Proteins In Mammary Epithelial Cells Research Articles

Mesenchymal Stem Cells Overexpressing ACE2 Favorably Ameliorate LPS-Induced Inflammatory Injury in Mammary Epithelial Cells.

Mesenchymal stem cells (MSCs) are capable of homing injury sites to exert anti-inflammatory as well as anti-damage effects and can be used as a vehicle for gene therapy. Angiotensin-converting enzyme 2 (ACE2) plays an important role in numerous inflammatory diseases, but fewer studies have been reported in animal mastitis. We hypothesized that MSCs overexpressing ACE2 is more effective in ameliorating lipopolysaccharide (LPS)-induced inflammatory injury in mammary epithelial cells compared to MSCs alone. The results showed that MSC-ACE2 inhibited the LPS induction by upregulation of TNF-α, IL-Iβ, IL-6, and iNOS mRNA expression levels in EpH4-Ev cells compared with MSCs. Furthermore, results showed that both MSC and MSC-ACE2 were significantly activated IL-10/STAT3/SOCS3 signaling pathway as well as inhibited TLR4/NF-κB and MAPK signaling pathways, but MSC-ACE2 had more significant effects. Meanwhile, MSC-ACE2 promoted the expression of proliferation-associated proteins and inhibited the expression of the apoptosis-associated proteins in EpH4-Ev cells. In addition, MSC and MSC-ACE2 reversed the LPS-induced downregulation expression levels of the tight junction proteins in mammary epithelial cells, indicating that both MSC as well as MSC-ACE2 could promote blood-milk barrier repair, and MSC-ACE2 was more effective. These results suggested that MSCs overexpressing ACE2 were more anti-inflammatory as well as anti-injurious action into LPS-induced inflammatory injury in the EpH4-Ev cells. Thus, MSCs overexpressing ACE2 is expected to serve as a potential strategy for mastitis treatment.

Substratum stiffness signals through integrin-linked kinase and β1-integrin to regulate midbody proteins and abscission during EMT.

Abscission is the final stage of cytokinesis during which the parent cell physically separates to yield two identical daughters. Failure of abscission results in multinucleation (MNC), a sign of genomic instability and a precursor to aneuploidy, enabling characteristics of neoplastic progression. Induction of epithelial-mesenchymal transition (EMT) causes MNC in mammary epithelial cells cultured on stiff microenvironments that have mechanical properties similar to those found in breast tumors, but not on soft microenvironments reminiscent of the normal mammary gland. Here we report that on stiff microenvironments, EMT signaling through Snail up-regulates the midbody-associated proteins septin-6, Mklp1, and anillin, leading to abscission failure and MNC. To uncover the mechanism by which stiff microenvironments promote MNC in cells undergoing EMT, we investigated the role of cell–matrix adhesion through β1-integrin and integrin-linked kinase (ILK). We found that ILK expression, but not kinase activity, is required for EMT-associated MNC in cells on stiff microenvironments. Conversely, increasing focal adhesions by expressing an autoclustering mutant of β1-integrin promotes MNC in cells on soft microenvironments. Our data suggest that signaling through focal adhesions causes failure of cytokinesis in cells actively undergoing EMT. These results highlight the importance of tissue mechanics and adhesion in regulating the cellular response to EMT inducers.

Impact of g-Load Shift on Temporal Expression Pattern of Apoptosis-linked Proteins in the Rat Mammary Gland

Abstract Alteration in gravitational load impacts homeorhetic response in rat dams which affects neonatal pup survival. However, the effects of hypergravity (HG) exposure on the abundance of apoptosis-associated proteins in mammary epithelial cells (MECs) have not been characterized. Therefore, we examined whether chronic exposure to HG from midpregnancy alters the abundance of proapoptotic proteins in MECs during the late pregnancy and early lactation. A group of pregnant Sprague Dawley rats were exposed to either HG (2g) or normo-gravity (1g: stationary control [SC]) from days 11 to 20 of gestation (G20). Another set of animals were investigated from day 11 of pregnancy through days 1 and 3 (P1 and P3, respectively) postpartum. Quantitative (pixels [px]/lobule) immunohistochemistry at G20 of Cleaved Caspase-3 (CC-3), Tumor Protein p53 (P53), and vitamin D receptor (VDR) revealed that all the three proteins were increased (p<0.01) in HG rats compared to SC animals. At P1, the HG group had twofold higher (p<0.001) expression of CC-3 relative to the SC group. Approximately, 50% (p<0.001) more VDR was detected in the HG cohorts than SC at P3. These results suggest that a shift in g-load upregulates the expression of key proapoptotic proteins during the pregnancy-to-lactation transition in the rat MECs.

Integrated analysis reveals that STAT3 is central to the crosstalk between HER/ErbB receptor signaling pathways in human mammary epithelial cells.

Human epidermal growth factor receptors (HER, also known as ErbB) drive cellular proliferation, pro-survival and stress responses by activating several downstream kinases, in particular ERK, p38 MAPK, JNK (SAPK), the PI3K/AKT, as well as various transcriptional regulators such as STAT3. When co-expressed, the first three members of HER family (HER1-3) can form homo- and hetero-dimers, and there is considerable evidence suggesting that the receptor dimers differentially activate intracellular signaling pathways. To better understand the interactions in this system, we pursued multi-factorial experiments where HER dimerization patterns and signaling pathways were rationally perturbed. We measured the activation of HER1-3 receptors and of the sentinel signaling proteins ERK, AKT, p38 MAPK, JNK, STAT3 as a function of time in a panel of human mammary epithelial (HME) cells expressing different levels of HER1-3 stimulated with various ligand combinations. We hypothesized that the HER dimerization pattern is a better predictor of downstream signaling than the total receptor activation levels. We validated this hypothesis using a combination of model-based analysis to quantify the HER dimerization patterns, and by clustering the activation data in multiple ways to confirm that the HER receptor dimer is a better predictor of the signaling through p38 MAPK, ERK and AKT pathways than the total HER receptor expression and activation levels. We then pursued combinatorial inhibition studies to identify the causal regulatory interactions between sentinel signaling proteins. Quantitative analysis of the collected data using the modular response analysis (MRA) and its Bayesian Variable Selection Algorithm (BVSA) version allowed us to obtain a consensus regulatory interaction model, which revealed that STAT3 occupies a central role in the crosstalk between the studied pathways in HME cells. Results of the BVSA/MRA and cluster analysis were in agreement with each other.

Abstract 2417: Oncogenic properties shared by all Snail family members

Abstract The epithelial to mesenchymal transition (EMT) is a latent embryonic process promoting cell migration by transiently affording epithelial cells a mesenchymal phenotype, motility and a survival advantage. The inappropriate activation of this program in adult cells is associated with several pathologies including tumor development where EMT promotes cancer cell dissemination and multidrug resistance development. We have recently demonstrated that EMT-inducers, beyond their prometastatic potential, facilitate the malignant transformation of human mammary epithelial cells in vitro and promote breast carcinogenesis in vivo. Although their ability to alleviate senescence and apoptosis failsafe program induction unquestionably contributes to their oncogenic potentials, we sought to investigate whether EMT inducers share additional properties. To this end, the activities of all three Snail members were examined. Likewise SNAI1 and SNAI2, SNAI3 expression is aberrantly activated in cancers, especially in mammary carcinomas. Ectopic expression of all three proteins in mammary epithelial cells induces their commitment into EMT, provides cells with a survival advantage in low adherent conditions and promotes their neoplastic transformation, with a similar gradient of efficiencies. Interestingly, this efficiency parallels with their ability to upregulate intracellular signaling pathways. We will update on the progress of our work aimed at understanding how these dysregulations impact on their oncogenic and prometastatic potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2417. doi:1538-7445.AM2012-2417

Characterisation of the potential SNARE proteins relevant to milk product release by mouse mammary epithelial cells

Casein micelles and fat globules are essential components of milk and are both secreted at the apical side of mammary epithelial cells during lactation. Milk fat globules are excreted by budding, being enwrapped by the apical plasma membrane, while caseins contained in transport vesicles are released by exocytosis. Nevertheless, the molecular mechanisms governing casein exocytosis are, to date, not fully deciphered. SNARE proteins are known to take part in cellular membrane trafficking and in exocytosis events in many cell types and we therefore attempted to identify those relevant to casein secretion. With this aim, we performed a detailed analysis of their expression by RT-PCR in both whole mouse mammary gland and in purified mammary acini at various physiological stages, as well as in the HC11 cell line. The expression of some regulatory proteins involved in SNARE complex formation such as Munc-13, Munc-18 and complexins was also explored. The amount of certain SNAREs appeared to be regulated depending on the physiological stage of the mammary gland. Co-immunoprecipitation experiments indicated that SNAP-23 interacted with syntaxin-6, -7 and -12, as well as with VAMP-3, -4 and -8 in mammary epithelial cells during lactation. Finally, the subcellular localisation of candidate SNAREs in these cells was determined both by indirect immunofluorescence and immunogold labelling. The present work provides important new data concerning SNARE proteins in mammary epithelial cells and points to SNAP-23 as a potential central player for the coupling of casein and milk fat globule secretion during lactation.

Temporal and spatial expression patterns for the tumor suppressor Maspin and its binding partner interferon regulatory factor 6 during breast development

Interferon regulatory factor 6 (IRF6) is a non-canonical member of the interferon regulatory factor family of transcription factors. We recently identified IRF6 as a novel Maspin-interacting protein in mammary epithelial cells. Maspin is a tumor suppressor in the breast and has also been implicated in mammary gland morphogenesis. To explore a possible role for IRF6 in conjunction with Maspin during mammary gland growth and differentiation, we examined the expression of IRF6 and Maspin during post-utero mammary gland development using a combination of in vitro and in vivo approaches. The data revealed that the expression of IRF6 and Maspin is temporally and spatially regulated throughout mammary gland development, with maximal expression of both proteins occurring in fully differentiated, lactating lobuloalveolar cells. We further show that IRF6 adopts a lumenal localization pattern following complete epithelial cell polarization and present new evidence for the secretion of IRF6 into the milk. These results support the hypothesis that IRF6 and Maspin are important for mammary epithelial cell differentiation, and advance our understanding of the Maspin-IRF6 partnership during normal mammary gland development.

Effects of amino acids on phosphorylation of S6 ribosomal protein in mammary epithelial cells

Protein synthesis is a highly regulated process responding to various signals including hormones and supply of energy and amino acids. The interactions of these signals have not been explored in bovine mammary epithelial (BME) cells. The objective of this study was to investigate the effects of individual amino acid deprivation on phosphorylation of ribosomal protein S6 which regulates protein synthesis. An established BME cell line (MAC‐T) was used as a model. Cells were grown to 85% confluence, serum starved for 12 h, and treated with serum‐free media containing all amino acids (Ctrl) or media singly deprived of Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, or Val for one hour. Then, cells were lysed, and cell lysates were subjected to Western immunoblotting using an antibody against phosphorylated S6 (rpS6235/236). Membranes were subsequently stripped and reprobed with an antibody against total S6. Results were expressed as ratios of pS6235/236: total S6 which is indicative of the S6 phosphorylation state. Deprivation of Arg, Val, and Ile reduced (P < 0.05) S6 phosphorylation by 96%, 90%, and 85%, respectively, compared to Ctrl. Deprivation of Phe and Leu reduced (P < 0.10) S6 phosphorylation by 45% and 30%, respectively. Branched chain amino acids, Arg, and Phe appeared to have key roles in regulation of protein translation initiation in BME cells.

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.

Expression of an engrailed-LMO4 fusion protein in mammary epithelial cells inhibits mammary gland development in mice.

LIM domain factors and associated cofactors are important developmental regulators in pattern formation and organogenesis. In addition, overexpression of two LIM-only factors (LMOs) causes acute lymphocytic leukemia. The more recently discovered LMO factor LMO4 is highly expressed in proliferating epithelial cells, and frequently overexpressed in breast carcinoma. Here we show that while LMO4 is expressed throughout mammary gland development, it is dramatically upregulated in mammary epithelial cells during midpregnancy. The LMO coactivator Clim2/Ldb1/NLI showed a similar expression pattern, consistent with the idea that LMO4 and Clim2 act as a complex in mammary epithelial cells. In MCF-7 cells, LMO4 transcripts were upregulated by heregulin, an activator of ErbB receptors that are known to be important in mammary gland development and breast cancer. To test the hypothesis that LMO4 plays roles in mammary gland development, we created an engrailed-LMO4 fusion protein. This fusion protein maintains the ability to interact with Clim2, but acts as a dominant repressor of both basal and activated transcription when recruited to a DNA-regulatory region. When the engrailed-LMO4 fusion protein was expressed under control of the MMTV promoter in transgenic mice, both ductular development in virgin mice and alveolar development in pregnant mice were inhibited. These results suggest that LMO4 plays a role in promoting mammary gland development.

Roads taken by milk proteins in mammary epithelial cells

Mammary cell secretory pathways are now well known: milk proteins initially appear over the endoplasmic reticulum, transiently associate with elements of the Golgi complex, then concentrate in post-Golgi secretory vesicles where caseins are detectable in aggregated form (casein micelles). Mechanisms controlling the transport of milk proteins between these different organelles are less well known. Study of transport of caseins in mammary cells from goats naturally deficient in α s1-casein has made it possible to show that quality control is associated with the forward transport of caseins out of the endoplasmic reticulum. Transport of caseins between endoplasmic reticulum, Golgi saccules, trans Golgi network (TGN) and secretory vesicles is under the control of protein kinase A and phospholipase D. Last steps of exocytosis of milk proteins are stimulated by prolactin-induced arachidonic acid release suggesting a role of PLA 2. The mammary epithelial cell also internalises plasma-borne proteins (hormones, growth factors, transferrin, immunoglobulins) in part via clathrin-coated vesicles, and carries many of them, to milk, by transcytosis. As shown in rodents and rabbits, sorting occurs, for the different ligands internalised, in endosomes. Transferrin is mainly recycled to the basal membrane, markers of the basal membrane are rapidly carried to the Golgi region and prolactin is transcytosed across late endosomes and secretory vesicles. Visualisation of the intracellular pathway of prolactin, in mammospheres from lactating rabbits, reveals that transcytosis occurs via a tubulo-vesicular network across the cell and that the roads of endocytosis and exocytosis are interconnected.

Differentiational regulation and phosphorylation of the fatty acid-binding protein from rat mammary epithelial cells

From the soluble protein fraction of lactating rat mammary epithelial cells, fatty acid-binding protein (FABP) was isolated by immunoaffinity chromatography. After digestion with trypsin, peptides were characterized with time-of-flight mass spectrometry and revealed identity with corresponding peptides derived from the heart-type FABP isolated from rat heart. In addition, by electrospray mass spectrometry the molecular mass has been determined to 14683.9 ± 3 Da, further corroborating the identity. The content of FABP in mammary glands from virgin, pregnant and lactating rats was evaluated using two-dimensional gel electrophoresis and a FABP-specific immunosorbent assay. In the two-dimensional gels FABP was the apparently most abundant cytosolic protein in mammary epithelial cells from rats in late pregnancy as well as from lactating rats. The content of FABP was 59 ± 19 μg/ mg ( n = 11) of soluble proteins from the fully differentiated lactating mammary gland as determined by ELISA. This value represented an 80-fold increase compared with the FABP content of mammary gland from virgin rats, and is comparable with the level found in rat heart. Upon stimulation with insulin a small fraction of FABP was phosphorylated in lactating mammary epithelial cells. In conclusion, these findings indicate that the FABPs from rat mammary gland and heart are identical and further suggest that in mammary gland this FABP may play a role in signal transduction downstream from the insulin receptor.

DNA-binding proteins and their cis-acting sites controlling hormonal induction of a mouse β-casein::CAT fusion protein in mammary epithelial cells

Transcription of the mouse β-casein (βCAS)-encoding gene ( casB) is regulated by the synergistic actions of insulin, glucocorticoid and prolactin in the mammary gland (MG). To delineate its regulatory sequence(s), we examined the hormonal inducibility of various chimeric constructs containing the promoter sequence of casB and the cat reporter gene in primary MG epithelial cells. A DNA fragment from bp − 258 to + 7 of casB was sufficient for the hormonal induction. Using a series of 5'- and internal deletions of the casB promoter region, at least three DNA elements were found to be necessary for full induction. These were located at bp positions −258 to −180, −154 to −136, and −98 to −62. DNase I footprinting analysis with a partially purified extract from lactating MG cells detected at least seven protected sequences, I(−242 to −219), II(−213 to −202), III(−151 to −139), IV(−125 to −110), V(−98 to −90), VI(−79 to −70) and VII(−59 to −45). Regions I/II, III and V/VI were included in the three DNA elements required for the hormonal induction, and the IV region corresponded to the MG consensus sequences of several milk protein-encoding genes. Competition gel retardation assays using nuclear extracts of lactating mouse MG revealed the presence of specific binding proteins for regions I, II, IV and VI, as well as specific protein(s) binding to both regions III and V. The binding activities of these proteins, except that associated with region IV, were increased from the virgin to lactating periods. The activity of protein binding to region IV existed in all stages, but its pattern in gel retardation assays changed during the development of MG from the virgin to lactating periods. These results suggest that multiple DNA elements and their binding proteins are involved in the hormonal induction of mouse casB expression during MG development.

Collagen‐Binding proteins of mammary epithelial cells are related to Ca2+‐and phospholipid‐binding annexins

Three major proteins of 34, 36, and 38 kDa were isolated from membrane preparations of chemically induced mammary tumors of the rat by collagen type I affinity chromatography and therefore were termed collagen-binding proteins (CBP). Three proteins in the same molecular weight range isolated from cell extracts by precipitation with calcium, solubilization of the precipitate with EGTA, and chromatography on hydroxylapatite were demonstrated to be immunologically related to CBP. As shown by immunoblot analysis, an antiserum directed against the cluster of the 34-38 kDa proteins reacted strongly with porcine intestinal protein I, weakly with porcine lipocortin I, and very weakly with porcine intestinal protein II. Antiserum against the 34 kDa protein reacted weakly with protein I but strongly with protein II. All three CBP reacted with protein I/calpactin I-specific antiserum of immunoblots and in immunoprecipitation experiments. However, antisera directed against CBP failed to show cross-reaction with collagen-binding protein anchorin II from chicken chondrocytes. Conversely, antisera against anchorin II did not react with CBP. Antiserum AS/87 immunoprecipitated CBP of 38 kDa that was labeled in a lactoperoxydase-catalyzed iodination, suggesting that this polypeptide is associated with the cell surface. Further, all three CBP were found to be phosphorylated by incubating mammary cells with 32P-orthophosphate. CBP bound to epithelial cell membranes in a Ca2+ dependent manner (= Triton X 100 insoluble form). Fractionated extraction and immunofluorescence microscopy also show that another form of CBP (= Triton X 100 soluble form) exists in these cells and is associated with a granular fraction. We therefore conclude that mammary collagen-binding proteins represent members of a family of Ca2(+)-binding membrane proteins. The 38 kDa CBP seems closely related to the pp60src kinase substrate protein I/calpactin I monomer, the 34 kDa CBP seems to be related or equivalent to protein II, while the relationship of the 36 kDa CBP to other defined proteins is still unclear.

Monoclonal antibodies prepared against PAS-I butyrophilin and GP-55 from guinea-pig milk-fat-globule membrane bind specifically to the apical pole of secretory-epithelial cells in lactating mammary tissue

Monoclonal antibodies to the three major glycoproteins of guinea-pig milk-fat-globule membrane were isolated. The specificity of these antibodies was determined by solid-phase immunoassays and by immunoblotting and autoradiographic techniques after one-and two-dimensional gel electrophoresis. The antibodies bound to PAS-I, a sialoglycoprotein of M r ⩾200 000 and the glycoproteins butyrophilin and GP-55, of M r 63 000 and 55 000, respectively. Immunolocalization studies showed that all three proteins were highly concentrated in the apical pole of secretory-epithelial cells in mammary tissue during lactation. PAS-I, butyrophilin or GP-55, were not detected in either the basal cytoplasm of mammary epithelial cells or in myoepithelial cells, capillary endothelial cells or other cells found in the mammary gland. These proteins were either present in small amounts or were absent from mammary tissue taken in late pregnancy. The monoclonal antibodies characterized in this study will therefore be useful as probes for studies of the biogenesis of apical membrane proteins in mammary epithelial cells during lactation.