Newly characterized bovine mammary stromal region with epithelial properties supports representative epithelial outgrowth development from transplanted stem cells.

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.

H2AX Promoter Demethylation at Specific Sites Plays a Role in STAT5-Induced Tumorigenesis.

Deregulated STAT5 activity in the mammary gland of transgenic mice results in parity-dependent latent tumorigenesis. The trigger for cell transformation was previously associated with hyperactivation of the H2AX proximal promoter in a small basal cell population during pregnancy. The current study focuses on the latent activation of tumor development. H2AX was highly expressed in carcinoma and adenocarcinoma as compared to the multiparous mammary gland, whereas pSTAT5 expression decreased in a tumor type-dependent manner. In contrast to the pregnant gland, no positive correlation between H2AX and pSTAT5 expression could be defined in carcinoma and adenocarcinoma. Using targeted methylation analysis, the methylation profile of the H2AX promoter was characterized in the intact gland and tumors. Average H2AX promoter methylation in the tumors was relatively high (~90%), but did not exceed that of the multiparous gland; 5mC methylation was higher in the differentiated tumors and negatively correlated with its oxidative product 5hmC and H2AX expression. Individual analysis of 25 H2AX promoter-methylation sites revealed two consecutive CpGs at positions -77 and - 54 that were actively demethylated in the multiparous gland, but not in their age-matched virgin counterpart. The different methylation profiles at these sites distinguished tumor types and may assume a prognostic role. In-silico and ChIP analyses revealed overlapping methylation-independent SP1-binding and methylation-dependent p53-binding to these sites. We propose that interference with SP1-assisted p53-binding to these sites abrogates H2AX's ability to arrest the cell cycle upon DNA damage, and contributes to triggering latent development of STAT5-induced tumors in estrapausal multiparous mice.

Calorie restriction and rapamycin administration induce stem cell self-renewal and consequent development and production in the mammary gland.

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.

Enrichment for Repopulating Cells and Identification of Differentiation Markers in the Bovine Mammary Gland.

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 cells, were indicative of unipotent basal stem cells or committed progenitors. Sorting luminal cells according to E-cadherin revealed three distinct populations: luminal progenitors, and early- and late-differentiating cells. Finally, miR-200c expression was negatively correlated with differentiation levels in both the luminal and basal branches of the bovine mammary cell hierarchy. Together, these experiments provide further evidence for the presence of a regenerative entity in the bovine mammary gland and for the multistage differentiation process within the luminal lineage.

Xanthosine administration does not affect the proportion of epithelial stem cells in bovine mammary tissue, but has a latent negative effect on cell proliferation.

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 observed 11 weeks after xanthosine administration. Tumor development in mice was also limited by xanthosine administration. These effects may have resulted from an initial decrease in expression of the rate-limiting enzyme in guanine synthesis, IMPDH. The data indicate that caution should be exerted when considering xanthosine for stem cell manipulation.

Stat5 in breast cancer: potential oncogenic activity coincides with positive prognosis for the disease.

Nuclear localization of signal transducer and activator of transcription (Stat) 5 marks good prognosis in estrogen receptor/progesterone receptor-positive breast tumors. This positive characteristic is counteracted by studies in laboratory animals demonstrating that deregulated Stat5 activity may convert proper mammary development into a latent oncogenic process. Tumorigenesis is initiated during the parity cycles, most probably during pregnancy, when the activated Stat5 antagonizes or manipulates parity's protective mechanisms. For example, it can alter the differentiation/proliferation balance, induce growth hormone signaling, cause specific alteration in chromatin structure, inhibit tumor-suppressor activity and induce DNA damage that counteracts the enhanced DNA-damage response exerted by parity. Palpable tumors develop after a latent period from individual cells. This happens in the estropausal period in transgenic mice maintaining deregulated Stat5 activity in the mammary gland, or during involution, months after transplantation of transfected cells with constitutively active Stat5. Candidate vulnerable cells are those which maintain high nuclear Stat5 activity. Due to the hazardous outcome of deregulated Stat5 activity in these cells, such as induced DNA damage or high cyclin D1 activity, the gland is prone to transformation. The developing tumors are mostly adenocarcinomas or their subtypes. They are estrogen receptor-positive and maintain a specific Stat5 gene signature that allows tracking their inducer. From a clinical point of view, deregulated Stat5 activity represents a genuine risk factor for breast cancer. Monitoring Stat5 activity during vulnerable periods and developing specific tools for its suppression in breast epithelial cells could potentially limit new incidence of the disease.

Macrophage-Secreted CSF1 Transmits a Calorie Restriction-Induced Self-Renewal Signal to Mammary Epithelial Stem Cells.

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 to the sensing mechanism. Enhancing CSF1 signaling with recombinant protein and interrupting the interaction with its highly expressed receptor in the epithelial stem cells by neutralizing antibodies were both affected stem cell self-renewal. In conclusion, combined in vivo, in vitro and in silico studies identified macrophages and secreted CSF1 as the energy sensor and paracrine transmitter, respectively, of the calorie restriction-induced effect on mammary stem cell self-renewal.

Intramammary rapamycin administration to calves induces epithelial stem cell self-renewal and latent cell proliferation and milk protein expression.

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 ß-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 and progesterone administration, rapamycin significantly induced proliferation rate. Higher numbers of basal and luminal PCNA+ cells were detected in small ducts near the elongating sites as compared to large ducts, in which only luminal cells were affected. Rapamycin administration resulted in induction of individual milk protein genes' expression, which was negatively correlated to their endogenous levels. The inductive effect of rapamycin on luminal cell number was confirmed in organoid cultures, but milk protein expression decreased, probably due to lack of oscillation in rapamycin levels. In conclusion, intramammary rapamycin administration is an effective methodology to reduce mTOR activity in bovine mammary epithelial cells and consequently, induce stem cell self-renewal. The latent positive effect of rapamycin on epithelial cell proliferation and its potential to improve milk protein expression in calves may have beneficial implications for mature cows.

Forced activation of Stat5 subjects mammary epithelial cells to DNA damage and preferential induction of the cellular response mechanism during proliferation.

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 ß-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.

Conditional repression of STAT5 expression during lactation reveals its exclusive roles in mammary gland morphology, milk-protein gene expression, and neonate growth.

The role of Stat5 in maintaining adequate lactation was studied in Stat5a(-/-) mice expressing a conditionally suppressed transgenic STAT5 in their mammary glands. This system enables distinguishing STAT5's effects on lactation from its contribution to mammary development during gestation. Females were allowed to express STAT5 during their first pregnancy. After delivery, STAT5 levels were manipulated by doxycycline administration and withdrawal. In two lines of genetically modified mice, the absence of STAT5 expression during the first 10 days of lactation resulted in a decrease of 29% or 41% in newborn weight gain. The STAT5-dependent decrease in growth was recoverable, but not completely reversible, particularly when STAT5 expression was omitted for the first 4 days of lactation. Within the first 10 days of STAT5-omitted lactation, alveolar occupancy regressed by 50% compared to that measured at delivery. By Day 10, only 18% of the fat-pad area was involved in milk production. The alveolar regression caused by 4 days of STAT5 deficiency was reversible, but neonate growth remained delayed. STAT5 deficiency resulted in reduced estrogen receptor α and connexin 32 gene expression, accompanied by delayed induction of both anti- and pro-apoptotic Bcl-2 family members. An increase in Gata-3 expression may reflect an attempt to maintain alveolar progenitors. A decrease of 39% and 23% in WAP and α-lactalbumin expression, respectively, with no associated effects on ß-casein, also resulted from lack of STAT5 expression in the first 10 days of lactation. This deficiency enhances the major effect of alveolar regression on delayed weight gain in newborns.

Distinct gene-expression profiles characterize mammary tumors developed in transgenic mice expressing constitutively active and C-terminally truncated variants of STAT5.

BACKGROUND: Stat5 is a latent transcription factor that regulates essential growth and survival functions in normal cells. Constitutive activity of Stat5 and the involvement of its C-terminally truncated variant have been implicated in blood cell malignancies and mammary or breast cancer. To distinguish the individual contributions of the Stat5 variants to mammary tumorigenesis, global gene-expression profiling was performed on transgenic STAT5-induced tumors. RESULTS: We identified 364 genes exhibiting differential expression in mammary tumors developed in transgenic mice expressing constitutively active STAT5 (STAT5ca) vs. its C-terminally truncated variant (STAT5Delta750). These genes mediate established Stat5 effects on cellular processes such as proliferation and cell death, as well as yet-unrelated homeostatic features, e.g. carbohydrate metabolism. A set of 14 genes linked STAT5Delta750 expression to the poorly differentiated carcinoma phenotype and STAT5ca to the highly differentiated papillary adenocarcinoma.Specifically affected genes exhibited differential expression in an individual tumor set vs. its counterpart and the intact mammary gland: 50 genes were specifically affected by STAT5ca, and 94% of these were downregulated, the latter involved in suppression of tumor suppressors and proliferation antagonistics. This substantial downregulation distinguishes the STAT5ca-induced tumorigenic consequences from the relatively equal effect of the STAT5Delta750 on gene expression, which included significant elevation in the expression of oncogenes and growth mediators.STAT5Delta750 mRNA expression was below detection levels in the tumors and the amount of STAT5ca transcript was not correlated with the expression of its specifically affected genes. Interestingly, we identified several groups of three to eight genes affected by a particular STAT5 variant with significant correlated expression at distinct locations in the clustergram. CONCLUSION: The different gene-expression profiles in mammary tumors caused by the STAT5Delta750 and STAT5ca variants, corroborated by the absence of a direct link to transgenic STAT5 expression, imply distinct metabolic consequences for their oncogenic role which probably initiate early in tumor development. Tumorigenesis may involve induction of growth factor and oncogenes by STAT5Delta750 or suppression of tumor suppressors and growth antagonists by STAT5ca. The list of genes specifically affected by the STAT5 variants may provide a basis for the development of a marker set for their distinct oncogenic role.

Negative effects of the amino acids Lys, His, and Thr on S6K1 phosphorylation in mammary epithelial cells.

The role of essential amino acids (AA) on protein synthesis via the mTOR pathway was studied in murine mammary epithelial cells cultured under lactogenic conditions. Leu, Ile, and Val increased S6K1 phosphorylation compared to that measured in AA-deprived cells. Trp, Phe, and Met had no effect. Surprisingly, Lys, His, and Thr inhibited S6K1 phosphorylation in both murine and bovine mammary cells. Thr exhibited the most potent inhibition, being the only amino acid that competed with Leu's positive role. In non-deprived cells, there was no observable effect of Lys, His, or Thr on S6K1 phosphorylation at concentrations up to five times those in the medium. However, their addition as a mix revealed a synergistic negative effect. Supplementation of Lys, His, and Thr abrogated mTOR Ser 2448 phosphorylation, with no effect on Akt Ser 473-an mTORC2 target. This confirms specific mTORC1 regulation of S6K1 phosphorylation. The individual supplementation of Lys, His, and Thr maintained a low level of IRS-1 phosphorylation, which was dose-dependently increased by their combined addition. Thus, in parallel to inhibiting S6K1 activity, these AA may act synergistically to activate an additional kinase, phosphorylating IRS-1 via an S6K1-independent pathway. In cultures supplemented by Lys, His, and Thr, cellular protein synthesis decreased by up to 65%. A more pronounced effect was observed on beta-casein synthesis. These findings indicate that positive and negative signaling from AA to the mTOR pathway, combined with modulation of insulin sensitization, mediate the synthesis rates of total and specific milk proteins in mammary epithelial cells.

Different gene-expression profiles for the poorly differentiated carcinoma and the highly differentiated papillary adenocarcinoma in mammary glands support distinct metabolic pathways.

BACKGROUND: Deregulation of Stat5 in the mammary gland of transgenic mice causes tumorigenesis. Poorly differentiated carcinoma and highly differentiated papillary adenocarcinoma tumors evolve. To distinguish the genes and elucidate the cellular processes and metabolic pathways utilized to preserve these phenotypes, gene-expression profiles were analyzed. METHODS: Mammary tumors were excised from transgenic mice carrying a constitutively active variant of Stat5, or a Stat5 variant lacking s transactivation domain. These tumors displayed either the carcinoma or the papillary adenocarcinoma phenotypes. cRNAs, prepared from each tumor were hybridized to an Affymetrix GeneChip(R) Mouse Genome 430A 2.0 array. Gene-ontology analysis, hierarchical clustering and biological-pathway analysis were performed to distinct the two types of tumors. Histopathology and immunofluorescence staining complemented the comparison between the tumor phenotypes. RESULTS: The nucleus-cytoskeleton-plasma membrane axis is a major target for differential gene expression between phenotypes. In the carcinoma, stronger expression of genes coding for specific integrins, cytoskeletal proteins and calcium-binding proteins highlight cell-adhesion and motility features of the tumor cells. This is supported by the higher expression of genes involved in O-glycan synthesis, TGF-beta, activin, their receptors and Smad3, as well as the Notch ligands and members of the gamma-secretase complex that enable Notch nuclear localization. The Wnt pathway was also a target for differential gene expression. Higher expression of genes encoding the degradation complex of the canonical pathway and limited TCF expression in the papillary adenocarcinoma result in membranal accumulation of beta-catenin, in contrast to its nuclear translocation in the carcinoma. Genes involved in cell-cycle arrest at G1 and response to DNA damage were more highly expressed in the papillary adenocarcinomas, as opposed to favored G2/M regulation in the carcinoma tumors. CONCLUSION: At least six metabolic pathways support the morphological and functional differences between carcinomas and papillary adenocarcinomas. Differential gene-expression profiles favor cell adhesion, motility and proliferation in the carcinoma. Cell-cell contact, polarity, earlier cell-cycle arrest and DNA damage control are better displayed in the papillary adenocarcinoma.

High Expression of CD200 and CD200R1 Distinguishes Stem and Progenitor Cell Populations within Mammary Repopulating Units.

Aiming to unravel the top of the mammary epithelial cell hierarchy, a subset of the CD49fhighCD24med mammary repopulating units (MRUs) was identified by flow cytometry, expressing high levels of CD200 and its receptor CD200R1. These MRUCD200/CD200R1 repopulated a larger area of de-epithelized mammary fat pads than the rest of the MRUs, termed MRUnot CD200/CD200R1. MRUCD200/CD200R1 maintained a much lower number of divergently defined, highly expressed genes and pathways that support better cell growth, development, differentiation, and progenitor activity than their MRUnot CD200/CD200R1 counterparts. A defined profile of hierarchically associated genes supporting a single-lineage hypothesis was confirmed by in vitro mammosphere analysis that assembled 114 genes with decreased expression from MRUCD200/CD200R1 via MRUnot CD200/CD200R1 toward CD200+CD200R1- and CD200R1+CD200- cells. About 40% of these genes were shared by a previously published database of upregulated genes in mammary/breast stem cells and may represent the core genes involved in mammary stemness.

Luminal STAT5 mediates H2AX promoter activity in distinct population of basal mammary epithelial cells.

Deregulated STAT5 activity in the mammary gland causes parity-dependent tumorigenesis. Epithelial cell cultures transfected with constitutively active STAT5 express higher levels of the histone H2AX than their non-transfected counterparts. Higher H2AX expression may be involved in tumorigenesis. Here, we aimed to link high STAT5 activity to H2AX-GFP expression by looking for distinct types of mammary cells that express these proteins. In vitro and in transgenic mice, only 0.2 and 0.02%, respectively, of the cells expressed the H2AX-GFP hybrid gene. Its expression correlated with that of the endogenous H2AX gene, suggesting that detectable H2AX-GFP expression marks high levels of H2AX transcript. Methylation of the H2AX promoter characterized non-GFP-expressing H2AX-GFP cells and was inversely correlated with promoter activity. Administration of 5-azacytidine increased H2AX promoter activity in an activated STAT5-dependent manner. In transgenic mice, H2AX-GFP expression peaked at pregnancy. The number of H2AX-GFP-expressing cells and GFP expression decreased in a Stat5a-null background and increased in mice expressing the hyperactivated STAT5. Importantly, H2AX-GFP activity was allocated to basal mammary cells lacking stem-cell properties, whereas STAT5 hyperactivity was detected in the adjacent luminal cells. Knockdown of RANKL by siRNA suggested its involvement in signaling between the two layers. These results suggest paracrine activation of H2AX via promoter demethylation in specific populations of basal mammary cells that is induced by a signal from neighboring luminal cells with hyper STAT5 activity. This pathway provides an alternative route for the luminally confined STAT5 to affect basal mammary cell activity.

Low-energy laser irradiation enhances de novo protein synthesis via its effects on translation-regulatory proteins in skeletal muscle myoblasts.

Low-energy laser irradiation (LELI) drives quiescent skeletal muscle satellite cells into the cell cycle and enhances their proliferation, thereby promoting skeletal muscle regeneration. Ongoing protein synthesis is a prerequisite for these processes. Here, we studied the signaling pathways involved in the LELI regulation of protein synthesis. High levels of labeled [35S]methionine incorporation were detected in LELI cells as early as 20 min after irradiation, suggesting translation of pre-existing mRNAs. Induced levels of protein synthesis were detected up until 8 h after LELI implying a role for LELI in de novo protein synthesis. Elevated levels of cyclin D1, associated with augmented phosphorylation of the eukaryotic initiation factor 4E (eIF4E) and its inhibitory binding protein PHAS-I, suggested the involvement of LELI in the initiation steps of protein translation. In the presence of the MEK inhibitor, PD98059, eIF4E phosphorylation was abolished and levels of cyclin D1 were dramatically reduced. The LELI-induced PHAS-I phosphorylation was abolished after preincubation with the PI3K inhibitor, Wortmannin. Concomitantly, LELI enhanced Akt phosphorylation, which was attenuated in the presence of Wortmannin. Taken together, these results suggest that LELI induces protein translation via the PI3K/Akt and Ras/Raf/ERK pathways.

BLG-e1 - a novel regulatory element in the distal region of the beta-lactoglobulin gene promoter.

beta-Lactoglobulin (BLG) is a major ruminant milk protein. A regulatory element, termed BLG-e1, was defined in the distal region of the ovine BLG gene promoter. This 299-bp element lacks the established cis-regulatory sequences that affect milk-protein gene expression. Nevertheless, it alters the binding of downstream BLG sequences to histone H4 and the sensitivity of the histone-DNA complexes to trichostatin A treatment. In mammary cells cultured under favorable lactogenic conditions, BLG-e1 acts as a potent, position-independent silencer of BLG/luciferase expression, and similarly affects the promoter activity of the mouse whey acidic protein gene. Intragenic sequences upstream of BLG exon 2 reverse the silencing effect of BLG-e1 in vitro and in transgenic mice.

Overexpression and forced activation of stat5 in mammary gland of transgenic mice promotes cellular proliferation, enhances differentiation, and delays postlactational apoptosis.

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 transgenic mouse lines expressed the wild-type Stat5 construct (BLG/STAT5) and the constitutively active Stat5 variant (BLG/STAT5ca) exclusively in mammary epithelial cells during pregnancy and lactation. BLG/STAT5 mice exhibited larger alveoli at mid-pregnancy and a delayed onset of involution. Condensed alveoli, a high degree of cellular proliferation, and delayed involution were associated with STAT5ca expression. Elevated levels of beta-casein gene expression were found in BLG/STAT5 and STAT5ca transgenic mice during late pregnancy and lactation, indicating a limiting role for Stat5 under normal physiological conditions. This was accompanied by higher levels of beta-casein secretion into the milk and enhanced growth of pups. Transgenic animals expressing the BLG/STAT5ca transgene were predisposed to tumor formation in the mammary gland. This study extends the functional observations made in cultured mammary epithelial cells and in gene knockout mice. It identifies Stat5 as a multifunctional regulator of mammary cell proliferation, milk protein gene expression, and postlactational apoptosis.

Insulin and prolactin synergistically stimulate beta-casein messenger ribonucleic acid translation by cytoplasmic polyadenylation.

Previous studies have shown that the synthesis and stability of milk protein mRNAs are regulated by lactogenic hormones. We demonstrate here in cultured mouse mammary epithelial cells (CID 9) that insulin plus prolactin also synergistically increases the rate of milk protein mRNA translation. Insulin alone stimulates synthesis of both milk and nonmilk proteins, whereas prolactin alone has no effect, but insulin plus prolactin selectively stimulate synthesis of milk proteins more than insulin alone. The increase in beta-casein mRNA translation is also reflected in a shift to larger polysomes, indicating an effect on translational initiation. Inhibitors of the phosphatidylinositol 3-kinase, mammalian target of rapamycin, and MAPK pathways block insulin-stimulated total protein and beta-casein synthesis but not the synergistic stimulation. Conversely, cordycepin abolishes synergistic stimulation of protein synthesis without affecting insulin-stimulated translation. The poly(A) tract of beta-casein mRNA progressively increases from approximately 20 to about 200 A residues over 30 min of treatment with insulin plus prolactin. The 3'-untranslated region of beta-casein mRNA containing an unaltered cytoplasmic polyadenylation element is sufficient for the translational enhancement and mRNA-specific polyadenylation, based on transient transfection of cells with a reporter construct. Insulin and prolactin stimulate cytoplasmic polyadenylation element binding protein phosphorylation with no increase of cytoplasmic poly(A) polymerase activity.

Characterization of an epithelial cell line from bovine mammary gland.

Elucidation of the bovine mammary gland's unique characteristics depends on obtaining an authentic cell line that will reproduce its function in vitro. Representative clones from bovine mammary cell populations, differing in their attachment capabilities, were cultured. L-1 cells showed strong attachment to the plate, whereas H-7 cells detached easily. Cultures established from these clones were nontumorigenic upon transplantation to an immunodeficient host; they exhibited the epithelial cell characteristics of positive cytokeratin but not smooth muscle actin staining. Both cell lines depended on fetal calf serum for proliferation. They exhibited distinct levels of differentiation on Matrigel in serum-free, insulin-supplemented medium on the basis of their organization and beta-lactoglobulin (BLG) secretion. H-7 cells organized into mammospheres, whereas L-1 cells arrested in a duct-like morphology. In both cell lines, prolactin activated phosphorylation of the signal transducer and activator of transcription, Stat5-a regulator of milk protein gene transcription, and of PHAS-I-an inhibitor of translation initiation in its nonphosphorylated form. De novo synthesis and secretion of BLG were detected in differentiated cultures: in L-1 cells, BLG was dependent on lactogenic hormones for maximal induction but was less stringently controlled than was beta-casein in the mouse CID-9 cell line. L-1 cells also encompassed a near-diploid chromosomal karyotype and may serve as a tool for studying functional characteristics of the bovine mammary gland.