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PubMed2004

Synthetic heterotrimeric collagen peptides as mimics of cell adhesion sites of the basement membrane.

Collagen type IV forms a network in the basement membrane into which other constituents of the tissue are incorporated. It also provides cell-adhesion sites that are specifically recognized by cell-surface receptors, i.e., the integrins. Different from the ubiquitous sequential RGD adhesion motif found in most of the matrix proteins, in collagen type IV, the responsible binding sites for alpha1beta1 integrin have been identified as Asp461 of the two alpha1 chains and Arg461 of the alpha2 chain. Because of the heterotrimeric character of this collagen, the spatial geometry of the binding epitope depends not only on the triple-helical fold, but decisively even on the stagger of the chains. To investigate the effects of chain registration on the conformational properties and binding affinities of this adhesion epitope, two synthetic heterotrimeric collagen peptides consisting of the identical three chains were assembled by an artificial cystine knot in two different registers, i.e., in the most plausible alpha2alpha1alpha1' and less probable alpha1alpha2alpha1' chain alignment. A detailed conformational characterization of both trimers allowed to correlate their different binding affinities for alpha1beta1 integrin with the degree of local plasticity of the two different triple helices. Optimal local breathing of the rod-shaped collagens is apparently crucial for selective recognition by proteins interacting with these main components of the extracellular matrix.

Basement Membrane↗

PubMed2000

Laminin alpha4 and integrin alpha6 are upregulated in regenerating dy/dy skeletal muscle: comparative expression of laminin and integrin isoforms in muscles regenerating after crush injury.

The expression of laminin isoforms and laminin-binding integrin receptors known to occur in muscle was investigated during myogenic regeneration after crush injury. Comparisons were made between dystrophic 129ReJ dy/dy mice, which have reduced laminin alpha2 expression, and their normal littermates. The overall histological pattern of regeneration after crush injury was similar in dy/dy and control muscle, but proceeded faster in dy/dy mice. In vitro studies revealed a greater yield of mononuclear cells extracted from dy/dy muscle and a reduced proportion of desmin-positive cells upon in vitro cultivation, reflecting the presence of inflammatory cells and "preactivated" myoblasts due to ongoing regenerative processes within the endogenous dystrophic lesions. Laminin alpha1 was not detectable in skeletal muscle. Laminin alpha2 was present in basement membranes of mature myofibers and newly formed myotubes in control and dy/dy muscles, albeit weaker in dy/dy. Laminin alpha2-negative myogenic cells were detected in dy/dy and control muscle, suggesting the involvement of other laminin alpha chains in early myogenic differentiation, such as laminin alpha4 and alpha5 which were both transiently expressed in basement membranes of newly formed myotubes of dy/dy and control mice. Integrin beta1 was expressed on endothelial cells, muscle fibers, and peripheral nerves in uninjured muscle and broadened after crush injury to the interstitium where it occurred on myogenic and nonmyogenic cells. Integrin alpha3 was not expressed in uninjured or regenerating muscle, while integrin alpha6 was expressed mainly on endothelial cells and peripheral nerves in uninjured muscle. Upon crush injury integrin alpha6 increased in the interstitium mainly on nonmyogenic cells, including infiltrating leukocytes, endothelial cells, and fibroblasts. In dy/dy muscle, integrin alpha6 occurred on some newly formed myotubes. Integrin alpha7 was expressed on muscle fibers at the myotendinous junction and showed weak and irregular expression on muscle fibers. After crush injury, integrin alpha7 expression extended to the newly formed myotubes and some myoblasts. However, many myoblasts and newly formed myotubes were integrin alpha7 negative. No marked difference was observed in integrin alpha7 expression between dy/dy and control muscle, either uninjured or after crush injury. Only laminin alpha4 and integrin alpha6 expression patterns were notably different between dy/dy and control muscle. Expression of both molecules was more extensive in dy/dy muscle, especially in the interstitium of regenerating areas and on newly formed myotubes. In view of the faster myogenic regeneration observed in dy/dy mice, the data suggest that laminin alpha4 and integrin alpha6 support myogenic regeneration. However, whether these accelerated myogenic effects are a direct consequence of the reduced laminin alpha2 expression in dy/dy mice, or an accentuation of the ongoing regenerative events in focal lesions in the muscle, requires further investigation.

Animals↗

PubMed1998

Splice variants of the Drosophila PS2 integrins differentially interact with RGD-containing fragments of the extracellular proteins tiggrin, ten-m, and D-laminin 2.

Two new potential ligands of the Drosophila PS2 integrins have been characterized by functional interaction in cell culture. These potential ligands are a new Drosophila laminin alpha2 chain encoded by the wing blister locus and Ten-m, an extracellular protein known to be involved in embryonic pattern formation. As with previously identified PS2 ligands, both contain RGD sequences, and RGD-containing fragments of these two proteins (DLAM-RGD and TENM-RGD) can support PS2 integrin-mediated cell spreading. In all cases, this spreading is inhibited specifically by short RGD-containing peptides. As previously found for the PS2 ligand tiggrin (and the tiggrin fragment TIG-RGD), TENM-RGD induces maximal spreading of cells expressing integrin containing the alphaPS2C splice variant. This is in contrast to DLAM-RGD, which is the first Drosophila polypeptide shown to interact preferentially with cells expressing the alphaPS2 m8 splice variant. The betaPS integrin subunit also varies in the presumed ligand binding region as a result of alternative splicing. For TIG-RGD and TENM-RGD, the beta splice variant has little effect, but for DLAM-RGD, maximal cell spreading is supported only by the betaPS4A form of the protein. Thus, the diversity in PS2 integrins due to splicing variations, in combination with diversity of matrix ligands, can greatly enhance the functional complexity of PS2-ligand interactions in the developing animal. The data also suggest that the splice variants may alter regions of the subunits that are directly involved in ligand interactions, and this is discussed with respect to models of integrin structure.

Alternative Splicing↗

PubMed2002

Modulation of integrin expression on rat bone marrow cells by substrates with different surface characteristics.

Biomaterials have been shown to be able to influence the growth and differentiation of osteogenic cells cultured on the surface. Although the precise mechanisms by which the materials influence osteogenic cells are unclear, it is possible that the materials manipulate the expression of integrins by the cells. We therefore studied the expression of a number of integrins by rat bone marrow (RBM) cells, after culture on culture polystyrene, on machined and grit-blasted titanium, and on calcium phosphate-coated titanium. Integrin expression was studied by FACS analysis. We found a large variation in the expression of integrins by cells in replicate experiments. After culture on polystyrene for 7 days, cells expressed alpha1, alpha2, alpha3, alpha5, alpha6, beta1, and beta3, although some of the subunits were expressed only occasionally. The cells did not express the alpha4 subunit. After culture of RBM cells for 8 days on coated and noncoated titanium substrates, cells always expressed alpha3, alpha5, alpha6, and beta1. The alpha1 and beta3 subunits were only expressed in some of the experiments. Frequently, the expression of alpha5, alpha6, and beta1 was higher on the coated than on the noncoated titanium substrates. Based on our results, we conclude that the studied materials are capable of influencing the expression of integrins by RBM cells cultured on relevant implant materials.

Animals↗

PubMed2004

Beta1 integrins activate a MAPK signalling pathway in neural stem cells that contributes to their maintenance.

The emerging evidence that stem cells develop in specialised niches highlights the potential role of environmental factors in their regulation. Here we examine the role of beta1 integrin/extracellular matrix interactions in neural stem cells. We find high levels of beta1 integrin expression in the stem-cell containing regions of the embryonic CNS, with associated expression of the laminin alpha2 chain. Expression levels of laminin alpha2 are reduced in the postnatal CNS, but a population of cells expressing high levels of beta1 remains. Using neurospheres - aggregate cultures, derived from single stem cells, that have a three-dimensional architecture that results in the localisation of the stem cell population around the edge of the sphere - we show directly that beta1 integrins are expressed at high levels on neural stem cells and can be used for their selection. MAPK, but not PI3K, signalling is required for neural stem cell maintenance, as assessed by neurosphere formation, and inhibition or genetic ablation of beta1 integrin using cre/lox technology reduces the level of MAPK activity. We conclude that integrins are therefore an important part of the signalling mechanisms that control neural stem cell behaviour in specific areas of the CNS.

Animals↗

PubMed1996

Structural requirements for alpha 1 beta 1 and alpha 2 beta 1 integrin mediated cell adhesion to collagen V.

A large variety of cells adhere to and spread on specific regions within the triple helix of collagens, mainly via alpha 1 beta 1 and alpha 2 beta 1 integrins. Disruption of collagen triple helical integrity generally affects the efficiency of cell adhesion on different collagens including collagen V. This report addresses the question of the importance of the linear sequence of the constitutive alpha-chains versus the triple helical conformation in the recognition of collagen V binding sites. To investigate this question, in vitro renaturation of the isolated alpha 1 (V) and alpha 2 (V) chains was performed according to the annealing procedure and formation of the triple helix was monitored by rotary shadowing and by mild trypsin digestion followed by electrophoretic analysis. The results indicate that the alpha 1 (V) and alpha 2 (V) homotrimeric reassociation can occur up to a full-length triple helix but intermediate forms of 50-200 nm long rod-like segments are also observed. We have previously shown that alpha 1 beta 1 and alpha 2 beta 1 integrins, the major collagen receptors, are also involved in cell adhesion to native collagen V. Therefore we chose the following two different cell lines for this study: HT1080 (a human fibrosarcoma cell line) expressing alpha 2 beta 1 and HBL100 (a human mammary epithelial cell line) containing significant amounts of alpha 1 beta 1 and alpha 2 beta 1 integrins. We showed that both alpha 1 (V) and alpha 2(V) homotrimers induced cell adhesion but refolded alpha2(V) chains were more efficient and promoted cell adhesion as well as native collagen V. Thermal stability of refolded alpha-chains was monitored by adhesion promoting activity and showed that cell adhesion was dependent on triple helical conformation of the substrates. Adhesion in all cases was strongly Mg2+ and Mn(2+)-dependent and Ca2+ ions alone were ineffective. Antibodies against alpha 2 and beta 1 integrin subunits completely inhibited HT1080 cell adhesion to all substrates. Moreover, addition of cyclic RGD peptides, which had been shown to interact with alpha 2 beta 1, dramatically affected HT1080 cell adhesion to native collagen V and to the refolded alpha-chains. Antibody to beta 1 subunits abolished HBL100 cell adhesion to all substrates. A complete inhibition of HBL100 cell adhesion to native collagen V was achieved only by simultaneous addition of function-blocking specific monoclonal antibodies against alpha 1 and alpha 2 integrin subunits. However, only alpha 2 beta 1 was engaged obviously in HBL100 cell adhesion to refolded alpha-chains. These data indicate that triple helical conformation is particularly critical for alpha 2 beta 1- and alpha 1 beta 1-dependent adhesion and that the integrin alpha 2 beta 1 is a dominant functional receptor for refolded alpha-chains. We conclude that alpha 2 beta 1-dependent adhesion seems to involve multiple different conformational binding sites while alpha 1 beta 1-dependent adhesion is more restricted to the heterotrimeric native form of the molecule.

Animals↗

PubMed2000

Regulation of inflammation by collagen-binding integrins alpha1beta1 and alpha2beta1 in models of hypersensitivity and arthritis.

Adhesive interactions play an important role in inflammation by promoting leukocyte attachment and extravasation from the vasculature into the peripheral tissues. However, the importance of adhesion molecules within the extracellular matrix-rich environment of peripheral tissues, in which cells must migrate and be activated, has not been well explored. We investigated the role of the major collagen-binding integrins, alpha1beta1 and alpha2beta1, in several in vivo models of inflammation. mAb's against murine alpha1 and alpha2 were found to significantly inhibit effector phase inflammatory responses in animal models of delayed-type hypersensitivity (DTH), contact hypersensitivity (CHS), and arthritis. Mice that were alpha1-deficient also showed decreased inflammatory responses in the CHS and arthritis models when compared with wild-type mice. Decreased leukocyte infiltration and edema formation accompanied inhibition of antigen-specific models of inflammation, as nonspecific inflammation induced by croton oil was not inhibited. This study demonstrates the importance in vivo of alpha1beta1 and alpha2beta1, the collagen-binding integrins, in inflammatory diseases. The study also extends the role of integrins in inflammation beyond leukocyte attachment and extravasation at the vascular endothelial interface, revealing the extracellular matrix environment of peripheral tissues as a new point of intervention for adhesion-based therapies.

Animals↗

PubMed2003

Cell adhesion and cytotoxicity studies over polyanionic collagen surfaces with variable negative charge and wettability.

This work describes the cytotoxicity, and the cell adhesion behavior of K562 cell line from human erythroleukemia transfected with the DNA for the alpha(2)beta(1) integrin over type-I collagen matrices with variable degree of carboxyl group and wettability. The results showed that type-I collagen materials with variable degree of carboxyl group prepared by selective hydrolysis of carboxyamide side chains of Asn and Gln residues present in the protein, independently from the extent of side chain hydrolysis, was characterized by preserved triple helix structure for materials with a carboxyl group content up to 87 +/- 17. Imbibition and wettability increased linearly with increasing carboxyl group content from 46 +/- 12 to 87 +/- 17, and no signs of cytotoxicity were detected. Nevertheless, in comparison to native collagen, K562 cell adhesion to PACMs was significantly improved by factors ranging from 1.60 to 1.47x, with the reduction in cell adhesion observed with increasing carboxyl content attributed to a balance between the inhibition of increasing negative charge and the stimulation by increased wettability. On the other hand, the overall improvement of K562 cell adhesion to polyanionic collagen was attributed to the introduction of new distinct motifs described as the minimal active recognition sequence for alpha(2)beta(1) integrins binding with type-I collagen produced as a result of Asn-Gly Glu-Ala alpha2(I)294-297, and Gly Gln-Arg-Gly Val-Val carboxyamide side chains hydrolysis.

Animals↗

PubMed2000

Integrins alpha2beta1 and alpha4beta1 can mediate SA11 rotavirus attachment and entry into cells.

Most mammalian rotaviruses contain tripeptide amino acid sequences in outer capsid proteins VP4 and VP7 which have been shown to act as ligands for integrins alpha2beta1 and alpha4beta1. Peptides containing these sequences and monoclonal antibodies directed to these integrins block rotavirus infection of cells. Here we report that SA11 rotavirus binding to and infection of K562 cells expressing alpha2beta1 or alpha4beta1 integrins via transfection is increased over virus binding to and infection of cells transfected with alpha3 integrin or parent cells. The increased binding and growth were specifically blocked by a monoclonal antibody to the transfected integrin subunit but not by irrelevant antibodies. In our experiments, integrin activation with phorbol ester did not affect virus binding to cells. However, phorbol ester treatment of K562 parent and transfected cells induced endogenous gene expression of alpha2beta1 integrin, which was detectable by flow cytometry 16 h after treatment and quantitatively correlated with the increased level of SA11 virus growth observed after this time. Virus binding to K562 cells treated with phorbol ester 24 h previously and expressing alpha2beta1 was elevated over binding to control cells and was specifically blocked by the anti-alpha2 monoclonal antibody AK7. Virus growth in alpha4-transfected K562 cells which had also been induced to express alpha2beta1 integrin with phorbol ester occurred at a level approaching that in the permissive MA104 cell line. We therefore have demonstrated that two integrins, alpha2beta1 and alpha4beta1, are capable of acting as cellular receptors for SA11 rotavirus.

Antibodies, Monoclonal↗

PubMed1999

Expression of beta-integrin adhesion molecules in non-Hodgkin's lymphoma: correlation with clinical and evolutive features.

PURPOSE: To analyze beta-integrin expression in non-Hodgkin's lymphomas (NHLs) in order to assess its distribution among histologic subtypes and correlate with clinical features and outcome. PATIENTS AND METHODS: The expression of alpha2 through alpha6 and beta1 common chains of very late activation antigen (VLA ) molecules and alphaL (CD11a) and beta2 common (CD18) chains of leukocyte function-associated antigen 1 molecule were studied in 137 patients with NHL. Immunostaining was performed by a streptavidin-biotin alkaline phosphatase method, and integrin expression was semiquantitatively assessed. Correlation with clinical features was analyzed in 80 patients consecutively diagnosed as having immunocytoma (five cases), follicular lymphoma (19 cases), mantle-cell lymphoma (MCL; four cases), diffuse large-cell lymphoma (DLCL; 40 cases), lymphoblastic lymphoma (LL; six cases), anaplastic Ki-1-positive lymphoma (one case), and other peripheral T-cell lymphoma (five cases). RESULTS: MCL cells did not show alpha2 and alpha6 expression, whereas most expressed weak to moderate levels of alpha3, alpha4, and alpha5. LL mostly showed alpha2 to alpha5 expression, whereas alpha6 was observed in seven of 11 cases (higher proportion than that shown in other subgroups). Alpha chains of VLA molecules were present more frequently in T-cell than in B-cell lymphomas. Patients with moderate/strong alpha4, CD11a, and beta2 common chain expression presented more frequently with advanced stage and bone marrow infiltration. Moderate/strong alpha4, alpha5, and beta1 common chain expression correlated with extranodal involvement. In the subset of B-cell DLCL patients, negative/weak expression of alpha3 and alpha4 chains was related to a higher complete response rate. Moreover, negative or weak expression of alpha2, alpha3, alpha4, and beta1( )common chain had favorable significance for overall and failure-free survivals. CONCLUSION: In NHL, beta-integrin expression is related to histologic subtype. The expression pattern of these molecules probably influences disease dissemination and patients' prognoses.

CD18 Antigens↗

PubMed2003

Studies of the local conformational properties of the cell-adhesion domain of collagen type IV in synthetic heterotrimeric peptides.

Collagen type IV is a specialized form of collagen that is found only in basement membranes. It is involved in integrin-mediated cell-adhesion processes, and the responsible binding sites for the alpha1beta1 integrin cell receptor have been identified as Asp461 of the two alpha1 chains and Arg461 of the alpha2 chain. In the most plausible stagger of native collagen type IV the alpha2 chain is the tailing one. This has recently been confirmed by the differentiated binding affinities of synthetic heterotrimeric collagen peptides in which the chains were staggered in this native register as well as in the less plausible alpha1alpha2alpha1' register with an artificial cystine knot. In the present work, two heterotrimeric collagen peptides with chain registers identical to the previous ones were synthesized for fluorescence resonance energy transfer and emission anisotropy measurements, exploiting the native Phe464 in the alpha2 chain as donor and an Ile467Tyr mutation in the alpha1' chain as acceptor fluorophore. This fluorophore pair allowed extraction of more detailed information on the conformational properties of the cell-adhesion epitope incorporated into the central part of the trimeric collagen model peptides. A comparison of the experimentally derived values of the interfluorophore distance and of the orientation factor kappa(2) with the values extracted from the molecular model of the trimer in the native stagger confirmed a triple-helical structure of the adhesion-site portion at low temperature. The thermal unfolding of this central domain was specifically monitored by emission anisotropy, allowing unambiguous assignment of the three structural domains of the trimeric collagen molecules detected by microcalorimetry, with the integrin binding site as the portion of weakest triple-helical stability flanked by two more stable triple-helical regions. The results are consistent with the picture of a conformational microheterogeneity as the responsible property for selective recognition of collagens by interacting proteins.

Amino Acid Sequence↗

PubMed2001

Evidence that integrins contribute to multiple stages in the consolidation of long term potentiation in rat hippocampus.

Three structurally distinct groups of antagonists were used to test the hypothesis that integrin adhesion receptors play an essential role in consolidating (stabilizing) long term potentiation of the Schaffer collaterals in rat hippocampus. Comparisons were made of percent potentiation at antagonist-treated versus control sites within CA1 stratum radiatum of the same hippocampal slice. Function blocking antibodies against the alpha5 subunit of the fibronectin receptor had no effect on baseline responses or initial potentiation but resulted in a >30% reduction, relative to within-slice control long term potentiation, 45 min later. Larger reductions were recorded in separate experiments continued for 4 h after the induction of potentiation. Alpha(v) and alpha2 subunit antibodies did not reliably affect the stabilization of potentiation. An antagonist peptide with preference for beta1 integrins produced a slowly developing decline of the type seen with alpha5 antibodies. A cyclic peptide antagonist reduced potentiation within 10 min of induction and caused an almost 40% decrease over 45 min. Two disintegrins (snake toxins that potently block integrins) were very effective in preventing the consolidation of long term potentiation: echistatin reduced potentiation by >70%, while triflavin caused approximately 50% decrease. The suppressing effects of echistatin were concentration-dependent, obtained with treatment after induction, and much more rapid than the effects of antibodies. Rapid declines in potentiation were particularly evident when the two disintegrins were applied together. These results indicate that hippocampal fibronectin receptors (alpha5/beta1 integrin) contribute importantly to a slowly developing phase of long term potentiation consolidation. They also suggest that other integrins are critical to aspects of consolidation occurring in the first few minutes after induction.

Animals↗

PubMed2003

Distribution of alpha and beta integrin subunits in the adult rat hippocampus after pilocarpine-induced neuronal cell loss, axonal reorganization and reactive astrogliosis.

Integrins are alphabeta-heterodimers that act as cell-extracellular matrix (ECM) and cell-cell adhesion molecules. During development, they are involved in axonal guidance, synaptogenesis, and in astrocytic maturation and migration. Here, we have examined the potential role of the integrin subunits alpha1-alpha5 and beta1-beta5 in axonal sprouting, synaptogenesis and reactive astrogliosis in the adult rat brain caused by pilocarpine-induced status epilepticus (SE). Strong hippocampal immunoreactivity of alpha1-alpha5, beta1, beta3, beta4, and beta5 was observed in the pia mater, in vascular endothelia, and in astrocytes at the pial surface. beta2 immunoreactivity was found exclusively in vascular endothelia. Pyramidal cells and interneurons of CA3-CA1, as well as hilar neurons revealed moderate alpha5 labeling in their cell bodies. Mossy fibers were immunoreactive for alpha2, beta4, and beta5. After pilocarpine-induced SE, strong immunoreactivity for alpha1, alpha2, alpha4, alpha5, beta1, beta3, and beta4 was observed in reactive astrocytes. Our results show that members of the integrin family are differently distributed in cellular and subcellular compartments of the hippocampus and undergo specific patterns of regulation, which may be important for lesion-induced reactive changes in the adult brain.

Animals↗

PubMed1999

Integrin subunit gene expression is regionally differentiated in adult brain.

Integrins are a diverse family of heterodimeric (alphabeta) adhesion receptors recently shown to be concentrated within synapses and involved in the consolidation of long-term potentiation. Whether neuronal types or anatomical systems in the adult rat brain are coded by integrin type was studied in the present experiments by mapping the relative densities of mRNAs for nine alpha and four beta subunits. Expression patterns were markedly different and in some regions complementary. General results and areas of notable labeling were as follows: alpha1-limited neuronal expression, neocortical layer V, hippocampal CA3; alpha3 and alpha5-diffuse neuronal and glial labeling, Purkinje cells, hippocampal stratum pyramidale, locus coeruleus (alpha3); alpha4- discrete limbic regions, olfactory cortical layer II, hippocampal CA2; alpha6-most prominently neuronal, neocortical subplate, endopiriform, subiculum; alpha7-discrete, all neocortical layers, hippocampal granule cells and CA3, cerebellar granule and Purkinje cells, all efferent cranial nerve nuclei; alpha8-discrete neuronal, deep cortex, hippocampal CA1, basolateral amygdala, striatum; alphaV-all cortical layers, striatum, Purkinje cells; beta4-dentate gyrus granule cells; beta5-broadly distributed, neocortex, medial amygdala, cerebellar granule and Purkinje cells, efferent cranial nerve nuclei; alpha2, beta2, and beta3-mRNAs not detected. These results establish that brain subfields express different balances of integrin subunits and thus different integrin receptors. Such variations will determine which matrix proteins are recognized by neurons and the types of intraneuronal signaling generated by matrix binding. They also could generate important differences in synaptic plasticity across brain systems.

Animals↗

PubMed2000

Structural and functional analysis of the recombinant G domain of the laminin alpha4 chain and its proteolytic processing in tissues.

The C-terminal G domains of laminin alpha chains have been implicated in various cellular and other interactions. The G domain of the alpha4 chain was now produced in transfected mammalian cells as two tandem arrays of LG modules, alpha4LG1-3 and alpha4LG4-5. The recombinant fragments were shown to fold into globular structures and could be distinguished by specific antibodies. Both fragments were able to bind to heparin, sulfatides, and the microfibrillar fibulin-1 and fibulin-2. They were, however, poor substrates for cell adhesion and had only a low affinity for the alpha-dystroglycan receptor when compared with the G domains of the laminin alpha1 and alpha2 chains. Yet antibodies to alpha4LG1-3 but not to alpha4LG4-5 clearly inhibited alpha(6)beta(1) integrin-mediated cell adhesion to laminin-8, indicating the participation of alpha4LG1-3 in a cell-adhesive structure of higher complexity. Proteolytic processing within a link region between the alpha4LG3 and alpha4LG4 modules was shown to occur during recombinant production and in endothelial and Schwann cell culture. Cleavage could be attributed to three different peptide bonds and is accompanied by the release of the alpha4LG4-5 segment. Immunohistology demonstrated abundant staining of alpha4LG1-3 in vessel walls, adipose, and perineural tissue. No significant staining was found for alpha4LG4-5, indicating their loss from tissues. Immunogold staining demonstrated an association of the alpha4 chain primarily with microfibrillar regions rather than with basement membranes, while laminin alpha2 chains appear primarily associated with various basement membranes.

Adipose Tissue↗

PubMed1999

alpha6beta1 integrin expression in hepatocarcinoma cells: regulation and role in cell adhesion and migration.

Liver carcinogenesis is associated with striking changes in the integrin repertoire of hepatocytes, including the overexpression of the laminin and collagen receptors alpha1beta1 and the de novo induction of the laminin receptor alpha6beta1. Our aim was to analyze the role of pro-inflammatory cytokines, interferons and fibrogenic cytokines TGF-beta and FGF2 in the regulation of the expression of beta1 integrins by neoplastic hepatocytes. The 2 human hepatocellular cell lines HepG2 and Hep3B were used as models. Integrin expression was assessed by qualitative methods (immunocytochemistry, Western blotting) and semi-quantitative techniques (FACS, cellular ELISA), before and after stimulation by TNFalpha, IL1-beta, TGF-beta, FGF2, interferon gamma and interferon alpha-2b. HepG2 and Hep3B constitutively expressed alpha1, alpha2, alpha6 and beta1 chains. A 24 to 48-hr stimulation with pro-inflammatory cytokines, TGF-beta and FGF2 induced a significant increase in the concentrations of all integrin chains. The maximum induction was registered for beta1 chain, which presented increases amounting up to 3, 4 and 7 times the control values in the presence of, respectively, TNF alpha/IL1-beta, TGF-beta and FGF2. Interferons had no direct effect on integrin expression and partially antagonized the effects of TNF alpha and TGF-beta. The increased concentrations of integrin chains were associated with an increased membrane expression of the corresponding dimers and with an increased adhesion of stimulated hepatocytes to laminin, which was antagonized by neutralizing anti-beta1 and anti-alpha6 antibodies. Finally, anti-alpha6 antibody inhibited the migration of HepG2 and Hep3B cells in reconstituted basement membrane. Our results suggest that the stimulation of alpha6beta1 integrin expression in hepatocarcinoma cells is essential for cell adhesion and migration.

Carcinoma, Hepatocellular↗

PubMed1997

Up-regulated beta1-integrin expression in autoimmune thyroid disorders.

Lymphocytic infiltration of the thyroid gland in autoimmune thyroid disorders requires, as a first step, their attachment to endothelial cells (EC) and, subsequently, interaction with thyrocytes and extracellular matrix proteins. Recent studies have focused on the pathophysiologic role of beta1-integrins as adhesion receptors for extracellular matrix proteins and as cell-to-cell adhesion receptors. In this study, we examine by flow cytometry and immunohistochemical techniques the differences in expression of beta1-integrins in thyrocytes and EC between normal thyroids and thyroid glands from patients with Graves' disease (GD) and Hashimoto's thyroiditis (HT). Remarkably, we found an up-regulated de novo expression of very late antigen (VLA)-alpha6 subunit in thyrocytes in close proximity to lymphocyte infiltrates in GD and HT thyroid glands, with no reactivity in control thyroids. Moreover, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and IL-1beta produced a significant enhancement of VLA-alpha6 expression in vitro in thyrocytes in culture. In addition, an up-regulated expression of VLA-alpha5 and beta1 subunits was found in thyrocytes from GD and HT glands, specifically in those areas more severely inflamed. VLA-alpha2 was basally expressed in middle size and large vessels in control glands, with an increased expression in vessels of all sizes in HT and GD glands. Dendritic cells in thyroid lymphoid follicles were also positive for VLA-beta1, alpha2 and alpha6 subunits. These results indicate the existence of an up-regulatory process in the expression of beta1-integrins, particularly the alpha6 subunit, in several cell types from inflamed GD and HT thyroid glands, suggesting that these integrins could play a relevant role in localizing and perpetuating the autoimmune response in the thyroid gland in autoimmune thyroid disorders.

Adult↗

PubMed2002

Quantitative analysis of adhesion molecules on cellular constituents of the human uterine microenvironment under the influence of estrogen and progesterone.

The uterus contains all the components of a tertiary lymphoid compartment. We hypothesize that specific leukocyte recruitment to the endometrium during the secretory phase of the menstrual cycle and early pregnancy limits the type of immunocyte that gains access. The present study utilized flow cytometry to define and quantify adhesion molecules possibly used by decidual infiltrating lymphocytes (DIL) as homing receptors, uterine microvascular myometrial endothelial cells (UtMVE-Myo) as addressins, and secretory endometrial stroma cells (STO) as retainment factors. Human umbilical cord vein endothelial cells and peripheral blood lymphocytes were used as control cells for comparison studies. DIL were composed of predominantly lymphocyte function-associated antigen (LFA)-1+, intercellular adhesion molecule (ICAM)-1+, LFA-2+, LFA-3+, gp150,95+, alpha1beta1+, Hermes cell adhesion molecule (H-CAM)+, and neural cell adhesion molecule (N-CAM)+ (CD56(bright)) memory/effector natural killer cells. A significant number of UtMVEC-Myo expressed platelet endothelial cell adhesion molecule (PECAM)-1, a percentage were uniquely LFA-3+, and alpha4 integrin expression was uniquely high. An increased number of STO uniquely expressed alpha3, beta3, and LFA-3, whereas alpha2, alpha4, alphaVbeta3, and H-CAM were significantly increased. Possible unique adhesions of DIL:UtMVEC-Myo included SLe(x):PECAM, vascular cell adhesion molecule-1:alpha4, and LFA-2:LFA-3, whereas DIL:STO included LFA-2:LFA-3 and N-CAM:N-CAM. Unique molecules on DIL may also associate with extracellular matrix (ECM) or complement on UtMVEC-Myo or STO to form gp150,95:fibrinogen/iC3b/C3dg, alpha1beta1:laminin (LM)/collagen (CO), and ICAM-1:fibronectin (FN) interactions. Bridges of ECM may also form between DIL and UtMVEC-Myo adhesion molecules including ICAM-1:FN:ICAM-1 and alpha4beta1:FN:alpha4beta1. DIL:ECM:STO interactions may involve alpha2beta1:CO:alpha2beta1, alpha3beta1:LM/CO/FN:alpha3beta1, alphaVbeta3:VN:alphaVbeta3, and H-CAM:hyaluronate:H-CAM. It is likely that many adhesion molecules play a role in the recruitment and retainment of specialized lymphocytes within the uterine microenvironment. (Mackay et al., 1990).

Adult↗