scholarly journals TRPV4 Increases the Expression of Tight Junction Protein-Encoding Genes via XBP1 in Mammary Epithelial Cells

Animals ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 1174
Author(s):  
Md Aminul Islam ◽  
Moeko Mizusawa ◽  
Mst Mamuna Sharmin ◽  
Satoko Hayashi ◽  
Shinichi Yonekura
Keyword(s):  
Gene Expression ◽  
Heat Treatment ◽  
Mammary Gland ◽  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Transcript Level ◽  
Mammary Epithelial ◽  
Mrna Levels ◽  
Mild Heat ◽  
Protein Encoding

Mild heat stress (39 °C–40 °C) can positively regulate cell proliferation and differentiation. Indeed, mild heat treatment at 39 °C enhances the less-permeable tight junctions (TJs) formation and milk production in mammary epithelial cells. However, the molecular mechanisms of this response have not yet been delineated. In this study, the involvement of temperature-sensitive transient receptor potential vanilloid 4 (TRPV4) in the increase of β-casein and TJ protein-encoding gene expression in response to mild heat treatment (39 °C) has been explored using HCll mouse mammary epithelial cells. Severe heat treatment (41 °C) induced the transcriptional level of Chop (C/EBP homologous protein; proapoptotic marker) and reduced the cell viability. It is speculated that the difference in unfolded protein response (UPR) gene expression upon stimulation at 39 °C vs. 41 °C controls cell survival vs. cell death. The accumulation of Trpv4 mRNA was significantly higher in 39 °C heat treatment cells. The β-casein, Zo-1 (zona occludens-1), Ocln (occludin), and Cldn3 (claudin 3) transcript levels were significantly increased in response to the addition of a selective TRPV4 channel agonist (GSK1016790A) at 37 °C. TRPV4 stimulation with GSK1016790A also increased the X-box-binding protein 1 splicing form (Xbp1s) at the transcript level. The increase in the mRNA levels of β-casein, Zo-1, Ocln, and Cldn3 in response to 39 °C heat treatment was suppressed by XBP1 knockdown. Moreover, the transcript level of Trpv4 was significantly increased at Day 15 of gestation, and its expression declined after 1 day of lactation. TRPV4 is activated not only by temperature but also by mechanical forces, such as cell stretching and shear stress, which guide mammary epithelial development in a normal mammary gland. These findings provide new insights of the possible function of TRPV4 in mammary gland development.

scholarly journals Review of: Metalloproteinase axes increase β-catenin signaling in primary mouse mammary epithelial cells lacking TIMP3

2007 ◽  
Vol 10 (8) ◽  
Author(s):  
D. S. Salomon
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Target Genes ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Specific Effects ◽  
Primary Mouse ◽  
Ductal Elongation

Citation of original article:C. V. Hojilla, I. Kim, Z. Kassiri, J. E. Fat, H. Fang, R. Khokha. Journal of Cell Science 2007; 120(6): 1050–1060.Abstract of the original article:Multiple cancers exhibit mutations in β-catenin that lead to increased stability, altered localization or amplified activity. β-Catenin is situated at the junction between the cadherin-mediated cell adhesion and Wnt signaling pathways, and TIMP3 functions to alter β-catenin signaling. Here we demonstrate that primary mouse embryonic fibroblasts (MEFs) and mammary epithelial cells (MECs) deficient in Timp3 have increased β-catenin signaling. Functionally, the loss of TIMP3 exerted cell-type-specific effects, with Timp3−/− MEFs being more sensitive and Timp3−/− MECs more resistant to EGTA-induced cell detachment than the wild type. Timp3−/− MECs had higher dephosphorylated β-catenin levels and increased β-catenin transcriptional activity as measured by TCF/LEF-responsive reporter assays. Real-time PCR analysis of β-catenin target genes in MEFs and MECs showed no alteration in Myc, decreased Ccnd1 (cyclin D1) and increased Mmp7 mRNA levels upon loss of TIMP3, with the latter occurring only in epithelial cells. Recombinant TIMP3 and synthetic metalloproteinase inhibitors reverted the increase in dephosphorylated β-catenin, decrease in Ccnd1 gene expression and increase in Mmp7 gene expression. Physiologically, Timp3−/− mammary glands displayed accelerated mammary ductal elongation during pubertal morphogenesis. Gain-of-function studies using slow-release TIMP-containing pellets revealed distinct effects of individual TIMPs on ductal morphogenesis. Recombinant TIMP1, TIMP3 and TIMP4 inhibited ductal elongation whereas TIMP2 promoted this process.

Gene expression and hormonal regulation of adiponectin and its receptors in bovine mammary gland and mammary epithelial cells

2010 ◽  
Vol 82 (1) ◽  
pp. 99-106 ◽  
Author(s):  
Yoshihisa OHTANI ◽  
Tomo YONEZAWA ◽  
Sang-Houn SONG ◽  
Tatsuyuki TAKAHASHI ◽  
Astrid ARDIYANTI ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Epithelial Cells ◽  
Hormonal Regulation ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Bovine Mammary Gland

Milk fat globule is an alternative to mammary epithelial cells for gene expression analysis in buffalo

2016 ◽  
Vol 83 (2) ◽  
pp. 202-208 ◽  
Author(s):  
Qiuming Chen ◽  
Yanjun Wu ◽  
Mingyuan Zhang ◽  
Wenwen Xu ◽  
Xiaoping Guo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Epithelial Cells ◽  
Expression Analysis ◽  
Milk Fat ◽  
Gene Expression Analysis ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Milk Fat Globule ◽  
Fat Globule

Owing to the difficulty in obtaining mammary gland tissue from lactating animals, it is difficult to test the expression levels of genes in mammary gland. The aim of the current study was to identify if milk fat globule (MFG) in buffalo milk was an alternative to mammary gland (MG) and milk somatic cell (MSC) for gene expression analysis. Six buffalos in late lactation were selected to collect MFG and MSC, and then MG was obtained by surgery. MFG was stained with acridine orange to successfully visualise RNA and several cytoplasmic crescents in MFG. The total RNA in MFG was successfully isolated and the integrity was assessed by agarose gel electrophoresis. We analysed the cellular components in MFG, MG and MSC through testing the expression of cell-specific genes by qRT-PCR. The results showed that adipocyte-specific gene (AdipoQ) and leucocyte-specific genes (CD43, CSF1 and IL1α) in MFG were not detected, whereas epithelial cell marker genes (Keratin 8 and Keratin 18) in MFG were higher than in MSC and lower than in MG, fibroblast marker gene (vimentin) in MFG was significantly lower than in MG and MSC, milk protein genes (LALBA, BLG and CSN2) and milk fat synthesis-related genes (ACC, BTN1A1, FABP3 and FAS) in MFG were higher than in MG and MSC. In conclusion, the total RNA in MFG mainly derives from mammary epithelial cells and can be used to study the functional gene expression of mammary epithelial cells.

Differential expression of the growth hormone receptor and growth hormone-binding protein in epithelia and stroma of the mouse mammary gland at various physiological stages

1999 ◽  
Vol 161 (1) ◽  
pp. 77-87 ◽  
Author(s):  
YN Ilkbahar ◽  
G Thordarson ◽  
IG Camarillo ◽  
F Talamantes
Keyword(s):  
Growth Hormone ◽  
Mammary Gland ◽  
Epithelial Cells ◽  
Growth Hormone Receptor ◽  
Mammary Epithelial Cells ◽  
Late Pregnancy ◽  
Mammary Glands ◽  
Mouse Mammary Gland ◽  
Mammary Epithelial ◽  
Mrna Levels

Increasing evidence suggests that GH is important in normal mammary gland development. To investigate this further, we studied the distribution and levels of growth hormone receptor (GHR) and GH-binding protein (GHBP) in the mouse mammary gland. At three weeks of age, the epithelial component of the right fourth inguinal mammary gland of female mice was removed. These animals were then either maintained as virgins until they were killed or they were mated. One group of the mated mice was killed on day 18 of pregnancy and the remaining mated animals were allowed to carry their pups until term and were killed on day 6 of lactation. At the time of death, both the intact left and the de-epithelialized right mammary glands were collected from all three groups. Some of the intact glands served as a source of epithelial cells, free of stroma. The mRNA levels for GHR and GHBP were measured in intact glands, epithelia-cleared fat pads, and isolated mammary epithelial cells. GHR and GHBP mRNAs were expressed in both the mammary epithelium and stroma. However, the levels of both GHR and GHBP mRNAs were significantly higher in the stroma as compared with the epithelium component. This increase for both mRNAs was from 3- to 12-fold at each physiological state examined. In the intact gland, both GHR and GHBP transcripts were highest in virgins, declined during late pregnancy, and the lowest levels were found in the lactating gland. GHBP and GHR protein concentrations were also assessed in intact glands and epithelia-free fat pads. Similar to the mRNAs, GHR and GHBP protein levels (means+/-s.e.m.) in intact glands were highest in virgin mice (0.891+/-0.15 pmoles/mg protein and 0.136+/-0.26 pmoles/mg protein respectively), declined during late pregnancy (0. 354+/-0.111 pmoles/mg protein and 0.178+/-0.039 pmoles/mg protein respectively), and were lowest during lactation (0.096+0.037 pmoles/mg protein and 0.017+0.006 pmoles/mg protein respectively). Immunocytochemistry utilizing specific antisera against mouse (m) GHR and mGHBP revealed that the two proteins are localized to both the stroma and parenchyma of mouse mammary glands, with similar patterns of immunostaining throughout the different physiological stages analyzed. GHR immunolocalized to the plasma membrane and cytosol of mammary epithelial cells and adipocytes, whereas the GHBP immunostaining was nuclear and cytosolic. In conclusion, we report here that GHR and GHBP mRNAs and proteins are expressed in both the epithelium and the stroma of mammary glands of virgin, pregnant, and lactating mice. In intact glands, GHR and GHBP proteins, as well as their transcripts are higher in abundance in virgin relative to lactating mice. At all physiological stages, GHR and GHBP mRNA levels are higher in the stroma compared with the parenchyma. These findings indicate that the actions of GH in the mammary gland are both direct through its binding to the epithelia, and indirect by binding to the stroma and stimulation of IGF-I production which, in turn, affects mammary epithelial development.

scholarly journals Immunolocalization of Antibacterial Peptide S100A7 in Goat Mammary Gland and Lipopolysaccharide Induces the Expression and Secretion of S100A7 in Goat Mammary Gland Epithelial Cells via TLR4/NFκB Signal Pathway

2021 ◽  
Author(s):  
Yutong Yan ◽  
Mingzhen Fan ◽  
Kunyuan Zhu ◽  
Yuyang Miao ◽  
Xiaoe Zhao ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Epithelial Cells ◽  
Squamous Epithelium ◽  
Mammary Epithelial Cells ◽  
Antimicrobial Activities ◽  
Mammary Epithelial ◽  
Regulation Mechanism ◽  
Mrna Levels ◽  
Protein Levels ◽  
Stratified Squamous Epithelium

Abstract Background: The antimicrobial peptide (AMP) S100A7, with antimicrobial activities for a broad spectrum of bacteria, have attracted more and more attention for the prevention and treatment of mastitis. However, in goat mastitis, there is little information about the expression and regulation mechanism of S100A7. In present study, the immunolocalization of S100A7 in healthy and mastitis goat udder were compared. In order to further explore the regulatory mechanism of S100A7 expression in mammary epithelial cells (MECs), goat MECs were isolated and treated by 2.5, 5, 10 and 20 μg/mL lipopolysaccharide (LPS) respectively for different time.Results: Both in healthy and mastitis goat teat, S100A7 was mainly expressed in stratified squamous epithelium of teat skin and streak canal. In healthy goat mammary gland, weakly S100A7 immunoreactivity was present in the alveolus. But in the collapsed alveolus of mastitis goat mammary gland, densely S100A7 immunoreactivity could be observed.The goat MECs were treated by 2.5, 5, 10 and 20 μg/mL LPS respectively for different time. For all of these four groups, after treatment for 3 h, increase in S100A7 mRNA expression and protein secretion were detected compared to control (p<0.05). For 10 and 20μg/mL LPS groups, after treatment for 6 h, the mRNA and secreted protein levels of S100A7 were remarkably up-regulated compared to control(p<0.01). For all of these four groups, the secretion level of S100A7 descended after 48 h treatment. Moreover, after treatment with LPS, the mRNA levels of Toll-like receptor 4(TLR4) and MyD88 were up-regulated, and the phosphorylation of p65 was up-regulated markedly compared to control. However, adding TLR4 inhibitor TAK-242 or/and NF-κB inhibitor QNZ significantly suppressed the phosphorylation of p-65，and then inhibited the expression and secretion of S100A7 induced by LPS treatment.Conclusions: S100A7 was mainly expressed in stratified squamous epithelium of teat skin and streak canal. In mastitis goat mammary gland alveolus, the expression level of S100A7 was up-regulated compared to that in healthy goat. LPS induced the expression and secretion of S100A7 in goat MECs depended on concentration and treatment duration. Moreover, LPS induced the expression and secretion of S100A7 in goat MECs via TLR4/NF-κB signaling pathway.

scholarly journals Characterization of Gene Expression Signatures for the Identification of Cellular Heterogeneity in the Developing Mammary Gland

2021 ◽  
Author(s):  
Samantha Henry ◽  
Marygrace C. Trousdell ◽  
Samantha L. Cyrill ◽  
Yixin Zhao ◽  
Mary. J. Feigman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Epithelial Cells ◽  
Single Cell ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Cellular Heterogeneity ◽  
Molecular Signatures ◽  
Mammary Cells ◽  
Human Breast

AbstractThe developing mammary gland depends on several transcription-dependent networks to define cellular identities and differentiation trajectories. Recent technological advancements that allow for single-cell profiling of gene expression have provided an initial picture into the epithelial cellular heterogeneity across the diverse stages of gland maturation. Still, a deeper dive into expanded molecular signatures would improve our understanding of the diversity of mammary epithelial and non-epithelial cellular populations across different tissue developmental stages, mouse strains and mammalian species. Here, we combined differential mammary gland fractionation approaches and transcriptional profiles obtained from FACS-isolated mammary cells to improve our definitions of mammary-resident, cellular identities at the single-cell level. Our approach yielded a series of expression signatures that illustrate the heterogeneity of mammary epithelial cells, specifically those of the luminal fate, and uncovered transcriptional changes to their lineage-defined, cellular states that are induced during gland development. Our analysis also provided molecular signatures that identified non-epithelial mammary cells, including adipocytes, fibroblasts and rare immune cells. Lastly, we extended our study to elucidate expression signatures of human, breast-resident cells, a strategy that allowed for the cross-species comparison of mammary epithelial identities. Collectively, our approach improved the existing signatures of normal mammary epithelial cells, as well as elucidated the diversity of non-epithelial cells in murine and human breast tissue. Our study provides a useful resource for future studies that use single-cell molecular profiling strategies to understand normal and malignant breast development.

scholarly journals Evidence of Rab3A Expression, Regulation of Vesicle Trafficking, and Cellular Secretion in Response to Heregulin in Mammary Epithelial Cells

2000 ◽  
Vol 20 (23) ◽  
pp. 9092-9101 ◽  
Author(s):  
Ratna K. Vadlamudi ◽  
Rui-An Wang ◽  
Amjad H. Talukder ◽  
Liana Adam ◽  
Randy Johnson ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Vesicle Trafficking ◽  
Mammary Epithelial ◽  
Coated Vesicle ◽  
Human Breast ◽  
Breast Epithelial Cells ◽  
Breast Epithelial ◽  
Stimulation Of

ABSTRACT Heregulin β1 (HRG), a combinatorial ligand for human growth factor receptors 3 and 4, is a regulatory polypeptide that promotes the differentiation of mammary epithelial cells into secretory lobuloalveoli. Emerging evidence suggests that the processes of secretory pathways, such as biogenesis and trafficking of vesicles in neurons and adipose cells, are regulated by the Rab family of low-molecular-weight GTPases. In this study, we identified Rab3A as a gene product induced by HRG. Full-length Rab3A was cloned from a mammary gland cDNA library. We demonstrated that HRG stimulation of human breast cancer cells and normal breast epithelial cells induces the expression of Rab3A protein and mRNA in a cycloheximide-independent manner. HRG-mediated induction of Rab3A expression was blocked by an inhibitor of phosphatidylinositol 3-kinase but not by inhibitors of mitogen-activated protein kinases p38MAPK and p42/44MAPK. Human breast epithelial cells also express other components of regulated vesicular traffic, such as rabphilin 3A, Doc2, and syntaxin. Rab3A was predominantly localized in the cytosol, and HRG stimulation of the epithelial cells also raised the level of membrane-bound Rab3A. HRG treatment induced a profound alteration in the cell morphology in which cells displayed neuron-like membrane extensions that contained Rab3A-coated, vesicle-like structures. In addition, HRG also promoted the secretion of cellular proteins from the mammary epithelial cells. The ability of HRG to modify exocytosis was verified by using a growth hormone transient-transfection system. Analysis of mouse mammary gland development revealed the expression of Rab3A in mammary epithelial cells. Furthermore, expression of the HRG transgene in Harderian tumors in mice also enhanced the expression of Rab3A. These observations provide new evidence of the existence of a Rab3A pathway in mammary epithelial cells and suggest that it may play a role in vesicle trafficking and secretion of proteins from epithelial cells in response to stimulation by the HRG expressed within the mammary mesenchyma.

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