Alpha 3 beta 1 integrin is moved into focal contacts in kidney mesangial cells

1993 ◽  
Vol 105 (3) ◽  
pp. 739-751 ◽  
Author(s):  
H. Grenz ◽  
S. Carbonetto ◽  
S.L. Goodman
Keyword(s):  
Mesangial Cells ◽  
Cell Attachment ◽  
Divalent Cations ◽  
Expression Patterns ◽  
Collagen Type I ◽  
Type I ◽  
Focal Contacts ◽  
Kidney Glomerulus ◽  
The Matrix ◽  
Alpha V Beta 3

The movement of integrins into focal adhesive structures accompanies cell attachment to extracellular matrix. The kinetics of incorporation of integrins into focal contacts was studied during attachment to matrix of mesangial cells of the kidney glomerulus. On collagen, fibronectin, laminin and vitronectin, the number and intensity of talin-focal contacts increased with time. Talin-containing focal contacts were present in mesangial cells within 2 h of plating and in control cells (HT1080 and Rugli) within 1 h. Integrin alpha-chains colocalized with talin, dependent on the matrix substrate. The attachment, spreading and organization of integrin into focal contacts was not affected when endogenous protein synthesis was suppressed with cycloheximide. In Rugli, alpha 1 beta 1 organized into focal contacts on collagen and laminin, while in HT1080 alpha 2 beta 1 organized on collagen type I, alpha 5 beta 1 on fibronectin, alpha 6 beta 1 on laminin, and alpha 3 beta 1 and alpha 4 beta 1 were diffusely distributed on all substrates. These distributions mirrored the usage and expression patterns previously established for integrins in these cells and was as predicted from the literature. In mesangial cells, however, alpha 3 beta 1 was also organized into prominent focal contact arrays on collagen, fibronectin, EHS and human placental laminins, but not on vitronectin, while alpha 6 beta 1 was not organized. Initial attachment and spreading of mesangial cells was absolutely dependent on divalent cations. Mg2+ and Mn2+ supported attachment on all substrates, while Ca2+ stimulated attachment on laminin (E8), fibronectin and vitronectin. The data suggest that the functional integrins on mesangial cells include alpha 1 beta 1 (on collagen and laminin) alpha 2 beta 1 (on collagen), alpha 5 beta 1 (on fibronectin) and alpha V beta 3 (on vitronectin). However, mesangial cells do not use alpha 6 beta 1 on laminin, and the data support a role for alpha 3 beta 1 as putative receptor for fibronectin, collagen and laminin.

Changes in extracellular matrix composition regulate cyclooxygenase-2 expression in human mesangial cells

2011 ◽  
Vol 300 (4) ◽  
pp. C907-C918 ◽  
Author(s):  
Matilde Alique ◽  
Laura Calleros ◽  
Alicia Luengo ◽  
Mercedes Griera ◽  
Miguel Ángel Iñiguez ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Collagen Type ◽  
Mesangial Cells ◽  
Collagen Type I ◽  
Collagen I ◽  
Type I ◽  
Camp Response Element ◽  
Response Element ◽  
Human Mesangial Cells ◽  
Cox 2

Glomerular diseases are characterized by a sustained synthesis and accumulation of abnormal extracellular matrix proteins, such as collagen type I. The extracellular matrix transmits information to cells through interactions with membrane components, which directly activate many intracellular signaling events. Moreover, accumulating evidence suggests that eicosanoids derived from cyclooxygenase (COX)-2 participate in a number of pathological processes in immune-mediated renal diseases, and it is known that protein kinase B (AKT) may act through different transcription factors in the regulation of the COX-2 promoter. The present results show that progressive accumulation of collagen I in the extracellular medium induces a significant increase of COX-2 expression in human mesangial cells, resulting in an enhancement in PGE2 production. COX-2 overexpression is due to increased COX-2 mRNA levels. The study of the mechanism implicated in COX-2 upregulation by collagen I showed focal adhesion kinase (FAK) activation. Furthermore, we observed that the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway by collagen I and collagen I-induced COX-2 overexpression was abolished by PI3K and AKT inhibitors. Additionally, we showed that the cAMP response element (CRE) transcription factor is implicated. Finally, we studied COX-2 expression in an animal model, NG-nitro-l-arginine methyl ester hypertensive rats. In renal tissue and vascular walls, COX-2 and collagen type I content were upregulated. In summary, our results provide evidence that collagen type I increases COX-2 expression via the FAK/PI3K/AKT/cAMP response element binding protein signaling pathway.

scholarly journals A durable and biocompatible ascorbic acid-based covalent coating method of polydimethylsiloxane for dynamic cell culture

2017 ◽  
Vol 14 (132) ◽  
pp. 20170318 ◽  
Author(s):  
Joni Leivo ◽  
Sanni Virjula ◽  
Sari Vanhatupa ◽  
Kimmo Kartasalo ◽  
Joose Kreutzer ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Cell Attachment ◽  
Collagen Type I ◽  
Contact Angle Measurement ◽  
Angle Measurement ◽  
Type I ◽  
Simple Method ◽  
Dynamic Conditions ◽  
Coating Method ◽  
Cross Linker

Polydimethylsiloxane (PDMS) is widely used in dynamic biological microfluidic applications. As a highly hydrophobic material, native PDMS does not support cell attachment and culture, especially in dynamic conditions. Previous covalent coating methods use glutaraldehyde (GA) which, however, is cytotoxic. This paper introduces a novel and simple method for binding collagen type I covalently on PDMS using ascorbic acid (AA) as a cross-linker instead of GA. We compare the novel method against physisorption and GA cross-linker-based methods. The coatings are characterized by immunostaining, contact angle measurement, atomic force microscopy and infrared spectroscopy, and evaluated in static and stretched human adipose stem cell (hASC) cultures up to 13 days. We found that AA can replace GA as a cross-linker in the covalent coating method and that the coating is durable after sonication and after 6 days of stretching. Furthermore, we show that hASCs attach and proliferate better on AA cross-linked samples compared with physisorbed or GA-based methods. Thus, in this paper, we provide a new PDMS coating method for studying cells, such as hASCs, in static and dynamic conditions. The proposed method is an important step in the development of PDMS-based devices in cell and tissue engineering applications.

Bacterial Endotoxin Inhibits Migration, Attachment, and Orientation of Human Gingival Fibroblasts in vitro and Delays Collagen Gel Contraction

1987 ◽  
Vol 66 (9) ◽  
pp. 1449-1455 ◽  
Author(s):  
S. Pitaru ◽  
M. Soldinger ◽  
D. Madgar ◽  
Z. Metzger
Keyword(s):  
Collagen Type ◽  
Cell Attachment ◽  
Collagen Gel ◽  
Collagen Type I ◽  
Human Gingival Fibroblasts ◽  
Bacterial Endotoxin ◽  
Type I ◽  
Gingival Fibroblasts ◽  
Collagen Gels

The purpose of this study was to assess the effect of endotoxin adsorbed to dental surfaces and to collagen type I on the migration, attachment, and orientation of human gingival fibroblasts (HGF). Transversely cut porcine tooth root slices (RS), 200 μm thick, were prepared. Half of the RS obtained were partially demineralized in EDTA. Half of the demineralized and non-demineralized RS were incubated with 400 μg/mL of endotoxin for 24 hr, whereas the other half were maintained in PBS and served as controls. Experimental and control RS were placed on confluent layers of HFG and cultured for six days. Cell migration toward and cell attachment to the periphery of the RS and the formation of oriented cell sheets were assessed by means of photographic techniques. Additionally, six-day-old cultures were fixed and processed for SEM observation. In separate experiments, the effect of endotoxin on cell attachment to collagen type I and on contraction of three-dimensional collagen gels was assessed. It was found that: (i) bacterial endotoxin inhibited migration and attachment of HGF to both demineralized and non-demineralized cementum and interfered with the development of oriented cellular structure ; (ii) the inhibitory effect was significantly more pronounced for non-demineralized than for demineralized cementum; (iii) the morphology of HGF attached to endotoxin-treated dental surfaces was altered compared with that of their controls; and (iv) bacterial endotoxin inhibited cell attachment to collagen type I and delayed the contraction of collagen gel.

scholarly journals Laminin-5 and type I collagen promote adhesion and osteogenic differentiation of animal serum-free expanded human mesenchymal stromal cells

2012 ◽  
Vol 4 (4) ◽  
pp. 36 ◽  
Author(s):  
Falk Mittag ◽  
Eva-Maria Falkenberg ◽  
Alexandra Janczyk ◽  
Marco Götze ◽  
Tino Felka ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Type I Collagen ◽  
Cell Attachment ◽  
Expression Patterns ◽  
Marker Genes ◽  
Type I ◽  
Laminin 5 ◽  
Laminin 1

Mesenchymal stromal cells (MSC) are differentiation competent cells and may generate, among others, mature osteoblasts or chondrocytes<em> in vitro</em> and <em>in vivo</em>. Laminin-5 and type I collagen are important components of the extracellular matrix. They are involved in a variety of cellular and extracellular activities including cell attachment and osteogenic differentiation of MSC. MSC were isolated and expanded using media conforming good medical practice (GMP)-regulations for medical products. Cells were characterized according to the defined minimal criteria for multipotent MSC. MTT- and BrdU-assays were performed to evaluate protein-dependent (laminin-5, laminin-1, type I collagen) metabolic activity and proliferation of MSC. MSC-attachment assays were performed using protein-coated culture plates. Osteogenic differentiation of MSC was measured by protein-dependant mineralization and expression of osteogenic marker genes (osteopontin, alkaline phophatase, Runx2) after three, seven and 28 days of differentiation. Marker genes were identified using quantitative reverse-transcription polymerase chain reaction. Expansion of MSC in GMP-conforming media yielded vital cells meeting all minimal criteria for MSC. Attachment assay revealed a favorable binding of MSC to laminin-5 and type I collagen at a protein concentration of 1-5 fmol/mL. Compared to plastic, osteogenic differentiation was significantly increased by laminin-5 after 28 days of culture (P&lt;0.04). No significant differences in gene expression patterns were observed. We conclude that laminin-5 and type I collagen promote attachment, but laminin-1 and laminin-5 promote osteogenic differentiation of MSC. This may influence future clinical applications.

scholarly journals Adhesion of platelets to laminin in the absence of activation.

1984 ◽  
Vol 99 (6) ◽  
pp. 2140-2145 ◽  
Author(s):  
C R Ill ◽  
E Engvall ◽  
E Ruoslahti
Keyword(s):  
Type I Collagen ◽  
Cell Attachment ◽  
Human Platelets ◽  
Type I ◽  
Initial Event ◽  
Serotonin Secretion ◽  
Platelet Binding ◽  
The Matrix ◽  
Subendothelial Matrix ◽  
Adhesion Of Platelets

The binding of platelets to components in the subendothelial matrix is an initial event in hemostasis and thrombosis. The glycoprotein components of the matrix are considered important in this interaction. Of these, collagen binds and activates platelets and induces their aggregation. In this study we demonstrate that substrate-bound laminin causes time- and concentration-dependent adherence of human platelets to the substrate. The binding of platelets to laminin was found to be similar in some respects, but different in others, to their binding to surfaces coated with fibronectin or collagen. The binding of platelets to laminin or fibronectin was not associated with their activation under conditions in which type I collagen activates the platelets as measured by [14C]serotonin secretion. Platelets bound to laminin and fibronectin differed in their appearance; they remained rounded on laminin whereas they flattened completely on fibronectin. Binding of platelets to fibronectin, but not laminin, is inhibited by a recently described peptide (Pierschbacher, M., and E. Ruoslahti, 1984, Nature (Lond.), 309:30-33) containing the cell-attachment tetrapeptide sequence of fibronectin, which suggests that separate receptors exist for laminin and fibronectin. These studies establish laminin as a platelet-binding protein and suggest that laminin can contribute to the adhesiveness of exposed tissue matrices to platelets. Since laminin and fibronectin do not activate platelets, whereas collagen does, and laminin differs from fibronectin in that it does not induce spreading of the attached platelets, all three proteins appear to confer different signals to the platelets. Some of these may be related to platelet functions other than those necessary for the formation of a hemostatic plug.

scholarly journals Identification of stromal cell products that interact with pre-B cells.

1996 ◽  
Vol 134 (3) ◽  
pp. 771-782 ◽  
Author(s):  
K Oritani ◽  
P W Kincade
Keyword(s):  
B Cells ◽  
Fusion Proteins ◽  
Stromal Cell ◽  
Divalent Cations ◽  
Transmembrane Protein ◽  
Collagen Type I ◽  
Type I ◽  
Interleukin 7 ◽  
A Cell ◽  
B Lineage

Our understanding of lympho-hematopoietic microenvironments is incomplete, and a new cloning strategy was developed to identify molecules that bind to B lineage lymphocyte precursors. A cell sorting procedure was used for initial enrichment of cDNAs from stromal cell mRNA that contained signal sequences and were therefore likely to encode transmembrane or secreted proteins. A second step involved expression of the library as soluble Ig fusion proteins. Finally, pools representing these proteins were screened for the ability to recognize pre-B cells. This approach resulted in the cloning of biglycan, syndecan 4, collagen type I, clusterin, matrix glycoprotein sc1, osteonectin, and one unknown molecule (designated SIM). The full-length cDNA of SIM revealed that it is a type I transmembrane protein, and its intracellular domain has weak homology with myosin heavy chain and related proteins. Staining of established cell lines and freshly isolated hematopoietic cells with the Ig fusion proteins revealed distinct patterns of reactivity and differential dependence on divalent cations. Biglycan-, sc1-, and SIM-Ig fusion proteins selectively increased interleukin 7-dependent proliferation of pre-B cells. Overexpression of the entire SIM protein affected the morphology of 293T cells, while expression of just the extracellular portion was without effect. Thus, a series of stromal cell surface molecules has been identified that interact with blood cell precursors. Three of them promoted the survival and/or proliferation of pre-B cells in culture, and all merit further study in relation to lympho-hematopoiesis.

scholarly journals Schwann cells use a novel collagen-dependent mechanism for fibronectin fibril assembly

1998 ◽  
Vol 111 (18) ◽  
pp. 2763-2777 ◽  
Author(s):  
M.A. Chernousov ◽  
R.C. Stahl ◽  
D.J. Carey
Keyword(s):  
Extracellular Matrix ◽  
Schwann Cells ◽  
Collagen Type ◽  
Collagen Type I ◽  
Collagen Type Iv ◽  
Type I ◽  
Type Iv ◽  
The Matrix ◽  
Fibronectin Fibrils ◽  
Dependent Mechanism

Cultured rat Schwann cells were stimulated to deposit fibrillar extracellular matrix by treatment with ascorbic acid in the absence of nerve cells. Immunofluoresence staining of the matrix showed that it contains collagens types I and IV, fibronectin and perlecan but not laminin. Collagen type IV, fibronectin and perlecan co-distributed completely in the matrix fibrils, whereas collagen type I was present in only a subset of these fibrils. Time course studies indicated that collagen type I fibrils appear at late stages of matrix formation. Digestion of Schwann cell extracellular matrix with collagenase effectively disrupted most of the matrix including fibronectin fibrils. This was in contrast with fibroblasts, where collagenase treatment removed collagen with no visible effect on fibronectin fibrils. alpha5 integrin was expressed on the cell surface of Schwann cells and partially codistributed with fibronectin-containing fibrils. This suggests that the inability of Schwann cells to deposit fibronectin-containing matrix through a conventional, collagen-independent mechanism was not due to the lack of fibronectin-binding integrins on their cell surface. Polyclonal anti-fibronectin antibodies inhibited the deposition of fibronectin into the matrix fibrils, whereas collagen type IV fibrils were generally unaffected. Growth of Schwann cells on collagen type IV-coated substrate in the absence of ascorbate induced deposition of fine fibronectin fibrils. These results suggest that Schwann cells use an apparently novel, collagen type IV-dependent mechanism for the deposition of fibronectin into their extracellular matrix.

Behavior of fetal intestinal organ culture explanted onto a collagen substratum

Development ◽  
1990 ◽  
Vol 110 (2) ◽  
pp. 353-370
Author(s):  
G.G. Altmann ◽  
A. Quaroni
Keyword(s):  
Smooth Muscle ◽  
Epithelial Cells ◽  
Organ Culture ◽  
Smooth Muscle Cells ◽  
Collagen Type ◽  
Collagen Type I ◽  
Type I ◽  
The Matrix ◽  
P Cells

A model of organ culture of 18 day old fetal rat intestine (Quaroni, 1985) was modified and characterized in the present work with the purpose of developing an in vitro model for the study of intestinal epithelial cell behaviour. Fragments of this intestine were kept in suspension culture for 7 days and then explanted onto collagen (type I) matrix. Within a day, the fragments became anchored to the substratum and a circular monolayer grew out to about 1 cm diameter. In the fragments, an outer layer of absorptive epithelial cells came to enclose a stroma, which was polarized into a loose (mesenchymal) and a dense portion. The dense portion contained a mixture of smooth muscle cells and primitive stem-type epithelial cells (‘p-cells’). After explantation, at the contact point with the matrix, the epithelium broke up and the mesenchyme grew into the matrix and anchored the fragment. The epithelial edges now became continuous with the developing monolayer. Radioautography with tritiated thymidine indicated a constant cell renewal in epithelium and monolayer apparently from foci of p-cells, a reserve population of which was seen to be sequestered among the smooth muscle cells. Activated stem cells could differentiate into three mature epithelial phenotypes, each differentiation pathway apparently being determined by the type of underlying stroma. Immunohistochemistry using gold- and fluorescein-labeled monoclonal antibodies indicated that adult differentiation-specific markers (e.g. brush border enzymes) were present in the fragment epithelium but not in the monolayer cells. On the other hand, the monolayer cells could be induced to express some of these markers by contact with mesenchymal cells or by co-culturing with fibroblastic cell lines. Matrigel substratum mixed with collagen (type I) supported the appearance in monolayer of strands positive for amino-peptidase and lactase. The model thus appears to be suitable for the in vitro study of epithelial renewal and differentiation, and it has already provided some results in this respect.

Drosophila PS integrins recognize vertebrate vitronectin and function as cell-substratum adhesion receptors in vitro

Development ◽  
1991 ◽  
Vol 113 (3) ◽  
pp. 1007-1016 ◽  
Author(s):  
S. Hirano ◽  
K. Ui ◽  
T. Miyake ◽  
T. Uemura ◽  
M. Takeichi
Keyword(s):  
Cell Attachment ◽  
Collagen Type I ◽  
Matrix Proteins ◽  
Type I ◽  
Rgd Peptides ◽  
Drosophila Cell ◽  
Type Iv ◽  
And Function

Using the Drosophila cell line MLDmBG-1, a monoclonal antibody aBG-1 that can inhibit not only cell clumping but also cell spreading was generated. This antibody immunoprecipitates a complex of molecules consisting of a major 120 × 10(3) Mr and other components. To characterize the 120 × 10(3) Mr component, we purified it, generated antibodies to it, and cloned its cDNA. Sequencing of this cDNA suggests that the 120 × 10(3) Mr molecule is identical to PS beta, a beta chain of Drosophila integrins. The other components immunoprecipitated included two alpha chains of Drosophila integrins, PS1 alpha and PS2 alpha, as revealed using specific antibodies to these molecules. These suggest that aBG-1 recognizes the PS beta associated with PS1 alpha or PS2 alpha. However, immunostaining of embryos and larvae with aBG-1 showed that the staining pattern is similar to that for PS2 alpha but not for PS beta, suggesting that the antibody preferentially recognizes the PS beta associated with particular alpha chains in situ. We then attempted to characterize the ligands for these integrin complexes, using culture dishes coated with various vertebrate matrix proteins. These cells spread very well on dishes coated with vitronectin and, to a lesser extent, on those with fibronectin. This spreading was partially inhibited by aBG-1, but not by other control antibodies or RGD peptides. The cell attachment to these substrata was not affected by the antibody. The cells also can attach to dishes coated with laminin but without spreading, and this attachment was not inhibited by aBG-1. Furthermore, they do not attach to dishes coated with collagen type I, type IV, and fibrinogen. These results indicate that Drosophila PS integrins can recognize vertebrate vitronectin, and also fibronectin with a weaker affinity, at sites other than RGD sequences, and thus can function in cell-substratum adhesion.

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