Miz1 deficiency in the mammary gland causes a lactation defect by attenuated Stat5 expression and phosphorylation.
ABSTRACT: Miz1 is a zinc finger transcription factor with an N-terminal POZ domain. Complexes with Myc, Bcl-6 or Gfi-1 repress expression of genes like Cdkn2b (p15(Ink4)) or Cdkn1a (p21(Cip1)). The role of Miz1 in normal mammary gland development has not been addressed so far. Conditional knockout of the Miz1 POZ domain in luminal cells during pregnancy caused a lactation defect with a transient reduction of glandular tissue, reduced proliferation and attenuated differentiation. This was recapitulated in vitro using mouse mammary gland derived HC11 cells. Further analysis revealed decreased Stat5 activity in Miz1?POZ mammary glands and an attenuated expression of Stat5 targets. Gene expression of the Prolactin receptor (PrlR) and ErbB4, both critical for Stat5 phosphorylation (pStat5) or pStat5 nuclear translocation, was decreased in Miz1?POZ females. Microarray, ChIP-Seq and gene set enrichment analysis revealed a down-regulation of Miz1 target genes being involved in vesicular transport processes. Our data suggest that deranged intracellular transport and localization of PrlR and ErbB4 disrupt the Stat5 signalling pathway in mutant glands and cause the observed lactation phenotype.
Project description:Xbp1, a key mediator of the unfolded protein response (UPR), is activated by IRE1?-mediated splicing, which results in a frameshift to encode a protein with transcriptional activity. However, the direct function of Xbp1 in epithelial cells during mammary gland development is unknown. Here we report that the loss of Xbp1 in the mammary epithelium through targeted deletion leads to poor branching morphogenesis, impaired terminal end bud formation, and spontaneous stromal fibrosis during the adult virgin period. Additionally, epithelial Xbp1 deletion induces endoplasmic reticulum (ER) stress in the epithelium and dramatically inhibits epithelial proliferation and differentiation during lactation. The synthesis of milk and its major components, ?/?-casein and whey acidic protein (WAP), is significantly reduced due to decreased prolactin receptor (Prlr) and ErbB4 expression in Xbp1-deficient mammary epithelium. Reduction of Prlr and ErbB4 expression and their diminished availability at the cell surface lead to reduced phosphorylated Stat5, an essential regulator of cell proliferation and differentiation during lactation. As a result, lactating mammary glands in these mice produce less milk protein, leading to poor pup growth and postnatal death. These findings suggest that the loss of Xbp1 induces a terminal UPR which blocks proliferation and differentiation during mammary gland development.
Project description:The development of the lactating mammary gland is a complex multifactorial process occurring in mammals during pregnancy. We show here that this process requires NHERF1/EBP50 (Na/H exchanger regulatory factor 1/ERM-binding phosphoprotein 50) expression and that successful lactation depends on NHERF1 allele copy number, with rates of 50 and 20% in NHERF1(+/-) and (-/-) mice, respectively. The prolactin receptor (PRLR)-STAT5 signaling provides the central axis triggering the differentiation of secretory mammary alveolar cells. In successfully lactating glands, NHERF1 is massively upregulated and forms complexes with PRLR, but also with ?-catenin, E-cadherin and ezrin at the alveolar basal membrane, establishing basal polarity. In NHERF1-deficient glands, the basal polarity is disrupted, the PRLR levels and basal membrane localization are abolished, and the downstream STAT5 activation collapses with consequent reduction of milk protein synthesis. NHERF1/EBP50, a protein deregulated in breast cancer, thus emerges as an important physiological mediator of milk secretion, by engagement of PRLR in multimeric complexes at the alveolar basal membrane with subsequent network activation leading to cell differentiation.
Project description:Obesity reduces breastfeeding success and lactation performance in women. However, the mechanisms involved are not entirely understood. In the present study, female C57BL/6 mice were chronically exposed to a high-fat diet to induce obesity and subsequently exhibited impaired offspring viability (only 15% survival rate), milk production (33% reduction), mammopoiesis (one-third of the glandular area compared to control animals) and postpartum maternal behaviors (higher latency to retrieving and grouping the pups). Reproductive experience attenuated these defects. Diet-induced obese mice exhibited high basal pSTAT5 levels in the mammary tissue and hypothalamus, and an acute prolactin stimulus was unable to further increase pSTAT5 levels above basal levels. In contrast, genetically obese leptin-deficient females showed normal prolactin responsiveness. Additionally, we identified the expression of leptin receptors specifically in basal/myoepithelial cells of the mouse mammary gland. Finally, high-fat diet females exhibited altered mRNA levels of ERBB4 and NRG1, suggesting that obesity may involve disturbances to mammary gland paracrine circuits that are critical in the control of luminal progenitor function and lactation. In summary, our findings indicate that high leptin levels are a possible cause of the peripheral and central prolactin resistance observed in obese mice which leads to impaired lactation performance.
Project description:Pten is a tumor suppressor gene regulating many cellular processes, including growth, adhesion, and apoptosis. In the aim of investigating the role of Pten during mammary gland development and lactation of dairy cows, we analyzed Pten expression levels in the mammary glands of dairy cows by using western blotting, immunohistochemistry, and quantitative polymerase chain reaction (qPCR) assays. Dairy cow mammary epithelial cells (DCMECs) were used to study the function of Pten in vitro. We determined concentrations of ?-casein, triglyceride, and lactose in the culture medium following Pten overexpression and siRNA inhibition. To determine whether Pten affected DCMEC viability and proliferation, cells were analyzed by CASY-TT and flow cytometry. Genes involved in lactation-related signaling pathways were detected. Pten expression was also assessed by adding prolactin and glucose to cell cultures. When Pten was overexpressed, proliferation of DCMECs and concentrations for ?-casein, triglyceride, and lactose were significantly decreased. Overexpression of Pten down-regulated expression of MAPK, CYCLIN D1, AKT, MTOR, S6K1, STAT5, SREBP1, PPAR?, PRLR, and GLUT1, but up-regulated 4EBP1 in DCMECs. The Pten siRNA inhibition experiments revealed results that opposed those from the gene overexpression experiments. Introduction of prolactin (PRL) increased secretion of ?-casein, triglyceride, and lactose, but decreased Pten expression levels. Introduction of glucose also increased ?-casein and triglyceride concentrations, but did not significantly alter Pten expression levels. The Pten mRNA and protein expression levels were decreased 0.3- and 0.4-fold in mammary glands of lactating cows producing high quality milk (milk protein >3.0%, milk fat >3.5%), compared with those cows producing low quality milk (milk protein <3.0%, milk fat <3.5%). In conclusion, Pten functions as an inhibitor during mammary gland development and lactation in dairy cows. It can down-regulate DCMECs secretion of ?-casein, triglyceride, and lactose, and plays a critical role in lactation related signaling pathways.
Project description:In the mammary gland, genetic circuits controlled by estrogen, progesterone, and prolactin, act in concert with pathways regulated by members of the epidermal growth factor family to orchestrate growth and morphogenesis during puberty, pregnancy and lactation. However, the precise mechanisms underlying the crosstalk between the hormonal and growth factor pathways remain poorly understood. We have identified the CUB and zona pellucida-like domain-containing protein 1 (CUZD1), expressed in mammary ductal and alveolar epithelium, as a novel mediator of mammary gland proliferation and differentiation during pregnancy and lactation. Cuzd1-null mice exhibited a striking impairment in mammary ductal branching and alveolar development during pregnancy, resulting in a subsequent defect in lactation. Gene expression profiling of mammary epithelium revealed that CUZD1 regulates the expression of a subset of the EGF family growth factors, epiregulin, neuregulin-1, and epigen, which act in an autocrine fashion to activate ErbB1 and ErbB4 receptors. Proteomic studies further revealed that CUZD1 interacts with a complex containing JAK1/JAK2 and STAT5, downstream transducers of prolactin signaling in the mammary gland. In the absence of CUZD1, STAT5 phosphorylation in the mammary epithelium during alveologenesis was abolished. Conversely, elevated expression of Cuzd1 in mammary epithelial cells stimulated prolactin-induced phosphorylation and nuclear translocation of STAT5. Chromatin immunoprecipitation confirmed co-occupancy of phosphorylated STAT5 and CUZD1 in the regulatory regions of epiregulin, a potential regulator of epithelial proliferation, and whey acidic protein, a marker of epithelial differentiation. Collectively, these findings suggest that CUZD1 plays a critical role in prolactin-induced JAK/STAT5 signaling that controls the expression of key STAT5 target genes involved in mammary epithelial proliferation and differentiation during alveolar development.
Project description:During pregnancy, as the mammary gland prepares for synthesis and delivery of milk to newborns, a luminal mammary epithelial cell (MEC) subpopulation proliferates rapidly in response to systemic hormonal cues that activate STAT5A. While the receptor tyrosine kinase ErbB4 is required for STAT5A activation in MECs during pregnancy, it is unclear how ErbB3, a heterodimeric partner of ErbB4 and activator of phosphatidyl inositol-3 kinase (PI3K) signaling, contributes to lactogenic expansion of the mammary gland.We assessed mRNA expression levels by expression microarray of mouse mammary glands harvested throughout pregnancy and lactation. To study the role of ErbB3 in mammary gland lactogenesis, we used transgenic mice expressing WAP-driven Cre recombinase to generate a mouse model in which conditional ErbB3 ablation occurred specifically in alveolar mammary epithelial cells (aMECs).Profiling of RNA from mouse MECs isolated throughout pregnancy revealed robust Erbb3 induction during mid-to-late pregnancy, a time point when aMECs proliferate rapidly and undergo differentiation to support milk production. Litters nursed by ErbB3 KO dams weighed significantly less when compared to litters nursed by ErbB3 WT dams. Further analysis revealed substantially reduced epithelial content, decreased aMEC proliferation, and increased aMEC cell death during late pregnancy. Consistent with the potent ability of ErbB3 to activate cell survival through the PI3K/Akt pathway, we found impaired Akt phosphorylation in ErbB3 KO samples, as well as impaired expression of STAT5A, a master regulator of lactogenesis. Constitutively active Akt rescued cell survival in ErbB3-depleted aMECs, but failed to restore STAT5A expression or activity. Interestingly, defects in growth and survival of ErbB3 KO aMECs as well as Akt phosphorylation, STAT5A activity, and expression of milk-encoding genes observed in ErbB3 KO MECs progressively improved between late pregnancy and lactation day 5. We found a compensatory upregulation of ErbB4 activity in ErbB3 KO mammary glands. Enforced ErbB4 expression alleviated the consequences of ErbB3 ablation in aMECs, while combined ablation of both ErbB3 and ErbB4 exaggerated the phenotype.These studies demonstrate that ErbB3, like ErbB4, enhances lactogenic expansion and differentiation of the mammary gland during pregnancy, through activation of Akt and STAT5A, two targets crucial for lactation.
Project description:Peptidylarginine deiminases (PADs) post-translationally convert arginine into neutral citrulline residues. Our past work shows that PADs are expressed in the canine and murine mammary glands; however, the mechanisms regulating PAD expression and the function of citrullination in the normal mammary gland are unclear. Therefore, the first objective herein was to investigate regulation of PAD expression in mammary epithelial cells. We first examined PAD levels in CID-9 cells, which were derived from the mammary gland of mid-pregnant mice. PAD3 expression is significantly higher than all other PAD isoforms and mediates protein citrullination in CID-9 cells. We next hypothesized that prolactin regulates PAD3 expression. To test this, CID-9 cells were stimulated with 5 ?g/mL of prolactin for 48 hours which significantly increases PAD3 mRNA and protein expression. Use of a JAK2 inhibitor and a dominant negative (DN)-STAT5 adenovirus indicate that prolactin stimulation of PAD3 expression is mediated by the JAK2/STAT5 signaling pathway in CID-9 cells. In addition, the human PAD3 gene promoter is prolactin responsive in CID-9 cells. Our second objective was to investigate the expression and activity of PAD3 in the lactating mouse mammary gland. PAD3 expression in the mammary gland is highest on lactation day 9 and coincident with citrullinated proteins such as histones. Use of the PAD3 specific inhibitor, Cl4-amidine, indicates that PAD3, in part, can citrullinate proteins in L9 mammary glands. Collectively, our results show that upregulation of PAD3 is mediated by prolactin induction of the JAK2/STAT5 signaling pathway, and that PAD3 appears to citrullinate proteins during lactation.
Project description:BACKGROUND: Insulin like growth factors (IGFs) and their binding proteins (IGFBPs) are secreted peptides that play major roles in regulating the normal development and maturation of mammary gland. While Igfbp7 has been shown to decrease breast tumor growth, its role in regulating the normal mammary gland development has not been studied. To this end, we generated Igfbp7-null mice and examined the development and maturation of mammary glands in the virgin, pregnant and lactating animals. RESULTS: We report here that loss of Igfbp7 significantly retards mammary gland development in the virgin animals. More significantly, the pregnant Igfpb7-null glands contained fewer alveolar structures and that during lactation these glands exhibit the morphological changes that are associated with involution. The transcriptome profile of the Igfbp7-null glands on the lactation day 3 revealed a distinct involution-related gene signature compared to the lactating WT glands. Interestingly, we found that the lactating Igfbp7-null glands exhibit increased expression of Stat3 and enhanced activation of (phosphorylated) Stat3, combined with decreased expression of Stat5 suggesting that the absence of Igfbp7 accelerates the onset of involution. We also found that in absence of Igfpb7, the lactating glands contain increased Igfbp5 protein along with decreased expression of IGF-1 Receptor and Akt activation. Finally, we show that during the normal course of involution, Igfbp7 expression is significantly decreased in the mammary gland. CONCLUSION: Our data suggest that loss of Igfbp7 induces precocious involution possibly through diminished cell survival signals. Our findings identify Igfbp7 as major regulator of involution in the mammary gland.
Project description:Although growth factors have been shown to influence mammary gland development, the nature of downstream effectors remains elusive. In this study, we show that the expression of p21-activated kinase (Pak)1, a serine/threonine protein kinase, is activated in mammary glands during pregnancy and lactation. By targeting an ectopic expression of a kinase-dead Pak1 mutant under the control of ovine beta-lactoglobulin promoter, we found that the mammary glands of female mice expressing kinase-dead Pak1 transgene revealed incomplete lobuloalveolar development and impaired functional differentiation. The expression of whey acidic protein and beta-casein and the amount of activated Stat5 in the nuclei of epithelial cells in transgenic mice were drastically reduced. Further analysis of the underlying mechanisms revealed that Pak1 stimulated beta-casein promoter activity in normal mouse mammary epithelial cells and also cooperated with Stat5a. Pak1 directly interacted with and phosphorylated Stat5a at Ser 779, and both COOH-terminal deletion containing Ser 779 of Stat5a and the Ser 779 to Ala mutation completely prevented the ability of Pak1 to stimulate beta-casein promoter. Mammary glands expressing inactive Pak1 exhibited a reduction of Stat5a Ser 779 phosphorylation. These findings suggest that Pak1 is required for alveolar morphogenesis and lactation function, and thus, identify novel functions of Pak1 in the mammary gland development.
Project description:The prolactin receptor (PRLR), its associated Janus kinase 2 (Jak2) and the signal transducer and activator of transcription 5 (Stat5) are essential for normal mammary gland development. Owing to the upregulation of the PRLR and the local synthesis of its ligand in neoplastic cells, it has been proposed that PRL can act as a local growth factor in human breast cancers. This notion is supported by experimental evidence in transgenic mice, which showed that the mammary-specific expression of PRL contributes to carcinogenesis in vivo. To assess the importance of Jak2/Stat5 signaling during mammary cancer initiation and progression, we generated a PRL-induced mammary cancer model that allows the functional ablation of the Jak2 gene in the mammary epithelium before and after neoplastic transformation. Collectively, the results of this study show that the functional ablation of Jak2 protects against the onset of PRL-induced mammary tumorigenesis, suggesting that targeting this kinase is a relevant strategy for mammary cancer prevention. Surprisingly, Jak2 deficiency did not affect the growth and survival of PRL-induced mammary cancer cells in culture and in vivo. Consequently, Jak2 cannot be a sole therapeutic target to treat the established disease. PRL-induced mammary cancers exhibited an upregulation of ErbB2 and other ErbB receptor tyrosine kinases that may supersede the functionality of PRLR signaling through Jak2.