also restored total protein synthesis. This restoration was associated with a more pronounced effect on 4E-BP1 and S6K1 phosphorylation in bovine versus murine cells. Rapamycin specifically reduced Leu- and AA-stimulated BLG translation initiation in a dose-dependent manner. A further reduction was observed for Leu-treated cells in the presence of LY294002, a PI3K (phosphatidylinositol 3-kinase) inhibitor, which also reduced total protein synthesis. These findings suggest that direct signaling from AA to the translational machinery is involved in determining the rates of milk protein synthesis in mammary epithelial cells.
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Members of the signal transducers and activators of transcription (Stat) family regulate essential cellular growth and survival functions in normal cells and have also been implicated in tumorigenesis. We have studied the potential role... more
Members of the signal transducers and activators of transcription (Stat) family regulate essential cellular growth and survival functions in normal cells and have also been implicated in tumorigenesis. We have studied the potential role of Stat5 in mammary tumorigenesis by targeting Stat5 variants to the mammary gland of transgenic mice using regulatory sequences of the beta-lactoglobulin gene. Mammary-directed expression of the wild-type Stat5, constitutively activated Stat5 and carboxyl-terminally truncated dominant negative Stat5 forms resulted in mammary tumors with incidence rates of up to 22% and latency periods of 8-12 months. Undifferentiated carcinomas most frequently occurred in mice expressing the carboxyl-terminally truncated Stat5. The more differentiated papillary and micropapillary adenocarcinomas were primarily found in mice overexpressing the native and constitutively active transgenes. Higher levels of translation initiation factor 4E (eIF4E) and cyclin D1 expression but lower levels of activated Stat3 were found in tumors of mice expressing the constitutively active Stat5 when compared to mice expressing the wild-type or truncated forms. A higher expression of the estrogen receptor (ERalpha) was observed in carcinomas compared to other phenotypes. The ability of both forms of Stat5, the transactivating form and the dominant negative form, to participate in oncogenesis indicates that there is more than one mechanism by which Stat5 contributes to this process. The transactivation function of Stat5 is involved in the determination of tumors with a more differentiated phenotype.
Research Interests:
Research Interests:
Research Interests: Genetics, Biology, Medicine, Gene expression, Extracellular Matrix, and 15 moreMolecular Mechanics, Cell Culture, Transgenic Mice, Growth Hormone, Cell line, Humans, Sheep, Female, Animals, Epithelial cells, Mammary gland, Transfection, Serum albumin, Human serum albumin, and Paediatrics and reproductive medicine
We compared the developmental pattern of expression of the sheep beta-lactoglobulin (BLG), the chimeric BLG/human serum albumin (HSA), and the endogenous murine beta-casein genes in the mammary gland of virgin, pregnant and lactating... more
We compared the developmental pattern of expression of the sheep beta-lactoglobulin (BLG), the chimeric BLG/human serum albumin (HSA), and the endogenous murine beta-casein genes in the mammary gland of virgin, pregnant and lactating transgenic mice, both at the RNA (expression) and protein (synthesis and secretion) levels. The BLG and casein genes were expressed at very low levels in virgin animals and during early stages of pregnancy. The increase in the expression of these genes started at the second half of pregnancy and reached a peak between the end of pregnancy and day 10 of lactation. The accumulation of their RNA coincided with that of the corresponding proteins, indicating a transcriptional control of expression of these genes. The expression and secretion patterns of the endogenous casein gene in transgenic and nontransgenic mice were indistinguishable. The hybrid BLG/HSA gene constructs displayed distinct patterns of expression in virgin animals and at early stage of pregnancy, from that of the BLG transgene or the endogenous mouse milk protein gene. High levels of expression (17-60% of that on day 18 of pregnancy) were detected in the mammary gland of virgin animals. At day 5 of pregnancy there was a dramatic decrease in HSA synthesis and secretion in all transgenic strains tested. The down-regulation, revealed by immunoprecipitation and immunohistochemical studies, demonstrated that at that stage of pregnancy only 10-18% of ductal structures contained HSA expressing cells in contrast to the majority of ducts expressing HSA in virgin animals. These morphological studies also demonstrated that the down-regulation in HSA synthesis and secretion was correlated with the transition from ducts comprised of a single layer of epithelial cells (characteristic of the virgin state) to ducts composed of multilayers of such cells. In two of the three transgenic strains tested, the down-regulation at the protein level was associated with a similar decrease in HSA transcripts. In the exceptional strain no. 23, HSA transcripts continued accumulating even at this stage. The differences in the control of expression at the RNA level between these transgenic strains were also confirmed by in situ hybridization. Our results suggest the involvement of at least two regulatory mechanisms effective at early stages of gestation in the control of expression/secretion of the HSA transgene targeted for expression in the mammary gland by the BLG milk protein promoter. These putative mechanisms may play key roles in the interplay between normal mammogenesis and lactogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)