Differential expression of laminin A and B chains during development of embryonic mouse organs

Development ◽  
1990 ◽  
Vol 110 (3) ◽  
pp. 823-837 ◽  
Author(s):  
G. Klein ◽  
M. Ekblom ◽  
L. Fecker ◽  
R. Timpl ◽  
P. Ekblom
Keyword(s):  
Cell Types ◽  
Northern Blotting ◽  
Basement Membranes ◽  
Mouse Tumor ◽  
Cell Binding ◽  
Embryonic Mouse ◽  
Developing Heart ◽  
Epithelial Sheets ◽  
A Chain ◽  
Polypeptide Chains

Laminin is a large glycoprotein of basement membranes. The best described laminin from a mouse tumor contains three polypeptide chains (A, B1 and B2), but there is recent evidence that some cell types produce laminin isoforms lacking the A chain. We have here studied the occurrence of the isoforms during mouse organogenesis. In all tissues studied, the A chain mRNA and polypeptide were more weakly expressed than those of the B chains. Laminin A chain polypeptides showed a much more restricted tissue distribution than the B chains. Laminin A chain polypeptide was mainly detected in basement membranes of epithelial cells, suggesting that this chain is important for morphogenesis of epithelial sheets. Most endothelial basement membranes and all embryonic mesenchyme matrices studied seemed to lack the A chain even though they contained B chains. Several of the cells producing laminin devoid of A chain seem to produce other polypeptides that become complexed to the B chains. With an anti-laminin antiserum, which in immunoblots reacts only with A and B polypeptide chains, additional polypeptides of 160 and 190 × 10(3) Mr were co-precipitated from all tissues studied. In developing heart, a polypeptide of 300 × 10(3) Mr was co-precipitated in addition. Our data suggest that these laminin-associated polypeptides are not formed by a differential splicing of the known A chain mRNA. Northern blotting of poly (A)+ RNA showed only 10kb A chain transcripts but no truncated forms. We conclude that several cell types in the mouse embryo produce laminin variants that lack the 400 × 10(3) Mr A chain. Since a major cell binding site of laminin contains parts of the A chain, the variants should differ in biological function from laminin containing this A chain.

Identification of laminin-10/11 as a strong cell adhesive complex for a normal and a malignant human epithelial cell line

1999 ◽  
Vol 112 (1) ◽  
pp. 1-10 ◽  
Author(s):  
M. Ferletta ◽  
P. Ekblom
Keyword(s):  
Adult Stage ◽  
Cell Attachment ◽  
Basement Membranes ◽  
Epithelial Cell Line ◽  
Cell Binding ◽  
Human Epithelial Cell ◽  
Integrin Beta1 ◽  
Cell Adhesive ◽  
Epithelial Sheets ◽  
Laminin 1

Laminins are heterotrimeric proteins of basement membranes. More than 50 different trimers may exist. Laminin-10 (alpha5beta1gamma1 rather than laminin-1 (alpha1beta1gamma1) could be the most abundant isoform in the adult stage, and laminin-10 is made by several developing epithelial sheets. We show here that a much used commercial human preparation contains laminin-10 (alpha5beta1gamma1), some laminin-11 (alpha5beta2gamma1), but no laminin-1. Moreover, the laminin-10/11 mixture was found to be a strong adhesive for two human cell lines derived from epithelia. Antibodies against integrin beta1, alpha6 or alpha3 (at 50 microgram/ml) or dystroglycan did not inhibit cell attachment to laminin-10/11, although lower concentrations of anti-dystroglycan and integrin alpha6 antibodies inhibited cell binding to laminin-1.

scholarly journals Recognition of the laminin E8 cell-binding site by an integrin possessing the alpha 6 subunit is essential for epithelial polarization in developing kidney tubules.

1990 ◽  
Vol 111 (3) ◽  
pp. 1265-1273 ◽  
Author(s):  
L Sorokin ◽  
A Sonnenberg ◽  
M Aumailley ◽  
R Timpl ◽  
P Ekblom
Keyword(s):  
Epithelial Cells ◽  
Binding Site ◽  
Apical Cell ◽  
Cell Types ◽  
Metanephric Mesenchyme ◽  
Kidney Tubule ◽  
Cell Binding ◽  
Developing Kidney ◽  
A Chain ◽  
Integrin Alpha 6

It has been previously shown that A-chain and domain(E8)-specific antibodies to laminin that inhibit cell adhesion also interfere with the establishment of epithelial cell polarity during kidney tubule development (Klein, G., M. Langegger, R. Timpl, and P. Ekblom. 1988. Cell. 55:331-341). A monoclonal antibody specific for the integrin alpha 6 subunit, which selectively blocks cell binding to E8, was used to study the receptors involved. Immunofluorescence staining of embryonic kidneys and of organ cultures of metanephric mesenchyme demonstrated coappearance of the integrin alpha 6 subunit and the laminin A-chain in regions where nonpolarized mesenchymal cells convert into polarized epithelial cells. Both epitopes showed marked colocalization in basal areas of tubules, while an exclusive immunostaining for alpha 6 was observed in lateral and apical cell surfaces of the tubular epithelial cells. Organ culture studies demonstrated a consistent inhibition of kidney epithelium development by antibodies against the alpha 6 subunit. The data suggest that the recognition of E8 cell-binding site of laminin by a specific integrin is crucial for the formation of kidney tubule epithelium from undifferentiated mesenchymal stem cells. In some other cell types (endothelium, some ureter cells) an exclusive expression of alpha 6 with no apparent colocalization of laminin A-chain in the corresponding basement membrane was seen. Thus, in these cells, integrins possessing the alpha 6 subunit may bind to laminin isoforms that differ from those synthesized by developing tubules.

Glycosylation of developing human glomeruli: lectin binding sites during cell induction and maturation.

1987 ◽  
Vol 35 (1) ◽  
pp. 33-37 ◽  
Author(s):  
H Holthöfer ◽  
I Virtanen
Keyword(s):  
Binding Sites ◽  
Mesangial Cells ◽  
Ricinus Communis ◽  
Lectin Binding ◽  
Helix Pomatia ◽  
Cell Types ◽  
Basement Membranes ◽  
Phenotypic Change ◽  
Neuraminidase Treatment ◽  
Nephron Development

Expression of cellular glycoconjugates during differentiation of human fetal kidney was studied using fluorochrome-labeled lectins. Each lectin revealed a characteristic binding pattern during the phenotypic change of the nephrogenic mesenchyme and during distinct stages of nephron development. The uninduced mesenchymal cells were positive for Pisum sativum (PSA), Concanavalin A (ConA), Wistaria floribunda (WGA), and Ricinus communis (RCA-I) lectins. However, these lectins failed to react with the uninduced cells of the S-shaped bodies, whereas Maclura pomifera (MPA), Triticum vulgaris (WGA) and, after neuraminidase treatment, Arachis hypogaea (PNA) agglutinins bound intensely to the presumptive podocytes. During later stages of nephrogenesis, MPA positively on the podocytes weakened and could not be observed in adult kidney glomeruli. Binding sites for Helix pomatia (HPA) agglutinin in glomeruli were also expressed only transiently during nephrogenesis. During further development PSA, ConA, WFA, and RCA-I reacted with mesangial cells in addition to the glomerular basement membranes. The segment-specific lectin binding patterns of the tubuli emerged in parallel with the appearance of brush border and Tamm-Horsfall antigens of the proximal and distal tubuli. The results show that nephron site-specific saccharides appear in a developmentally regulated manner and in parallel with morphologic maturation of the nephron. Lectins therefore appear to be useful tools for study of induction and maturation of various nephron cell types.

scholarly journals Inhibition of Vesicular Secretion in Both Neuronal and Nonneuronal Cells by a Retargeted Endopeptidase Derivative ofClostridium botulinum Neurotoxin Type A

2000 ◽  
Vol 68 (5) ◽  
pp. 2587-2593 ◽  
Author(s):  
John A. Chaddock ◽  
John R. Purkiss ◽  
Lorna M. Friis ◽  
Janice D. Broadbridge ◽  
Michael J. Duggan ◽  
...  
Keyword(s):  
Neurotransmitter Release ◽  
Botulinum Neurotoxin ◽  
Type A ◽  
Cell Types ◽  
Snare Complex ◽  
Cell Binding ◽  
Vesicle Docking ◽  
Botulinum Neurotoxin Type A ◽  
Clostridial Neurotoxins ◽  
Dose Dependent

ABSTRACT Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types by a mechanism that involves cleavage of specific components of the vesicle docking/fusion complex, the SNARE complex. A derivative of the type A neurotoxin fromClostridium botulinum (termed LHN/A) that retains catalytic activity can be prepared by proteolysis. The LHN/A, however, lacks the putative native binding domain (HC) of the neurotoxin and is thus unable to bind to neurons and effect inhibition of neurotransmitter release. Here we report the chemical conjugation of LHN/A to an alternative cell-binding ligand, wheat germ agglutinin (WGA). When applied to a variety of cell lines, including those that are ordinarily resistant to the effects of neurotoxin, WGA-LHN/A conjugate potently inhibits secretory responses in those cells. Inhibition of release is demonstrated to be ligand mediated and dose dependent and to occur via a mechanism involving endopeptidase-dependent cleavage of the natural botulinum neurotoxin type A substrate. These data confirm that the function of the HC domain of C. botulinumneurotoxin type A is limited to binding to cell surface moieties. The data also demonstrate that the endopeptidase and translocation functions of the neurotoxin are effective in a range of cell types, including those of nonneuronal origin. These observations lead to the conclusion that a clostridial endopeptidase conjugate that can be used to investigate SNARE-mediated processes in a variety of cells has been successfully generated.

scholarly journals Differential Requirement for TANK-binding Kinase-1 in Type I Interferon Responses to Toll-like Receptor Activation and Viral Infection

2004 ◽  
Vol 199 (12) ◽  
pp. 1651-1658 ◽  
Author(s):  
Andrea K. Perry ◽  
Edward K. Chow ◽  
Julia B. Goodnough ◽  
Wen-Chen Yeh ◽  
Genhong Cheng
Keyword(s):  
Viral Infection ◽  
Nuclear Translocation ◽  
Sendai Virus ◽  
Cell Types ◽  
Receptor Activation ◽  
Northern Blotting ◽  
Type I ◽  
Toll Like Receptor ◽  
Embryonic Fibroblasts ◽  
Iκb Kinase

TANK-binding kinase-1 (TBK1) and the inducible IκB kinase (IKK-i) have been shown recently to activate interferon (IFN) regulatory factor-3 (IRF3), the primary transcription factor regulating induction of type I IFNs. Here, we have compared the role and specificity of TBK1 in the type I IFN response to lipopolysaccharide (LPS), polyI:C, and viral challenge by examining IRF3 nuclear translocation, signal transducer and activator of transcription 1 phosphorylation, and induction of IFN-regulated genes. The LPS and polyI:C-induced IFN responses were abolished and delayed, respectively, in macrophages from mice with a targeted disruption of the TBK1 gene. When challenged with Sendai virus, the IFN response was normal in TBK1−/− macrophages, but defective in TBK1−/− embryonic fibroblasts. Although both TBK1 and IKK-i are expressed in macrophages, only TBK1 but not IKK-i was detected in embryonic fibroblasts by Northern blotting analysis. Furthermore, the IFN response in TBK1−/− embryonic fibroblasts can be restored by reconstitution with wild-type IKK-i but not a mutant IKK-i lacking kinase activity. Thus, our studies suggest that TBK1 plays an important role in the Toll-like receptor–mediated IFN response and is redundant with IKK-i in the response of certain cell types to viral infection.

Effects of polystyrene surface chemistry on the biological activity of solid phase fibronectin and vitronectin, analysed with monoclonal antibodies

1993 ◽  
Vol 104 (3) ◽  
pp. 793-803 ◽  
Author(s):  
P.A. Underwood ◽  
J.G. Steele ◽  
B.A. Dalton
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Adhesion ◽  
Solid Phase ◽  
Divalent Cations ◽  
Biological Activities ◽  
Optimal Growth ◽  
Cell Types ◽  
Cell Binding ◽  
Urea Treatment ◽  
Bhk Cells

The conformation and biological activities of fibronectin (FN) and vitronectin (VN) coated on different plastic surfaces were investigated using cell adhesion and a panel of domain-specific monoclonal antibodies (mAbs). The adhesion of BHK fibroblasts was markedly better on FN coated on hydrophilic tissue culture polystyrene (TCPS) than on hydrophobic, untreated polystyrene (PS). mAbs A17 and 3E3, which inhibit the binding of BHK cells to the RGD-containing sequence within the cell binding region of FN, also bound preferentially to FN on TCPS. In contrast, two anti-FN mAbs, which have no effect on cell adhesion (A35 and A3), bound preferentially to the conformation of FN on the more hydrophobic PS. Mouse melanoma cells utilise an additional cell-binding site in the Hep II domain of FN and their preference for FN coated on TCPS was less marked than that of BHK cells. This reduced preference was again mimicked by the binding of a mAb, A32, which inhibits the binding of B16 cells to the Hep II domain of FN. In contrast, BHK cell adhesion to VN did not display a preference for TCPS over PS. The cell-binding activity of adsorbed VN was matched by the binding of a cell adhesion-inhibitory mAb, A18, which, unlike mAbs A17 and A32, displayed slightly increased binding to VN coated on PS, rather than TCPS. When the denaturating effect of coating FN and VN to PS in the presence of urea was investigated, similar correlations between BHK cell adhesion and the binding of inhibitory mAbs were observed. Urea treatment of FN significantly reduced both BHK cell adhesion and the binding of both cell adhesion-inhibitory mAbs, whereas the binding of A35 and A3 was unaffected. There was no significant effect of urea treatment of VN upon either BHK cell adhesion or mAb binding. A larger panel of anti-FN mAbs was used, together with the anti-VN mAbs, to determine whether there were differences in mAb recognition of FN and VN adsorbed on three different brands of TCPS. The mAbs segregated into four reactivity patterns, of which A17, A32, A35 and A18 respectively were representative. Significant differences were observed in mAb recognition of FN and VN adsorbed to different brands of TCPS. These may reflect differences in the ability of these surfaces to support optimal growth of different cell types. The effect of divalent cations upon adsorbed FN and VN was also investigated.(ABSTRACT TRUNCATED AT 400 WORDS)

Abstract 92: Platelet Derived Growth Factor Receptor Alpha Signaling is Required for Cardiac Fibroblast Survival and Proliferation

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Malina J Ivey ◽  
Michelle D Tallquist
Keyword(s):  
Preliminary Data ◽  
Cardiac Fibrosis ◽  
Heart Function ◽  
Cardiac Fibroblasts ◽  
Growth Factor Receptor ◽  
Cell Types ◽  
Fibroblast Proliferation ◽  
Artery Ligation ◽  
Developing Heart ◽  
Factor Receptor Alpha

Cardiac fibrosis contributes significantly to heart disease and is a hallmark of decreased cardiac function. Currently, there are no treatments that attenuate fibrosis, but identification of signaling pathways required for fibroblast function would provide some potential targets. PDGFRα is a receptor tyrosine kinase that is required for fibroblast formation in the developing heart, and preliminary data indicates that it is also required for maintenance of resident fibroblasts and expansion of activated fibroblasts after injury. Preliminary experiments demonstrate that loss of PDGFRα expression in adult cardiac fibroblasts results in 50% reduction in the number of the resident fibroblasts by 4 days after gene deletion. This was further validated using an independent fibroblast marker, collagen1a1GFP. Based on the low basal level of fibroblast proliferation, we hypothesize that PDGFRα signaling is essential for fibroblast survival and that fibroblasts undergo rapid turnover in the absence of PDGFRα signaling. Future studies will determine the exact mechanism of this loss. We have also begun to elucidate which PDGFRα downstream signals promote fibroblast maintenance. Using a PDGFRα-dependent-PI3K-deficient mouse model, preliminary data indicates that PDGFRα-dependent PI3K signaling is essential for cell survival. We are also investigating the role of PDGFRα signaling after myocardial infarction. Using recently described genetic tools to follow fibroblasts after injury, we have determined that fibroblasts reach their peak of proliferation within a week after permanent left anterior descending artery ligation. This injury-induced proliferation is reduced by 50% after deletion of PDGFRα. Therefore, we have demonstrated that PDGFRα has a role in fibroblast maintenance in the healthy heart, as well as a role in fibroblast proliferation after injury. Our studies will continue to illuminate additional roles for PDGFRα in the fibroblast, as well as the implications of fibroblast loss on other cell types and overall heart function.

STUDIES ON SMALL MOLECULAR WEIGHT ADHESION PROTEINS (SAPs) FROM CONNECTIVE TISSUES

1987 ◽  
Author(s):  
S Wasi ◽  
P Alles ◽  
D Gauthier ◽  
U Bhargava ◽  
J Farsi ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Polyclonal Antibody ◽  
Type I Collagen ◽  
Cell Attachment ◽  
Binding Capacity ◽  
Guanidine Hydrochloride ◽  
Cell Types ◽  
Type I ◽  
Cell Binding ◽  
Connective Tissues

We have identified a family of low molecular weight proteins with cell attachment properties in a variety of soft and mineralised connective tissues (Wong et al., Biochem. J. 232, 119, 1985). For further characterisation of these proteins we extracted porcine bones with 4 M guanidine hydrochloride and purified the proteins on a series of gel filtration columns The purifed SAPs comprise three bands with Mr -14 000 -17 000. All three proteins bound to heparin-sepahrose in both the presence and absence of 4M urea, and when eluted with 2 M NaCl they retained their cell binding capacity. These proteins promoted the adhesion and spreading of a variety of cell types, including normal fibroblasts, osteoblasts, and epithelial cells, and tumour (osteosarcoma) cells. On Western blotting SAPs did not cross-react with antibodies against fibronectin, laminin or type I collagen; however, they were recognised by a monoclonal antibody to human vitronectin, a polyclonal antibody to bovine vitronectin and polyclonal antibody to human somatomedin B. Dose response experiments indicated that maximum attachment of human gingival fibroblasts occurred in the presence or absence of fetal bovine serum on wells precoated with 2.5 μg/cm2 of SAPs. Attachment of cells to these proteins was partially inhibited by the synthetic pentapeptide Gly-Arg-Gly-Asp-Ser. Utilising the nitrocellulose cell binding assay of Hayman et al (J. Cell. Biol. 95, 20, 1982), the cell attachment to these proteins could be completely inhibited by heparin (100 units/mL) whereas up to 1000 units/mL of heparin had no inhibitory effect on cell attachment to fibronectin and vitronectin. The occurrence of these proteins in a variety of connective tissues and their recognition by different cell types may reflect their general biological role in adhesive mechanisms in both hard and soft connective tissues. Currently, we are investigating the relationship between SAPs and vitronectin, since it is possible that SAPs represent a tissue-processed form of vitronectin or may be novel attachment proteins with regions of homology with vitronectin

Diverse patterns of cell-specific gene expression in response to glucocorticoid in the developing small intestine

2006 ◽  
Vol 291 (6) ◽  
pp. G1041-G1050 ◽  
Author(s):  
Murat B. Yaylaoglu ◽  
Barbara M. Agbemafle ◽  
Thomas J. Oesterreicher ◽  
Milton J. Finegold ◽  
Christina Thaller ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Northern Blotting ◽  
Specific Gene ◽  
Cellular Expression ◽  
Intestinal Segments ◽  
Transcript Gene ◽  
Microarray Studies ◽  
Glucocorticoid Action

Although glucocorticoids are known to elicit functional maturation of the gastrointestinal tract, the molecular mechanisms of glucocorticoid action on the developing intestine have not been fully elucidated. Our previous microarray studies identified 66 transcripts as being rapidly induced in the jejunum following dexamethasone (Dex) administration to suckling mice. Now we report the specific cellular location of a subset of these transcripts. Mouse pups at P8 received Dex or vehicle and intestinal segments were collected 3–4 h later. Robotic-based in situ hybridization (ISH) was performed with digoxygenin-labeled riboprobes. Transcripts studied included Ndrg1, Sgk1, Fos, and two unknown genes ( Gene 9 and Gene 36). As predicted, ISH revealed marked diversity of cellular expression. In small intestinal segments, Sgk1 mRNA was in all epithelial cells; Fos mRNA was confined to epithelial cells at the villus tip; and Ndrg1 and Gene 36 mRNAs were localized to epithelial cells of the upper crypt and villus base. The remaining transcript ( Gene 9) was induced modestly in villus stroma and strongly in the muscle layers. In the colon, Ndrg1, Sgk1, and Gene 36 were induced in all epithelial cells; Gene 9 was in muscle layers only; and Fos was not detectable. For jejunal segments, quantitation of ISH signals in tissue from Dex-treated and vehicle-treated mice demonstrated mRNA increases very similar to those measured by Northern blotting. We conclude that glucocorticoid action in the intestine reflects diverse molecular mechanisms operating in different cell types and that quantitative ISH is a valuable tool for studying hormone action in this tissue.

The relation between connective tissue cells and intercellular substances, including basement membranes

1975 ◽  
Vol 271 (912) ◽  
pp. 363-377 ◽  
Keyword(s):  
Endothelial Cells ◽  
Basement Membrane ◽  
Immune Complexes ◽  
Hydrolytic Enzymes ◽  
Inflammatory Cells ◽  
Cell Types ◽  
Cellular Level ◽  
Basement Membranes ◽  
The Body ◽  
Pathological Changes

Basement membranes are distributed widely in the body forming an extracellular matrix for epithelial and endothelial cells. The collagenous and glycoprotein constituents of basement membranes are synthesized by these two cell types. Disturbance of the interactions between basement membranes and their associated epithelial and endothelial cells can lead to the pathological changes seen in diseases involving basement membranes. These changes are illustrated here by reference to glomerulonephritis induced by the deposition of immune complexes in the glomerulus of the kidney, and chronic inflammatory changes occurring in the lung after inhalation of asbestos. In these diseases basement membrane changes can occur in several ways. Hydrolytic enzymes released from inflammatory cells degrade basement membranes while other factors released from these cells may stimulate synthesis of basement membrane constituents by epithelial and endothelial cells. Alternatively the physical separation of epithelial and endothelial cells from their basement membranes by space-occupying substances such as immune complexes can interfere with feedback mechanisms leading to synthesis of basement membrane constituents and cell proliferation. Studies of these pathological changes at a cellular level should shed new light on the ways in which cells interact with their pericellular environment.

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