Subcellular location of serum- and glucocorticoid-induced kinase-1 in renal and mammary epithelial cells

2007 ◽  
Vol 292 (5) ◽  
pp. C1971-C1981 ◽  
Author(s):  
Emily Cordas ◽  
Anikó Náray-Fejes-Tóth ◽  
Géza Fejes-Tóth
Keyword(s):  
Subcellular Localization ◽  
Mammary Epithelial Cells ◽  
Collecting Duct ◽  
Subcellular Location ◽  
Mammary Epithelial ◽  
Live Cells ◽  
Epithelial Cell Line ◽  
Cortical Collecting Duct ◽  
Mitochondrial Marker ◽  
Amino Acid Region

Serum- and glucocorticoid-induced kinase-1 (SGK1) is involved in aldosterone-induced Na+ reabsorption by increasing epithelial Na+ channel (ENaC) activity in cortical collecting duct (CCD) cells, but its exact mechanisms of action are unknown. Although several potential targets such as Nedd4-2 have been described in expression systems, endogenous substrates mediating SGK1's physiological effects remain to be identified. In addition, subcellular localization studies of SGK1 have provided controversial results. We determined the subcellular location of SGK1 using SGK1-autofluorescent protein (AFP) fusion proteins. Rabbit CCD (RCCT-28A) cells were transiently transfected with a construct encoding for SGK1-AFP and were stained or cotransfected with markers for various subcellular compartments. In live cells, transiently expressed SGK1-AFP clearly colocalized with the mitochondrial marker rhodamine 123. Similarly, SGK1-AFP colocalized with the mitochondrial marker MitoTracker when stably expressed using a retroviral system in either RCCT-28A cells or the mammary epithelial cell line MCF10A. To determine which region of SGK1 is responsible for this subcellular localization, we generated RCCT-28A cell lines stably expressing SGK1 mutants. The results indicate that the NH2-terminal 60-amino acid region of SGK1 is necessary and sufficient for its subcellular localization. Localization of SGK1 to the mitochondria raises the possibility that SGK1 may play a role in regulating energy metabolism.

Repression of Wnt-5a impairs DDR1 phosphorylation and modifies adhesion and migration of mammary cells

2001 ◽  
Vol 114 (11) ◽  
pp. 2043-2053 ◽  
Author(s):  
Marzieh Jönsson ◽  
Tommy Andersson
Keyword(s):  
Mammary Epithelial Cells ◽  
Human Mammary Epithelial Cell ◽  
Mammary Epithelial ◽  
Epithelial Cell Line ◽  
Collagen Binding ◽  
Collagen Matrices ◽  
Endogenous Expression ◽  
Human Mammary Epithelial ◽  
Antisense Approach ◽  
And Migration

The Wnt-5a gene encodes a secreted protein that controls several normal processes during embryogenesis and development of adult tissues by as yet unknown mechanisms. Endogenous expression of Wnt-5a mRNA is known to occur in both mouse and human mammary cell lines. To investigate the biological role of Wnt-5a in the human mammary epithelial cell line HB2, we used an antisense approach to repress endogenous expression of Wnt-5a protein. We also generated a cell population that constitutively overexpresses this protein. We found that overexpression of Wnt-5a protein enhanced cell-to-collagen binding and abolished hepatocyte growth factor-stimulated migration of HB2 transfectants through collagen matrices. Conversely, repression of Wnt-5a protein led to cell scattering, impaired cell-collagen interaction and enhanced cell motility. As we were searching for modified collagen receptors in antisense cells, we discovered that the collagen-binding discoidin domain receptor 1 (DDR1) failed to undergo phosphorylation. In reciprocal experiments, phosphorylation of DDR1 was consistently enabled by expression of Wnt-5a-HA protein in non-Wnt-5a-producing MCF-7 breast cancer cells. Activation of the Wnt/β-catenin signalling pathway did not influence or mimic the Wnt-5a-mediated effect on DDR1 phosphorylation. These data demonstrate that Wnt-5a protein participates in regulation of adhesion to and migration through collagen and is also a co-factor necessary for collagen-induced activation of DDR1 receptors in mammary epithelial cells.

Immunocytochemical and immunoelectron microscopic localization of α-, β-, and γ-ENaC in rat kidney

2001 ◽  
Vol 280 (6) ◽  
pp. F1093-F1106 ◽  
Author(s):  
Henrik Hager ◽  
Tae-Hwan Kwon ◽  
Anna K. Vinnikova ◽  
Shyama Masilamani ◽  
Heddwen L. Brooks ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Confocal Microscopy ◽  
Subcellular Localization ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Cortical Collecting Duct ◽  
Principal Cells ◽  
Medullary Collecting Duct ◽  
Epithelial Sodium Channel Enac

Epithelial sodium channel (ENaC) subunit (α, β, and γ) mRNA and protein have been localized to the principal cells of the connecting tubule (CNT), cortical collecting duct (CCD), and outer medullary collecting duct (OMCD) in rat kidney. However, the subcellular localization of ENaC subunits in the principal cells of these cells is undefined. The cellular and subcellular localization of ENaC subunits in rat kidney was therefore examined. Immunocytochemistry demonstrated the presence of all three subunits in principal cells of the CNT, CCD, OMCD, and IMCD. In cortex and outer medulla, confocal microscopy demonstrated a difference in the subcellular localization of subunits. α-ENaC was localized mainly in a zone in the apical domains, whereas β- and γ-ENaC were found throughout the cytoplasm. Immunoelectron microscopy confirmed the presence of ENaC subunits in both the apical plasma membrane and intracellular vesicles. In contrast to the labeling pattern seen in cortex, α-ENaC labeling in IMCD cells was distributed throughout the cytoplasm. In the urothelium covering pelvis, ureters, and bladder, immunoperoxidase and confocal microscopy revealed differences the presence of all ENaC subunits. As seen in CCD, α-ENaC was present in a narrow zone near the apical plasma membrane, whereas β- and γ-ENaC were dispersed throughout the cytoplasm. In conclusion, all three subunits of ENaC are expressed throughout the collecting duct (CD), including the IMCD as well as in the urothelium. The intracellular vesicular pool in CD principal cells suggests ENaC trafficking as a potential mechanism for the regulation of Na+ reabsorption.

New acceptor cell for transfected genomic DNA: oncogene transfer into a mouse mammary epithelial cell line

1985 ◽  
Vol 5 (1) ◽  
pp. 268-272
Author(s):  
N E Hynes ◽  
R Jaggi ◽  
S C Kozma ◽  
R Ball ◽  
D Muellener ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Line ◽  
Nude Mice ◽  
Genomic Dna ◽  
Mammary Epithelial Cells ◽  
Human Tumor ◽  
Mammary Epithelial ◽  
Epithelial Cell Line ◽  
Exogenous Dna ◽  
Stable Transfectants

A line of mouse mammary epithelial cells (NMuMG) has been characterized for its ability to be stably transfected with exogenous DNA. A transfection frequency of at least 1 cell per 1,000 was obtained with the pSV2neo plasmid. Several thousand G418-resistant NMuMG cell clones can easily be generated in cotransfection of genomic DNA and pSV2neo. The NMuMG cells were isolated from normal mammary glands and do not form malignant lesions when injected into nude mice. We have cotransfected NMuMG cells with pSV2neo and genomic DNA from the human EJ bladder carcinoma line, a cell line which contains an activated c-rasH oncogene. When a pool of 4,700 G418-resistant colonies was injected into nude mice, tumors were obtained. These tumors contain a transfected human rasH gene. Genomic DNA transfection into a line of mouse epithelial cells, in combination with the selection of stable transfectants and tumor induction in nude mice, can be used to screen human tumor DNA for the presence of activated oncogenes.

scholarly journals GIRK1 triggers multiple cancer-related pathways in the benign mammary epithelial cell line MCF10A

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Gebhard Schratter ◽  
Susanne Scheruebel ◽  
Sonja Langthaler ◽  
Katja Ester ◽  
Brigitte Pelzmann ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Cellular Migration ◽  
Epithelial Cell Line ◽  
Survival Times ◽  
Mcf7 Cells ◽  
Multiple Cancer ◽  
Cellular Pathways ◽  
Vital Parameters

AbstractExcessive expression of subunit 1 of GIRK1 in ER+ breast tumors is associated with reduced survival times and increased lymph node metastasis in patients. To investigate possible tumor-initiating properties, benign MCF10A and malign MCF7 mammary epithelial cells were engineered to overexpress GIRK1 neoplasia associated vital parameters and resting potentials were measured and compared to controls. The presence of GIRK1 resulted in resting potentials negative to the controls. Upon GIRK1 overexpression, several cellular pathways were regulated towards pro-tumorigenic action as revealed by comparison of transcriptomes of MCF10AGIRK1 with the control (MCF10AeGFP). According to transcriptome analysis, cellular migration was promoted while wound healing and extracellular matrix interactions were impaired. Vital parameters in MCF7 cells were affected akin the benign MCF10A lines, but to a lesser extent. Thus, GIRK1 regulated cellular pathways in mammary epithelial cells are likely to contribute to the development and progression of breast cancer.

scholarly journals New acceptor cell for transfected genomic DNA: oncogene transfer into a mouse mammary epithelial cell line.

1985 ◽  
Vol 5 (1) ◽  
pp. 268-272 ◽  
Author(s):  
N E Hynes ◽  
R Jaggi ◽  
S C Kozma ◽  
R Ball ◽  
D Muellener ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Line ◽  
Nude Mice ◽  
Genomic Dna ◽  
Mammary Epithelial Cells ◽  
Human Tumor ◽  
Mammary Epithelial ◽  
Epithelial Cell Line ◽  
Exogenous Dna ◽  
Stable Transfectants

A line of mouse mammary epithelial cells (NMuMG) has been characterized for its ability to be stably transfected with exogenous DNA. A transfection frequency of at least 1 cell per 1,000 was obtained with the pSV2neo plasmid. Several thousand G418-resistant NMuMG cell clones can easily be generated in cotransfection of genomic DNA and pSV2neo. The NMuMG cells were isolated from normal mammary glands and do not form malignant lesions when injected into nude mice. We have cotransfected NMuMG cells with pSV2neo and genomic DNA from the human EJ bladder carcinoma line, a cell line which contains an activated c-rasH oncogene. When a pool of 4,700 G418-resistant colonies was injected into nude mice, tumors were obtained. These tumors contain a transfected human rasH gene. Genomic DNA transfection into a line of mouse epithelial cells, in combination with the selection of stable transfectants and tumor induction in nude mice, can be used to screen human tumor DNA for the presence of activated oncogenes.

scholarly journals Dandelion Extract Alleviated Lipopolysaccharide-Induced Oxidative Stress through the Nrf2 Pathway in Bovine Mammary Epithelial Cells

Toxins ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 496 ◽  
Author(s):  
Yawang Sun ◽  
Yongjiang Wu ◽  
Zili Wang ◽  
Juncai Chen ◽  
You Yang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Epithelial Cells ◽  
Signaling Pathway ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Epithelial Cell Line ◽  
Related Factor ◽  
Nrf2 Pathway ◽  
Bovine Mammary Epithelial Cells ◽  
Nrf2 Signaling Pathway

In practical dairy production, cows are frequently subjected to inflammatory diseases, such as high-grain diet-induced subacute ruminal acidosis (SARA) as well as mastitis and metritis. Under the circumstances, lipopolysaccharide (LPS) induces oxidative stress within the cow and in the mammary epithelial cells. It has implications in practical production to alleviate oxidative stress and to optimize the lactational function of the mammary epithelial cells. This study thus aimed to investigate the antioxidative effects of dandelion aqueous extract (DAE) on LPS-induced oxidative stress and the mechanism of DAE as an antioxidant to alleviate oxidative stress through the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in the bovine mammary epithelial cell line MAC-T cells. The cells were cultured for 48 h in six treatments including control (without LPS and DAE), LPS (100 ng/mL), DAE10 (100 ng/mL LPS and 10 μg/mL DAE), DAE50 (100 ng/mL LPS and 50 μg/mL DAE), DAE100 (100 ng/mL LPS and 100 μg/mL DAE), and DAE200 (100 ng/mL LPS and 200 μg/mL DAE), respectively. The results showed that cell viability was reduced by LPS, and the adverse effect of LPS was suppressed with the supplementation of DAE. Lipopolysaccharide-induced oxidative stress through enhancing reactive oxygen species (ROS) production, resulted in increases in oxidative damage marker concentrations, while 10 and 50 μg/mL DAE alleviated the LPS-induced oxidative stress via scavenging cellular ROS and improving antioxidant enzyme activity. The upregulation of antioxidative gene expression in DAE treatments was promoted through activating the Nrf2 signaling pathway, with DAE at a concentration of 50 μg/mL exhibiting the highest effect. Overall, DAE acted as an effective antioxidant to inhibit LPS-induced oxidative stress and as a potential inducer of the Nrf2 signaling pathway.

Fibronectin fragments induce MMP activity in mouse mammary epithelial cells: evidence for a role in mammary tissue remodeling

2000 ◽  
Vol 113 (5) ◽  
pp. 795-806 ◽  
Author(s):  
P. Schedin ◽  
R. Strange ◽  
T. Mitrenga ◽  
P. Wolfe ◽  
M. Kaeck
Keyword(s):  
Extracellular Matrix ◽  
Mammary Gland ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Protease Activity ◽  
Mammary Epithelial Cells ◽  
Cell Loss ◽  
Mammary Epithelial ◽  
Epithelial Cell Line ◽  
Fibronectin Fragments

Mammary gland form and function are regulated by interactions between epithelium and extracellular matrix. Major glycoprotein components of extracellular matrix have been identified that give survival, proliferation and differentiation signals to mammary epithelial cells. We provide evidence that proteolytic fragments of the extracellular matrix glycoprotein, fibronectin, suppress growth and can promote apoptosis of mouse mammary epithelial cells. During mammary gland involution, total fibronectin and fibronectin fragment levels are increased. The peak levels of fibronectin protein and fragments are observed 4–6 days post-weaning, coincident with the peak in epithelial cell death. Using a model for hormone withdrawal-induced death of mammary epithelium, elevated levels of fibronectin proteolytic fragments were associated with apoptosis in TM-6 cells, a tumorigenic mouse mammary epithelial cell line. Treatment of TM-6 cells with exogenous fibronectin fragments (FN120) reduced cell number, and induced apoptosis and matrix degrading protease activity. Inhibition of matrix protease activity rescued TM-6 cell viability, indicating that FN120-induced cell loss is mediated through matrix protease activity. In a three-dimensional model for mammary gland development, FN120 reduced alveolar-like and promoted ductal-like development by a matrix protease-dependent mechanism. These data suggest that during post-lactational involution, fibronectin fragments may contribute to epithelial cell loss and dissolution of mammary alveoli by inducing matrix degrading proteinases.

Receptor binding and growth-promoting activity of insulin-like growth factor-I in a bovine mammary epithelial cell line (MAC-T3)

1992 ◽  
Vol 134 (2) ◽  
pp. 307-312 ◽  
Author(s):  
X. Zhao ◽  
B. W. McBride ◽  
I. Politis ◽  
H. T. Huynh ◽  
R. M. Akers ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Mammary Epithelial Cell ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Epithelial Cell Line ◽  
Mammary Epithelial Cell Line ◽  
Bovine Mammary Epithelial Cells ◽  
Bovine Mammary Epithelial Cell ◽  
Igf I

ABSTRACT Insulin-like growth factor-I (IGF-I) has been known to be mitogenic to a variety of cell types, although a growth-regulatory role for IGF-I on bovine mammary epithelial cells has not been fully investigated. In the present study, we examined the receptor binding of IGF-I and its effect on growth in a bovine mammary epithelial cell line (MAC-T3). Specific receptors for IGF-I were detected on cultured bovine mammary epithelial cells. Competitive binding revealed that half-maximal inhibition of 125I-labelled IGF-I binding by IGF-I was approximately 3 μg/l. Dissociation rate constant of the IGF-I receptor was 3·10±0·06 nmol/l (s.e.m.) with a receptor site concentration of 366 ± 8 fmol/mg protein for the average of three experiments. IGF-I exerted a positive mitogenic effect on MAC-T3 cells according to both direct DNA assay and thymidine incorporation assay. Moreover, the mitogenic effect of IGF-I on MAC-T3 cells was enhanced by the addition of fetal calf serum in the culture media. The present results suggest that the bovine mammary epithelial cell line (MAC-T3) provides a useful model system with which to study the biological actions of insulin-like growth factors on the bovine mammary secretory tissue in vitro. Journal of Endocrinology (1992) 134, 307–312

scholarly journals Cross-talk between Stat5b and estrogen receptor-alpha and -beta in mammary epithelial cells

2001 ◽  
Vol 27 (1) ◽  
pp. 93-106 ◽  
Author(s):  
L Bjornstrom ◽  
E Kilic ◽  
M Norman ◽  
MG Parker ◽  
M Sjoberg
Keyword(s):  
Estrogen Receptor ◽  
Cross Talk ◽  
Estrogen Receptor Alpha ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Epithelial Cell Line ◽  
Receptor Alpha ◽  
Cell Extracts ◽  
C Terminus

Both 17beta-estradiol and prolactin play important roles in the mammary gland, raising the possibility of functional cross-talk between the two signaling pathways. Here, we demonstrate that estrogen receptor-alpha (ERalpha) and -beta (ERbeta) are both able to potentiate transcription from a Stat5-responsive promoter when activated by prolactin. Potentiation was observed not only in the presence of 17beta-estradiol, but also in the presence of anti-estrogens such as tamoxifen and ICI 182,780. The magnitude of the response was dependent on cell-type: in the HC11 mouse mammary epithelial cell line ERbeta potentiates transcription efficiently whereas ERalpha showed low activity. Conversely, in COS-7 cells, both estrogen receptors were active. We show that activation domains in the N-terminus (AF-1) and the C-terminus (AF-2) of the ERs are dispensable for potentiation. The effects are dependent on the presence of an intact DNA-binding/hinge domain, which we show is capable of interacting with Stat5b in vitro and in HC11 cell extracts. We conclude that ERalpha and ERbeta act as coactivators for Stat5b through a mechanism which is independent of AF-1 and AF-2.

scholarly journals Maspin Is an Intracellular Serpin That Partitions into Secretory Vesicles and Is Present at the Cell Surface

1997 ◽  
Vol 45 (12) ◽  
pp. 1697-1706 ◽  
Author(s):  
Philip A. Pemberton ◽  
A. Rene Tipton ◽  
Nadine Pavloff ◽  
Jason Smith ◽  
James R. Erickson ◽  
...  
Keyword(s):  
Cell Surface ◽  
Subcellular Localization ◽  
Mammary Epithelial Cells ◽  
Myoepithelial Cells ◽  
Mammary Epithelial ◽  
In Vitro Assays ◽  
Secretory Vesicles ◽  
Mammary Tumor Cells ◽  
Secretion Signal

The tumor suppressor maspin (mammary serpin) was originally identified as a component of human mammary epithelial cells that is downregulated as mammary tumor cells progress from the benign to the invasive and metastatic states. Maspin inhibits cellular invasion, motility, and proliferation, but its mechanism of action is currently unknown. Because the cellular machinery responsible for these processes is cytoplasmic, we have reexamined the tissue distribution and subcellular localization of maspin. We find that maspin, or a maspin-like protein, is present in many human organs, in which it localizes to epithelia. In cultured human mammary myoepithelial cells, maspin is predominantly a soluble cytoplasmic protein that associates with secretory vesicles and is present at the cell surface. In vitro assays show that the vesicle association is due to the existence of an uncleaved facultative secretion signal that allows small amounts of maspin to partition into the endoplasmic reticulum. These results demonstrate that maspin is more widespread than previously believed. The subcellular localization studies indicate that soluble intracellular and vesicle-associated maspin probably play an important role in controlling the invasion, motility, and proliferation of cells expressing it, whereas extracellular maspin may also regulate these processes in adjacent cells.

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