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PubMed1998

Glomerular extracellular matrix components and integrins.

It has become apparent that extracellular matrix components and their cellular receptors, the integrins, are important regulators of glomerular development and function. In this rapidly evolving field we studied the production of extracellular matrix components and integrins by rat glomerular visceral epithelial and mesangial cells, using molecular probes and antibodies that have recently become available. Special attention was paid to laminin isoforms and to splice variants of the integrin subunits alpha 3 and alpha 6. Results were compared to the in vivo expression in human fetal, newborn and adult kidneys. The mesangial cells were found to produce laminin-1, nidogen and two as yet unidentified laminin isoforms with putative alpha chains of about 395 (alpha x) and of 375 kDa (alpha y), tentatively described before as bovine kidney laminin. Furthermore, they expressed the integrins alpha 1 beta 1, alpha 2 beta 1, alpha 3A beta 1, alpha 5 beta 1, alpha v beta 3, alpha v beta 5, and small amounts of alpha 6A beta 1 and alpha 6B beta 1. The glomerular visceral epithelial cells produced the two new laminin isoforms mentioned above, laminin-5, but no laminin-1 or nidogen. The integrins alpha 2 beta 1, alpha 3A beta 1, alpha 6A beta 4, alpha 6B beta 4 and the integrin subunit alpha v were found to be expressed. We show that during nephrogenesis, the laminin alpha 1 chain disappears and is replaced by another alpha chain, possibly one of the two as yet unidentified alpha chains mentioned above. The laminin beta 1 chain is replaced by the beta 2 chain somewhat later in glomerular development. In general, the integrins found to be expressed in glomeruli of adult kidney were consistent with those found in cultured glomerular visceral epithelial and mesangial cells. No splice variant switch of the integrin alpha 3 or alpha 6 subunits could be demonstrated during nephrogenesis. Our results suggest an important role for the mesangial cell in providing nidogen as a crucial component of the supramolecular structure of the glomerular basement membrane. Furthermore our results indicate that laminin alpha x beta 2 gamma 1 and alpha y beta 2 gamma 1 isoforms are important in the glomerulus of adult kidney and that the integrin alpha 3A beta 1 is the main integrin receptor for laminin isoforms on glomerular visceral epithelial and mesangial cells, both in vitro and in vivo.

Adult↗

PubMed2000

alpha6 integrin expression in esophageal carcinoma.

One of the genes that the authors have isolated by a differential display of hepatocellular carcinoma compared to adjacent liver is the alpha6 integrin. alpha6 integrin is the major adhesion receptor for laminin and is suggested to be involved in tumor cell invasion and metastasis. To our knowledge, however, there are no reports on alpha6 integrin expression in esophageal carcinoma. We thus conducted a study to determine its clinicopathologic significance in human esophageal carcinoma. The tumor/normal (T/N) ratio of alpha6 integrin expression was calculated by Northern hybridization in 45 cases of esophageal carcinoma. In selected cases the expression of the alpha6 integrin variants A and B was also investigated by reverse transcriptase-polymerase chain reaction, and immunostaining for alpha6 integrin was performed. The expression levels of alpha6 integrin mRNA in the tumor tissue were greater than those of the corresponding normal tissue in 39 of 45 cases (87%). The overexpression of alpha6 integrin was also recognized by immunostaining. Fifteen cases with a high T/N ratio demonstrated a deeper invasion into the esophageal wall than the 30 cases with a low T/N ratio. Although there was no significant difference, the 15 cases with a high T/N ratio had a tendency for a worse prognosis. The ratio of the two variants (alpha6A/alpha6B) did not show any relationship to survival. The findings imply that alpha6 integrin is overexpressed in human esophageal carcinomas and that alpha6 integrin may play an important role in esophageal tumor invasion.

Adult↗

PubMed2002

Alpha 4 integrin signaling activates phosphatidylinositol 3-kinase and stimulates T cell adhesion to intercellular adhesion molecule-1 to a similar extent as CD3, but induces a distinct rearrangement of the actin cytoskeleton.

Dynamic regulation of beta(2) integrin-dependent adhesion is critical for a wide array of T cell functions. We previously showed that binding of high-affinity alpha(4)beta(1) integrins to VCAM-1 strengthens alpha(L)beta(2) integrin-mediated adhesion to ICAM-1. In this study, we compared beta(2) integrin-mediated adhesion of T cells to ICAM-1 under two different functional contexts: alpha(4) integrin signaling during emigration from blood into tissues and CD3 signaling during adhesion to APCs and target cells. Cross-linking either alpha(4) integrin or CD3 on Jurkat T cells induced adhesion to ICAM-1 of comparable strength. Adhesion was dependent on phosphatidylinositol (PI) 3-kinase but not p44/42 mitogen-activated protein kinase (extracellular regulated kinase 1/2), because it was inhibited by wortmannin and LY294002 but not U0126. These data suggest that PI 3-kinase is a ubiquitous regulator of beta(2) integrin-mediated adhesion. A distinct morphological change consisting of Jurkat cell spreading and extension of filopodia was induced by alpha(4) integrin signaling. In contrast, CD3 induced radial rings of cortical actin polymerization. Inhibitors of PI 3-kinase and extracellular regulated kinase 1/2 did not affect alpha(4) integrin-induced rearrangement of the actin cytoskeleton, but treatment with ionomycin, a Ca(2+) ionophore, modulated cell morphology by reducing filopodia and promoting lamellipodia formation. Qualitatively similar morphological and adhesive changes to those observed with Jurkat cells were observed following alpha(4) integrin or CD3 stimulation of human peripheral blood T cells.

Actins↗

PubMed1999

Ovarian carcinoma regulation of matrix metalloproteinase-2 and membrane type 1 matrix metalloproteinase through beta1 integrin.

Culturing DOV 13 ovarian carcinoma cells on three-dimensional collagen lattice but not on thin-layer collagen induces processing of promatrix metalloproteinase (MMP)-2 to a M(r) 62,000 form, suggesting that multivalent integrin aggregation may participate in proteinase regulation. To address the role of collagen-binding integrins in this event, we treated DOV 13 cells with soluble beta1 integrin antibodies (clones P4C10 or 21C8) or beta1 integrin antibodies immobilized on latex beads to promote integrin aggregation. Divalent ligation of beta1 integrins with soluble P4C10 antibodies stimulated expression of pro-MMP-2 and its inhibitor, tissue inhibitor of metalloproteinase-2, whereas soluble 21C8 antibodies had no effect. Aggregation of beta1 integrins with immobilized 21C8 or P4C10 antibodies stimulated MMP-dependent pro-MMP-2 activation and accumulation of a M(r) 43,000 form of membrane type 1 MMP (MT1-MMP), a cell surface activator of pro-MMP-2, in cell extracts. beta1 integrin-mediated MMP-2 activation required protein synthesis and tyrosine kinase signaling and was reduced by an inhibitor of gene transcription. Treatment of control cells with concanavalin A stimulated MMP-dependent pro-MMP-2 activation and accumulation of M(r) 55,000 and 43,000 forms of MT1-MMP in cell extracts. Addition of either the MMP inhibitor GM-6001-X or exogenous tissue inhibitor of metalloproteinase-2 to concanavalin A-treated cells resulted in loss of the M(r) 43,000 form of MT1-MMP and accumulation of the M(r) 55,000 form of the enzyme in cell extracts, suggesting that the M(r) 43,000 form is a product of MMP-dependent M(r) 55,000 MT1-MMP proteolysis. Together, these data suggest that beta1 integrin stimulation of pro-MMP-2 activation involves MT1-MMP posttranslational processing and requires multivalent integrin aggregation.

Collagen↗

PubMed1999

Towards a structural model of an integrin.

Integrins are currently viewed as the principal family of extracellular matrix receptors. The interactions mediated by integrins are responsible for certain typical properties of adhesive cells, such as attachment and migration, but these molecules are also recognized to contribute to intracellular signalling processes, either by transducing signals themselves or by enabling and/or coordinating signalling via other receptor systems. As yet, the structural basis of integrin function is unknown, although detailed computer-based predictions have suggested working models for integrin tertiary structure. In this chapter, I will review this information and discuss recent studies examining the molecular basis of integrin regulation using stimulatory and inhibitory monoclonal antibodies (mAbs). Through the use of sensitive isolated integrin-binding assays, stimulatory mAbs have been found to function either by inducing shape changes in integrins or by selectively recognizing and stabilizing active and ligand-occupied conformations of integrins, while blocking mAbs were found to be allosteric inhibitors of ligand binding that report specific ligand engagement events. This information has improved our understanding of the composition of the integrin ligand-binding pocket and the structural basis of integrin activation.

Antibodies, Monoclonal↗

PubMed2002

Aberrant expression of integrin and erbB subunits in breast cancer cell lines.

Integrin and growth factor receptors play an important role in cell functions and their aberrant expressions are implicated in breast cancer malignancy. Recent studies have shown that integrins physically and functionally associate with growth factor receptors suggesting the cooperative regulation of these two signals. We studied the expression of integrin and erbB subunits by flow cytometer in human normal mammary epithelial (HME) cell, non-metastatic (MCF-7, ZR-75-1, MDA-MB453) and metastatic tumor cell lines (MDA-MB231, MDA-MB435). Compared with HME cells, all of non-metastatic and metastatic cell lines showed decreased expressions of alpha2 and beta4 integrin subunits. Two metastatic cell lines, but not three non-metastatic tumor cell lines, expressed alpha5 and alpha6 comparable to HME cells. There was no correlation of erbB2 expression with integrin expressions. We isolated MDA-MB435 subpopulations expressing lower amount of alpha6 integrin and found that alpha5, but not alpha2 and alphav integrins, was concomitantly decreased while erbB family was not affected. Then we transfected erbB2 gene into MDA-MB435 and found the induction of erbB3 expression but not erbB1 and erbB4. However, erbB2 transfection had no effect on the expression of alpha6 and beta4 integrin subunits. These data suggest that the expression of alpha5 and alpha6 integrins may contribute to metastasis, and that the regulation of erbB2 and alpha6 integrin expressions is independent in breast cancer cells.

Breast Neoplasms↗

PubMed2003

Integrin-mediated suppression of endotoxin-induced DNA damage in lung endothelial cells is sensitive to poly(ADP-ribose) polymerase-1 gene deletion.

Endotoxin (LPS) is a cause of adult respiratory distress syndrome (ARDS), a disease which is preceded by acute lung injury involving the pulmonary endothelium. Experimentally, LPS causes acute DNA strand breakage in mouse lung endothelial cells (MLEC). Engagement of integrin cell adhesion receptors inhibits acute DNA breakage, which could be of use in reducing lung injury. Because integrins presumably inhibit DNA damage or activate repair, we hypothesized that the DNA-damage response protein, poly(ADP-ribose) polymerase-1 (PARP-1), regulates the protective action of integrins, as well as sensitivity to LPS. Therefore, the effect of LPS on MLEC cultured from wild-type and PARP-1 knockout mice was determined. Fluorescence microscopic measures were used to assess plasma membrane integrity, PARP activity, DNA strand breakage and DNA repair in attached cells. LPS caused a concentration-dependent increase in the permeability of wild-type MLEC. Engagement of beta1 integrins with an antibody protected wild-type MLEC from this LPS-induced injury. Wild-type cells treated with the PARP-inhibitor, 3-aminobenzamide, and PARP-1 knockout MLEC were also resistant. LPS caused acute DNA breakage in both wild-type and knockout MLEC, but PARP was activated only in wild-type cells. LPS-induced DNA breakage was inhibited by 3-aminobenzamide, but not by knockout. Anti-beta1 integrin antibody also inhibited the DNA breakage and PARP activation caused by LPS in wild-type MLEC. However, integrin engagement did not prevent DNA breakage in PARP-1 knockout cells, despite a similar level of beta1 integrin in wild-type and knockout cells. Thus, integrin engagement, 3-aminobenzamide, and PARP-1 deletion protected MLEC from increases in membrane permeability caused by LPS. PARP-1 deletion also impaired the ability of integrin engagement to inhibit LPS-induced DNA breakage, suggesting that knockout may affect nuclear factors necessary for integrin-mediated suppression of LPS-induced DNA breakage.

Animals↗

PubMed2003

Transcriptional regulation of the beta1C integrin splice variant in human prostate adenocarcinoma.

Members of the integrin family of cell adhesion receptors influence several important aspects of cancer cell behaviour, including motility and invasiveness, cell growth and cell survival. beta1C integrin, an alternatively spliced variant of the human beta1 integrin, has been shown to inhibit cell proliferation. We have previously demonstrated that beta1C integrin mRNA and protein are present in normal prostate and are down-regulated in prostate adenocarcinoma. To explore some of the molecular mechanisms regulating beta1C integrin gene expression, we have analysed the transcriptional activity of the beta1 integrin gene in neoplastic and normal human prostate tissue. Run-on analysis demonstrates that the transcription rate of the beta1 integrin gene is significantly reduced in prostate cancer specimens compared to normal prostate, thus accounting for the reduction in mRNA levels of the beta1 integrin variants. Moreover, the decrease in transcriptional activity of the beta1 integrin gene directly correlates to the reduction of beta1C integrin steady-state mRNA levels (r=0.78).

Aged↗

PubMed2004

[The changes of beta 1-integrin and its mRNA distribution in renal tubular epithelial cells during kidney ischemia/reperfusion injury in neonatal rats].

OBJECTIVE: By studying the distribution and expression of beta 1-integrin and its mRNA in renal tubular epithelial cell at different ischemia/reperfusion time phases we probe the role of beta 1-integrin in I/R injury of neonatal animals. METHODS: The neonatal SD rat ischemia/reperfusion model was set up. Immunofluorescent staining and in situ hybridization (ISH) were used to show the distribution and expression of beta 1-integrin with its mRNA in renal tubular epithelial cells and the quantities of beta 1-integrin and its mRNA were counted by ImagePlus-Pro system. RESULTS: 1. beta 1-integrin was located at the basal plasma membrane in normal renal tubular epithelial cells. After ischemia for 0.5 h, the redistribution was not significant. After reperfusing for 0.5 h, beta 1-integrin began to move onto the lateral and apex surfaces of cells. This kind of change was most apparent after reperfusion for 2 h and was accompanied with reduced expression and destruction of renal tubular. No positive dying was seen in the lumen. The regeneration started 24 hours post-ischemia. beta 1-integrin was redistributed onto the basal plasma membrane. After reperfusion for 120 hours, the regeneration ended, but the expression did not gain the normal level. 2. The mRNA of beta 1-integrin was mainly expressed in the cell plasma. Its expression was increased while reperfusion for 0.5 h to 2 h and was still above normal level after 24 h to 120 h of reperfusion. CONCLUSION: The distribution of beta 1-integrin was "depolarized" and its expression was reduced. But the level of mRNA was raised, which indicated that during ischemia/reperfusion injury, there were mechanisms that influenced the distribution and synthesis of beta 1-integrin at the same time.

Animals↗

PubMed2004

Evaluation of sarcoglycans, vinculin-talin-integrin system and filamin2 in alpha- and gamma-sarcoglycanopathy: an immunohistochemical study.

The sarcoglycan subcomplex (SGC) is a well-known system of interaction between extracellular matrix and sarcolemma-associated cytoskeleton in skeletal and cardiac muscle. The SGC is included in the DGC made up of sarcoplasmic subcomplex and a dystroglycan subcomplex. Recent developments in molecular genetics have demonstrated that the mutation of each single sarcoglycan gene, causes a series of recessive autosomal muscular dystrophies, dystrophin-positive, called sarcoglycanopathies or limb girdle muscular dystrophies. Our recent studies have demonstrated that costameres are a proteic machinery made up of DGC and vinculin-talin-integrin system, also revealing the colocalization of sarcoglycans and integrins in adult human skeletal muscle. These results may support the hypothesis of the existence of a bidirectional signalling between sarcoglycans and integrins in cultured L6 myocytes. The hypothesis of bidirectional signalling between sarcoglycans and integrins could be supported by the identification of a skeletal and cardiac muscle filamin2 as a gamma-sarcoglycan, delta-sarcoglycan and, hypothetically, beta1 integrin interacting protein. Our results, acquired with an immunofluorescence study on adult human skeletal muscle affected by LGMD type 2D and 2C, showed that in LGMD2D: a) alpha-sarcoglycan staining is severely reduced; b) the beta-gamma-delta-sarcoglycan subunit and all tested integrins staining are clearly detectable; c) filamin2 is normal and shows a costameric distribution. In LGMD2C: a) alpha-sarcoglycan staining is preserved; b) the beta-gamma-delta-sarcoglycan subunit staining is severely reduced; c) the alpha7B-integrin is slightly reduced and beta1D-integrin is severely reduced; d) filamin2 is severely reduced. Other tested proteins of the two systems show a normal staining pattern in both sarcoglycanopathies. Our study seems to confirm, for the first time on adult human skeletal muscle of subjects affected by LGMDs, the hypo-theses of: a) the existence, in mouse myotubes in culture, of two distinct subunits in sarcoglycans subcomplex; b) the presence of a bidirectional signalling between sarcoglycans and integrins, previously demonstrated on rat cultured L6 myocytes; c) the interaction of FLN2 with both sarcoglycans and integrins. These results may stimulate the search of yet unidentified common interactors of both fiber-extracellular matrix interaction systems.

Biopsy↗

PubMedDate not supplied

Differential modulation of integrin expression in chondrocytes during expansion for tissue engineering.

Cartilage tissue engineering has an important role to play in the generation of graft material for reconstructive surgery. In cultured chondrocytes, the dedifferentiation of cells seems unavoidable for multiplication. Dedifferentiated cells produce matrix of less quality. Normal cartilage is composed of chondrocytes, which are embedded within an extracellular matrix (ECM). The ECM plays a key role in controlling cellular characteristics and contains the integrins as a large family of heterodimeric cell adhesion receptors involved in cell-cell and cell-matrix interactions. In this study, the characteristic changes of integrin expression and expression of matrix proteins during the course of dedifferentiation of chondrocytes in cell culture for 1, 6 and 21 days, analyzed at the mRNA level by microarray analysis and at the protein level by immunohistochemistry, are described. The components of the fibronectin receptor, integrin beta1,alpha5, in conjunction with the ligand fibronectin, were up-regulated during dedifferentiation. Integrin beta3 was expressed in the grey area. The components of the vitronectin-receptor, integrin alpha2b, alpha v, as well as integrin beta5, were activated on day 21, but neither vitronectin nor osteopontin were expressed by the cells. With ongoing dedifferentiation, activation of the GPIIb/GPIIIa receptor was found. The integrins beta2, beta4, beta6, beta8 and alpha2, alpha4, alpha6, alpha7 and alpha11 were never expressed. ILK, CD47 and ICAP1, as components of the intracellular signalling cascade of several integrins, were activated with ongoing dedifferentiation. In conclusion, a candidate for signal transmission during dedifferentiation is the fibronectin receptor (integrin alpha5beta1) in conjunction with its ligand fibronectin. Other receptors, e.g. for vitronectin and osteopontin (alphaVbeta3) or laminin (alpha6beta1) or their ligands, do not seem to be involved in signal transmission for dedifferentiation. In addition, the GPIIb/IIIa-receptor seems to assist the process of dedifferentiation. Intracellularly, ILK, ICAP1 and CD47 might assist the transduction of the integrin-dependent signals.

Cell Culture Techniques↗

PubMed1990

Distribution of beta 1 and beta 3 integrins in human fetal and adult kidney.

The distribution of beta 1 and beta 3 integrins was studied in fetal and adult human kidneys by indirect immunofluorescence microscopy. In the developing kidney, the cells of the undifferentiated metanephric blastema displayed strong cell surface-confined beta 1 integrin immunoreactivity, whereas the cells of primary vesicles and comma- and S-shaped bodies reacted more weakly. In mature fetal as well as adult glomeruli, beta 1 integrins were distinctly localized, apparently confining to the basal cell surfaces of endothelial cells and podocytes abutting the glomerular basement membrane. In adult proximal tubules, beta 1 integrin immunoreactivity was strictly confined to the basal aspect of the epithelial cells, being absent laterally, which is unusual for membrane proteins of polarized epithelial cells. A more diffuse overall immunoreactivity was seen in distal tubules and collecting ducts. The epithelial cells of developing proximal and distal tubules displayed an overall distribution of beta 1 integrins. In each case, talin immunoreactivity followed that of beta 1 integrins. Compared with beta 1 integrins, beta 3 integrins showed a more restricted distribution, and differences were seen in the reactions of mono- and polyclonal antibodies. In developing glomeruli, beta 3 integrin immunoreactivity was prominently seen in the cells of Bowman's capsule, possibly revealing the presence of vitronectin receptor. Solitary cells, that reacted also with antibodies to the platelet glycoprotein IIb, were consistently detected in fetal glomeruli, suggesting the presence of megakaryocytes. The results show that during nephrogenesis, beta 1 integrins become distinctly polarized both in glomerular endothelial cells and podocytes, as well as in the epithelial cells of proximal tubules.

Adult↗

PubMed2006

[The role of integrins in the physiologic and pathogenic processes].

Cell adhesion molecules (CAM) are a numerous, diverse group of cell surface proteins, which are both receptors and ligands for receptors. Their functions include adhesion, recognition, cell-cell interaction, and communication between mediate cells and extracellular matrix. The following groups of CAM can be distinguished: seletins, integrins, cadherins and other isoforms, including CD 44. Integrins are heterodimers formed from the alpha and beta chains. The a subclass is responsible for a specific bond with ligands. It defines the specificity of integrins. The 8 chain participates in the integration with cytoskeleton ptoteins. It determines the functions of the integrin receptor. The best recognized integrins include: integrin beta1, beta2 and beta3. The expression and activity of integrins have been found to be affected by a variety of factors being either activators or inhibitors. Adhesion molecules (including integrins) play a significant role in both physiological processes (embryogenesis, organogenesis, the normal growth and tissue development) and pathogenic ones. In the latter case, they are particularly involved in inflammatory, allergic and neoplastic diseases. The role of integrins is also emphasized in organ response to trauma and in skin lesion redevelopment. The knowlegde of the integrin molecular basis and that of other adhesion molecules can contribute significantly to the creation of new diagnostic and therapeutic perspectives. An adequate modification of cellular adhesion constitutes a promising way of the pathogenic processes control.

Animals↗

PubMed1990

Interaction of integrins alpha v beta 3 and glycoprotein IIb-IIIa with fibrinogen. Differential peptide recognition accounts for distinct binding sites.

Glycoprotein (GP) IIb-IIIa is the major fibrinogen receptor on platelets and participates in platelet aggregation at the site of a wound. Integrin alpha v beta 3, which contains an identical beta-subunit, is expressed on endothelial cells and also serves as a fibrinogen receptor. Here, we demonstrate by several criteria that purified GPIIb-IIIa and integrin alpha v beta 3 bind to distinct sites on fibrinogen. First, a plasmin-generated fragment of fibrinogen lacking the RGD sequence at residues 572-574 retained the ability to bind GPIIb-IIIa, but failed to bind integrin alpha v beta 3. Second, a monoclonal antibody which exclusively recognizes the RGD sequence at fibrinogen A alpha chain residues 572-574 abolished interaction between integrin alpha v beta 3 and fibrinogen, but had only a minimal effect on fibrinogen binding to GPIIb-IIIa. Finally, we show that the difference in recognition of sites on fibrinogen by these two integrins is probably a consequence of their remarkably different ligand binding properties. Peptides corresponding to fibrinogen gamma chain residues 400-411 effectively blocked RGD sequence and fibrinogen binding by GPIIb-IIIa, but had no effect on the ability of integrin alpha v beta 3 to bind these ligands. We also show that integrin alpha v beta 3 has a higher affinity than GPIIb-IIIa for a synthetic hexapeptide containing the RGD sequence. In fact, this RGD-containing peptide was 150-fold more effective at blocking fibrinogen binding to integrin alpha v beta 3 than to GPIIb-IIIa. Collectively, our results demonstrate that integrins alpha v beta 3 and GPIIb-IIIa display qualitative and quantitative differences in their ligand binding properties, as is evident by their ability to interact with synthetic peptides. The ultimate result of these differences is the recognition of distinct sites on fibrinogen by the two integrins. These observations may have relevance in the processes of hemostasis and wound healing.

Amino Acid Sequence↗

PubMed1989

Integrins isolated from Rous sarcoma virus-transformed chicken embryo fibroblasts.

Avian integrins are a complex of three integral membrane glycoproteins that are thought to have a role both in anchoring the cytoskeleton to the plasma membrane and establishing linkages to the extracellular matrix. We previously demonstrated that bands 2 and 3 of integrin are phosphorylated on both tyrosine and serine residues in chicken embryo fibroblasts (CEF) transformed with Rous Sarcoma virus (RSV) and other oncogenic retroviruses. The effects of RSV transformation on integrins from chick cells are now further characterized. The major site of tyrosine phosphorylation on band 3 in RSV transformed CEF has been identified as tyrosine 788. We also demonstrate that the product of the RSV oncogene, pp60v-src, can phosphorylate integrin in vitro, at the same residue. Tryptic peptide mapping and tunicamycin treatment indicates that a previously observed 4-8 k increase in the Mr of integrins from RSV-transformed cells can be attributed to an alteration in a post-translational modification such as glycosylation. Equilibrium gel filtration assays were used to test the ability of integrins from RSV-transformed CEF to interact with talin and fibronectin. Tyrosine phosphorylated integrins showed a decreased ability to interact with both these ligands in vitro. Conversely, integrins isolated from RSV-transformed CEF metabolically labeled in the absence of phosphatase inhibitor contained only low levels of phosphotyrosine and showed an almost normal ability to interact with ligands. Competition experiments indicated that the region of integrin containing tyrosine 788 is also important for talin binding to integrins.

Animals↗

PubMed1995

Direct interaction of filamin (ABP-280) with the beta 2-integrin subunit CD18.

beta 2-Integrins mediate leukocyte extravasation into inflamed tissues, phagocytosis, and target cell killing, functions that require an intact actin cytoskeleton. Previous studies have focused on elucidating interactions of the cytoplasmic tails of integrins with the cytoskeleton at focal contacts in stationary cells. As integrins are also located at other types of cell-substratum junctions, such as the leading edge of migrating cells, additional cytosolic proteins abundant at these sites may also interact with integrins. In this study, we have identified the actin-binding protein, filamin (ABP-280), as a major cytoskeletal protein that binds to the cytoplasmic tail of the beta 2-integrin subunit CD18. Filamin bound to cytoplasmic CD18 directly and specifically, co-immunoprecipitated with beta 2-integrins in detergent cell lysate and co-immunolocalized with cross-linked beta 2-integrins in intact cells. The filamin binding site in CD18 was localized to the N-terminal (amino acids (aa) 724 to 747) but not to the C-terminal (aa 743 to 769) half of cytoplasmic CD18. Filamin did not bind to the major alpha-actinin binding site (aa 733 to 742), however, suggesting that these two cytoskeletal proteins bind to distinct but overlapping sites. Given the conservation of the filamin binding region among beta-integrin subunits, these findings suggest the presence of similar associations between filamin and other integrins. These associations may be important in the spreading and extension of lamellipodia at the leading edge during cell movement and, if interrupted, may contribute to the dramatic decrease in cell locomotion observed in genetic deletions involving filamin or integrins.

Actinin↗

PubMed1995

Distribution of integrin cell adhesion molecules in endometrial cancer.

Integrins are ubiquitous cell adhesion molecules that are involved in maintaining normal tissue morphology and have been implicated in the behavior of certain malignancies. We examined the expression of nine integrin subunits in 38 endometrial adenocarcinomas using immunohistochemistry. The pattern of integrin expression in the cancers was compared with that seen in the endometrium of 20 normal cycling women and 7 postmenopausal women. Integrin expression was correlated with grade, stage, nodal status, depth of invasion, steroid receptor status, and histological pattern. In endometrial cancers there was an inverse relationship between the number of integrins expressed and histological grade (P = 0.011). Of the normally expressed, constitutive endometrial epithelial integrin subunits (alpha 2, alpha 3, alpha 6, and beta 4), the least frequently seen in the cancers was the alpha 3 subunit (44.7%) and the most frequently found was alpha 6 (81.6%). The alpha 5 beta 1 integrin, a fibronectin receptor normally found only on endometrial stromal cells, was seen in 17.8% of cases of these epithelial cancers. In addition, a significant association was found between the loss of the alpha 2 beta 1 integrin and the presence of lymph node metastases (P < 0.001). These data suggest that a decline in integrin expression occurs more frequently in poorly differentiated endometrial cancers and that the loss of specific integrins may be associated with metastatic nodal spread.

Adenocarcinoma↗

PubMed1994

Differential activation-dependent regulation of integrin function in cultured human T-leukemic cell lines.

T lymphocytes isolated from human peripheral blood express beta 1 (VLA) and LFA-1 integrins, but strong binding to integrin ligands occurs only after the delivery of an activation stimulus to the T cell. To gain further insight into activation-dependent regulation of integrin function, we have analyzed integrin activity on three different T-leukemic cell lines: Jurkat, CEM, and H9. This analysis shows important mechanistic differences in integrin regulation. First, phorbol ester treatment results in increased beta 1 integrin-dependent adhesion of both Jurkat and CEM cells to fibronectin, but decreased adhesion of H9 cells. Second, certain activation stimuli that upregulate beta 1 integrin activity in peripheral T cells are nonfunctional in these T-cell lines. Third, analysis of a panel of Jurkat mutants lacking surface expression of CD2 and/or CD3 shows that CD2-mediated upregulation of beta 1 integrin activity is dependent on expression of CD3, whereas CD28-mediated upregulation is not dependent on either CD2 or CD3 expression. Fourth, all T-cell lines tested show an inability to adhere to purified ICAM-1 via LFA-1. The selective alterations in integrin regulation in these cell lines relative to peripheral blood T cells provide important insights into the intracellular processes involved in integrin activation.

Antigens, CD↗