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.

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 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 Enteral arginase II provides ornithine for citrulline synthesis

2011 ◽  
Vol 300 (1) ◽  
pp. E188-E194 ◽  
Author(s):  
Juan C. Marini ◽  
Bettina Keller ◽  
Inka Cajo Didelija ◽  
Leticia Castillo ◽  
Brendan Lee
Keyword(s):  
Small Intestine ◽  
Stable Isotope ◽  
Transgenic Mice ◽  
Small Intestinal Mucosa ◽  
Wild Type ◽  
Proline Utilization ◽  
Small Intestinal ◽  
Arginase Ii ◽  
Citrulline Synthesis

The synthesis of citrulline from arginine in the small intestine depends on the provision of ornithine. To test the hypothesis that arginase II plays a central role in the supply of ornithine for citrulline synthesis, the contribution of dietary arginine, glutamine, and proline was determined by utilizing multitracer stable isotope protocols in arginase II knockout (AII−/−) and wild-type (WT) mice. The lack of arginase II resulted in a lower citrulline rate of appearance (121 vs. 137 μmol·kg−1·h−1) due to a reduced availability of ornithine; ornithine supplementation was able to restore the rate of citrulline production in AII−/− to levels comparable with WT mice. There were significant differences in the utilization of dietary citrulline precursors. The contribution of dietary arginine to the synthesis of citrulline was reduced from 45 to 10 μmol·kg−1·h−1 due to the lack of arginase II. No enteral utilization of arginine was observed in AII−/− mice (WT = 25 μmol·kg−1·h−1), and the contribution of dietary arginine through plasma ornithine was reduced in the transgenic mice (20 vs. 13 μmol·kg−1·h−1). Dietary glutamine and proline utilization were greater in AII−/− than in WT mice (20 vs. 13 and 1.4 vs. 3.7 μmol·kg−1·h−1, respectively). Most of the contribution of glutamine and proline was enteral rather than through plasma ornithine. The arginase isoform present in the small intestinal mucosa has the role of providing ornithine for citrulline synthesis. The lack of arginase II results in a greater contribution of plasma ornithine and dietary glutamine and proline to the synthesis of citrulline.

Loss of the INK4A Locus Accelerates tal1/scl-Induced Leukemia in Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3495-3495
Author(s):  
Jennifer A. Calvo ◽  
Michelle Kelliher
Keyword(s):  
Cell Cycle ◽  
T Cell ◽  
Transgenic Mice ◽  
Median Survival ◽  
S Phase ◽  
Bhlh Transcription Factor ◽  
Human Leukemia ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Wild Type

Abstract Tal1/scl, a basic helix-loop-helix (bHLH) transcription factor essential for hematopoiesis and vasculogenesis, is also found misexpressed in patients with T cell acute lymphoblastic leukemia (T-ALL). In our mouse model of tal1/scl-induced disease, mice develop T cell leukemia after a long latency, suggesting that additional mutations are required for leukemogenesis. In T-ALL patients, the INK4a locus, which encodes both p14ARF and p16INK4a, is often found mutated or methylated, suggesting that loss of p16INK4A/p14ARF expression contributes to the development of T-ALL. Similarly, we fail to detect p16INK4A/p19ARF expression in our mouse tal1/scl leukemic cells. To determine if the loss of INK4a cooperates with tal1/scl to induce leukemia, we mated our tal1/scl transgenic mice to INK4a −/− mice to generate a cohort of tal1/scl/INK4a+/− mice. Mice heterozygous for INK4a are healthy and are not predisposed to the development of leukemia. In contrast, tal1/scl/INK4a+/− mice develop T cell leukemia at an accelerated rate, within a median survival period of 110 days, compared to a median survival of 352 days for the tal1/scl transgenic mice, which are wild type with respect to INK4a. Taken together, these data suggest that mutation of the INK4a locus contributes to tal1/scl-mediated leukemogenesis in mice, similar to the widespread deletion of the INK4a/ARF locus observed in TAL1/SCL-expressing human leukemia (Ferrando, 2002). BrdU labeling of preleukemic tal1/scl thymocytes reveals a 41% increase in the percentage of thymocytes in S phase of the cell cycle, suggesting that tal1/scl may stimulate cell cycle progression. In contrast to wild type thymocytes which exhibit 14.5% (±1.5) of cells in S phase, 24.5% (± 2.9) of preleukemic tal1/scl thymocytes are found in S phase (p<0.05, n=4). Moreover, an increase in the sub-G1 population is also observed in the tal1/scl preleukemic thymus. Preliminary cell cycle analysis of tal1/scl/INK4a−/− thymocytes reveals a decrease in the sub-G1 population, suggesting that the absence of the INK4a locus may contribute to tal1/scl leukemogenesis, in part, by inhibiting the tal1/scl-induced apoptosis.

scholarly journals HMG Box Transcriptional Repressor HBP1 Maintains a Proliferation Barrier in Differentiated Liver Tissue

2001 ◽  
Vol 21 (17) ◽  
pp. 5723-5732 ◽  
Author(s):  
Heather H. Shih ◽  
Mei Xiu ◽  
Stephen P. Berasi ◽  
Ellen M. Sampson ◽  
Andrew Leiter ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transgenic Mice ◽  
Partial Hepatectomy ◽  
Cell Cycle Progression ◽  
Early Response ◽  
S Phase ◽  
Subsequent Work ◽  
Hmg Box ◽  
Cell Cycle Reentry ◽  
The Impact

ABSTRACT We previously isolated HBP1 as a target of the retinoblastoma (RB) and p130 family members and as the first of the HMG box transcriptional repressors. Our subsequent work demonstrated that HBP1 coordinates differentiation in cell culture models. In the present study, we show that HBP1 regulates proliferation in a differentiated tissue of an animal. Using transgenic mice in which HBP1 expression was specifically increased in hepatocytes under control of the transthyretin promoter, we determined the impact of HBP1 on synchronous cell cycle reentry following partial hepatectomy. Modest overexpression of HBP1 yielded a detectable cell cycle phenotype. Following a mitogenic stimulus induced by two-thirds partial hepatectomy, mice expressing the HBP1 transgene showed a 10- to 12-h delay in progression through G1 to the peak of S phase. There was a concomitant delay in mid-G1events, such as the induction of cyclin E. While the delay in G1 and S phases correlated with the slight overexpression of transgenic HBP1, the level of the endogenous HBP1 protein itself declined in S phase. In contrast, the onset of the immediate-early response following partial hepatectomy was unchanged in HBP1 transgenic mice. This observation indicated that the observed delay in S phase did not result from changes in signaling pathways leading into the G0-to-G1 transition. Finally, transgenic mice expressing a mutant HBP1 lacking the N-terminal RB interacting domain showed a stronger S-phase response following partial hepatectomy. These results provide the first evidence that HBP1 can regulate cell cycle progression in differentiated tissues.

scholarly journals Isolation of Glomerular Podocytes by Cationic Colloidal Silica-coated Ferromagnetic Nanoparticles

2016 ◽  
Vol 9 (1) ◽  
pp. 67-87
Author(s):  
Andreas Blutke
Keyword(s):  
Transgenic Mice ◽  
Transgenic Animals ◽  
Dominant Negative ◽  
Ferromagnetic Nanoparticles ◽  
Wild Type ◽  
Isolated Cells ◽  
Specific Expression ◽  
Specific Antibodies ◽  
Isolated Glomeruli ◽  
Fluorescent Markers

Background: Podocyte homeostasis plays a crucial role for the maintenance of physiological glomerular function and podocyte injury is regarded as a major determinant of development and progression of renal disease. Objective: Investigation of podocytes requires appropriate methods for their isolation. Previously reported methods use podocyte specific antibodies or transgenic mice with podocyte specific expression of fluorescent markers for isolation of podocytes by magnetic or fluorescence activated cell sorting. Method: Here, a novel, antibody-free method for isolation of podocyte protein and RNA from mouse glomeruli is described. Preparations of isolated glomeruli were added to a suspension of cationic silica-coated colloidal ferromagnetic nanoparticles. The nanoparticles bound to the negatively charged cell surfaces of podocytes residing on the outer surface of the isolated glomeruli. After enzymatic and mechanical dissociation of glomerular cells, nanoparticle-coated podocytes were isolated in a magnetic field. The method was tested in adult wild-type mice without renal lesions and in mice of two nephropathy models (Growth hormone (GH)-transgenic mice and transgenic mice expressing a dominant negative receptor for the glucose dependent insulinotropic polypeptide, GIPRdn) displaying albuminuria, glomerular hypertrophy and evidence for a reduced negative cell surface charge of podocytes. Results: The isolated cells displayed typical morphological and ultrastructural properties of podocytes. On average, 182,000 ± 37,000 cells were counted in the podocyte isolates harvested from ~10,000-12,000 glomeruli per mouse. On the average, the purity of podocyte isolates of these mice accounted for ~63 ± 18 % and the podocyte isolates displayed high mRNA and protein expression abundances of podocyte markers (nephrin and WT1), whereas the expression of endothelial (Cd31) and mesangial markers (Serpinb7) was significantly decreased in podocyte isolates, as compared to samples of isolated glomeruli. The numbers of cells isolated from GH- transgenic and GIPRdn-transgenic mice were not markedly different from that of wild-type mice. Conclusion: The described method represents an alternative for podocyte isolation, particularly in experiments where podocyte specific antibodies or transgenic animals with podocyte specific expression of fluorescent markers are not applicable.

Impaired pericyte recruitment and abnormal retinal angiogenesis as a result of angiopoietin-2 overexpression

2007 ◽  
Vol 97 (01) ◽  
pp. 99-108 ◽  
Author(s):  
Yuxi Feng ◽  
Franziska vom Hagen ◽  
Frederick Pfister ◽  
Snezana Djokic ◽  
Sigrid Hoffmann ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Transgenic Animals ◽  
Mouse Retina ◽  
Vascular Density ◽  
Wild Type ◽  
Transgenic Mouse Line ◽  
Oxygen Induced Retinopathy ◽  
Genes Encoding ◽  
Angiopoietin 2 ◽  
The Impact

SummaryAngiopoietin-2 (Ang2) is among the relevant growth factors induced by hypoxia and plays an important role in the initiation of retinal neovascularizations. Ang2 is also involved in incipient diabetic retinopathy, as it may cause pericyte loss. To investigate the impact of Ang2 on developmental and hypoxia-induced angiogenesis, we used a transgenic mouse line overexpressing human Ang2 in the mouse retina. Transgenic mice displayed a reduced coverage of capillaries with pericytes (-14 %; p<0.01) and a 46% increase of vascular density of the capillary network at postnatal day 10 compared to wild type mice. In the model of oxygen-induced retinopathy (OIR), Ang2 overexpression resulted in enhanced preretinal (+103%) and intraretinal neovascularization (+29%). Newly formed intraretinal vessels in OIR were also pericyte-deficient (-26 %; p<0.01). The total expression of Ang2 in transgenic mice was seven-fold, compared with wild type controls. Ang2 modulated expression of genes encoding VEGF (+65%) and Ang1 (+79%) in transgenic animals. These data suggest that Ang2 is involved in pericyte recruitment, and modulates intraretinal, and preretinal vessel formation in the eye under physiological and pathological conditions.

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.

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