Transferrin gene expression and transferrin immunolocalization in developing foetal rat lung

1991 ◽  
Vol 99 (3) ◽  
pp. 651-656 ◽  
Author(s):  
S.J. Skinner ◽  
C.E. Somervell ◽  
S. Buch ◽  
M. Post
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Chondroitin Sulphate ◽  
Primary Cultures ◽  
Basement Membranes ◽  
Lung Cells ◽  
Intense Staining ◽  
Tf Gene ◽  
Foetal Lung

In previous studies we have shown that transferrin (Tf) specifically stimulates dermatan- and chondroitin-sulphate proteoglycan accumulation around lung cells, and in the extracellular matrix of lung tissue, in vitro. The aim of this study was to determine whether the gene for Tf was activated in specific lung cells during development, and whether the protein product showed evidence of association with extracellular matrix. The expression of the gene in developing lung was shown by the hybridization of a Tf cDNA to a 2.4 kb (kilobase) mRNA species in total RNA extracts of foetal lung. The expression of the Tf gene in comparison to a control gene (GAPD, glyceraldehyde phosphate dehydrogenase) was greatest in 19, 20 and 21 day foetal lung, rising from low levels on day 18 and decreasing markedly at term (day 22). Extracts of RNA from primary cultures of mesenchymal fibroblasts and type II epithelial cells were also analysed for Tf mRNA. These experiments indicated that Tf gene expression was predominantly confined to the mesenchymal compartment. The presence of Tf in histological sections of foetal lung was demonstrated by immunohistochemistry and showed a distinct pattern, with intense staining of the alveolar and the capillary basement membranes. The matrix surrounding the mesenchymal fibroblasts was stained in a diffuse network while epithelial cells were unstained. The staining was low from days 12–16 of gestation, increased to a maximum at days 19–20 but decreased markedly toward term. The Tf staining did not co-localize with transferrin receptor, also demonstrated by immunohistochemistry. These results suggest that Tf is not only present at specific sites in the developing lung, but also is synthesized according to a strict developmental schedule of gene expression.

Abstract 28: Extracellular Matrix Coating Enhances Nanoparticle Uptake by Lung Epithelial Cells

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Primana Punnakitikashem ◽  
Priya Ravikumar ◽  
Jinglei Wu ◽  
Kytai Nguyen ◽  
Connie Hsia ◽  
...  
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Cellular Uptake ◽  
Lung Tissue ◽  
Protein Release ◽  
Lung Epithelial Cells ◽  
Lung Cells ◽  
Significant Difference ◽  
Lung Epithelial

Introduction: Rapid uptake of drug-loaded nanoparticles (NPs) by lung cells is critical for effective pulmonary delivery of therapeutic agents because of the rapid pulmonary clearance mechanisms. We tested the possibility that coating NPs with extracellular matrix (ECM) derived from lung tissue enhances nanoparticles uptake by lung cells. Methods and Results: Fresh adult porcine lung tissue obtained from a local slaughterhouse was decellularized using detergent (sodium dodecyl sulfate) and then enzymatically digested into a soluble solution. The double emulsion method was utilized to fabricate core-shell poly(lactic-co-glycolic) (PLGA) nanoparticles loaded with bovine serum albumin (BSA) for protein release studies, 6-coumarin for cellular uptake studies, or human erythropoietin receptor (hEPOR) cDNA co-expressing green fluorescent protein (GFP) for in vivo gene expression studies. The ECM was coated onto the nanoparticle surface by physical adsorption using the ECM solution (100 μg/ml). There is no significant difference in the diameter, blood compatibility or cell toxicity between coated and uncoated NPs. ECM-coated NPs showed slower protein release rate than uncoated NPs as the ECM coating hindered protein diffusion into the solution. ECM-coated NPs showed significantly higher cellular uptake by human lung epithelial cells than collagen-coated or uncoated NPs. In addition, ECM-coated and uncoated NPs loaded with hEPOR-GFP cDNA were aerosolized and delivered by inhalation into rat lung. Following single inhalation using uncoated NPs, GFP expression in lung tissue progressively increased for up to 21 days. Using the ECM-coated NPs EPOR expression peaked at 14 days, then declined thereafter. Conclusions: Coating NPs with lung-derived ECM markedly enhances NP uptake by lung cells, delays the release of encapsulated protein or DNA, and shortens the duration of peak tissue gene expression compared to uncoated NPs. This NP formulation may be useful where more precise timing of delayed payload release is desired.

Tenascin-C inhibits extracellular matrix-dependent gene expression in mammary epithelial cells. Localization of active regions using recombinant tenascin fragments

1995 ◽  
Vol 108 (2) ◽  
pp. 519-527 ◽  
Author(s):  
P.L. Jones ◽  
N. Boudreau ◽  
C.A. Myers ◽  
H.P. Erickson ◽  
M.J. Bissell
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Protein Synthesis ◽  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Active Regions ◽  
Mammary Epithelial ◽  
Dependent Manner ◽  
Beta Casein ◽  
Casein Protein

The physiological role of tenascin in vivo has remained obscure. Although tenascin is regulated in a stage and tissue-dependent manner, knock-out mice appear normal. When tenascin expression was examined in the normal adult mouse mammary gland, little or none was present during lactation, when epithelial cells actively synthesize and secrete milk proteins in an extracellular matrix/lactogenic hormone-dependent manner. In contrast, tenascin was prominently expressed during involution, a stage characterized by the degradation of the extracellular matrix and the subsequent loss of milk production. Studies with mammary cell lines indicated that tenascin expression was high on plastic, but was suppressed in the presence of the laminin-rich, Engelbreth-Holm-Swarm (EHS) tumour biomatrix. When exogenous tenascin was added together with EHS to mammary epithelial cells, beta-casein protein synthesis and steady-state mRNA levels were inhibited in a concentration-dependent manner. Moreover, this inhibition by tenascin could be segregated from its effects on cell morphology. Using two beta-casein promoter constructs attached to the chloramphenicol acetyltransferase reporter gene we showed that tenascin selectively suppressed extracellular matrix/prolactin-dependent transcription of the beta-casein gene in three-dimensional cultures. Finally, we mapped the active regions within the fibronectin type III repeat region of the tenascin molecule that are capable of inhibiting beta-casein protein synthesis. Our data are consistent with a model where both the loss of a laminin-rich basement membrane by extracellular matrix-degrading enzymes and the induction of tenascin contribute to the loss of tissue-specific gene expression and thus the involuting process.

scholarly journals Extracellular matrix of lymphoid tissues in the chick.

1989 ◽  
Vol 37 (5) ◽  
pp. 757-763 ◽  
Author(s):  
A Colombatti ◽  
A Poletti ◽  
A Carbone ◽  
D Volpin ◽  
G M Bressan
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Basement Membrane ◽  
Basement Membranes ◽  
Intestinal Lumen ◽  
Lymphoid Tissues ◽  
White Pulp ◽  
Extracellular Matrix Components ◽  
Coarse Fibers ◽  
Distribution Of Components

We describe the immunohistochemical distribution of components of the extracellular matrix of the chick lymphoid system. In the thymus, basement membranes of epithelial cells bordering the lobules were intensely stained by laminin antibodies; fibronectin antibodies labeled the capsule and the septal matrix, and similar reactivity was seen with tropoelastin and gp 115 antibodies. No positivity was detected with any of the antibodies within the cortical parenchymal cells. Laminin was not detected in the medullary parenchyma, whereas fibronectin was present as coarse fibers. Tropoelastin and gp 115 appeared as a finer and more diffuse meshwork. In the bursa, laminin antibodies outlined the epithelial cells separating the cortex from the medulla. Fibronectin, tropoelastin, and gp 115 antibody stained the interfollicular septa and the cortical matrix, although to a different extent. Laminin was also detected in association with the interfollicular epithelium (IFE) basement membrane, whereas no staining was found underneath the follicle-associated epithelium (FAE). FAE cells not only lack a proper basement membrane but are also not separated from medullary lymphocytes by any of the other extracellular matrix components were investigated. Consequently, medullary lymphocytes are not sequestered, and can come easily into contact with antigens present in the intestinal lumen. All four antibodies stained the spleen capsule and spleen blood vessels, tropoelastin and gp 115 antibodies giving the strongest reactivity. A fine trabecular staining pattern was detected with gp 115 antibodies in the white pulp.

scholarly journals Expression of extracellular matrix components is regulated by substratum.

1990 ◽  
Vol 110 (4) ◽  
pp. 1405-1415 ◽  
Author(s):  
C H Streuli ◽  
M J Bissell
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Basement Membrane ◽  
Type I Collagen ◽  
Basement Membranes ◽  
Type I ◽  
Collagen Gels ◽  
Extracellular Matrix Components ◽  
Matrix Components ◽  
Cells Cultured

Reconstituted basement membranes and extracellular matrices have been demonstrated to affect, positively and dramatically, the production of milk proteins in cultured mammary epithelial cells. Here we show that both the expression and the deposition of extracellular matrix components themselves are regulated by substratum. The steady-state levels of the laminin, type IV collagen, and fibronectin mRNAs in mammary epithelial cells cultured on plastic dishes and on type I collagen gels have been examined, as has the ability of these cells to synthesize, secrete, and deposit laminin and other, extracellular matrix proteins. We demonstrate de novo synthesis of a basement membrane by cells cultured on type I collagen gels which have been floated into the medium. Expression of the mRNA and proteins of basement membranes, however, are quite low in these cultures. In contrast, the levels of laminin, type IV collagen, and fibronectin mRNAs are highest in cells cultured on plastic surfaces, where no basement membrane is deposited. It is suggested that the interaction between epithelial cells and both basement membrane and stromally derived matrices exerts a negative influence on the expression of mRNA for extracellular matrix components. In addition, we show that the capacity for lactational differentiation correlates with conditions that favor the deposition of a continuous basement membrane, and argue that the interaction between specialized epithelial cells and stroma enables them to create their own microenvironment for accurate signal transduction and phenotypic function.

scholarly journals Dystroglycan receptor is involved in integrin activation in intestinal epithelia

2006 ◽  
Vol 290 (6) ◽  
pp. G1228-G1242 ◽  
Author(s):  
Adel Driss ◽  
Laetitia Charrier ◽  
Yutao Yan ◽  
Vivienne Nduati ◽  
Shanthi Sitaraman ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Basolateral Membrane ◽  
Basement Membranes ◽  
Intestinal Epithelial ◽  
Integrin Activation ◽  
Intestinal Epithelia ◽  
Laminin 1

The dystroglycans (α-DG and β-DG), which play important roles in the formation of basement membranes, have been well studied in skeletal muscle and nerve, but their expression and localization in intestinal epithelial cells has not been previously investigated. Here, we demonstrated that the DG complex, composed of α-DG, β-DG, and utrophin, is specifically expressed in the basolateral membrane of the Caco-2-BBE monolayer. The DG complex coprecipitated with β1-integrin, suggesting a possible interaction among these proteins. In addition, we observed that activation of DG receptors by laminin-1 enhanced the interaction between β1-integrin and laminin-1, whereas activation of DG receptors by laminin-2 reduced the interaction between β1-integrin and laminin-2. Finally, we demonstrated that the intracellular COOH-terminal tail of β-DG and its binding to the DG binding domain of utrophin are crucial for the interactions between laminin-1/-2 and β1-integrin. Collectively, these novel results indicate that dystroglycans play important roles in the regulation of interactions between intestinal epithelial cells and the extracellular matrix.

Cell-specific involvement of HNF-1β in α1-antitrypsin gene expression in human respiratory epithelial cells

2002 ◽  
Vol 282 (4) ◽  
pp. L757-L765 ◽  
Author(s):  
Chaobin Hu ◽  
David H. Perlmutter
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Cell Line ◽  
Primary Cultures ◽  
Synergistic Action ◽  
Intestinal Epithelial ◽  
Hep G2 ◽  
Respiratory Epithelial Cells ◽  
Human Respiratory Epithelial Cells ◽  
Respiratory Epithelial

The synergistic action of hepatocyte nuclear factor (HNF)-1α and HNF-4 plays an important role in expression of the α1-antitrypsin (α1-AT) gene in human hepatic and intestinal epithelial cells. Recent studies have indicated that the α1-AT gene is also expressed in human pulmonary alveolar epithelial cells, a potentially important local site of the lung antiprotease defense. In this study, we examined the possibility that α1-AT gene expression in a human pulmonary epithelial cell line H441 was also directed by the synergistic action of HNF-1α and HNF-4 and/or by the action of HNF-3, which has been shown to play a dominant role in gene expression in H441 cells. The results show that α1-AT gene expression in H441 cells is predominantly driven by HNF-1β, even though HNF-1β has no effect on α1-AT gene expression in human hepatic Hep G2 and human intestinal epithelial Caco-2 cell lines. Expression of α1-AT and HNF-1β was also demonstrated in primary cultures of human respiratory epithelial cells. HNF-4 has no effect on α1-AT gene expression in H441 cells, even when it is cotransfected with HNF-1β or HNF-1α. HNF-3 by itself has little effect on α1-AT gene expression in H441, Hep G2, or Caco-2 cells but tends to have an upregulating effect when cotransfected with HNF-1 in Hep G2 and Caco-2 cells. These results indicate the unique involvement of HNF-1β in α1-AT gene expression in a cell line and primary cultures derived from human respiratory epithelium.

Characterization of connective tissue growth factor expression in primary cultures of human tubular epithelial cells: modulation by hypoxia

2010 ◽  
Vol 298 (3) ◽  
pp. F796-F806 ◽  
Author(s):  
Sven Kroening ◽  
Emily Neubauer ◽  
Bernd Wullich ◽  
Jan Aten ◽  
Margarete Goppelt-Struebe
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Epithelial Cells ◽  
Connective Tissue ◽  
Connective Tissue Growth Factor ◽  
Transforming Growth Factor ◽  
Primary Cultures ◽  
Tissue Growth ◽  
Tubular Epithelial Cells ◽  
Mesenchymal Phenotype

Tubular epithelial cells secrete connective tissue growth factor (CTGF, CCN2), which contributes to tubulointerstitial fibrosis. However, the molecular regulation of CTGF in human primary tubular epithelial cells (hPTECs) is not well defined. Therefore, CTGF expression was characterized in hPTECs isolated from healthy parts of tumor nephrectomies, with special emphasis on the regulation by transforming growth factor-β (TGF-β) and hypoxia, essential factors in the development of fibrosis. CTGF synthesis was strongly dependent on cell density. High CTGF levels were detected in sparse cells, whereas CTGF expression was reduced in confluent cells. Concomitantly, stimulation of CTGF by TGF-β or the histone deacetylase inhibitor trichostatin was prevented in dense cells. Exposure of hPTECs to low oxygen tension (1% O2) or the hypoxia mimetic dimethyl-oxalylglycine for 24 h reduced CTGF gene expression in most of the 17 preparations analyzed. Preincubation of the cells under hypoxic conditions significantly reduced TGF-β-mediated upregulation of CTGF. In line with these data, CTGF mRNA was only induced in interstitial cells, but not in tubular cells in kidneys of mice exposed to hypoxia. Longer exposure to hypoxia or TGF-β (up to 72 h) did not induce hPTECs to adopt a mesenchymal phenotype characterized by upregulation of α-smooth muscle actin, downregulation of E-cadherin, or increased sensitivity of the cells in terms of CTGF expression. Sensitivity was restored by inhibition of DNA methylation. Taken together, our data provide evidence that exposure to hypoxia decreased CTGF gene expression. Furthermore, hypoxia per se was not sufficient to induce a mesenchymal phenotype in primary tubular epithelial cells.

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