beta1-Integrin mediates cell adhesion, haptotaxis, and chemokinesis of hepatoma cells.
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Journal Article↗
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Role of the beta1-integrin subunit in the adhesion, extravasation and migration of T24 human bladder carcinoma cells.
The abilities of tumor cells to extravasate from the blood vessel system and to migrate through the connective tissue are prerequisites in metastasis formation. Both processes are chiefly mediated by integrins, which mediate both cell-cell and cell-matrix interactions. We investigated the role of integrin subunits in the adhesion, extravasation and migration of the highly invasive human bladder carcinoma cell line T24. Here we show that inhibition of the beta(1)-integrin subunit using the specific beta(1)-integrin blocking antibody 4B4 significantly reduces the adhesion to HUVEC and transmigratory activity of T24 cells. The blockade of the beta(1)-integrin subunit also resulted in a significantly reduced locomotory activity of T24 cells. A detailed cell migration analysis on a single cell level revealed that blockade of the beta(1)-integrin subunit leads to an altered migration pattern of single cells but does not influence migration per se. Migration parameters such as time active, velocity and distance migrated were significantly reduced as compared to untreated control cells. Our observations strongly suggest a central role for the beta(1)-integrin subunit in forming the cell-cell and cell-matrix bonds necessary for adhesion, extravasation and migration.
Carcinoma↗
TGFbeta2 influences alpha5-beta1 integrin distribution in human lens cells.
TGFbeta plays a central role in posterior capsule opacification, in which cell proliferation and matrix deposition, accompanied by capsular wrinkling, are largely responsible for the increased light scatter involved. Human FHL124 cells were plated onto uncoated glass coverslips to form circular patches so that the central cells reached confluency while the peripheral cells grew outwards. Cell patches were exposed to serum free (SF) EMEM (control) or TGFbeta supplemented (10 ng ml(-1)) EMEM. Fibronectin (Fn), alpha5beta1 integrin and F-actin were localized by immunofluorescence techniques and analysed by confocal microscopy. In the confluent, central cells in SF medium alpha5beta1 showed a punctate distribution while Fn was present in strongly staining fibres. TGFbeta had no effect on integrin or Fn distribution in confluent cells. In the peripheral, motile cells of the patches in SF conditions alpha5beta1 was localized in well-defined focal adhesion plaques at the ends of actin stress fibres, while Fn was distributed in a punctate perinuclear pattern. TGFbeta had a profound dispersing effect on the integrin causing a widespread distribution of alpha5beta1 in the membrane with no apparent association with the actin filaments. The cells had a more fibroblastic morphology with increased deposition of Fn near the nucleus. All the TGFbeta-induced changes were inhibited by the TGFbeta antibody CAT152 (Cambridge Antibody Technology). Culture with a function-blocking alpha5 antibody or Fn antibody resulted in detachment of the peripheral cells from the patches, but the central cells remained intact. The patch culture method therefore provides a convenient means of investigating the differences between confluent and growing lens cells both in terms of the patterns of alpha5beta1 integrin and Fn and also in the response of the molecular arrangements of both to TGFbeta2.
Actins↗
Structural requirements of MLD-containing disintegrins for functional interaction with alpha 4 beta 1 and alpha 9 beta1 integrins.
Three non-RGD-containing disintegrins, VLO5, EO5, and EC3, belong to the heterodimeric family of these snake venom-derived proteins. They are potent inhibitors of certain leukocyte integrins such as alpha4beta1, alpha4beta7, and alpha9beta1, and act through the MLD motif present in one of their subunits. However, the selectivity of these disintegrins to interact with integrins is related to the amino acid composition of the integrin-binding loop in the MLD-containing subunit. The most important amino acid is that preceding the MLD motif. In vitro experiments in adhesion and ELISA assays revealed that the TMLD-containing disintegrins, VLO5 and EO5, appeared to be very potent inhibitors of human alpha4beta1 and alpha9beta1 and less effective in inhibition of the alpha4beta7 integrin. The reverse effect was observed for the AMLD-containing disintegrin, EC3. The data with native disintegrins were confirmed by experiments with synthetic peptides displaying TMLD and AMLD motifs. The MLD-containing disintegrins showed differential activities to inhibit human and murine alpha4beta1 integrin. EC3 was a weaker inhibitor of human integrin, whereas VLO5 and EO5 less actively inhibited murine alpha4beta1. These data describe a useful set of potent and selective integrin antagonists and suggest conformational requirements of human and mouse integrins for interaction with ligands.
Amino Acid Motifs↗
Expression of alpha3 beta1 integrin in suprabasal layers of the epidermis of melanocytic tumors is due to epithelial hyperproliferation.
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Cell Division↗
The tetraspan protein epithelial membrane protein-2 interacts with beta1 integrins and regulates adhesion.
The growth arrest-specific-3 (GAS3)/PMP22 proteins are members of the four-transmembrane (tetraspan) superfamily. Although the function of these proteins is poorly understood, GAS3/PMP22 proteins have been implicated in the control of growth and progression of certain cancers. Epithelial membrane protein-2 (EMP2), a GAS3/PMP22 family member, was recently identified as a putative tumor suppressor gene. Here, we addressed the normal function of EMP2 by testing the prediction that it influences integrin-related cell functions. We observed that EMP2 associates with the beta(1) integrin subunit. Co-immunoprecipitation and immunodepletion experiments indicated that approximately 60% of beta(1) integrins and EMP2 can be isolated in common protein complexes. Whereas this association between EMP2 and beta(1) integrin may be direct or indirect, it has features of integrin heterodimer selectivity. Thus, by laser confocal microscopy, EMP2 colocalized with alpha(6)beta(1) but not alpha(5)beta(1) integrin. Increased expression of EMP2 also influenced the integrin heterodimer repertoire present on the plasma membrane. EMP2 specifically increased the surface expression of the alpha(6)beta(1) integrin while decreasing that of the alpha(5)beta(1) protein. Reciprocally, reduction in EMP2 expression using a specific ribozyme decreased surface expression of alpha(6)beta(1) integrin. Accordingly, these EMP2-mediated changes resulted in a dramatic alteration in cellular adhesion to extracellular matrix proteins. This study demonstrates for the first time the interaction of a GAS3/PMP22 family member with an integrin protein and suggests that such interactions and their functional consequences are a physiologic role of GAS3/PMP22 proteins.
3T3 Cells↗
Human ether-a-go-go-related gene 1 channels are physically linked to beta1 integrins and modulate adhesion-dependent signaling.
Adhesive receptors of the integrin family are primarily involved in cell-extracellular matrix adhesion. Additionally, integrins trigger multiple signaling pathways that are involved in cell migration, proliferation, survival, and differentiation. We previously demonstrated that the activation of integrins containing the beta(1) subunit leads to a selective increase in potassium currents carried by the human ether-a-go-go-related gene (hERG) channels in neuroblastoma and leukemia cells; this current activation modulates adhesion-dependent differentiation in these cells. We hypothesized that the cross-talk between integrins and hERG channels could be traced back to the assembly of a macromolecular signaling complex comprising the two proteins. We tested this hypothesis in both SH-SY5Y neuroblastoma cells and in human embryonic kidney 293 cells stably transfected with hERG1 and, therefore, expressing only the full-length hERG1 protein on the plasma membrane. The beta(1) integrin and hERG1 coprecipitate in these cells and colocalize in both intracellular and surface membrane compartments. The two proteins also coprecipitate with caveolin-1, suggesting the localization of the complex in lipid rafts/caveolae. hERG1-transfected cells undergo an activation of hERG currents after beta(1) integrin-mediated adhesion to fibronectin; concomitant with this activation, the focal adhesion kinase associates with the hERG1 protein and becomes tyrosine phosphorylated. Using hERG1-specific inhibitors, we show that the tyrosine phosphorylation of focal adhesion kinase is strictly dependent on hERG channel activity. Similarly, the activity of the small GTPase Rac1 turned out to be dependent on hERG currents. On the whole, these data indicate that the hERG1 protein associates with beta(1) integrins and modulates adhesion receptor signaling.
Cell Adhesion↗
[Beta1 integrins and edema formation in acute inflammation--new therapeutic possibilities?].
BACKGROUND: The role of the intestitium (the extracellular and extravascular tissue) with regard to transcapillary fluid balance and control of interstitial fluid volume has normally been considered to be a "passive controller". MATERIAL AND METHODS: This review is based upon literature collected through the authors' own studies and through Medline searches. RESULTS: Recent studies, however, indicate that the connective tissue cells can actively modulate physical properties of the interstitial matrix, so that it becomes an "active" participant in transcapillary fluid exchange and thereby interstitial fluid homeostasis. The beta 1-integrin system seems to provide a common pathway by which the cells can raise and lower interstitial fluid pressure and thereby regulate the tissue fluid volume. INTERPRETATION: Experiments in which a new anti-inflammatory agent (alpha-trinositol), platelet-derived growth factor (PDGF), and a prostagladin F2 alpha-analog (latanoprost) modulate interstitial fluid pressure and oedema generation in acute inflammation, suggest that the extracellular matrix can be a target for pharmacological intervention during inflammatory processes.
Acute Disease↗
Influence of beta1 integrins on epidermal squamous cell carcinoma formation in a transgenic mouse model: alpha3beta1, but not alpha2beta1, suppresses malignant conversion.
Although aberrant integrin expression has been documented in many epithelial tumors, little is known about how integrins influence neoplastic progression. To examine this issue, transgenic mice in which the alpha2beta1 or alpha3beta1 integrin was expressed in the suprabasal epidermal layers via the involucrin promoter were subjected to skin carcinogenesis. Equal numbers of benign squamous papillomas were observed in transgenic and wild-type animals. However, the frequency of conversion of papillomas to malignant squamous cell carcinomas was much lower in alpha3beta1 transgenic than in alpha2beta1 transgenic and wild-type mice. No differences were observed in apoptosis or in the expression of endogenous integrins in transgenic and wild-type papillomas. However, alpha3beta1 transgenic papillomas displayed a diminished proliferative capacity and were more highly differentiated as judged by BrdUrd incorporation and keratin 10 expression, respectively, than alpha2beta1 transgenic and wild-type papillomas. Two proteins that associate with alpha3beta1 and not alpha2beta1 are extracellular matrix metalloproteinase inducer and CD81. Extracellular matrix metalloproteinase inducer expression correlated inversely with the degree of differentiation in normal epidermis and in transgenic and wild-type papillomas. Up-regulation of CD81 was observed in 100% of wild-type and 88% of alpha2beta1 transgenic papillomas but in only 25% of alpha3beta1 transgenic papillomas. CD81 was undetectable in untreated epidermis and strongly expressed in all transgenic and wild-type squamous cell carcinomas. Our results demonstrate that the alpha3beta1 integrin can suppress malignant conversion, and that the mechanism may involve CD81.
9,10-Dimethyl-1,2-benzanthracene↗
The effect of alpha4 beta1-integrin binding sequences of fibronectin on growth of cells from human hematopoietic progenitors.
Highly regulated interactions between adhesion receptors on progenitor cells and their extracellular matrix ligands are essential for the control of hematopoiesis in bone marrow stroma. We have examined the relationship between alpha4beta1-integrin-mediated adhesion and growth of CD34(+) cells by assessing their adhesive and migratory patterns of proliferation in a mixture of hematopoietic growth factors in the presence of different recombinant fragments of the HepII/IIICS region of fibronectin. CD34(+) cells were isolated from cord blood and placed in culture wells containing serum-free medium and growth factors. Wells were precoated with either the H120 fragment of fibronectin, which contains three alpha4beta1-integrin binding sites, or the H0 fragment, which lacks the two highest affinity alpha4beta1 binding sequences. Proliferation of single cells of CD34(+)38(+)DR+ and CD34(+)38(-)DR+ phenotypes occurred in contact with the H120 substrate and was associated with migration. Larger numbers of cells were used to quantitate proliferative responses. Cells growing in wells coated with H120 formed attachments to the base of the wells throughout the culture period. Higher total cell counts were consistently found in wells coated with H120 compared with H0 and bovine serum albumin controls. The difference was first apparent at day 8 of culture and reached a maximum at days 11 through 13, when expansion with H120 was a mean of 1.8-fold higher than that seen with H0 (P</= .0001). The greatest expansion (2.25-fold) with H120 compared with H0 was seen when the growth factor concentrations were reduced to 1/16 of the standard levels (P </= .001). The increase in total cell numbers was not at the expense of CD34(+) cells as numbers of these were similar in H120 and control cultures. These results provide evidence for synergy between growth factors and integrins that may be relevant to understanding hematopoiesis in marrow stroma.
ADP-ribosyl Cyclase↗
Use of a beta1 integrin-deficient human T cell to identify beta1 integrin cytoplasmic domain sequences critical for integrin function.
T cell activation rapidly and transiently regulates the functional activity of integrin receptors. Stimulation of CD3/T cell receptor, CD2 or CD28, as well as activation with phorbol esters, can induce within minutes an increase in beta1 integrin-mediated adhesion of T cells to fibronectin. In this study, we have produced and utilized a mutant of the Jurkat T cell line, designated A1, that lacks protein and mRNA expression of the beta1 integrin subunit but retains normal levels of CD2, CD3, and CD28 on the cell surface. Activation-dependent adhesion of A1 cells to fibronectin could be restored upon transfection of a wild-type human beta1 integrin cDNA. Adhesion induced by phorbol 12-myristate 13-acetate-, CD3-, CD2-, and CD28 stimulation did not occur if the carboxy-terminal five amino acids of the beta1 tail were truncated or if either of two well-conserved NPXY motifs were deleted. Scanning alanine substitutions of the carboxy-terminal five amino acids demonstrated a critical role for the tyrosine residue at position 795. The carboxy-terminal truncation and the NPXY deletions also reduced adhesion induced by direct stimulation of the beta1 integrin with the activating beta1 integrin-specific mAb TS2/16, although the effects were not as dramatic as observed with the other integrin-activating signals. These results demonstrate a vital role for the amino-terminal NPXY motif and the carboxy-terminal end of the beta1 integrin cytoplasmic domain in activation-dependent regulation of integrin-mediated adhesion in T cells. Furthermore, the A1 cell line represents a valuable new cellular reagent for the analysis of beta1 integrin structure and function in human T cells.
Amino Acid Sequence↗
Identification and isolation of candidate human colonic clonogenic cells based on cell surface integrin expression.
BACKGROUND & AIMS: The surface epithelium of the colon is being replaced constantly with cells derived from the stem cells of the crypt. Although the location of the stem cells is known, there are no markers for these cells. This study tested the hypothesis that colonic stem cells might be isolated and cultured on the basis of specific integrin expression patterns in normal human colonic epithelium. METHODS: Integrin expression in normal human colonic mucosa was determined by using indirect immunofluorescence. Crypt cells were then isolated as single cells from normal colon tissues and the expression pattern of integrins was analyzed by flow cytometry. Based on the specific expression of integrin beta1 in colonic crypts, the cells were sorted by using a flow cytometer, and colony assays in soft agar were performed to evaluate the clonogenicity of the sorted cells. RESULTS: By immunofluorescence, the cells located in the lower one third of crypts expressed higher levels of beta1-integrin than the cells in the remainder of the crypt. When isolated crypt cells were stained with the beta1-integrin antibody and examined in a flow cytometer, there were 2 peaks of fluorescence. Sorting of crypt cells based on staining with anti-beta1 integrin antibody produced a cell population with a significantly enhanced ability to form colonies. CONCLUSIONS: beta1-integrin is a candidate surface marker for the proliferative zone of the human colonic crypt. Our in vitro culture system for the clonal growth of a single colonic crypt cell suspension could facilitate the identification of other candidate stem cell markers.
Cell Adhesion↗
Integrin beta1-chain residues involved in substrate recognition and specificity of binding to invasin.
A highly conserved 14-amino-acid region of the chicken beta1 integrin chain (beta1chk residues 151-168) hypothesized to be involved in divalent cation coordination was analysed to determine whether invasin uses the same structural determinants as fibronectin (Fn) to recognize receptors. For the most part, both proteins required similar beta1 chain residues for integrin recognition, although the relative preference of the integrin for the two substrates could be inverted mutationally. Substitution mutations in the amino terminal residues of this region resulted in defective binding to both substrates by the receptor, while substitutions at the carboxyl-terminal end of this region were better tolerated. A derivative carrying the double substitution (KDD160RDV) had the unique phenotype of maintaining Fn binding while abolishing invasin binding, indicating that this region of the receptor may determine substrate specificity. These data indicate that the integrin beta1 chain possesses a ligand binding site shared by invasin and Fn that can be altered by mutation to allow greater preference for the normally lower affinity substrate Fn than for invasin. It was also established that the region analysed has the ability to bind divalent cations.
Adhesins, Bacterial↗
CD44 variants but not CD44s cooperate with beta1-containing integrins to permit cells to bind to osteopontin independently of arginine-glycine-aspartic acid, thereby stimulating cell motility and chemotaxis.
The expression of osteopontin (OPN), CD44 variants, and integrins has been correlated with tumorigenesis and metastasis. Here we show that these proteins cooperate to enhance cell motility. First, we demonstrate that several different CD44 variants bind to OPN in an arginine-glycineaspartic acid-independent manner, but that the standard form of CD44 does not. These CD44 variants bind to both the amino- and COOH-terminal portions of OPN independently of the arginine-glycine-aspartic acid sequence, suggesting that multiple domains on OPN can be bound by the CD44 variants. Antibodies directed against the integrin beta1 subunit are able to inhibit this binding. The binding of CD44 variants to OPN is significantly augmented by both anti-CD44s and anti-CD44v antibodies. This augmentation by anti-CD44 antibodies is OPN specific and, again, can be blocked by anti-beta1 antibodies. Finally, we show that OPN binding by CD44 variants/beta1-containing integrins promotes cell spreading, motility, and chemotactic behavior.
Animals↗
Colorectal cancer cell adhesion attenuates Ad-E2F-1 mediated apoptosis.
BACKGROUND: The complex interplay of cell adhesion molecules, intracellular signaling, and tumor growth behavior have important implications for the failure of most conventional cancer therapies. Cell adhesion to the basement membrane has been shown to promote tumor cell survival. We hypothesize that the presence of matrix substrate contributes to chemoresistance through signaling via cell adhesion molecule. MATERIALS AND METHODS: RKO colorectal cancer cells express integrin beta1 to adhere to substrate. We measured apoptosis of the cells after infection with adenovirus vector containing the transgene E2F-1 (Ad-E2F-1), a potent tumor suppressor gene, and Ad-LacZ (as control), both under the control of the cytomegalovirus promoter. Cells were plated on Matrigel, an extracellular substrate similar to basement membrane and compared to tissue culture plastic. Apoptosis was assessed by flow cytometry-based TUNEL assay and cell proliferation was assessed by WST-1 assay. E2F-1 expression was confirmed by Western blot analysis. A function-blocking anti-beta1 integrin antibody was used to assess the contribution of beta1 on cell survival. RESULTS: At 120 h postinfection of RKO cells with 50 multiplicity of infection, cells plated on plastic underwent marked apoptosis in response to Ad-E2F-1 compared with Ad-LacZ control-treated cells (53% vs 1% apoptosis, respectively). However, when cells were plated on Matrigel, the same dose of E2F-1 was ineffective at inducing apoptosis (3% vs 1% apoptosis, comparing Ad-E2F-1 with Ad-LacZ control). The cell proliferation assay showed >3-fold cell survival in E2F-1-infected cells on Matrigel vs plastic (P < 0.004). By Western blot analysis, attenuation of apoptosis may be a result of reduction in transduction efficiency on Matrigel and function-blocking anti-beta1 integrin antibody does not abolish the decrease in apoptosis afforded by Matrigel. CONCLUSIONS: These data suggest that escape from adenoviral E2F-1-mediated apoptosis, at least in part, is related to reduction of intracellular E2F-1 expression. Interactions involving cellular adhesion via beta1 integrin to matrix proteins does not seem to contribute toward gene therapy resistance. Further studies will investigate other specific receptor-ligand interactions after gene and/or chemotherapy.
Adenoviridae↗
The C-terminal domain V of perlecan promotes beta1 integrin-mediated cell adhesion, binds heparin, nidogen and fibulin-2 and can be modified by glycosaminoglycans.
Domain V of the major basement-membrane proteoglycan perlecan, a domain which consists of three laminin type G (LG) and four epidermal-growth-factor-like (EG) modules, was obtained in recombinant form by transfecting embryonic kidney cells with an episomal expression vector. A major 90-kDa fragment V was obtained together with fragments Va (74 kDa) and Vb (26 kDa) which were generated by endogenous proteolysis in front of the most C-terminal LG module. All three fragments bound to a heparin affinity column and could be displaced at a moderate (0.2 M) NaCl concentration. Rotary-shadowing electron microscopy demonstrated a three-globule structure for fragment V. Fragment V also showed a strong immunological cross-reaction with tissue-derived perlecan, indicating that it was folded into a native structure. A further, larger fragment, Vc, was apparently substituted with heparan sulphate and/or chondroitin sulphate chains and failed to bind to heparin. Fragment V but not fragment Vc promoted a distinct adhesion of several cell lines and this could be blocked by antibodies against the integrin beta1 chain. This domain may, however, represent only one of several cell-adhesive sites of perlecan. The recombinant perlecan fragment V bound in surface plasmon resonance assays to fibulin-2, laminin-nidogen complex, nidogen and two nidogen fragments. This indicated two different nidogen-binding epitopes on perlecan domain V with about a 10-fold difference in their affinities (Kd = 0.05-0.2 microM and about 2 microM). Perlecan domain V therefore seems to participate in the supramolecular assembly and cell connections of basement membranes.
Animals↗
Two pathways for induction of apoptosis by ultraviolet radiation in cultured human keratinocytes.
Loss of attachment may induce apoptosis in epithelial cells, but it is unclear whether substrate adhesion modulates apoptosis triggered by genotoxic agents such as ultraviolet radiation (UV). To investigate this issue, we plated neonatal human keratinocytes on different substrates and irradiated them with UVB. DNA strand breaks were nick-labeled to identify apoptotic nuclei. Keratinocytes grown in monolayers were less susceptible to UV-induced apoptosis than were cells freshly seeded on glass (ED50 2130 +/- 96 J per m2, mean +/- SD, versus 131 +/- 96 J per m2, mean +/- SD, respectively). This phenomenon depended on differences in integrin-mediated adhesion, because blocking of integrin beta1 with a monoclonal antibody increased sensitivity of keratinocyte monolayers to UV and an increase in beta1 integrin receptor occupancy by plating on fibronectin, type IV collagen, or keratinocyte-derived extracellular matrix diminished the UV-dependent apoptosis. Down-regulation of p53 with an anti-sense oligonucleotide did not affect apoptosis in glass-plated keratinocytes but effectively suppressed apoptosis in keratinocytes adhering via beta1 integrin. Thus, in addition to the known p53-dependent pathway, UV was able to induce a p53-independent apoptosis that could be blocked by integrin-mediated cell attachment (the integrin-sensitive pathway). The susceptibility to the p53-dependent apoptosis, but not to the integrin-sensitive process, varied among keratinocytes of different clonogenic potential: transit amplifying cells > stem cells > terminally differentiated cells. The p53-independent integrin-sensitive apoptotic pathway may provide an additional mechanism counteracting UV carcinogenesis in the skin.
Adhesins, Bacterial↗
Integrins of the beta1 family influence keratinocyte-lymphocyte interaction.
Data from the literature indicate that ICAM-1 molecules play an important role in keratinocyte interactions with lymphocytes via the lymphocyte function-associated-1 lymphocyte-adhesion molecule. We examined the role of beta1 integrins in keratinocyte-lymphocyte adhesion under different activation conditions. Among the beta1 integrins expressed on keratinocytes and lymphocytes detected by indirect immunofluorescence microscopy and flow cytofluorometry, primarily the alpha2 and the alpha3 subunits on both cell types were involved in keratinocyte-lymphocyte adhesion. Moreover, the highest adhesion level was observed when both cell types were activated by IFN-gamma for keratinocytes and phorbol 12-myristate 13-acetate for lymphocytes, suggesting that the former involved the protein kinase C pathway. Keratinocyte activation, characterized by the expression of ICAM-1, a decrease of beta1 integrins, and the absence of alpha5beta1 integrin, was required for optimal lymphocyte adhesion. Thus, beta1 integrins remaining at the surface of IFN-gamma-treated keratinocytes could be activated by this cytokine, and could synergize with ICAM-1 and lymphocyte function-associated-1 molecules to consolidate keratinocyte-lymphocyte adhesion.
Cell Adhesion↗