Mammary gland morphogenesis is inhibited in transgenic mice that overexpress cell surface beta1,4-galactosyltransferase

Development ◽  
1996 ◽  
Vol 122 (9) ◽  
pp. 2859-2872 ◽  
Author(s):  
H.J. Hathaway ◽  
B.D. Shur
Keyword(s):  
Mammary Gland ◽  
Cell Surface ◽  
Transgenic Animals ◽  
Mammary Epithelial ◽  
Wild Type ◽  
Cell Matrix ◽  
Matrix Interactions ◽  
Mammary Gland Morphogenesis ◽  
Gland Morphogenesis ◽  
Cell Matrix Interactions

Mammary gland morphogenesis is facilitated by a precise sequence of cell-cell and cell-matrix interactions, which are mediated in part through a variety of cell surface receptors and their ligands (Boudreau, N., Myers, C. and Bissell, M. J. (1995). Trends in Cell Biology 5, 1–4). Cell surface beta1,4-galactosyltransferase (GalTase) is one receptor that participates in a variety of cell-cell and cell-matrix interactions during fertilization and development, including mammary epithelial cell-matrix interactions (Barcellos-Hoff, M. H. (1992). Exp. Cell Res. 201, 225–234). To analyze GalTase function during mammary gland morphogenesis in vivo, we created transgenic animals that overexpress the long isoform of GalTase under the control of a heterologous promoter. As expected, mammary epithelial cells from transgenic animals had 2.3 times more GalTase activity on their cell surface than did wild-type cells. Homozygous transgenic females from multiple independent lines failed to lactate, whereas transgenic mice overexpressing the Golgi-localized short isoform of GalTase lactated normally. Glands from transgenic females overexpressing surface GalTase were characterized by abnormal and reduced ductal development with a concomitant reduction in alveolar expansion during pregnancy. The phenotype was not due to a defect in proliferation, since the mitotic index for transgenic and wild-type glands was similar. Morphological changes were accompanied by a dramatic reduction in the expression of milk-specific proteins. Immunohistochemical markers for epithelia and myoepithelia demonstrated that both cell types were present. To better understand how overexpression of surface GalTase impairs ductal morphogenesis, primary mammary epithelial cultures were established on basement membranes. Cultures derived from transgenic mammary glands were unable to form anastomosing networks of epithelial cells and failed to express milk-specific proteins, unlike wild-type mammary cultures that formed epithelial tubules and expressed milk proteins. Our results suggest that cell surface GalTase is an important mediator of mammary cell interaction with the extracellular matrix. Furthermore, perturbing surface GalTase levels inhibits the expression of mammary-specific gene products, implicating GalTase as a component of a receptor-mediated signal transduction pathway required for normal mammary gland differentiation.

scholarly journals Cyclin-Dependent Kinase Inhibitor P27Kip1 Is Required for Mouse Mammary Gland Morphogenesis and Function

2001 ◽  
Vol 153 (5) ◽  
pp. 917-932 ◽  
Author(s):  
Rebecca S. Muraoka ◽  
Anne E.G. Lenferink ◽  
Jean Simpson ◽  
Dana M. Brantley ◽  
L. Renee Roebuck ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Cyclin D1 ◽  
Mammary Epithelial Cells ◽  
Mammary Glands ◽  
Mammary Epithelial ◽  
Cyclin Dependent Kinase ◽  
Wild Type ◽  
Mammary Gland Morphogenesis ◽  
Ductal Branching ◽  
Gland Morphogenesis

We have studied the role of the cyclin-dependent kinase (Cdk) inhibitor p27Kip1 in postnatal mammary gland morphogenesis. Based on its ability to negatively regulate cyclin/Cdk function, loss of p27 may result in unrestrained cellular proliferation. However, recent evidence about the stabilizing effect of p27 on cyclin D1–Cdk4 complexes suggests that p27 deficiency might recapitulate the hypoplastic mammary phenotype of cyclin D1–deficient animals. These hypotheses were investigated in postnatal p27-deficient (p27−/−), hemizygous (p27+/−), or wild-type (p27+/+) mammary glands. Mammary glands from p27+/− mice displayed increased ductal branching and proliferation with delayed postlactational involution. In contrast, p27−/− mammary glands or wild-type mammary fat pads reconstituted with p27−/− epithelium produced the opposite phenotype: hypoplasia, low proliferation, decreased ductal branching, impaired lobuloalveolar differentiation, and inability to lactate. The association of cyclin D1 with Cdk4, the kinase activity of Cdk4 against pRb in vitro, the nuclear localization of cyclin D1, and the stability of cyclin D1 were all severely impaired in p27−/− mammary epithelial cells compared with p27+/+ and p27+/− mammary epithelial cells. Therefore, p27 is required for mammary gland development in a dose-dependent fashion and positively regulates cyclin D–Cdk4 function in the mammary gland.

scholarly journals Matricellular Proteins as Modulators of Cell–Matrix Interactions: Adhesive Defect in Thrombospondin 2-null Fibroblasts is a Consequence of Increased Levels of Matrix Metalloproteinase-2

2000 ◽  
Vol 11 (10) ◽  
pp. 3353-3364 ◽  
Author(s):  
Zhantao Yang ◽  
Themis R. Kyriakides ◽  
Paul Bornstein
Keyword(s):  
Molecular Mechanisms ◽  
Fold Increase ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Matrix Metalloproteinase 2 ◽  
Wild Type ◽  
Matricellular Proteins ◽  
Altered Expression ◽  
Cell Matrix ◽  
Matrix Interactions ◽  
Cell Matrix Interactions

Thrombospondin 2 (TSP2)-null mice, generated by disruption of theThbs2 gene, display a variety of connective tissue abnormalities, including fragile skin and the presence of abnormally large collagen fibrils with irregular contours in skin and tendon. In this study we demonstrate that TSP2-null skin fibroblasts show a defect in attachment to a number of matrix proteins, and a reduction in cell spreading. To investigate the molecular mechanisms responsible for these abnormal cell–matrix interactions, we compared the levels of matrix metalloproteinases (MMPs) in wild-type and mutant fibroblasts. Isolation and analysis of gelatinases from conditioned media by gelatin-agarose affinity chromatography and gelatinolytic assays demonstrated that TSP2-null fibroblasts produce a 2-fold increase in gelatinase A (MMP2) compared with wild-type cells. The adhesive defect was corrected by treatment of TSP2-null fibroblasts with soluble TSP2, with the MMP inhibitors BB94 and tissue inhibitor of metalloproteinase-2, and with a neutralizing antibody to MMP2. Moreover, stable transfection of TSP2-null fibroblasts with mouse TSP2 cDNA corrected both the adhesive defect and the altered expression of MMP2. Finally, MMP2 was shown to interact with TSP2 in a direct-binding plate assay. We conclude that TSP2 plays an important role in cell–matrix interactions, and that a deficiency in the protein results in increased levels of MMP2 that contribute to the adhesive defect in TSP2-null fibroblasts and could play a role in the complex phenotype of TSP2-null mice.

scholarly journals Bi-transgenic Mice Reveal that K-rasVal12 Augments a p53-independent Apoptosis When Small Intestinal Villus Enterocytes Reenter the Cell Cycle

1997 ◽  
Vol 138 (1) ◽  
pp. 167-179 ◽  
Author(s):  
Craig M. Coopersmith ◽  
Chitra Chandrasekaran ◽  
M. Shane McNevin ◽  
Jeffrey I. Gordon
Keyword(s):  
Cell Cycle ◽  
Transgenic Mice ◽  
Transgenic Animals ◽  
Wild Type ◽  
Activating Mutations ◽  
Cell Cycle Reentry ◽  
Cell Matrix ◽  
Small Intestinal ◽  
Small Intestinal Villus ◽  
Cell Matrix Interactions

Studies in cell culture systems have indicated that oncogenic forms of Ras can affect apoptosis. Activating mutations of Ras occur in ∼30% of all human tumors and 50% of colorectal carcinomas. Since these mutations appear at early or intermediate stages in multistep journeys to neoplasia, an effect on apoptosis may help determine whether initiated cells progress towards a more neoplastic state. We have tested the effects of K-rasVal12 on apoptosis in transgenic mice. A lineage-specific promoter was used to direct expression of human K-rasVal12, with or without wild-type (wt) or mutant SV-40 T antigens (TAg), in postmitotic villus enterocytes, the principal cell type of the small intestinal epithelium. Enterocytes can be induced to reenter the cell cycle by TAgWt. Reentry is dependent upon the ability of TAg to bind pRB and is associated with a p53-independent apoptosis. Analyses of K-rasVal12 × TAgWt bi-transgenic animals indicated that K-rasVal12 can enhance this apoptosis threefold but only in cycling cells; increased apoptosis does not occur when K-rasVal12 is expressed alone or with a TAg containing Glu107,108→ Lys107,108 substitutions that block its ability to bind pRB. Analysis of bi-transgenic K-rasVal12 × TAgWt mice homozygous for wild-type or null p53 alleles established that the enhancement of apoptosis occurs through a p53-independent mechanism, is not attributable to augmented proliferation or to an increase in abortive cell cycle reentry (compared to TAgWt mice), and is not associated with detectable changes in the crypt–villus patterns of expression of apoptotic regulators (Bcl-2, Bcl-xL, Bak, and Bax) or mediators of epithelial cell–matrix interactions and survival (e.g., α5β1 integrin and its ligand, fibronectin). Coexpression of K-rasVal12 and TAgWt produces dysplasia. The K-rasVal12-augmented apoptosis is unrelated to this dysplasia; enhanced apoptosis is also observed in cycling nondysplastic enterocytes that produce K-rasVal12 and a TAg with a COOH-terminal truncation. The dysplastic epithelium of K-rasVal12 × TAgWt mice does not develop neoplasms. Our results are consistent with this finding: (a) When expressed in initiated enterocytes with a proliferative abnormality, K-rasVal12 facilitates progression to a dysplastic phenotype; (b) by diminishing cell survival on the villus, the oncoprotein may impede further progression; and (c) additional mutations may be needed to suppress this proapoptotic response to K-rasVal12.

Vitamin D3 receptor ablation alters mammary gland morphogenesis

Development ◽  
2002 ◽  
Vol 129 (13) ◽  
pp. 3067-3076
Author(s):  
Glendon Zinser ◽  
Kathryn Packman ◽  
JoEllen Welsh
Keyword(s):  
Mammary Gland ◽  
Vitamin D3 ◽  
Stromal Cells ◽  
Knockout Mice ◽  
Wild Type ◽  
Dihydroxyvitamin D3 ◽  
Mammary Gland Morphogenesis ◽  
Gland Morphogenesis ◽  
Gland Development

Postnatal mammary gland morphogenesis is achieved through coordination of signaling networks in both the epithelial and stromal cells of the developing gland. While the major proliferative hormones driving pubertal mammary gland development are estrogen and progesterone, studies in transgenic and knockout mice have successfully identified other steroid and peptide hormones that impact on mammary gland development. The vitamin D3 receptor (VDR), whose ligand 1,25-dihydroxyvitamin D3 is the biologically active form of vitamin D3, has been implicated in control of differentiation, cell cycle and apoptosis of mammary cells in culture, but little is known about the physiological relevance of the vitamin D3 endocrine system in the developing gland. In these studies, we report the expression of the VDR in epithelial cells of the terminal end bud and subtending ducts, in stromal cells and in a subset of lymphocytes within the lymph node. In the terminal end bud, a distinct gradient of VDR expression is observed, with weak VDR staining in proliferative populations and strong VDR staining in differentiated populations. The role of the VDR in ductal morphogenesis was examined in Vdr knockout mice fed high dietary Ca2+ which normalizes fertility, serum estrogen and neonatal growth. Our results indicate that mammary glands from virgin Vdr knockout mice are heavier and exhibit enhanced growth, as evidenced by higher numbers of terminal end buds, greater ductal outgrowth and enhanced secondary branch points, compared with glands from age- and weight-matched wild-type mice. In addition, glands from Vdr knockout mice exhibit enhanced growth in response to exogenous estrogen and progesterone, both in vivo and in organ culture, compared with glands from wild-type mice. Our data provide the first in vivo evidence that 1,25-dihydroxyvitamin D3 and the VDR impact on ductal elongation and branching morphogenesis during pubertal development of the mammary gland. Collectively, these results suggest that the vitamin D3 signaling pathway participates in negative growth regulation of the mammary gland.

Lack of ADAM15 in mice is associated with increased osteoblast function and bone mass

2011 ◽  
Vol 392 (10) ◽  
pp. 877-885 ◽  
Author(s):  
Marilena Marzia ◽  
Victor Guaiquil ◽  
William C. Horne ◽  
Carl P. Blobel ◽  
Roland Baron ◽  
...  
Keyword(s):  
Bone Mass ◽  
Cell Activation ◽  
Nuclear Translocation ◽  
Wild Type ◽  
Cell Matrix ◽  
Matrix Interactions ◽  
Skeletal Homeostasis ◽  
Cortical Bone Mass ◽  
And Function ◽  
Cell Matrix Interactions

Abstract The ADAMs (a disintegrin and metalloprotease) contribute to various biological functions including the development of tissues by taking part in cell-cell and cell-matrix interactions. We previously found that ADAM15 is prominently expressed in osteoblasts and to a lesser extent in osteoclasts. The aim of this study was to investigate a possible function of ADAM15 in bone. Adult ADAM15-/- mice displayed an increase in bone volume and thickness with an increase in the number and activity of osteoblasts, whereas osteoclasts were apparently unaffected. We found an increase in proliferation, alkaline phosphatase (ALP) staining and nodule deposition, and mineralization in cultures of ADAM15-/- osteoblasts compared to wild-type osteoblasts. We also observed an increase in β-catenin immunoreactivity in the nucleus of ADAM15-/- osteoblasts compared to wild-type, whereas β-catenin in the membrane/cytoplasm compartment appeared to undergo increased degradation. Furthermore, cyclin D1 and c-Jun, known downstream targets of β-catenin and effectors of cell activation, were found up-regulated in absence of ADAM15. This study indicates that ADAM15 is required for normal skeletal homeostasis and that its absence causes increased nuclear translocation of β-catenin in osteoblasts leading to increased osteoblast proliferation and function, which results in higher trabecular and cortical bone mass.

scholarly journals Essential function of Wnt-4 in mammary gland development downstream of progesterone signaling

2000 ◽  
Vol 14 (6) ◽  
pp. 650-654 ◽  
Author(s):  
Cathrin Brisken ◽  
Anna Heineman ◽  
Tony Chavarria ◽  
Brian Elenbaas ◽  
Jian Tan ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Mammary Epithelial Cells ◽  
Ectopic Expression ◽  
Mammary Epithelium ◽  
Reproductive Hormones ◽  
Mammary Epithelial ◽  
Mammary Gland Morphogenesis ◽  
Mammary Epithelia ◽  
Gland Morphogenesis ◽  
Gland Development

Female reproductive hormones control mammary gland morphogenesis. In the absence of the progesterone receptor (PR) from the mammary epithelium, ductal side-branching fails to occur. We can overcome this defect by ectopic expression of the protooncogene Wnt-1. Transplantation of mammary epithelia fromWnt-4−/− mice shows that Wnt-4 has an essential role in side-branching early in pregnancy. PR andWnt-4 mRNAs colocalize to the luminal compartment of the ductal epithelium. Progesterone induces Wnt-4 in mammary epithelial cells and is required for increased Wnt-4 expression during pregnancy. Thus, Wnt signaling is essential in mediating progesterone function during mammary gland morphogenesis.

scholarly journals Increased recycling of (alpha)5(beta)1 integrins by lung endothelial cells in response to tumor necrosis factor

2000 ◽  
Vol 113 (2) ◽  
pp. 247-257 ◽  
Author(s):  
B. Gao ◽  
T.M. Curtis ◽  
F.A. Blumenstock ◽  
F.L. Minnear ◽  
T.M. Saba
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Immunofluorescence Microscopy ◽  
Tnf Alpha ◽  
Cell Matrix ◽  
Endothelial Monolayers ◽  
Matrix Interactions ◽  
Cell Matrix Interactions ◽  
Protein Permeability ◽  
Necrosis Factor

Tumor necrosis factor (alpha) (TNF-(alpha) can change the interaction of lung endothelial cell monolayers with their extracellular matrix in association with an increase in endothelial monolayer protein permeability. Using immunofluorescence microscopy and flow cytometry, we determined if exposure of calf pulmonary artery endothelial monolayers to TNF-(alpha) may influence cell-matrix interactions by altering the clustering as well as internalization of the (α)5(beta)1 integrins (or fibronectin receptors) on the surface of endothelial cells. Immunofluorescence microscopy revealed that TNF-(alpha) caused an increase in the intracellular staining of (alpha)5(alpha)1 integrins within structures similar to endocytic vesicles as well as an increase in antibody-induced clustering of the integrins at the cell periphery. Flow cytometric analysis of endothelial cells incubated at 37 degrees C after antibody-labeling of their surface (alpha)5(beta)1 integrins at 4 degrees C confirmed an increase in the rate of (alpha)5(beta)1 integrin internalization which was at least 3 times greater after TNF-(α) exposure, based on the half-life for antibody-labeled surface integrins to reach equilibrium with non-labeled integrins within the intracellular pool. Interestingly, the total cell surface expression of (alpha)5(beta)1 integrins was relatively constant after TNF-(alpha) exposure despite the enhanced rate of internalization, suggesting an accelerated recycling of the internalized (alpha)5(beta)1 integrins back to the cell surface. This response was confirmed by the measurement of labeled integrin recycling, which showed a significant (P<0.01) increase in the rate of recycling of the internalized integrins in TNF-treated endothelial cells. Enhanced internalization and subsequent recycling of (alpha)5(beta)1 integrins by endothelial monolayers exposed to TNF-(alpha) may facilitate the redistribution of cell-surface integrins in response to this inflammatory cytokine and may also modify cell-matrix interactions leading to reduced integrity and increased protein permeability of the lung endothelial monolayers.

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