Metalloproteinase axes increase beta-catenin signaling in primary mouse mammary epithelial cells lacking TIMP3

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.

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Role of Toll-like receptor 2 against Streptococcus uberis infection in primary mouse mammary epithelial cells

International Immunopharmacology ◽

10.1016/j.intimp.2019.106142 ◽

2020 ◽

Vol 79 ◽

pp. 106142 ◽

Cited By ~ 1

Author(s):

Zhixin Wan ◽

Xudong Wang ◽

Ming Liu ◽

Jiakun Zuo ◽

Yuanyuan Xu ◽

...

Keyword(s):

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Toll Like Receptor ◽

Streptococcus Uberis ◽

Primary Mouse ◽

Toll Like Receptor 2

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The EGF-CFC family: novel epidermal growth factor-related proteins in development and cancer.

Endocrine Related Cancer ◽

10.1677/erc.0.0070199 ◽

2000 ◽

pp. 199-226 ◽

Cited By ~ 83

Author(s):

D S Saloman ◽

C Bianco ◽

A D Ebert ◽

N I Khan ◽

M De Santis ◽

...

Keyword(s):

Mammary Gland ◽

Growth Factor ◽

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Branching Morphogenesis ◽

Mammary Epithelial ◽

Biologically Active ◽

Beta Catenin ◽

Mesenchymal Transition ◽

Related Proteins

The EGF-CFC gene family encodes a group of structurally related proteins that serve as important competence factors during early embryogenesis in Xenopus, zebrafish, mice and humans. This multigene family consists of Xenopus FRL-1, zebrafish one-eyed-pinhead (oep), mouse cripto (Cr-1) and cryptic, and human cripto (CR-1) and criptin. FRL-1, oep and mouse cripto are essential for the formation of mesoderm and endoderm and for correct establishment of the anterior/posterior axis. In addition, oep and cryptic are important for the establishment of left-right (L/R) asymmetry. In zebrafish, there is strong genetic evidence that oep functions as an obligatory co-factor for the correct signaling of a transforming growth factor-beta (TGFbeta)-related gene, nodal, during gastrulation and during L/R asymmetry development. Expression of Cr-1 and cryptic is extinguished in the embryo after day 8 of gestation except for the developing heart where Cr-1 expression is necessary for myocardial development. In the mouse, cryptic is not expressed in adult tissues whereas Cr-1 is expressed at a low level in several different tissues including the mammary gland. In the mammary gland, expression of Cr-1 in the ductal epithelial cells increases during pregnancy and lactation and immunoreactive and biologically active Cr-1 protein can be detected in human milk. Overexpression of Cr-1 in mouse mammary epithelial cells can facilitate their in vitro transformation and in vivo these Cr-1-transduced cells produce ductal hyperplasias in the mammary gland. Recombinant mouse or human cripto can enhance cell motility and branching morphogenesis in mammary epithelial cells and in some human tumor cells. These effects are accompanied by an epithelial-mesenchymal transition which is associated with a decrease in beta-catenin function and an increase in vimentin expression. Expression of cripto is increased several-fold in human colon, gastric, pancreatic and lung carcinomas and in a variety of different types of mouse and human breast carcinomas. More importantly, this increase can first be detected in premalignant lesions in some of these tissues. Although a specific receptor for the EGF-CFC proteins has not yet been identified, oep depends upon an activin-type RIIB and RIB receptor system that functions through Smad-2. Mouse and human cripto have been shown to activate a ras/raf/MAP kinase signaling pathway in mammary epithelial cells. Activation of phosphatidylinositol 3-kinase and Akt are also important for the ability of CR-1 to stimulate cell migration and to block lactogenic hormone-induced expression of beta-casein and whey acidic protein. In mammary epithelial cells, part of these responses may depend on the ability of CR-1 to transactivate erb B-4 and/or fibroblast growth factor receptor 1 through an src-like tyrosine kinase.

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IL-4 and IL-13 Promote Proliferation of Mammary Epithelial Cells through STAT6 and IRS-1

International Journal of Molecular Sciences ◽

10.3390/ijms222112008 ◽

2021 ◽

Vol 22 (21) ◽

pp. 12008

Author(s):

Wan-Ju Wu ◽

Sue-Hong Wang ◽

Chun-Chi Wu ◽

Yi-An Su ◽

Chin-Yin Chiang ◽

...

Keyword(s):

Cell Proliferation ◽

Epithelial Cells ◽

Tyrosine Phosphorylation ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Prolonged Treatment ◽

Signaling Crosstalk ◽

Promote Cell Proliferation ◽

Primary Mouse ◽

Cytokine Milieu

T helper (Th)2 cytokines such as interleukin (IL)-4 and IL-13 control immune function by acting on leukocytes. They also regulate multiple responses in non-hematopoietic cells. During pregnancy, IL-4 and IL-13 facilitate alveologenesis of mammary glands. This particular morphogenesis generates alveoli from existing ducts and requires substantial cell proliferation. Using 3D cultures of primary mouse mammary epithelial cells, we demonstrate that IL-4 and IL-13 promote cell proliferation, leading to enlargement of mammary acini with partially filled lumens. The mitogenic effects of IL-4 and IL-13 are mediated by STAT6 as inhibition of STAT6 suppresses cell proliferation and improves lumen formation. In addition, IL-4 and IL-13 stimulate tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). Prolonged treatment with these cytokines leads to increased IRS-1 abundance, which, in turn, amplifies IL-4- and IL-13-stimulated IRS-1 tyrosine phosphorylation. Through signaling crosstalk between IL-4/IL-13 and insulin, a hormone routinely included in mammary cultures, IRS-1 tyrosine phosphorylation is further enhanced. Lowering IRS-1 expression reduces cell proliferation, suggesting that IRS-1 is involved in IL-4- and IL-13-stimulated cell proliferation. Thus, a Th2-dominant cytokine milieu during pregnancy confers mammary gland development by promoting cell proliferation.

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Cytodifferentiation of mouse mammary epithelial cells cultured on a reconstituted basement membrane reveals striking similarities to development in vivo

Journal of Cell Science ◽

10.1242/jcs.99.2.407 ◽

1991 ◽

Vol 99 (2) ◽

pp. 407-417 ◽

Cited By ~ 3

Author(s):

J. Aggeler ◽

J. Ward ◽

L.M. Blackie ◽

M.H. Barcellos-Hoff ◽

C.H. Streuli ◽

...

Keyword(s):

Epithelial Cells ◽

Basement Membrane ◽

Milk Fat ◽

Milk Protein ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Milk Protein Gene ◽

Primary Mouse ◽

Beta Casein

In the present study we provide evidence that the cytodifferentiation of primary mouse mammary epithelial cells within the alveolar-like structures formed after culture on a reconstituted basement membrane resembles development in vivo during late pregnancy and early lactation. During the first two days in culture on a basement membrane gel in the presence of lactogenic hormones, epithelial cells isolated from mid-pregnant mice are disorganized and central lumina are largely absent. Levels of mRNA for the milk proteins, beta-casein and transferrin, are dramatically reduced. By the second or third day in culture, cytoplasmic polarization becomes evident and prominent apical junctional complexes are formed. Synthesis of both mRNA and milk protein is reinitiated at this time. By day 4, well-defined lumina appear, and abundant synthesis and secretion of casein and lipid is observed. A striking feature of this differentiation in culture is the specific localization of milk protein gene expression (beta-casein mRNA) to luminal epithelial cells in the alveolar-like structures. At the ultrastructural level, increased milk protein synthesis and secretion are paralleled by a fourfold increase in rough ER that resembles the dramatic increase in the ER observed in vivo following parturition. One indication of tissue-specific differentiation observed in later cultures (days 4–11) is the synthesis and secretion of abundant casein micelles. A second characteristic of lactating mammary epithelial cells in vivo that has not previously been observed in culture is the secretion of milk fat globules. Taken together, these observations indicate that mammary epithelial cells plated onto a reconstituted basement membrane differentiate to the lactating phenotype in culture.

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Casitas B-cell lymphoma (Cbl) proteins protect mammary epithelial cells from proteotoxicity of active c-Src accumulation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1615677113 ◽

2016 ◽

Vol 113 (51) ◽

pp. E8228-E8237 ◽

Cited By ~ 4

Author(s):

Chandrani Mukhopadhyay ◽

Aleata Triplett ◽

Tom Bargar ◽

Carol Heckman ◽

Kay-Uwe Wagner ◽

...

Keyword(s):

Cell Death ◽

Epithelial Cells ◽

B Cell ◽

Kinase Inhibitor ◽

Mammary Epithelial Cells ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Mammary Epithelial ◽

Protein Aggregate ◽

Primary Mouse

Casitas B-cell lymphoma (Cbl) family ubiquitin ligases negatively regulate tyrosine kinase-dependent signal transduction by promoting degradation of active kinases. We and others previously reported that loss of Cbl functions caused hyperproliferation in lymphoid and hematopoietic systems. Unexpectedly, Cbl deletion in Cbl-b–null, Cbl-c–null primary mouse mammary epithelial cells (MECs) (Cbl triple-deficiency) induced rapid cell death despite enhanced MAP kinase and AKT activation. Acute Cbl triple-deficiency elicited distinct transcriptional and biochemical responses with partial overlap with previously described cellular reactions to unfolded proteins and oxidative stress. Although the levels of reactive oxygen species were comparable, detergent-insoluble protein aggregates containing phosphorylated c-Src accumulated in Cbl triple-deficient MECs. Treatment with a broad-spectrum kinase inhibitor dasatinib blocked protein aggregate accumulation and restored in vitro organoid formation. This effect is most likely mediated through c-Src because Cbl triple-deficient MECs were able to form organoids upon shRNA-mediated c-Src knockdown. Taking these data together, the present study demonstrates that Cbl family proteins are required to protect MECs from proteotoxic stress-induced cell death by promoting turnover of active c-Src.

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