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PubMed1996

Circulating leukocyte integrin expression in Wegener's granulomatosis.

Leukocyte adhesion and infiltration are important in the pathogenesis of Wegener's granulomatosis (WG). We tested the hypothesis that the expression of the beta 1-chain integrin VLA-4 (CD49d/CD29) and the beta 2-chain integrins LFA-1 (CD11a/CD18), Mac-1 (CD11a/CD18), and gp150,95 (CD11c/CD18) is increased on leukocytes in patients with active WG. Fifteen patients with active WG as defined by positive antineutrophil cytoplasmic autoantibody (cANCA) titers and biopsy, 30 patients with WG in remission as defined by negative cANCA titers and/or immunosuppressive therapy, 25 normal control subjects, and 12 patients with other inflammatory renal and systemic diseases were studied. Surface expression of LFA-1, Mac-1, p150, 95, and VLA-4 on neutrophils, lymphocytes, and monocytes was measured by fluorescent antibody cell sorting with monoclonal antibodies against CD11a, CD11b, CD11c, CD18, CD49d, and CD29 respectively. Immunocytochemistry and confocal microscopy were also utilized. beta 1 (CD29) and beta 2 (CD18) integrin subunit expression on neutrophils, monocytes, and lymphocytes from patients with acute WG was significantly increased compared with healthy persons and compared with patients with treated vasculitis. Furthermore, the alpha-integrin subunit CD11b expression was increased on granulocytes and monocytes, but not on lymphocytes. Finally, the alpha-integrin subunit CD11a expression was increased on monocytes. Immunocytochemistry showed that the increased immunoreactivity on neutrophils was evenly distributed on the plasma membrane and in the cytosol. Immunosuppression resulted in decreased expression of the beta 1 and beta 2-integrin subunits. It was concluded that the integrin adhesion molecules, particularly Mac-1 (CD11b/CD18), are upregulated on leukocytes in active WG. This finding suggests a role for integrin expression in the pathogenesis of WG and a possible clue for treatment.

Follow-Up Studies↗

PubMed2000

Role of intracellular chloride in the reversible activation of neutrophil beta 2 integrins: a lesson from TNF stimulation.

The process of beta(2) integrin activation, which enhances the interaction of these heterodimers with ligands, plays a crucial role in the adherence-dependent neutrophilic polymorphonuclear leukocytes' (PMN) responses to TNF. Our previous observation, showing that a marked decrease of the high basal Cl(-) content (Cl(-)(i)) is an essential step in the TNF-induced activation of PMN, stimulated this study, which investigates the role of alterations of Cl(-)(i) in the activation of beta(2) integrins triggered by TNF. Here we show that TNF enhances the expression of activation-specific neoepitopes of beta(2) integrins, namely, epitope 24, a unique epitope present on all three leukocyte integrin alpha subunits, and epitope CBRM1/5, localized to the I domain on the alpha-chain of Mac-1 (CD11bCD18). Moreover, we demonstrate that the conformational changes underlying the expression of the neoepitopes are dependent on a drop in Cl(-)(i) because 1) inhibition of Cl(-)(i) decrease is invariably accompanied by inhibition of beta(2) integrin activation, 2) Cl(-)(i) decrease induced by means other than agonist stimulation, i.e., by placing PMN in Cl(-)-free buffers, activates beta(2) integrins, and 3) restoration of the original Cl(-)(i) levels is accompanied by deactivation of beta(2) integrins. We also show that Cl(-)(i) decrease is required for TNF-induced cytoplasmic alkalinization, but such a rise in pH(i) does not seem to be relevant for beta(2) integrin activation. The results of our study emphasize the role of Cl(-) as a new PMN "second messenger."

Antiporters↗

PubMed2001

Role of p190RhoGAP in beta 2 integrin regulation of RhoA in human neutrophils.

We found that engagement of beta(2) integrins on human neutrophils induced activation of RhoA, as indicated by the increased ratio of GTP:GTP + GDP recovered on RhoA and translocation of RhoA to a membrane fraction. The clustering of beta(2) integrins also induced a time-dependent increase in GDP bound to RhoA, which correlated with beta(2) integrin-induced activation of p190RHOGAP: The activation of p190RhoGAP was completely blocked by [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] (PP1), a selective inhibitor of Src family tyrosine kinases. However, clustering of beta(2) integrins did not increase the basal tyrosine phosphorylation of p190RhoGAP, nor did it affect the amount of p120RasGAP bound to p190RHOGAP: Instead, the beta(2) integrin-induced activation of p190RhoGAP was accompanied by increased tyrosine phosphorylation of a p190RhoGAP-associated protein, p120RasGAP, and accumulation of both p120RasGAP and p190RhoGAP in a membrane fraction. PP1 blocked the beta(2) integrin-induced phosphorylation of p120RasGAP, as well as the translocation of p190RhoGAP and p120RasGAP, but it did not affect the accumulation of RhoA in the membrane fraction. In agreement with the mentioned findings, PP1 also increased the GTP:GTP + GDP ratio recovered on RhoA immunoprecipitated from beta(2) integrin-stimulated cells. Thus, in neutrophils, beta(2) integrin-induced activation of p190RhoGAP requires a signal from a Src family tyrosine kinase, but it does not occur via the signaling pathway responsible for activation of RHOA:

CD18 Antigens↗

PubMed2004

TNF-induced beta2 integrin activation involves Src kinases and a redox-regulated activation of p38 MAPK.

We previously demonstrated that the TNF-alpha-induced inside-out signaling leading to beta(2) integrin activation is redox regulated. To identify kinases involved in this pathway, the effects of kinase inhibitors on the expression of beta(2) integrin activation neoepitope (clone 24) were investigated. We show that both p38 MAPK (inhibited by SB203580) and Src kinases (inhibited by PP2) are involved in beta(2) integrin activation by TNF and oxidants in human neutrophils. Src kinases appeared constitutively active in resting neutrophils and not further activated by TNF or oxidants in nonadherent conditions. However, PP2 blocked both TNF-induced expression of the 24 epitope and cell adhesion promoted by the integrin activating anti-CD18 KIM185 mAb, showing that both the inside-out and the outside-in signaling involve Src kinases. p38 MAPK was activated by TNF and oxidants in nonadherent conditions i.e., with 10 mM EDTA. This activation in EDTA resulted in CD11b, CD35 and CD66 up-regulation and in an oxidative response, all blocked by SB203580 and PP2. p38 MAPK was not activated upon direct integrin activation by KIM185 mAb. Thus, p38 activation allows the study to distinguish the initial transduction pathway leading to beta(2) integrin activation from the signaling resulting from integrin engagement. Finally, p38 MAPK activation by TNF was blocked by diphenylene iodonium, an inhibitor of flavoprotein oxidoreductase, and by the free radical scavenger N-acetylcystein. Taken together, these results demonstrate, for the first time, that constitutively activated Src tyrosine kinases and a redox-regulated activation of p38 MAPK are involved in TNF inside-out signaling leading to beta(2) integrin activation.

CD18 Antigens↗

PubMed2001

Comparative specificity of platelet alpha(IIb)beta(3) integrin antagonists.

Several platelet alpha(IIb)beta(3) integrin antagonists have been designed as preventive agents against the formation of arterial thrombi. Although the potency of these compounds in inhibiting platelet aggregation is in the nanomolar range, their specificity on other integrins that can bind ligands through an arginine-glycine-aspartic acid (RGD) motif is far from being well established. For instance, some cyclic RGD peptides can also interact with alpha(v)beta(3) integrin. We used a novel pharmacological assay, based on SDS-stable interaction between (125)I-echistatin and RGD-dependent integrins, to evaluate the specificity of several RGD compounds on integrins present on rat cardiac fibroblasts and human skin fibroblasts. None of the RGD peptidomimetics tested (L-734,217, lamifiban, Ro 44-3888, SR 121566A, BIBU-52, XV459) could interact with either alpha(v)beta(3) and alpha(8)beta(1) on rat fibroblasts or with alpha(v)beta(3) and alpha(v)beta(1) on human fibroblasts. Cyclic RGD peptides showed some potency (3-80 microM) on rat and human integrins with an alpha(v) subunit. We also compared the potency of these compounds on platelets. All RGD compounds demonstrated IC(50) between 0.6 and 530 nM on basal human platelets. Activation of the receptor with thrombin resulted in a 2- to 60-fold increase in potency, with L-734,217 and BIBU-52 showing the largest difference. On basal and thrombin-activated rat platelets, only eptifibatide, DMP728, and XJ735 could displace (125)I-echistatin (IC(50) approximately 0.1-1.5 microM). These results indicate that RGD peptidomimetics have a specificity limited to alpha(IIb)beta(3) integrin, whereas cyclic RGD peptides can also interact with other RGD-dependent integrins, particularly those of the alpha(v) subunit family.

Animals↗

PubMed2001

A role for beta2 integrin (CD11/CD18)-mediated tyrosine signaling in extravasation of human polymorphonuclear neutrophils.

During the recruitment of human polymorphonuclear neutrophils (PMN) to sites of inflammation, leukocyte adhesion molecules of the beta2 integrin (CD11/CD18) family mediate firm adhesion of these cells to the endothelial cell monolayer lining the vessel wall. This process is a prerequisite for shape change and spreading of PMN on the endothelium which eventually allows PMN emigration into the extravascular space. In order to elucidate the molecular mechanisms which mediate this sequence of events, intracellular protein tyrosine signaling was studied subsequent to beta2 integrin-mediated ligand binding. Using western blotting technique, beta2 integrin-mediated adhesion was found to induce tyrosine phosphorylation of different proteins. The effect was absent in PMN derived from CD18-deficient mice which lack any beta2 integrin expression on the cell surface demonstrating the specificity of the observed response. Inhibition of beta2 integrin-mediated tyrosine signaling by herbimycin A almost completely inhibited adhesion, shape change, and subsequent spreading of PMN. Herbimycin A also diminished chemotactic migration of these cells in response to the soluble mediator N-formyl-Met-Leu-Phe (fMLP). In contrast, treatment of PMN with cytochalasin D had no substantial effect on beta2 integrin-mediated signaling or adhesion but inhibited shape change, spreading, and chemotactic migration of PMN. This suggests that the signaling capacity exerted by beta2 integrins upon ligand binding was independent of an intact cytoskeleton. Moreover, the beta2 integrin-mediated activation of intracellular signal transduction pathways was critical for firm adhesion of PMN, the prerequisite subsequent shape change and spreading, which allows emigration of PMN into the extravascular space.

Animals↗

PubMed2002

Protein kinase A regulates beta2 integrin avidity in neutrophils.

The adhesive phenotype of neutrophils (PMN) depends largely on activating and deactivating intracellular signals regulating beta2 integrin avidity for ligand. Our hypothesis is that PKA is a negative regulator of beta2 integrin avidity. In this work, we examined the role of PKA in PMN alphaMbeta2 integrin activation. Elevation of cAMP inhibited alphaMbeta2 integrin-dependent adhesion of PMN to immune complexes (IC), but not PMA-induced adhesion. The PKA inhibitor KT5720 reversed the ability of cAMP to suppress adhesion to IC. Moreover, inhibition of PKA activity was sufficient to activate alphaMbeta2 integrin-dependent adhesion and increase beta2 integrin expression and binding of the monoclonal antibody CBRM1/5, which recognizes activated alphaMbeta2 specifically. However, PKA activity was necessary for sustained adhesion. Disruption of A kinase-anchoring, protein-PKA binding with a cell-permeant peptide derived from the AKAP Ht31 also activated adhesion. Unlike pharmacologic inhibition of PKA, AKAP peptide-induced adhesion was PKC dependent and did not affect beta2 integrin expression or CBRM1/5 binding. These data demonstrate that PKA appears to have a dual role in the mechanism regulating alphaMbeta2 integrin avidity and adhesion.

CD18 Antigens↗

PubMed2001

[Low Concentrations of STI571 Enhances beta1 Integrin Mediated Inhibitory Effect on Proliferation of Myeloid Progenitors in Ph(+)Chronic Myeloid Leukemia]

To investigate whether ABL specific tyrosine kinase specific inhibitor STI571 can restore beta1 integrin mediated negative effect on Ph(+) chronic myeloid leukemia(CML), the inhibitory effect of beta 1 integrin activator (beta1 integrin activating antibody 8A2, cytokines such as GM-CSF, G-CSF and SCF) and/or FN on the granulocyte-macrophage colony forming unit (CFU-GM) from 16 patients with Ph(+)CML and 13 normal individuals were examined; the bone marrow mononuclear cells (BMMNC) before and after ABL kinase specific inhibitor STI571 pretreatment (0.1 micro mol/L for 30-60 minutes) were target cells in this study. The roles which VLA4 and VLA5 played in this process were evaluated through blocking assay. The results showed: (1) beta1 integrin activator(s) or FN alone have no effect on CFU-GM from CML or normal bone marrow mononuclear cells before or after STI571 pretreatment, nor STI571 pretreatment itself. (2) The inhibitory effect of beta1 integrin activator(s) plus FN on CML CFU-GM are significantly lower than that on normal CFU-GM. (3) The inhibitory effect of beta1 integrin activator(s) plus FN on CML CFU-GM after STI571 pretreatment is comparable to that on normal CFU-GM. (4) Monoclonal antibody to VLA4 and VLA5 or to total beta1 integrins almost completely abrogate the above effect of STI571. The results suggested enhancing beta1 integrin mediated negative effect on myeloid progenitors in Ph(+)CML is one of the therapeutic mechanisms of STI571 on Ph(+)CML.

Journal Article↗

PubMed1996

Integrin alpha3beta1 can promote adhesion and spreading of metastatic breast carcinoma cells on the lymph node stroma.

We have reported that metastatic human melanoma cells utilize the alpha (v)beta3 integrin to adhere to lymph node vitronectin (VN). In the present study, the adhesion of human and rat breast carcinoma cells to lymph node tissue was analyzed. We have previously shown a correlation between the metastatic potential of breast carcinoma cells and an RGD-mediated adhesion to cryostat sections of peripheral lymph nodes; this adhesion could be blocked by an antibody to the integrin beta1 subunit. Here, we show that the metastatic breast carcinoma cells were significantly more adherent to fibronectin (FN) expressed by lymph node-derived stromal cells than non-metastatic cells. Metastatic cells also spread more rapidly than non-metastatic cells on FN-coated substrates. Using a combination of immunofluorescence microscopy, immunoprecipitation and blocking assays with integrin-specific antibodies, we found (i) that expression of the alpha3beta1 integrin on metastatic mammary carcinoma cells was specifically increased in comparison to non-metastatic cells and (ii) that the alpha3beta1 receptor was involved in the increased adhesion of metastatic cells to lymph node FN and in cell spreading on FN-coated substrates. Our data also suggest that the alpha5beta1 integrin, which is also expressed on the metastatic cells, did not contribute to this increase in adhesion. Our data implicate the alpha3beta1 integrin in adhesion to lymph node stromal cell FN and suggest that metastatic cells of different tissue origins (e.g., melanoma and breast carcinoma) may utilize distinct integrin-ligand combinations to colonize the same target organ.

Animals↗

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↗

PubMed1995

Identification of Sp1-binding sites in the CD11c (p150,95 alpha) and CD11a (LFA-1 alpha) integrin subunit promoters and their involvement in the tissue-specific expression of CD11c.

The leukocyte integrins LFA-1 (CD11a/CD18) and p150,95 (CD11c/CD18) mediate cell-cell and cell-extracellular matrix interactions during inflammatory responses and signal transduction into the cytoplasm. While the CD11a integrin subunit is expressed on all leukocytes, CD11c is almost exclusively expressed on cells of the myeloid lineage and on activated B lymphocytes. Its expression is regulated during cell activation and differentiation by transcriptional mechanisms. We have previously demonstrated that the proximal region of the CD11c promoter directs tissue-restricted and developmentally-regulated expression of reporter genes. Structural studies by electrophoretic mobility shift assays have demonstrated the presence of two Sp1-binding sites at -70 (Sp1-70) and -120 (Sp1-120) which mediate the Sp1 transactivation of the CD11c promoter in Sp1-defective SL2 cells, and which are involved in cell lineage-specific DNA-protein interactions, as demonstrated by footprinting in vivo. More importantly, mutation of either Sp1 site inhibited the activity of the CD11c promoter both in myeloid U937 cells and the CD11c-expressing B lymphoblastoid JY cell line, while the opposite effect was observed in the CD11c-negative epithelial HeLa cell line, demonstrating the involvement of both Sp1-binding sites in the basal and the tissue-restricted expression of the CD11c integrin subunit gene. Interestingly, the analysis of the CD11a proximal promoter also revealed the existence of an Sp1-binding site at -70, indicating a common role for these cis-acting elements in the transcription of the leukocyte integrin alpha subunit genes. The binding of Sp1 to the regulatory regions of the leukocyte integrin genes raises the possibility that the retinoblastoma susceptibility gene product is implicated in integrin expression through its functional interaction with Sp1, thus establishing a link between integrin-dependent leukocyte adhesiveness and the state of cellular differentiation/proliferation.

Base Sequence↗

PubMed1996

Modulation of integrin-mediated intercellular adhesion during the interaction of thymocytes with stromal cells expressing VLA-4 and LFA-1 ligands.

Mature peripheral T cells closely regulate their intercellular interactions by modulating integrin adhesion functions. The ability of members of the integrin family to mediate intercellular adhesion is dependent on signals from within the cells (inside-out signaling) that increase the avidity of integrins for their ligands. These changes in avidity are independent of the quantitative changes on the number of receptors, and there is evidence to suggest that phosphorylation events play a predominant role in the regulation of the avidity state of the integrins. Whether such regulatory mechanisms are operative during T cell development had hitherto been an opened question. In the present work, we have used an in vitro adhesion assay between thymocytes and target cells expressing VLA-4 and LFA-1 counter ligands to determine how thymocytes can discriminate between integrin-specific signals during T cell development. Our findings are that VLA-4, but not LFA-1, is constitutively expressed in its high-avidity state during the early stages of T cell development, and that the high-avidity state of thymocytes for VCAM-1-expressing cells is closely regulated by signaling through protein kinase C and protein tyrosine kinase pathways. At later stages of development, mature thymocytes prior to leaving the thymus turn off both VLA-4 and LFA-1 adhesion functions. Our results show that the low-affinity state of integrins on peripheral mature T cells is established before mature thymocytes leave the thymus. Only when mature T cells recognize antigenic peptides in the context of major histocompatibility complex in the periphery will they turn on the adhesion function of VLA-4 and/or LFA-1 integrins.

Animals↗

PubMed1993

Altered glycosylation of beta 1 integrins associated with reduced adhesiveness to fibronectin and laminin.

The carbohydrate structures of the beta 1 integrins obtained from a mouse metastatic melanoma B16 F1 and its weakly metastatic wheat-germ agglutinin-resistant mutant Wa4-b1 were studied comparatively. The results indicated that the integrins from both cells contain high mannose-type and bi-, tri- and tetra-antennary complex-type sugar chains. No significant difference was found in the outer chain branching between both integrins, but sialylation of the sugar chains of the mutant's integrin was markedly decreased and almost all the outer chain moieties of tri- and tetra-antennary oligosaccharides of the mutant's integrin were fucosylated, resulting in the formation of X-antigenic determinants, Gal beta 1-->4 (Fuc alpha 1-->3) GlcNAc. In contrast, the integrin from parental cell contained no X-antigenic determinant. These structural differences found in the integrin are thought to account for the reduction in the metastatic potential of the mutant which also shows reduced adhesion to fibronectin and laminin as compared with the parental cell.

Animals↗

PubMed1994

Loss of polarity and de novo expression of the beta 1 family of integrins in thyroid tumors.

The expression and cell-membrane distribution of the beta 1 family of integrins (very-late-activation antigens, VLA) were investigated in benign and malignant human thyroid tumors. We compared tissue samples of normal glands, nodular goiters, adenomas and carcinomas. We also examined 3 thyroid-carcinoma cell lines cultured in vitro. The expression of subunits of the beta 1 family of integrins was assessed by flow cytometry and specific antibodies in dispersed single-cell suspensions and by immunofluorescence on frozen tissue sections. In contrast to the heterogeneity of the expression of beta 1 integrins observed in other tumors, thyroid neoplastic lesions showed a remarkably constant VLA profile. In all tumors, benign as well as malignant, and in carcinoma cell lines, all sub-units of beta 1 integrins were expressed at high levels. While sub-units alpha 1, alpha 3, alpha 5, alpha 6 and occasionally alpha 2 were also present in a cell sub-set of normal glands and nodular goiters, expression of alpha 4 was restricted to neoplastic lesions; this integrin can be therefore considered an antigen associated with thyroid tumors. It has been reported that in normal glands and in nodular goiters, the expression of beta 1 integrins is restricted to the basal-cell membrane. Immunofluorescence on tissue sections showed instead that, in adenomas and carcinomas, the polarized distribution of these integrins on the cell membrane is lost.

Carcinoma, Papillary, Follicular↗

PubMed1995

Glycosylation of beta-1 integrins in B16-F10 mouse melanoma cells as determinant of differential binding and acquisition of biological activity.

Studying B16-F10 cells we could identify beta-1 integrins as laminin, fibronectin and collagen receptors. Gradient ionic strength elution analysis of affinity chromatography showed differential interactions between laminin-binding beta-1 integrins (two beta-1 polypeptides of 105 and 120 kDa) and fibronectin and collagen-binding beta-1 integrins (elution of one major beta-1 polypeptide of 120 kDa) and their respective ligands. To evaluate this diversity we submitted B16-F10 extracts to IEF and SDS-PAGE and found that one beta-1 integrin formed acidic and larger isoforms, while another formed basic and smaller isoforms. To study this difference we also submitted material eluted from WGA-Sepharose columns to IEF but now only the acidic beta-1 isoform was found. Extracts of B16-F10 treated with neuraminidase showed only the basic beta-1 isoform, suggesting that terminal sialic acid residues may be responsible for this acidic pattern, an interpretation supported by the fact that MAA (Maackia ammurensis agglutinin) reacts only with the acidic isoform. Differential glycosylation of beta-1 integrin isoforms in B16-F10 was also demonstrated since the smaller laminin-binding beta-1 integrin isoform reacted only with GNA (Galanthus nivalis agglutinin), whereas the mature larger form reacted with DSA (Datura stramonium agglutinin) and MAA; thus this heterogeneity of beta-1 chains is essentially due to variable glycosylation. Autoradiography and immunoblotting analysis of material separated by 2-dimensional electrophoresis show that only the processed forms of beta-1 integrins are expressed at the cell surface.

Animals↗

PubMed1995

Expression of beta 1 integrin receptors in transformed mouse epidermal keratinocytes: upregulation of alpha 5 beta 1 in spindle carcinoma cells.

The adhesive properties and the expression of extracellular matrix receptors of the beta 1-integrin subfamily were analyzed in transformed epidermal keratinocyte cell lines of different stages of mouse skin carcinogenesis. One- and two-dimensional analyses of the immunoprecipitates obtained with anti-beta 1- and specific anti-alpha-integrin subunits showed qualitative and quantitative changes in the expression of beta 1 integrins by the different cell lines. The polyvalent alpha 3 beta 1 integrin was expressed by all analyzed cell lines, although the levels detected in undifferentiated spindle CarC cells were lower than those present in the rest of keratinocyte cell lines. In contrast, spindle cells expressed high levels of the specific fibronectin receptor alpha 5 beta 1, whereas this integrin was absent or expressed at very reduced levels in the other epithelial cell lines. Expression of alpha 5 beta 1 integrin in spindle cells appeared organized in cell-substratum contact areas on spread cells. In addition, high and homogenous expression of alpha 5 beta 1 was detected in fully undifferentiated tumors induced in nude mice by three independent spindle cell lines. These results suggest that the expression of alpha 5 beta 1 integrin is upregulated during the development of spindle cell carcinomas that occur in the last stages of mouse skin carcinogenesis and can be associated with the acquisition of the fibroblastoid phenotype of spindle cells. On the other hand, expression of the collagen receptor alpha 2 beta 1 was demonstrated in a transformed cell line (PDV), and it was apparently also expressed in two other malignant keratinocyte cell lines (PDVC57 and HaCa4). The expression of alpha 2 beta 1 was correlated with the increased adhesion to collagen type I and collagen type IV exhibited by the tumorigenic cell lines.

Animals↗

PubMed1997

Up-regulation of integrin beta 3 expression by cyclic stretch in human umbilical endothelial cells.

The effect of uni-axial cyclic mechanical stretch on the expression of the adhesion protein integrin was investigated. Human umbilical endothelial cells (HUVECs) cultured on fibronectin coated silicon membranes were subjected to uni-axial cyclic stretch. The level of expression of integrin beta 3 mRNA was found to be increased and peaked at 4 hours in response to cyclic stretch using a semiquantitative RT-PCR method. The increased level of the integrin mRNA from stretched HUVECs remained higher than that from non-stretched controls. The amount of integrin beta 3 also increased and peaked at 12 hr. Immuno-fluorescent microscopy revealed that the amount of integrin beta 3 adhesions increased in stretched HUVECs compared with that in non-stretched HUVECs. These results suggest that uni-axial cyclic stretch up-regulates the expression of integrin beta 3. This increase in integrin beta 3 may enhance the adhesiveness to the substratum and contribute to the protection of HUVECs against being peeled off from the vessel wall.

Antigens, CD↗

PubMed1999

H-Ras is a negative regulator of alpha3beta1 integrin expression in ECV304 endothelial cells.

We have examined the role of Ras in integrin expression in ECV304 endothelial cells. Among the integrins examined in stable ECV304 transfectants expressing dominant active H-Ras (DAR-ECV), expression of alpha3beta1 integrin showed a prominent reduction in all the DAR-ECV clones when compared to the parental ECV304 cells. This implies that H-Ras negatively regulates the expression of alpha3beta1 integrin in ECV304 cells. When treated with inhibitors of the Ras downstream pathway (LY294002, PD98059, SB203580), the expression of alpha3beta1 integrin was up-regulated most significantly by LY294002, suggesting that among the downstream pathways of Ras, phosphatidylinositol 3-kinase is a major determinant. With the application of blocking antibody to alpha3beta1 integrin (2 - 2 x 10(4) nM), migration of ECV304 cells was enhanced to maximal (18%) at 20 nM. These results suggest that migration of endothelial cells could be modulated by H-Ras via alteration of the expression levels of alpha3beta1 integrin.

Antibodies↗