scholarly journals The Voltage-Gated Sodium Channel Beta4 Subunit Maintains Epithelial Phenotype in Mammary Cells

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1624
Author(s):  
Adélaïde Doray ◽  
Roxane Lemoine ◽  
Marc Severin ◽  
Stéphanie Chadet ◽  
Osbaldo Lopez-Charcas ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Morphological Changes ◽  
Three Dimensional ◽  
Mammary Epithelial ◽  
Mammary Cells ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Gene Coding ◽  
Epithelial Phenotype

The SCN4B gene, coding for the NaVβ4 subunit of voltage-gated sodium channels, was recently found to be expressed in normal epithelial cells and down-regulated in several cancers. However, its function in normal epithelial cells has not been characterized. In this study, we demonstrated that reducing NaVβ4 expression in MCF10A non-cancer mammary epithelial cells generated important morphological changes observed both in two-dimensional cultures and in three-dimensional cysts. Most notably, the loss of NaVβ4 induced a complete loss of epithelial organisation in cysts and increased proteolytic activity towards the extracellular matrix. Loss of epithelial morphology was associated with an increased degradation of β-catenin, reduced E-cadherin expression and induction of mesenchymal markers N-cadherin, vimentin, and α-SMA expression. Overall, our results suggest that Navβ4 may participate in the maintenance of the epithelial phenotype in mammary cells and that its downregulation might be a determining step in early carcinogenesis.

scholarly journals Phosphoinositide-3 Kinase–Rac1–c-Jun NH2-terminal Kinase Signaling Mediates Collagen I–induced Cell Scattering and Up-Regulation of N-Cadherin Expression in Mouse Mammary Epithelial Cells

2006 ◽  
Vol 17 (7) ◽  
pp. 2963-2975 ◽  
Author(s):  
Yasushi Shintani ◽  
Margaret J. Wheelock ◽  
Keith R. Johnson
Keyword(s):  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Morphological Changes ◽  
Three Dimensional ◽  
Mammary Epithelial ◽  
Collagen I ◽  
Jnk Signaling ◽  
Cell Scattering ◽  
Phosphoinositide 3 Kinase ◽  
Induced Changes

During epithelial-to-mesenchymal transitions (EMTs), cells must change their interactions with one another and with their extracellular matrix in a synchronized manner. To characterize signaling pathways cells use to coordinate these changes, we used NMuMG mammary epithelial cells. We showed that these cells become fibroblastic and scattered, with increased N-cadherin expression when cultured on collagen I. Rac1 and c-Jun NH2-terminal kinase (JNK) were activated when cells were plated on collagen I, and dominant inhibitory Rac1 (RacN17) or inhibition of JNK signaling prevented collagen I–induced morphological changes and N-cadherin up-regulation. Furthermore, inhibiting phosphoinositide-3 kinase (PI3K) activity prevented Rac1 and JNK activation as well as collagen I–induced N-cadherin up-regulation. These data implicate PI3K–Rac1–JNK signaling in collagen I–induced changes in NMuMG cells. To establish a role for N-cadherin in collagen I–induced cell scattering, we generated N-cadherin overexpressing and knockdown NMuMG cells and showed that knocking down N-cadherin expression prevented collagen I–induced morphological changes. Motility assays showed that cells overexpressing N-cadherin were significantly more motile than mock-transfected cells and that N-cadherin-mediated motility was collagen I dependent. In addition, we showed that cord formation and branching in three-dimensional culture (EMT-dependent events) required N-cadherin expression and PI3K–Rac1–JNK signaling.

Investigation of the role of microtubules in protein secretion from lactating mouse mammary epithelial cells

1992 ◽  
Vol 102 (2) ◽  
pp. 239-247 ◽  
Author(s):  
M.E. Rennison ◽  
S.E. Handel ◽  
C.J. Wilde ◽  
R.D. Burgoyne
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Protein Secretion ◽  
Secretory Pathway ◽  
Mammary Epithelial Cells ◽  
Early Stage ◽  
Major Effect ◽  
Vesicle Transport ◽  
Mammary Epithelial ◽  
Mammary Cells

Disruption of microtubules has been shown to reduce protein secretion from lactating mammary epithelial cells. To investigate the involvement of microtubules in the secretory pathway in these cells we have examined the effect of nocodazole on protein secretion from mammary epithelial cells derived from the lactating mouse. Mouse mammary cells have extensive microtubule networks and 85% of their tubulin was in a polymeric form. Treatment with 1 micrograms/ml nocodazole converted most of the tubulin into a soluble form. In a continuous labelling protocol it was found that nocodazole did not interfere with protein synthesis but over a 5 h period secretion was markedly inhibited. To determine whether the inhibition was at the level of early or late stages of the secretory pathway mammary cells were pulse-labelled for 1 h to label protein throughout the secretory pathway before nocodazole treatment. When secretion was subsequently assayed it was found to be slower and only partially inhibited. These findings suggest that the major effect of nocodazole is on an early stage of the secretory pathway and that microtubules normally facilitate vesicle transport to the plasma membrane. An involvement of microtubules in vesicle transport to the plasma membrane is consistent with an observed accumulation of casein vesicles in nocodazole-treated cells. Exocytosis stimulated by the calcium ionophore ionomycin was unaffected by nocodazole treatment. We conclude from these results that the major effect of nocodazole is at an early stage of the secretory pathway, one possible target being casein vesicle biogenesis in the trans-Golgi network.

scholarly journals MMTV Env encodes an ITAM responsible for transformation of mammary epithelial cells in three-dimensional culture

2005 ◽  
Vol 201 (3) ◽  
pp. 431-439 ◽  
Author(s):  
Elad Katz ◽  
Mohamed H. Lareef ◽  
John C. Rassa ◽  
Shannon M. Grande ◽  
Leslie B. King ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tyrosine Kinases ◽  
Mammary Epithelial Cells ◽  
Cell Transformation ◽  
Three Dimensional ◽  
Soft Agar ◽  
Mammary Epithelial ◽  
Matrigel Invasion ◽  
Tumor Virus

Expression of immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling proteins is normally restricted to hematopoietic tissues. The basal activity of ITAM-containing proteins is mediated through negative regulation by coreceptors restricted to hematopoietic tissues. We have identified an ITAM signaling domain encoded within the env gene of murine mammary tumor virus (MMTV). Three-dimensional structures derived in vitro from murine cells stably transfected with MMTV env display a depolarized morphology in comparison with control mammary epithelial cells. This effect is abolished by Y>F substitution within the Env ITAM, as well as inhibitors of Syk and Src protein tyrosine kinases. Env-expressing cells bear hallmarks of cell transformation such as sensitivity to apoptosis induced by tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) or TNFα, as well as down-regulation of E-cadherin and Keratin-18. Human normal mammary epithelial cells expressing MMTV Env also develop transformed phenotype, as typified by growth in soft agar and Matrigel invasion. These disruptions are abrogated by Y>F substitutions. We conclude that ITAM-dependent signals are generated through MMTV Env and trigger early hallmarks of transformation of mouse and human mammary epithelial cells. Therefore, these data suggest a heretofore unappreciated potential mechanism for the initiation of breast cancer and identify MMTV Env and ITAM-containing proteins in human breast tumors as probable oncoproteins.

scholarly journals Cyclic AMP regulates formation of mammary epithelial acini in vitro

2012 ◽  
Vol 23 (15) ◽  
pp. 2973-2981 ◽  
Author(s):  
Pavel I. Nedvetsky ◽  
Sang-Ho Kwon ◽  
Jayanta Debnath ◽  
Keith E. Mostov
Keyword(s):  
Epithelial Cells ◽  
Cyclic Amp ◽  
Mammary Epithelial Cells ◽  
Three Dimensional ◽  
Mammary Epithelial ◽  
Inhibitory Antibody ◽  
Proapoptotic Protein ◽  
Dependent Polarization ◽  
Dependent Protein

Epithelial cells form tubular and acinar structures notable for a hollow lumen. In three-dimensional culture utilizing MCF10A mammary epithelial cells, acini form due to integrin-dependent polarization and survival of cells contacting extracellular matrix (ECM), and the apoptosis of inner cells of acini lacking contact with the ECM. In this paper, we report that cyclic AMP (cAMP)-dependent protein kinase A (PKA) promotes acinus formation via two mechanisms. First, cAMP accelerates redistribution of α6-integrin to the periphery of the acinus and thus facilitates the polarization of outer acinar cells. Blocking of α6-integrin function by inhibitory antibody prevents cAMP-dependent polarization. Second, cAMP promotes the death of inner cells occupying the lumen. In the absence of cAMP, apoptosis is delayed, resulting in perturbed luminal clearance. cAMP-dependent apoptosis is accompanied by a posttranscriptional PKA-dependent increase in the proapoptotic protein Bcl-2 interacting mediator of cell death. These data demonstrate that cAMP regulates lumen formation in mammary epithelial cells in vitro, both through acceleration of polarization of outer cells and apoptosis of inner cells of the acinus.

scholarly journals Matrix Metalloproteinase Stromelysin-1 Triggers a Cascade of Molecular Alterations That Leads to Stable Epithelial-to-Mesenchymal Conversion and a Premalignant Phenotype in Mammary Epithelial Cells

1997 ◽  
Vol 139 (7) ◽  
pp. 1861-1872 ◽  
Author(s):  
André Lochter ◽  
Sybille Galosy ◽  
John Muschler ◽  
Neal Freedman ◽  
Zena Werb ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Keratinocyte Growth Factor ◽  
Mammary Epithelium ◽  
Mammary Epithelial ◽  
Neoplastic Progression ◽  
Molecular Alterations ◽  
Epithelial Phenotype ◽  
Inappropriate Expression ◽  
E Cadherin

Matrix metalloproteinases (MMPs) regulate ductal morphogenesis, apoptosis, and neoplastic progression in mammary epithelial cells. To elucidate the direct effects of MMPs on mammary epithelium, we generated functionally normal cells expressing an inducible autoactivating stromelysin-1 (SL-1) transgene. Induction of SL-1 expression resulted in cleavage of E-cadherin, and triggered progressive phenotypic conversion characterized by disappearance of E-cadherin and catenins from cell–cell contacts, downregulation of cytokeratins, upregulation of vimentin, induction of keratinocyte growth factor expression and activation, and upregulation of endogenous MMPs. Cells expressing SL-1 were unable to undergo lactogenic differentiation and became invasive. Once initiated, this phenotypic conversion was essentially stable, and progressed even in the absence of continued SL-1 expression. These observations demonstrate that inappropriate expression of SL-1 initiates a cascade of events that may represent a coordinated program leading to loss of the differentiated epithelial phenotype and gain of some characteristics of tumor cells. Our data provide novel insights into how MMPs function in development and neoplastic conversion.

scholarly journals A FACS-Free Purification Method to Study Estrogen Signaling, Organoid Formation, and Metabolic Reprogramming in Mammary Epithelial Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Aurélie Lacouture ◽  
Cynthia Jobin ◽  
Cindy Weidmann ◽  
Line Berthiaume ◽  
Dominic Bastien ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Metabolic Flux ◽  
Mammary Epithelial Cells ◽  
Aerobic Glycolysis ◽  
Three Dimensional ◽  
Two Dimensions ◽  
Mammary Epithelial ◽  
Metabolic Reprogramming ◽  
Phenotypic Characterization ◽  
Flux Analysis

Few in vitro models are used to study mammary epithelial cells (MECs), and most of these do not express the estrogen receptor α (ERα). Primary MECs can be used to overcome this issue, but methods to purify these cells generally require flow cytometry and fluorescence-activated cell sorting (FACS), which require specialized instruments and expertise. Herein, we present in detail a FACS-free protocol for purification and primary culture of mouse MECs. These MECs remain differentiated for up to six days with >85% luminal epithelial cells in two-dimensional culture. When seeded in Matrigel, they form organoids that recapitulate the mammary gland’s morphology in vivo by developing lumens, contractile cells, and lobular structures. MECs express a functional ERα signaling pathway in both two- and three-dimensional cell culture, as shown at the mRNA and protein levels and by the phenotypic characterization. Extracellular metabolic flux analysis showed that estrogens induce a metabolic switch favoring aerobic glycolysis over mitochondrial respiration in MECs grown in two-dimensions, a phenomenon known as the Warburg effect. We also performed mass spectrometry (MS)-based metabolomics in organoids. Estrogens altered the levels of metabolites from various pathways, including aerobic glycolysis, citric acid cycle, urea cycle, and amino acid metabolism, demonstrating that ERα reprograms cell metabolism in mammary organoids. Overall, we have optimized mouse MEC isolation and purification for two- and three-dimensional cultures. This model represents a valuable tool to study how estrogens modulate mammary gland biology, and particularly how these hormones reprogram metabolism during lactation and breast carcinogenesis.

Molecular Characterization of Epithelial to Mesenchymal Transition in Human Prostatic Epithelial Cells

2010 ◽  
Vol 1 (2) ◽  
Author(s):  
Victor K. Lin ◽  
Shih-Ya Wang ◽  
Lanxiao Wu ◽  
Smitha M. Rao ◽  
J. C. Chiao ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cancer Metastasis ◽  
Epithelial To Mesenchymal Transition ◽  
Mammary Epithelial Cells ◽  
Critical Role ◽  
Three Dimensional ◽  
Mammary Epithelial ◽  
Mesenchymal Transition ◽  
Enhanced Mobility ◽  
E Cadherin

Epithelial to mesenchymal transition (EMT) has been believed to play a critical role in cancer metastasis. TGFβ has been described as an inducer of EMT in normal mammary epithelial cells by signaling through receptor serine/threonine kinase pathways to regulate epithelial cell plasticity and invasion. In this study, we investigated the EMT cellular responses, including morphologic changes, phenotype switches, invasiveness enhancement, and cellular contraction alteration, in TGFβ stimulated human prostate normal epithelial cells (PZ-HPV-7). Migration of TGFβ treated PZ-HPV-7 cells across matrigel was measured in invasion chambers (8 μm pore size). The cells were treated with or without TGFβ (2 ng/ml) in PrEGM media for 3 days. Immunoblot assay was conducted and it was demonstrated that the induction of vimentin when stimulated by TGFβ was accompanied by a downregulation of E-cadherin, though p-cadherin level was not altered. It was also observed that there was a decrease in cytokaretin 5/6 expression associated with the downregulation of E-cadherin during the induction of EMT. In order to study the cell contraction, three-dimensional collage lattice assay was performed. It was demonstrated that TGFβ-stimulated PZ-HPV-7 cells gained contractility. Our results showed that TGFβ stimulation induced PZ-HPV-7 cells to undergo epithelial to mesenchymal transition. EMT characteristics such as acquisition of mesenchymal markers and loss of epithelial markers were evident in the induction of vimentin and downregulation of E-cadherin and cytokeratins, as well as phenotypic alterations including increased contraction and enhanced mobility were detected.

scholarly journals Bacteriophages isolated from dairy farm mitigated Klebsiella pneumoniae-induced inflammation in bovine mammary epithelial cells cultured in vitro

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yuxiang Shi ◽  
Wenpeng Zhao ◽  
Gang Liu ◽  
Tariq Ali ◽  
Peng Chen ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Klebsiella Pneumoniae ◽  
Inflammatory Responses ◽  
Mammary Epithelial Cells ◽  
Therapeutic Potential ◽  
Morphological Changes ◽  
Mammary Epithelial ◽  
Bovine Mammary Epithelial Cells ◽  
Tnf Α

Abstract Background Klebsiella pneumoniae, an environmental pathogen causing mastitis in dairy cattle, is often resistant to antibiotics. K. pneumoniae was used as the host bacteria to support bacteriophage replication; 2 bacteriophages, CM8-1 and SJT-2 were isolated and considered to have therapeutic potential. In the present study, we determined the ability of these 2 bacteriophages to mitigate cytotoxicity, pathomorphological changes, inflammatory responses and apoptosis induced by K. pneumoniae (bacteriophage to K. pneumoniae MOI 1:10) in bovine mammary epithelial cells (bMECs) cultured in vitro. Results Bacteriophages reduced bacterial adhesion and invasion and cytotoxicity (lactate dehydrogenase release). Morphological changes in bMECs, including swelling, shrinkage, necrosis and hematoxylin and eosin staining of cytoplasm, were apparent 4 to 8 h after infection with K. pneumoniae, but each bacteriophage significantly suppressed damage and decreased TNF-α and IL-1β concentrations. K. pneumoniae enhanced mRNA expression of TLR4, NF-κB, TNF-α, IL-1β, IL-6, IL-8, caspase-3, caspase-9 and cyt-c in bMECs and increased apoptosis of bMECs, although these effects were mitigated by treatment with either bacteriophage for 8 h. Conclusions Bacteriophages CM8-1 and SJT-2 mitigated K. pneumoniae-induced inflammation in bMECs cultured in vitro. Therefore, the potential of these bacteriophages for treating mastitis in cows should be determined in clinical trials.

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