Mouse Mammary Gland Epithelial Cells Research Articles

MiR-30a-3p Regulates Autophagy in the Involution of Mice Mammary Glands.

The mammary gland undergoes intensive remodeling during the lactation cycle, and the involution process of mammary gland contains extensive epithelial cells involved in the process of autophagy. Our studies of mice mammary glands suggest that miR-30a-3p expression was low during involution compared with its high expression in the mammary glands of lactating mice. Then, we revealed that miR-30a-3p negatively regulated autophagy by autophagy related 12 (Atg12) in mouse mammary gland epithelial cells (MMECs). Restoring ATG12, knocking down autophagy related 5 (Atg5), starvation, and Rapamycin were used to further confirm this conclusion. Overexpression of miR-30a-3p inhibited autophagy and altered mammary structure in the involution of the mammary glands of mice, which was indicative of alteration in mammary remodeling. Taken together, these results elucidated the molecular mechanisms of miR-30a-3p as a key induction mediator of autophagy by targeting Atg12 within the transition period between lactation and involution in mammary glands.

Development of Highly Sensitive Anti-Mouse HER2 Monoclonal Antibodies for Flow Cytometry

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer is an important target of monoclonal antibody (mAb) therapy such as trastuzumab. Due to the development of trastuzumab–deruxtecan, an antibody-drug conjugate, the targetable HER2-positive breast cancer patients have been expanded. To evaluate the developing modalities using anti-HER2 mAbs, reliable preclinical mouse models are required. Therefore, sensitive mAbs against mouse HER2 (mHER2) should be established. This study developed anti-mHER2 mAbs using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mHER2 mAbs, H2Mab-300 (rat IgG2b, kappa) and H2Mab-304 (rat IgG1, kappa), reacted with mHER2-overexpressed Chinese hamster ovary-K1 (CHO/mHER2) and endogenously mHER2-expressed cell line, NMuMG (a mouse mammary gland epithelial cell) via flow cytometry. Furthermore, these mAbs never recognized mHER2-knockout NMuMG cells. The kinetic analysis using flow cytometry indicated that the dissociation constant (KD) values of H2Mab-300 and H2Mab-304 for CHO/mHER2 were 1.2 × 10−9 M and 1.7 × 10−9 M, respectively. The KD values of H2Mab-300 and H2Mab-304 for NMuMG were 4.9 × 10−10 M and 9.0 × 10−10 M, respectively. These results indicated that H2Mab-300 and H2Mab-304 could apply to the detection of mHER2 using flow cytometry and may be useful to obtain the proof of concept in preclinical studies.

EMab-300 Detects Mouse Epidermal Growth Factor Receptor-Expressing Cancer Cell Lines in Flow Cytometry.

Epidermal Growth Factor Receptor (EGFR) overexpression or its mutation mediates the sustaining proliferative signaling, which is an important hallmark of cancer. Human EGFR-targeting monoclonal antibody (mAb) therapy such as cetuximab has been approved for clinical use in patients with colorectal cancers and head and neck squamous cell carcinomas. A reliable preclinical mouse model is essential to further develop the mAb therapy against EGFR. Therefore, sensitive mAbs against mouse EGFR (mEGFR) should be established. In this study, we developed a specific and sensitive mAb for mEGFR using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mEGFR mAb, EMab-300 (rat IgG1, kappa), reacted with mEGFR-overexpressed Chinese hamster ovary-K1 (CHO/mEGFR) and endogenously mEGFR-expressed cell lines, including NMuMG (a mouse mammary gland epithelial cell) and Lewis lung carcinoma cells, using flow cytometry. The kinetic analysis using flow cytometry indicated that the KD of EMab-300 for CHO/mEGFR and NMuMG was 4.3 × 10-8 M and 1.9 × 10-8 M, respectively. These results indicated that EMab-300 applies to the detection of mEGFR using flow cytometry and may be useful to obtain the proof of concept in preclinical studies.

Modulation of Cell-Cycle Progression by Hydrogen Peroxide-Mediated Cross-Linking and Degradation of Cell-Adhesive Hydrogels.

The cell cycle is known to be regulated by features such as the mechanical properties of the surrounding environment and interaction of cells with the adhering substrates. Here, we investigated the possibility of regulating cell-cycle progression of the cells on gelatin/hyaluronic acid composite hydrogels obtained through hydrogen peroxide (H2O2)-mediated cross-linking and degradation of the polymers by varying the exposure time to H2O2 contained in the air. The stiffness of the hydrogel varied with the exposure time. Human cervical cancer cells (HeLa) and mouse mammary gland epithelial cells (NMuMG) expressing cell-cycle reporter Fucci2 showed the exposure-time-dependent different cell-cycle progressions on the hydrogels. Although HeLa/Fucci2 cells cultured on the soft hydrogel (Young’s modulus: 0.20 and 0.40 kPa) obtained through 15 min and 120 min of the H2O2 exposure showed a G2/M-phase arrest, NMuMG cells showed a G1-phase arrest. Additionally, the cell-cycle progression of NMuMG cells was not only governed by the hydrogel stiffness, but also by the low-molecular-weight HA resulting from H2O2-mediated degradation. These results indicate that H2O2-mediated cross-linking and degradation of gelatin/hyaluronic acid composite hydrogel could be used to control the cell adhesion and cell-cycle progression.

Metabolic Reprogramming of Mammary Epithelial Cells during TGF-β-Induced Epithelial-to-Mesenchymal Transition.

The cytokine transforming growth factor-β (TGF-β) can induce normal breast epithelial cells to take on a mesenchymal phenotype, termed epithelial-to-mesenchymal transition (EMT). While the transcriptional and proteomic changes during TGF-β-induced EMT have been described, the metabolic rewiring that occurs in epithelial cells undergoing EMT is not well understood. Here, we quantitively analyzed the TGF-β-induced metabolic reprogramming during EMT of non-transformed NMuMG mouse mammary gland epithelial cells using nuclear magnetic resonance (NMR) spectroscopy. We found that TGF-β elevates glycolytic and tricarboxylic acid (TCA)-cycle activity and increases glutaminolysis. Additionally, TGF-β affects the hexosamine pathway, arginine-proline metabolism, the cellular redox state, and strongly affects choline metabolism during EMT. TGF-β was found to induce phosphocholine production. A kinase inhibitor RSM-93A that inhibits choline kinase α (CHKα) mitigated TGF-β-induced changes associated with EMT, i.e., increased filamentous (F)-actin stress fiber formation and N-Cadherin mesenchymal marker expression.

Low Intensity Ultrasound Induces Epithelial Cell Adhesion Responses.

In this study, we investigated the cellular mechanosensitive responses to a low intensity ultrasound (LIUS) stimulation (ISATA = 1 mW/cm2, pressure = 10 kPa). The dose and temporal effects at cell-substrate adhesion (CSA) at the basal level and cell-cell adhesion (CCA) at the apical level are reported in detail. A model of mouse mammary gland epithelial cells (EpH4) and the phosphorylation of mechanosensitive 130 kDa Crk-associated substrate (p130CAS) as an indicator for cellular responses were used. The intensity of phospho-p130CAS was found to be dependent on LIUS stress level, and the p130CAS was phosphorylated after 1 min stimulation at CSA. The phospho-p130CAS was also found to increase significantly at CCA upon LIUS stimulation. We confirmed that the cellular responses to ultrasound are immediate and dose dependent. Ultrasound affects not only CSA but also CCA. An E-cadherin knockout (EpH4ECad-/-) model also confirmed that phosphorylation of p130CAS at CCA is related to E-cadherins.

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells.

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation. The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins β-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed "domes". The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce β-casein and WAP. Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

MiR-142-3p Regulates Milk Synthesis and Structure of Murine Mammary Glands via PRLR-Mediated Multiple Signaling Pathways.

Murine mammary gland is an ideal model for studying the development and milk synthesis in dairy animals. MicroRNAs play an important role in milk synthesis and mammary gland development; however, the molecular mechanism of miR-142-3p continues to be poorly understood. Here, we knocked down miR-142-3p expression in vitro and vivo, increased the prolactin receptor expression and activated many downstream cellular proteins, such as mammalian target of rapamycin, sterol regulatory element-binding transcription factor 1, cyclin D1, and signal transducer and activator of transcription 5. Additionally, miR-142-3p knockdown in mouse mammary gland epithelial cells increased proliferation but not viability, induced cell cycle progression, decreased apoptosis, and increased the expression of triglycerides and β-casein. Moreover, miR-142-3p knockdown in murine mammary gland tissue in vivo affected the structure and function of the mammary gland, which showed an increased number of lobules and ducts and was more capable of producing milk. However, overexpression of miR-142-3p had the opposite effects. In summary, these data reveal that miR-142-3p regulates milk synthesis and the structure of murine mammary glands via PRLR-mediated multiple signaling pathways.

Design of an ultrasound chamber for cellular excitation and observation.

In this work, a design of integrating ultrasonic transduction with live cell imaging chamber is introduced. The principle of a metal-incident-glass-output acoustic path was used to deliver a uniform energy profile into the imaging/incubation chamber in the form of leaky Lamb waves. The design was applied to examine living mouse mammary gland epithelial cells (EpH4). Significant changes in intracellular activities were observed even at a very low energy intensity level (1 MHz, ISATA = 1 mW/cm2, continuous wave). Live imaging with ultrasonic stimulation provides a different paradigm to interrogate cellular mechanosensitive responses in real time.

Aqueous/Aqueous Micro Phase Separation: Construction of an Artificial Model of Cellular Assembly.

To artificially construct a three-dimensional cell assembly, we investigated the availability of long-duration microdroplets that emerged near a critical point in an aqueous two-phase system (ATPS) with the hydrophilic binary polymers, polyethylene glycol (PEG), and dextran (DEX), as host containers. We found that erythrocytes (horse red blood cells; RBCs) and NAMRU mouse mammary gland epithelial cells (NMuMG cells) were completely and spontaneously entrapped inside DEX-rich microdroplets. RBCs and NMuMG cells were located in the interior and at the periphery of the droplets at PEG/DEX = 5%:5%. In contrast, the cells exhibited opposite localizations at PEG/DEX = 10%:5%, where, interestingly, NMuMG cells apparently assembled to achieve cell adhesion. We simply interpreted such specific localizations by considering the alternative responses of these cells to the properties of the PEG/DEX interfaces with different gradients in polymer concentrations.

The histone demethylase Kdm3a is required for normal epithelial proliferation, ductal elongation and tumor growth in the mouse mammary gland.

Histone modification alters chromatin architecture to regulate gene transcription. KDM3A is a histone demethylase in the JmjC domain-containing protein family. It removes di- and mono- methyl residues from di- or mono-methylated lysine 9 of histone H3 (H3K9me2/me1). Recent studies have shown that Kdm3a plays an important role in self-renewal of embryonic stem cells, spermatogenesis, metabolism, sex determination and tumor angiogenesis. However, its role in mammary gland development and breast carcinogenesis remains unclear. In this study, we found that Kdm3a is expressed in the mouse mammary gland epithelial cells. Knockout of Kdm3a significantly increased H3K9me2/me1 levels in these epithelial cells, which correlated with markedly decreased mammary gland ductal elongation and branching in the intact knockout virgin mice. Furthermore, estrogen replacement in the ovariectomized Kdm3a knockout mice couldn’t rescue the retarded ductal growth. Moreover, transplantation of KO mammary gland pieces to wild type recipient mice showed slower ductal growth compared with that of WT gland pieces. Consistently, knockout of Kdm3a also reduced the proliferation rates and cyclin D1 expression in the mammary gland epithelial cells. In addition, Kdm3a knockout did not significantly change the latency of the polyoma middle T oncogene-induced mammary gland tumorigenesis. Tumor growth, however, was slowed which might be due to the decrease in cyclin D1 expression and tumor cell proliferation. We also found that Kdm3a binds and activates the cyclin D1 promoter. These results demonstrate that Kdm3a plays an important intrinsic role in promoting mammary gland ductal growth and tumor growth probably through enhancing cyclin D1 expression and cell proliferation.

Dynamics of chromatin accessibility during TGF-\u03b2-induced EMT of Ras-transformed mammary gland epithelial cells

Epithelial-mesenchymal transition (EMT) is induced by transforming growth factor (TGF)-β and facilitates tumor progression. We here performed global mapping of accessible chromatin in the mouse mammary gland epithelial EpH4 cell line and its Ras-transformed derivative (EpRas) using formaldehyde-assisted isolation of regulatory element (FAIRE)-sequencing. TGF-β and Ras altered chromatin accessibility either cooperatively or independently, and AP1, ETS, and RUNX binding motifs were enriched in the accessible chromatin regions of EpH4 and EpRas cells. Etv4, an ETS family oncogenic transcription factor, was strongly expressed and bound to more than one-third of the accessible chromatin regions in EpRas cells treated with TGF-β. While knockdown of Etv4 and another ETS family member Etv5 showed limited effects on the decrease in the E-cadherin abundance and stress fiber formation by TGF-β, gene ontology analysis showed that genes encoding extracellular proteins were most strongly down-regulated by Etv4 and Etv5 siRNAs. Accordingly, TGF-β-induced expression of Mmp13 and cell invasiveness were suppressed by Etv4 and Etv5 siRNAs, which were accompanied by the reduced chromatin accessibility at an enhancer region of Mmp13 gene. These findings suggest a mechanism of transcriptional regulation during Ras- and TGF-β-induced EMT that involves alterations of accessible chromatin, which are partly regulated by Etv4 and Etv5.

GosB Inhibits Triacylglycerol Synthesis and Promotes Cell Survival in Mouse Mammary Epithelial Cells.

It has been demonstrated that the activator protein related transcription factor Finkel-Biskis-Jinkins murine osteosarcoma B (GosB) is involved in preadipocyte differentiation and triacylglycerol synthesis. However, the role of GosB in regulating the synthesis of milk fatty acid in mouse mammary glands remains unclear. This research uncovered potentially new roles of GosB in suppressing milk fatty acid synthesis. Results revealed that GosB had the highest expression in lung tissue and showed a higher expression level during nonlactation than during lactation. GosB inhibited the expression of fatty acid synthase (FASN), stearoyl-CoA desaturase (SCD), fatty acid binding protein 4 (FABP4), diacylglycerol acyltransferase 1 (DGAT1), perilipin 2 (PLIN2), perilipin 3 (PLIN3), and C/EBPα in mouse mammary gland epithelial cells (MEC). In addition, GosB reduced cellular triglyceride content and the accumulation of lipid droplets; in particular, GosB enhanced saturated fatty acid concentration (C16:0 and C18:0). The PPARγ agonist, rosiglitazone (ROSI), promoted apoptosis and inhibited cell proliferation. GosB increased the expression of Bcl-2 and protected MEC from ROSI-induced apoptosis. Furthermore, MECs were protected from apoptosis through the GosB regulation of intracellular calcium concentrations. These findings suggest that GosB may regulate mammary epithelial cells milk fat synthesis and apoptosis via PPARγ in mouse mammary glands.

Identification of two candidate innate immune genes by transcriptional profiling and RNA interference in mouse mammary gland epithelial cells stimulated with lipopolysaccharide

Mammary epithelial cells (MECs) play an important role in immune responses and inflammatory diseases such as mastitis, which is mainly attributed to the activation of Toll-like receptors and the release of cytokines. However, the overall change of gene expression and biological pathways of MECs to microbial factors stimulation remains unknown. Here, we analyzed the gene expression profile in mouse MECs treated with lipopolysaccharide (LPS) for 24 h. Microarray analysis revealed that about 1548 genes differentially expressed, these genes mainly involved in 346 gene ontology terms and 128 molecular pathways, and particularly, some innate immune-associated pathways were significant. By analyzing data for pathway relation network, we prioritized differentially expressed genes with respect to LPS. The importance of changes, indicating that RNA interference-mediated inhibition of two genes identified in this analysis, transforming growth factor beta 1 (Tgf-β1) and platelet-derived growth factor B (Pdgfb), reduced interleukin (IL)-6 and tumor necrosis factor α production respectively, in gene expression was verified. These findings delineate mouse MECs gene response patterns induced by LPS and identify Tgf-β1 and Pdgfb that have been closely related to innate immunity.

Translational control of TGF-β-mediated Treg cell differentiation by hnRNP E1 (IRM15P.460)

Abstract CD4+Foxp3+ T cells are generated either in the thymus for natural regulatory T cells, or in periphery for inducible Treg cells (iTregs), which differentiate from naïve T cell precursors. Both Tregs express Foxp3 albeit they differ in their plasticity and in their expression of certain genes. Trans­forming growth factor-β (TGF-β) and interleukin-2 are important for iTreg generation and maintenance, whereas proinflammatory cytokines such as IL-4 and IL-6 impair Foxp3 induction. Much has been learned regarding the transcriptional roles of TGF-β in determining the fate of iTregs; however, the limited focus if any on a translational mechanism by which TGF-β regulates the fate of functional T cells is somewhat myopic. We previously showed a novel translational mechanism for TGF-β regulation of EMT in mouse mammary gland epithelial cells whereby hnRNP E1 (E1) binds to a structural, 33-nucleotide TGF-β-activated translation element in the 3’ UTR of target transcripts and represses their translation. In this study, we found that E1 knockdown (E1KD) in naïve CD4+ T cells induces TGF-β signaling and upregulates expression of multiple TGF-β target molecules implicated in iTreg cell differentiation. Furthermore, E1KD CD4+ T cells bypass the need for TGF-β and express Foxp3. This study adds to our knowledge on a hitherto unrecognized translational mechanism by which TGF-β controls iTreg cell fate.

Exosomes from breast cancer cells stimulate proliferation and inhibit apoptosis of CD133+ cancer cells in vitro.

Exosomes are a family of bioactive vesicles that are secreted from various types of cell, including tumor cells. Exosomes derived from breast cancer cells have been demonstrated to perform important functions in tumor progression in vitro and in vivo. However, few studies exist regarding the function of exosomes in CD133+ breast cancer cells. In the present study, exosomes from 4T1 mouse breast cancer cells and mouse mammary gland epithelial cells were purified. The exosome-associated markers CD63, CD9 and CD81 were detected, and the size distribution and ζ potential of the exosomes were determined. Exosome uptake by CD133+ and CD133-4T1 cells was confirmed by confocal microscopy. An ATPlite assay indicated that the proliferation of CD133+ cells was increased and the apoptosis was suppressed by exosomes from 4T1 cells. Collectively, the findings of the present study demonstrate a novel mechanism by which the action of exosomes on CD133+ 4T1 cells may contribute to breast cancer progression.

Abstract 3524: Unraveling the dichotomy of Runx1 in breast cancer

Abstract Each of the Runx family of transcription factors (Runx1, Runx2 and Runx3) is associated with specific human cancers. Recent genetic evidence suggests that Runx1 plays an important role in human breast cancer. It has been shown to be frequently mutated in certain breast cancer subtypes, and an inverse correlation between Runx1 expression and increasing tumor grade was established. Although Runx1 is considered to be a tumor suppressor, one study suggests otherwise, with loss of Runx1 in epithelial cancer cell lines impairing tumor growth. Thus, the dichotomous nature of Runx1 during breast cancer progression and metastasis remains ambiguous. Using the MMTV-PyMT transgenic mouse model of breast cancer and complementary in vitro models, we are further defining the role of Runx factors, particularly Runx1, in breast cancer progression. MMTV-PyMT mice and age- matched controls were sacrificed at multiple time points (4-15 weeks) representing all stages of disease progression. Mammary gland (primary site), tumor and lung (metastatic site) were excised and analyzed: Runx1 RNA and protein expression in the mammary gland was significantly increased over controls as early as 8 weeks. This increase persisted throughout the course of the disease with Runx1 levels being highest at late time points (13 and 15 weeks), concomitant with the presence of metastatic lesions in the lung. During tumor progression, Runx1 (and to a lesser extent, Runx2) levels were consistently elevated. In the lungs, Runx1 (and Runx2) protein expression significantly increased as a function of the number/size of metastatic lesions present. Furthermore, phenotypic analyses as well as relevant RNA/protein expression were determined in both normal mouse mammary gland epithelial cells (NMuMG) and an MMTV-PyMT tumor-derived cell line (MMTV): MMTV cells exhibited superior rates of growth, invasion and migration and expressed 3-fold more Runx1 (yet only half as much Runx2) than NMuMG cells; complementary knockdown studies were used to reveal the contribution of Runx1 to these processes. Additionally, analysis of microRNA expression using microRNA arrays and QPCR in both mouse and cell line models revealed the potential for specific microRNAs in the temporal regulation of Runx factors during breast cancer progression. Overall, our results support roles for Runx factors, particularly Runx1, in breast cancer. Notably, increased Runx1 expression is associated with disease progression and metastasis as well as a more aggressive phenotype in our models. Moreover, in our disease models potential regulation of Runx factors by specific microRNAs is demonstrated. Taken together, these data highlight Runx1 as a promising novel therapeutic target in breast cancer. Citation Format: Gillian Browne, Nicole M. Bishop, Jason R. Dobson, Sharath C. Madasu, Farina H. Nicholas, Maria L. La Porta, Dana Frederick, Jennifer L. Colby, Leslie M. Shaw, Justine Landis, Janet L. Stein, Jane B. Lian, Gary S. Stein. Unraveling the dichotomy of Runx1 in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3524. doi:10.1158/1538-7445.AM2014-3524

Transforming growth factor-β-induced lncRNA-Smad7 inhibits apoptosis of mouse breast cancer JygMC(A) cells.

Transforming growth factor (TGF)-β exhibits both pro-apoptotic and anti-apoptotic effects on epithelial cells in a context-dependent manner. The anti-apoptotic function of TGF-β is mediated by several downstream regulatory mechanisms, and has been implicated in the tumor-progressive phenotype of breast cancer cells. We conducted RNA sequencing of mouse mammary gland epithelial (NMuMG) cells and identified a long non-coding RNA, termed lncRNA-Smad7, which has anti-apoptotic functions, as a target of TGF-β. lncRNA-Smad7 was located adjacent to the mouse Smad7 gene, and its expression was induced by TGF-β in all of the mouse mammary gland epithelial cell lines and breast cancer cell lines that we evaluated. Suppression of lncRNA-Smad7 expression cancelled the anti-apoptotic function of TGF-β. In contrast, forced expression of lncRNA-Smad7 rescued apoptosis induced by a TGF-β type I receptor kinase inhibitor in the mouse breast cancer cell line JygMC(A). The anti-apoptotic effect of lncRNA-Smad7 appeared to occur independently of the transcriptional regulation by TGF-β of anti-apoptotic DEC1 and pro-apoptotic Bim proteins. Small interfering RNA for lncRNA-Smad7 did not alter the process of TGF-β-induced epithelial–mesenchymal transition, phosphorylation of Smad2 or expression of the Smad7 gene, suggesting that the contribution of this lncRNA to TGF-β functions may be restricted to apoptosis. Our findings suggest a complex mechanism for regulating the anti-apoptotic and tumor-progressive aspects of TGF-β signaling.

Altered N-Glycan expression profile in epithelial-to-mesenchymal transition of NMuMG cells revealed by an integrated strategy using mass spectrometry and glycogene and lectin microarray analysis.

Epithelial-to-mesenchymal transition (EMT) is an essential biological process that occurs in embryonic development, metastatic diseases, and cancer progression. Altered expression of glycans is known to be associated with cancer progression. No studies to date have presented global analysis of the precise variation of N-glycans in EMT. We describe here the profile of N-glycans and glycogene expression in the EMT process induced by transforming growth factor-β1 (TGFβ1) in a normal mouse mammary gland epithelial (NMuMG) cell model. An integrated strategy with a combination of mass spectrometry, glycogene microarray analysis, and lectin microarray analysis was applied, and results were confirmed by lectin histochemistry and quantitative real-time PCR. In TGFβ-induced EMT, levels of high-mannose-type N-glycans were enhanced, antennary N-glycans, and fucosylation were suppressed, and bisecting GlcNAc N-glycans were greatly suppressed. The expression of seven N-glycan-related genes was significantly changed. The products of glycogenes ALG9, MGAT3, and MGAT4B appeared to contribute to the observed alteration of N-glycans. The findings indicate that dysregulation of N-glycan synthesis plays a role in the EMT process. Systematic glycomic analysis based on the combination of techniques described here is expected to facilitate the discovery of the aberrant N-glycosylation in tumor progression and provide essential information in systems glycobiology.

Morphogen-induced Platelet Activation and Cell Signalling

Morphogens are signalling molecules that act directly on cells to produce specific cellular responses. This term was originally put forward by Turing (1952) who also described its chemical basis. Most of these secreted proteins signal between the cells. Wolpert (1969) first reported a role of morphogen in cell signalling. The roles of several morphogens (e.g., Transforming Growth Factor- (TGF- ), Hedgehog/ Sonic Hedgehog, Wingless (Wnt) signalling, Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), and Retinoic Acid (RA) in signal transduction have been studied by scientists in several cell lineages. Assoian et al. (1983) identified major storage sites of TGF- in human platelets. Xie et al. (2003) showed expression of TGF- regulated gene in a mouse mammary gland epithelial cell line. Iwasaki et al. (2000) reported that EGF stimulates growth of vascular smooth muscles that may be involved in genesis of human vascular disease. RA acts as a ligand and diffusible morphogen, the distribution and levels of which are well controlled in embryonic tissues by its regulated synthesis and breakdown. The occurrence of RA signalling in