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Results for “Integrin alpha3”

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PubMed1998

Integrin-mediated adhesion and signalling in ovarian cancer cells.

The integrins are a family of cell surface receptors which mediate cellular adhesion and signalling events. Our goal was to evaluate integrin function and signalling pathways in ovarian cancer cells. Ovarian cancer cell lines, NIH:OVCAR-3 and NIH:OVCAR-5, exhibited distinct extracellular matrix (ECM) binding preferences which were mediated primarily through beta1 integrin interactions. Western blot analysis was used to identify changes in cellular phosphotyrosine, focal adhesion kinase (FAK) and mitogen activated protein (MAP) kinase. Tyrosine phosphorylation of integrin-associated phosphoproteins was not enhanced in either cell type in response to adhesion onto ECM components or receptor crosslinking. FAK expression was greater in NIH:OVCAR-5 cells while MAP kinase activity was higher in NIH:OVCAR-3 cells. The data suggest that these two ovarian cancer cell lines exhibit specific ECM binding preferences and distinct differences in phosphotyrosine, focal adhesion and MAP kinase expression profiles.

Antigens, CD↗

PubMed1997

The expression of VLA integrins in the human tonsilla palatina.

The integrin receptors are a family of transmembrane glycoproteins comprising non-covalent heterodimers. They interact with a wide variety of ligands including extracellular matrix glycoproteins, complement and other cell, while their intracellular domains interact with the cytoskeleton. They participate in cell-matrix and cell-cell adhesion in many physiologically important processes including embryological development, hemostasis, thrombosis, wound healing, immune and nonimmune defense mechanisms, and in oncogenic transformation. This investigation was focused on the histological distribution of the beta 1-integrins in the human tonsil using an indirect immunoperoxidase method. Present data suggest that lymphocyte and antigen presenting cells (FDCs, IDCs, and macrophages) interact with each other following adhesion to extracellular matrix proteins (e.g. fibronectin) through their integrin receptors in order to carry out special immunological functions. In addition, stromal elements and epithelial components were shown to express VLA integrins providing interactions for tissue organization and compartmentalization.

Antibodies, Monoclonal↗

PubMed2003

An extracellular matrix-specific microarray allowed the identification of target genes downstream of discoidin domain receptors.

The two discoidin domain receptors, DDR1 and DDR2, are tyrosine kinases that are activated by collagen and are essential regulators of cell-matrix communication. However, the target genes downstream of activated DDRs and their physiological significance are largely unknown. Here, we describe a novel method to dissect signaling pathways induced by extracellular matrix (ECM) receptors. Using the doxycycline-inducible repression system (tet-off), we generated human fibrosarcoma and mouse fibroblast cell lines over-expressing DDR1 or DDR2. These cell lines were employed for gene expression analysis using microarrays specific for human and mouse genes coding for ECM proteins or ECM-interacting factors. We found that approximately 10% of the genes studied were up- or down-regulated more than twofold in response to signals generated by over-expressing DDRs. A common event downstream of DDR1 and DDR2 in human and mouse cells was the up-regulation of P-selectin glycoprotein ligand. Key target genes repressed upon DDR activation were agrin, syndecan-1 and alpha3 integrin. ECM-specific microarrays were found a valuable tool to dissect gene expression changes induced by collagen-receptor signaling pathways.

Animals↗

PubMedDate not supplied

Mesothelium expression of integrins in vivo and in vitro.

OBJECTIVE: To characterize the expression of alpha subunits of integrin adhesion molecules in peritoneal tissue in vivo and in vitro. METHOD: Peritoneum from the anterior abdominal wall (n = 22) and the serosa of the posterior uterus (n = 11) was obtained from women of reproductive age without endometriosis who were undergoing surgery for benign conditions. Immunohistochemical studies were performed on serial sections of peritoneum from the anterior abdominal wall, the uterine serosa, mesothelial monolayer cultures, and peritoneum explants from the abdominal wall using monoclonal antibodies to alpha subunits of integrin adhesion molecules. Electron microscopy was performed to localize these adhesion molecules in the mesothelium. RESULTS: The mesothelial expression of alpha integrin subunits was identical in the anterior peritoneum and uterine serosa. In vivo the mesothelium strongly expressed alpha 2 and alpha 3 and variably expressed alpha 6. In the monolayer cultures there was moderate/strong staining for alpha 2, alpha 3, and alpha 5; there was minimal expression of alpha v. In the explants there was moderate/strong expression of alpha 2, alpha 3, alpha 5, and alpha v; alpha 6 was variably expressed. The ultrastructure of the mesothelium was unique in the anterior peritoneum, uterine serosa, and the monolayer cultures. The integrin subunits were distributed throughout the cytoplasm, were expressed in the plasma membrane, and were present on the surface (i.e., towards the peritoneal cavity) of the mesothelium. CONCLUSION: Integrins are expressed by the mesothelium of the peritoneum. The mesothelium expression of integrins in vivo differs from that of the mesothelium integrin expression in monolayer culture and explant culture.

Antigens, CD↗

PubMed1999

Alpha 3 beta 1 adhesion to laminin-5 and invasin: critical and differential role of integrin residues clustered at the boundary between alpha 3 N-terminal repeats 2 and 3.

Integrin/ligand interaction is a therapeutic target for many diseases. We previously reported that residues critical for ligand binding are clustered in N-terminal repeat 3 (in the predicted 2-3 loop) of alpha 4, alpha 5 and alpha IIb. Here we have localized residues critical for ligand binding in the alpha 3 subunit of integrin alpha 3 beta 1 with distinct ligand specificity (laminin-5). We identified an alpha 3 epitope common to several function-blocking anti-alpha 3 antibodies at the boundary between repeats 1 and 2 (residues 75-80). We found that swapping the predicted 4-1 loop (residues 153-165) at the boundary between repeats 2 and 3 with the corresponding alpha 4 sequence and mutating Thr-162 and Gly-163 residues in this predicted loop block laminin-5 binding. Thr-162 and Gly-163 and the antibody epitope are separated in the primary structure; however, they are close to each other in the proposed beta-propeller model. Mutating residues recently reported to block (Tyr-186 and Trp-188) or enhance (Asp-122) laminin-5 binding to alpha 3 beta 1 [Krukonis, E. S., Dersch, P., Eble, J. A., and Isberg, R. R.(1998) J. Biol. Chem. 273, 31837-31843] did not affect laminin-5 binding under the assay conditions used. Thr-162 and Gly-163 are not critical for adhesion to invasin, indicating that laminin-5 and invasin may use different recognition mechanisms, and that mutation of Thr-162 and Gly-163 does not drastically affect the integrity of alpha 3 beta 1. These results suggest that residues critical for ligand binding may be similarly (but not identically) located in repeat 3 of the alpha subunit regardless of ligand specificity.

Adhesins, Bacterial↗

PubMed1998

Alterative expression of the collagenase and adhesion molecules in the highly metastatic clones of human colonic cancer cell lines.

Human colonic carcinoma cell lines, KM12C, KM12SM and KM12L4, were previously established and their in vivo metastatic potentials have been well evaluated. The highly metastatic cell lines KM12SM and KM12L4 were derived from the parental low metastatic cell line KM12C in vivo. To evaluate the metastatic behavior of these cell lines in vitro, we examined colony formation on monolayers of the pulmonary arterial endothelial (CPAE) cells. On day 4, the highly metastatic cell lines showed an approximately 2-fold increase in number of colonies on CPAE cell monolayers relative to the parental KM12C cell line. To investigate what evidence is correlated with their metastatic and invasive abilities, Northern blot analysis and flow cytometry were performed in all cell lines. According to the results of Northern blot analysis, the levels of matrix metalloproteinase (MMP)-2 and c-met mRNA expression were increased in highly metastatic cell lines as compared with the parental cell line. We also examined the cell-surface expression of several adhesion molecules by flow cytometry. The levels of expression of sialyl Lewisa antigen (sLe(a)) in KM12SM and KM12L4 were twice higher than that in KM12C. However, the levels of expression of E-cadherin in KM12SM and KM12L4 were decreased to half that in KM12C. The alterative expression of the collagenase and adhesion molecules might contribute to their metastatic/invasive abilities of these cell lines both in vivo and in vitro.

Antigens, CD↗

PubMed2001

Differential tissue expression of epitopes of the tetraspanin CD151 recognised by monoclonal antibodies.

CD151, a member of the tetraspanin family of cell membrane proteins, is widely expressed in epithelial, endothelial and muscle cells as well as platelets and megakaryocytes. Several monoclonal antibodies recognising CD151 in transfected cells and immunoprecipitating typical bands of 28 and 32 kDa from cell lysates have been produced. Surprisingly, these antibodies show different patterns of staining on tissue sections and on haemopoietic cells. Here we show that these differences are at least in part due to masking of certain epitopes in integrin/CD151 complexes. These data have important implications for the use of monoclonal antibodies in studies of the distribution and function of CD151. Of six monoclonal antibodies from four laboratories, 11B1 was found to be the most reliable for detection of CD151 in different cell and tissue contexts.

Animals↗

PubMed1997

Importance of the tubulointerstitium in human glomerulonephritis. II. Distribution of integrin chains beta 1, alpha 1 to 6 and alpha V.

Accumulation of extracellular matrix is important in the progression of glomerulonephritis. Since adherent cell types utilize integrins to bind and organize extracellular matrix proteins, we have assessed expression of the beta 1 integrins in sequential sections from 85 human renal biopsies and 4 normal kidneys by immunohistochemical staining. Our results demonstrate strong correlations between expression of the alpha 5 chain within the interstitium, the alpha V chain on proximal and distal tubular epithelium and the presence of chronic histological damage. Moreover, staining for interstitial alpha 5 and proximal and distal tubular alpha V were also strongly associated with expression of certain adhesion molecules (ICAM-1, VCAM-1, E-selectin and L-selectin) and the presence of macrophages within the interstitium, which have been linked, in an earlier study, with the degree of chronic histological damage and disease progression. However, in contrast to our earlier study of adhesion molecules, there were also associations between expression of integrin chains within the glomerulus and tubulointerstitium. For example, there were strong positive associations between staining for alpha 5 on glomerular endothelium and its expression on extraglomerular vascular endothelium and between both mesangial alpha 1 and podocyte alpha 3 and tubular staining for the common beta 1 subunit. While the functional significance of these associations is obscure, they suggest some kind of communication between cells in different sites in the kidney. There were also positive associations between staining for different integrins within the glomerulus, notably mesangial cell staining for alpha 2, glomerular endothelial cells staining for alpha 5 and glomerular epithelial cell alpha 3. These results suggest that there is a coordinated upregulation of integrin expression both within the tubulointerstitium and the glomerulus and that at least some of these integrins (interstitial alpha 5 and distal tubular alpha V) are associated with the expression of other adhesion molecules, macrophage infiltration and the presence of markers of disease progression (interstitial fibrosis and tubular atrophy).

Antigens, CD↗

PubMed2003

Expression of CD18, CD49b, CD49c and CD49e on lens anterior capsules in human cataracts.

PURPOSE: To examine the lens epithelial cells obtained from the anterior lens capsules removed during cataract surgery and detect various subclasses of the cell surface adhesion molecules known as integrins. METHODS: The circular sections of anterior capsules with attached lens epithelial cells (LECs) were obtained during cataract surgery from 28 patients. The lens capsules were immunohistochemically stained. RESULTS: CD49b CD49c, CD49e, and CD18 were detected in varying degrees in specimens obtained from human cataractous lenses. The positive percentages were 33, 75, 33, and 20%, respectively. The most striking feature was the increasing staining profiles towards the edges of the capsules (away from the central part of the lens capsules) for CD49c, suggesting that the LECs showed higher immunoreactivity for this antibody. The immunoreactivity for CD49b and CD49e was weaker. This was absent for CD18 in the central part of the lens capsules. CONCLUSION: The positive expression of antibodies suggests that specific integrin subunits were expressed in LECs of human cataracts. These results suggest that lens epithelial cells expressing CD49b, CD49c, CD49e, and CD18 might be precursors in the process of anterior lens epithelial cell (A cell) adhesion, and hence play a role in anterior capsule opacification or in subsequent migration and a possible role in posterior capsule opacification.

Aged↗

PubMed2003

Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization.

In spite of the attention given to the study of mesenchymal stem cells (MSCs) derived from the bone marrow (BM) of humans and other species, there is a lack of information about murine MSCs. We describe the establishment of conditions for the in vitro expansion of plastic-adherent cells from murine BM for over 50 passages, and provide their characterization regarding morphology, surface marker profile and growth kinetics. These cells were shown to differentiate along osteogenic and adipogenic pathways, and to support the growth and differentiation of haematopoietic stem cells, and were thus operationally defined as murine mesenchymal stem cells (mMSCs). mMSCs were positive for the surface markers CD44, CD49e, CD29 and Sca-1, and exhibited a homogeneous, distinctive morphology. Their frequency in the BM of adult BALB/c and C57Bl/6 mice, normal or knockout for the alpha-L-iduronidase (IDUA) gene, was preliminarily estimated to be 1 per 11,300-27,000 nucleated cells. The emergence of a defined methodology for the culture of mMSCs, as well as a comprehensive understanding of their biology, will make the development of cellular and genetic therapy protocols in murine models possible, and provide new perspectives in the field of adult stem cells research.

Animals↗

PubMed2001

Expression of beta 1 integrins in human dental pulp in vivo: a comparative immunohistochemical study on healthy and chronic marginal periodontitis samples.

AIM: The objective of this study was to determine the tissue distribution of beta 1 integrin chains in sound human dental pulps and to compare the findings with connective tissue compartments of other organs and to pulp tissue in teeth extracted due to periodontal disease. METHODOLOGY: Freshly frozen pulp tissue samples from teeth extracted for orthodontic reasons were examined and compared to samples from teeth extracted due to chronic (marginal) periodontitis. beta 1 integrin chains were determined using an indirect-immunoperoxidase technique. Seven monoclonal antibodies recognizing alpha 1, alpha 2, alpha 3, alpha 4, alpha 5, alpha 6 and beta 1 chains of Very Late Activation Antigen (VLA) integrins were used for this purpose. RESULTS: VLA-1, VLA-2, VLA-3 and VLA-5 were expressed by vascular endothelium and vascular smooth muscle in varying intensities in both groups. VLA-6 reactivity was observed in the basal surfaces of arterial, venous and capillary endothelia. Our results indicate that there was no significant difference in the expression of VLA integrins in sound pulp tissue when compared to the samples from chronic (marginal) periodontitis and the connective tissue compartments of other viscera. CONCLUSION: The present findings suggest that human dental pulp tissue is not different from other connective tissue compartments in the body with respect to VLA integrin expression, and chronic marginal periodontitis does not affect pulp tissue to a histopathologically detectable extent.

Antibodies, Monoclonal↗

PubMed1997

Expression of integrins during axolotl limb regeneration.

Limb regeneration in urodeles is achieved through the dedifferentiation of tissues at the amputation plane and through the production of the blastema. This tissue breakdown is possible by extensive alterations in molecules of the extracellular matrix. In this respect we describe the regulation of several integrins during such events. It was found that alpha 1 and beta 1 integrins were down-regulated as blastema formation proceeded. In contrast, the expression of alpha 3, alpha 6 and alpha v integrins were upregulated in the blastema. These data are consistent with the roles of integrins in developmental phenomena and are discussed in light of the mechanisms of dedifferentiation.

Ambystoma↗

PubMed2000

Cloning of rat nephrin: expression in developing glomeruli and in proteinuric states.

BACKGROUND: Nephrin is identified as a product of the gene mutated in a patient with congenital nephrotic syndrome of the Finnish type. However, its precise localization and function are not yet fully clarified. METHODS: To clone the rat homologue of nephrin, polymerase chain reaction (PCR) was employed. To elucidate the localization and expression of nephrin, immunohistological analysis with a specific antirat nephrin antibody, reverse transcription-PCR, and RNase protection assay were performed. RESULTS: Amino acid sequences of rat and human nephrin are highly homologous (82.2% identity). The domain structure of nephrin is also highly conserved between rats and humans. The rat nephrin was detected only in kidney glomeruli along glomerular capillary walls, and its localization was always identical to that of the anti-slit diaphragm monoclonal antibody (mAb) 5-1-6-recognized antigen in normal matured and fetal rat glomeruli and in the glomeruli of proteinuric states. The nephrin staining pattern was clearly distinguished from that of zonula occludens-1 (ZO-1), alpha3-integrin, or podocalyxin. mRNA expression for nephrin was first detected in the fetal rat kidneys at 18.5 embryonic days. Nephrin mRNA expression decreased just after injection of mAb 5-1-6 (47.4%) or puromycin aminonucleoside (51.2%), and the staining pattern of nephrin shifted from a linear to a granular pattern in both proteinuric states. CONCLUSIONS: Nephrin is localized in slit diaphragm in the matured glomeruli and is identical with mAb 5-1-6 antigen. Nephrin is involved in the development of proteinuria not only in mAb 5-1-6 nephropathy, but also in puromycin aminonucleoside nephropathy.

Amino Acid Sequence↗

PubMed2001

Role of alpha(3)beta(1) integrin in tubulogenesis of Madin-Darby canine kidney cells.

BACKGROUND: We isolated several Madin-Darby canine kidney (MDCK) subclones that exhibit different degrees of branching tubulogenesis in lower concentrations of collagen gel. The M634 clone formed cell aggregates in 0.3% collagen gel, but developed branching tubules vigorously in 0.1% collagen gel. In contrast, the Y224 clone formed cysts in 0.3% collagen gel and displayed fewer branching structures in 0.1% collagen gel. Morphologically, M634 cells exhibited higher levels of cell scattering as well as collagen-induced cell migration than Y224. We conducted this study to delineate the underlying mechanism of branching tubulogenesis in M634 cells. METHODS: Components of the focal contact machinery were analyzed in both cell lines, including the extracellular matrix glycoproteins fibronectin, laminin, and vitronectin; cytoskeleton-associated elements alpha-actinin, talin, and vinculin; and receptors for extracellular matrix and alpha(2), alpha(3), alpha(5), alpha(v), beta(1), and beta(3) integrins. Furthermore, we established several stable transfectants of alpha(3) integrin antisense RNA in M634 cells to examine the role of alpha(3)beta(1) integrin in branching morphogenesis directly. RESULTS: There were no obvious differences in levels of the focal adhesion complex proteins between M634 and Y224 cells, except that the content of the alpha(3) and beta1 integrins were 1.2- and 0.6-fold higher in M634 cells, respectively. The expression of alpha(3) integrin antisense RNA significantly lowered the levels of alpha(3) integrin mRNA and protein. The potential of cell scattering, migration, and branching tubulogenesis in M634 cells was inhibited according to the decrease in alpha(3) integrin expression. CONCLUSION: Our data indicate that expression of alpha(3)beta(1) integrin regulates cell scattering, migration, and branching tubulogenesis of MDCK cells, possibly via adhesion to or serving as a signaling molecule for type I collagen.

Animals↗

PubMed1997

Expression of beta-1 integrins in ganglionic and aganglionic segments of patients with Hirschsprung's disease.

An immunocytochemical study was performed to assess the role of beta-1 integrins in the pathogenesis of Hirschsprung's disease. Fresh tissue samples from both aganglionic and ganglionic segments of five patients who were undergoing surgery for Hirschsprung's disease were obtained. Samples were rapidly frozen in liquid nitrogen. Sections were cut and stained using anti alpha-1, 2, 3, 4, 5, 6 and beta-1 monoclonals according to the indirect immunoperoxidase method. The evaluation did not reveal any significant change of pattern in the distribution of beta-1 integrins in the non-neural elements of both aganglionic and ganglionic segments of colon, such as the epithelium, muscularis mucosa, muscularis externa, connective tissues and blood vessels. Nerve fibres in both aganglionic and ganglionic segments strongly expressed the alpha-6 chain of very late activation antigen which led to their increase in the aganglionic segment. In addition to revealing the increase, alpha-6 monoclonals also had in situ positive control due to their presence in non-neural elements. Hence, immunostaining of the suction biopsies with anti alpha-6 monoclonals may be employed as a new and simple method in the diagnosis of Hirschsprung's disease. On the other hand, beta-1 integrins do not seem to play a role in the defective migration of ganglion cells occurring in Hirschsprung's disease.

Antigens, CD↗

PubMed2000

Colocalization of integrin receptors and reelin in dendritic spine postsynaptic densities of adult nonhuman primate cortex.

The expression of telencephalic reelin (Reln) and glutamic acid decarboxylase mRNAs and their respective cognate proteins is down-regulated in postmortem brains of schizophrenia and bipolar disorder patients. To interpret the pathophysiological significance of this finding, immunoelectron microscopic experiments are required, but these cannot be carried out in postmortem human brains. As an alternative, we carried out such experiments in the cortex of rats and nonhuman primates. We found that Reln is expressed predominantly in layer I of both cortices and is localized to bitufted (double-bouquet), horizontal, and multipolar gamma-aminobutyric acid-ergic interneurons, which secrete Reln into extracellular matrix. Reln secretion is mediated by a constitutive mechanism that depends on the expression of a specific signal peptide present in the Reln carboxy-terminal domain. Extracellular matrix Reln is found to aggregate in proximity of postsynaptic densities expressed in apical dendrite spines, which include also the alpha(3) subunit of integrin receptors. Most pyramidal neurons of various cortical layers express the mouse-disabled 1 (Dab1) protein, which, after phosphorylation by a soluble tyrosine kinase, functions as an adapter protein, probably mediating a modulation of cytoskeleton protein expression. We hypothesize that the decrease of neuropil and dendritic spine density reported to exist in the neocortex of psychiatric patients may be related to a down-regulation of Reln-integrin interactions and the consequent decrease of cytoskeleton protein turnover.

Animals↗

PubMed1996

Domain-specific interactions between entactin and neutrophil integrins. G2 domain ligation of integrin alpha3beta1 and E domain ligation of the leukocyte response integrin signal for different responses.

Extracellular matrix proteins activate neutrophils to up-regulate many physiologic functions that are necessary at sites of tissue injury. To elucidate the ligand-receptor interactions that mediate these functions, we examined neutrophil activation by the basement membrane protein, entactin. Entactin is structurally and functionally organized into distinct domains; therefore, we utilized glutathione S-transferase -fusion proteins encompassing its four major domains, G1, G2, E, and G3, to assess interactions between entactin and neutrophil integrin receptors. We show that the E domain, which contains the single RGD sequence of entactin, is sufficient for ligation of the beta3-like integrin, leukocyte response integrin, and signaling for chemotaxis. Moreover, the G2 domain signals for stimulation of Fc receptor-mediated phagocytosis via ligation of alpha3beta1. This receptor-ligand interaction was revealed only after stimulation of neutrophil by immune complexes or phorbol esters. Interestingly, the E domain does not enhance phagocytosis, and the G2 domain is not chemotactic. Furthermore, cleavage of entactin with the matrix metalloproteinase, matrilysin, liberates peptides that retain E domain-mediated chemotaxis and G2 domain-mediated enhancement of phagocytosis. These studies indicate that multiple domains of entactin have the ability to ligate individual integrins expressed by neutrophils and to activate distinct functions.

Antigen-Antibody Complex↗

PubMed1999

Anchorage-dependent regulation of the mitogen-activated protein kinase cascade by growth factors is supported by a variety of integrin alpha chains.

Integrin cooperation with growth factor receptors to enable permissive signaling to the mitogen-activated protein (MAP) kinase pathway has important implications for cell proliferation, differentiation, and survival. Here we have sought to determine whether anchorage regulation of the MAP kinase pathway is specific to the alpha chain subunit of the integrins employed during adhesion. Human umbilical vein endothelial cells (HUVECs) anchored via endogenous alpha(2), alpha(3), or alpha(5) integrin subunits or NIH3T3 fibroblast cells lines anchored via ectopically expressed human integrin alpha(2) or alpha(5) subunits displayed comparable MAP kinase activation upon growth factor stimulation, regardless of the integrin alpha chain employed. In contrast, when either cell type was maintained in suspension, growth factor treatment inefficiently activated the MAP kinase pathway. The integrin-mediated enhancement of MAP kinase activation by growth factor correlated with the tyrosine phosphorylation of focal adhesion kinase but was independent of Shc. These data indicate that integrin modulation of the MAP kinase pathway is supported by a variety of integrin complexes and imply that other pathways may be required for the previously reported alpha chain-specific effects on cell cycle regulation and cell differentiation.

3T3 Cells↗