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PubMed2006

Fibronectin increases matrix metalloproteinase 9 expression through activation of c-Fos via extracellular-regulated kinase and phosphatidylinositol 3-kinase pathways in human lung carcinoma cells.

Enhanced expression of matrix metalloproteinase-9 (MMP-9) is associated with human lung tumor invasion and/or metastasis. We have demonstrated that fibronectin (FN), a matrix glycoprotein, stimulates human non-small cell lung carcinoma (NSCLC) cell proliferation. The current study examines the effect of FN on MMP-9 expression in NSCLC cells. We show that FN increases MMP-9 protein, mRNA expression, and gelatinolytic activity in NSCLC cells. The integrin alpha5beta1 mediated the effects of FN because alpha5 small interfering RNA blocked FN-stimulated MMP-9 protein expression, and also abrogated FN-induced phosphorylation of ERK and phosphatidylinositol 3-kinase (PI3K) signals. The inhibitor of ERK, PD98095, and of PI3K, wortmannin, but not that of protein kinase A, H89, of Rho kinase, Y-27632, of mTOR, rapamycin, or of JNK, SP600125, prevented FN-induced MMP-9 gelatinolytic activity and gene expression. FN enhanced MMP-9 gene promoter activity; however, there was no response to FN in DNA constructs with an AP-1 site mutation. FN increased AP-1 DNA binding activity, and this was abrogated by cyclic AMP response element decoy oligonucleotides, which also diminished FN-induced MMP-9 promoter activity. FN increased the expression of the AP-1 subunit c-Fos protein, but not in the presence of PD98095 and wortmannin. The AP-1 inhibitor, nordihydroguaiaretic acid, and a c-Fos small interfering RNA eliminated the effect of FN on MMP-9 expression. This study indicates that FN, by binding to the integrin alpha5beta1 receptor, stimulates the expression of MMP-9 through increased AP-1/DNA binding and c-Fos protein expression via ERK and PI3K signaling pathways. The data unveils a novel mechanism by which FN could promote NSCLC cell invasion and metastasis.

Carcinoma, Non-Small-Cell Lung↗

PubMed2003

Behavior of SaOS-2 cells cultured on different titanium surfaces.

Surface properties may affect the clinical outcome of titanium dental implants. The aim of the present study was to investigate the effects of 3 different titanium surfaces-smooth (S), sandblasted (SB), and titanium plasma-sprayed (TPS)-on proliferation, differentiation, and apoptosis of human osteoblast-like cells, SaOS-2. Cell proliferation was significantly (p < 0.05) higher on the S surface, and synthesis of extracellular matrix proteins was more abundant on TPS and SB than on S surfaces. Analysis of integrin receptors showed a higher expression of alpha2, alpha5, alphaVbeta3, and ss1 on TPS as compared with SB and S surfaces. An increase in alkaline phosphatase activity was detected only on SB and TPS surfaces. Analysis of cell apoptosis did not demonstrate any significant difference among the 3 different surfaces. The results indicate that titanium surface topography affects proliferation and differentiation of osteoblast-like SaOS-2 cells, suggesting that surface properties might be important for bone response around dental implants in vivo.

Alkaline Phosphatase↗

PubMed2004

Molecular regulation of osteoblasts for tissue engineered bone repair.

The use of biodegradable polymers in medicine and biomedical research is increasing. A key growth area has been the use of these materials in tissue engineering, especially for guided regeneration of bone and cartilage. Our interest has been in determining the mechanisms by which cellular attachment and growth occurs on these materials. In the current study, we examined human osteoblast cell adhesion, growth, and morphologic changes on polymeric scaffolds composed of polylactic-co-glycolic acid and polylactic acid materials. We examined these characteristics in association with measurements of levels of key adhesion integrin receptors in the presence and absence of antibodies against alpha2, alpha3, alpha4, alpha5, alpha6, and beta1 subunits, and the adhesion ligand peptides RGD (Arg-Gly-Asp) and RGE (Arg-Gly-Ser). At 2 hours, results showed initial cell adhesion was considerably decreased on polylactic-co-glycolic acid and polylactic acid in the presence of the alpha2 and beta1, antibodies with a 70% adhesion rate difference observed among the groups evaluated. Higher levels of inhibition were observed on polylactic-co-glycolic acid relative to polylactic acid, which may be correlated to a higher number of cells being able to interact with the surface initially. The presence of known competitive peptide (RGD) at 2 hours, revealed its ability to block cellular adhesion to these matrices relative to the control and noncompetitive peptide RGE on polylactic-co-glycolic acid matrices. Overall adhesion rate was affected by the presence of the integrin antibodies to the alpha2, alpha3, alpha4, alpha5, alpha6, and beta1 subunits with highest differences among polylactic-co-glycolic acid relative to its control, therefore suggesting that initial osteoblastic cell adhesion to commonly used biomaterials is regulated through integrin binding.

Biocompatible Materials↗

PubMed2005

Laminins 2 (alpha2beta1gamma1, Lm-211) and 8 (alpha4beta1gamma1, Lm-411) are synthesized and secreted by tooth pulp fibroblasts and differentially promote neurite outgrowth from trigeminal ganglion sensory neurons.

The tooth pulp innervation originates from the trigeminal ganglion (TG) and represents an illustrative example of tissue targeting by sensory nerves. Pulpal fibroblasts strongly promote neurite outgrowth from TG neurons in vitro. In the present study, we have investigated the possible participation of laminins (LNs), potent neuritogenic extracellular matrix components. Immunohistochemistry of human tooth pulp demonstrated expression of LN alpha1, alpha2, alpha4, alpha5, beta1 and gamma1, and laminin-binding integrin alpha3, alpha6, beta1 and beta4 chains in nerves. Though faintly stained for laminins in situ, pulpal fibroblasts reacted, once cultured and permeabilized, with antibodies to LN alpha2, alpha4, beta1 and gamma1 chains by flow cytometry. The cells also expressed the corresponding mRNAs and were able to assemble and secrete LN-2 (alpha2beta1gamma1, Lm-211) and LN-8 (alpha4beta1gamma1, Lm-411). LN-8 displayed a chondroitin sulphate (CS) modification in its alpha4 chain. In functional assays, mouse LN-1 (alpha1beta1gamma1, Lm-111) and recombinant human (rh) LN-8, but not native or rhLN-2, strongly promoted neurite outgrowth from TG neurons, mimicking the effect of cultured pulp fibroblast. Altogether, the results indicate that LN-2 and LN-8 are synthesized by tooth pulp fibroblasts and differentially promote neurite outgrowth from TG neurons. LN-8 may contribute to sensory innervation of teeth and other tissues during development and/or regeneration.

Adolescent↗

PubMed2005

CD34+ marrow progenitors from MDS patients with high levels of intramedullary apoptosis have reduced expression of alpha4beta1 and alpha5beta1 integrins.

Excessive intramedullary apoptosis is central in the pathogenesis of myelodysplastic syndromes (MDS). Growth-inhibiting cytokines, the Fas/FasLigand pathway, and autoreactive cytotoxic T-lymphocytes have been identified to be important proapoptotic factors in MDS. In normal hematopoiesis, alpha4beta1 and alpha5beta1 integrin-mediated interactions between progenitors and fibronectin are critical for progenitor cell survival. In this study, we have used flow cytometry to quantify the expression levels of members of the beta1 integrin family on CD34(+) marrow progenitors in 27 untreated patients with MDS, three with s-AML, and 25 control subjects. In MDS, we observed that nonapoptotic progenitors significantly downregulate cell surface expression levels of alpha4 and beta1 integrin chains compared with healthy controls. Downregulation of alpha4, beta1, and also alpha5 was present in MDS patients with > or =25% apoptotic progenitors, irrespective of their French, American, British subcategory. Reduced cell surface expression levels of alpha4, alpha5, and beta1 did also correlate with decreased in vitro adhesiveness to fibronectin fragments. Therefore, our observations suggest that downregulation of alpha4beta1 and alpha5beta1 integrins on CD34(+) progenitors could be a newly identified proapoptotic mechanism in MDS.

Aged↗

PubMed1998

Integrins involved in the adhesion of megakaryocytes to fibronectin and fibrinogen.

We studied integrins involved in the adhesion of resting and activated megakaryocytes (MK) to fibronectin (FN) and fibrinogen (FGN). Guinea pig MK were isolated and in some experiments were activated by thrombin. MK adhering to FN or FGN coated on coverslips were quantitated by a computerized image analysis program. The binding of soluble human FN to MK was detected by Western blotting. Anti-integrin antibodies, disintegrins, and cyclic RGD peptides were used to identify integrins involved in the adhesion of MK to FN or FGN. Resting MK adhered to coverslips with immobilized FN. The adhesion of MK to FN was primarily inhibited by an anti-alpha5 antibody and EMF-10, a distintegrin highly specific for alpha5 beta1. However, the adhesion of MK to FN was not blocked by agents that inhibit alphaIIb beta3, alphav beta3 or alpha4 beta1. A beta1 activating antibody increased the number of MK bound to FN due to the activation of alpha5 beta1. The binding of soluble FN was also primarily inhibited by agents that block alpha5 beta1. Resting MK did not adhere to FGN. However, MK activated by thrombin did adhere to FGN. This binding was mediated by alphaIIb beta3, because binding was inhibited by bitistatin, a disintegrin, and a cyclic RGD peptide that are known to block this integrin. The binding of thrombin-activated MK to FN was mediated by both alpha5 beta1 and alphaIIb beta3 based on the additive effect of agents that inhibit these integrins. The study indicates that resting MK bind to FN but not to FGN and that alpha5 beta1 is the major integrin involved in the binding of MK to FN. Activated MK bind to FGN primarily by alphaIIb beta3. However, the binding of activated MK to FN is due to both alpha5 beta1 and alphaIIb beta3. The demonstration that alpha5 beta1 and that alphaIIb beta3 are involved in MK adhesion indicates that these integrins may have a role in MK maturation and platelet production.

Animals↗

PubMed1999

Effect of all-trans-retinoic acid on integrin receptors of human cervical cancer (SiHa) cells.

Cell surface receptors have been the subject of intensive investigations over the past few decades. One very important group of receptors on the cell surface is the "integrin" receptors which bind to extracellular matrix (ECM) proteins. Because of integrin's importance in cellular growth, development, and morphology the role of integrin receptors in cellular transformation, malignant growth, and metastasis has received wide attention. In this article we report on the effect of all-trans-retinoic acid (ATRA) on (a) the integrin family of cell surface receptors, (b) collagenase enzyme activity, and (c) invasive potential in human cervical cancer (SiHa) cells. A comparative cell adhesion assay clearly showed that ATRA affects the cell surface integrin receptors against different ECM proteins in a dose- and time-dependent manner. The binding of SiHa cells to ECM proteins (fibronectin, vitronectin, laminin, collagen IV) was drastically reduced when cells were treated with ATRA at 10 microM for 96 h in culture. Interestingly, when ATRA-treated (10 microM, 96 h) SiHa cells were allowed to grow for 15 days in ATRA-free complete medium the binding of SiHa cells to fibronectin and vitronectin was inhibited, even after 15 days of drug withdrawal, whereas cell adhesion to laminin and collagen IV returned to normal within 3-7 days. The comparative immunoprecipitation of two cell surface integrin receptors (alpha5beta1 and alphavbeta3) shows the effect of ATRA on the expression of alpha5, alphav, and beta1 subunits. In ATRA-treated SiHa cells the cell surface expression of the alphav subunit (in alphavbeta3 receptor) is much less than in untreated SiHa cells. In the case of the alpha5beta1 integrin receptor ATRA treatment caused a significant reduction in the expression of both alpha5 and beta1 subunits on the cell surface. Comparative zymography clearly demonstrated the inhibitory effect of ATRA on collagenase enzyme activity. Interestingly, the effect was irreversible, even after 15 days of culture in ATRA-free medium. The assay of the invasive potential of ATRA-treated and untreated SiHa cells in Boyden's invasion chamber demonstrated that ATRA treatment (10 microM, 96 h) inhibits the invasive potential of SiHa cells. The effect was not reversible even after 15 days of culture in ATRA-free medium. In conclusion, our observations indicate that ATRA has an inhibitory effect on the expression of SiHa cell surface integrin receptors and collagenase enzyme activity. The effect of ATRA on cell surface integrin receptors and collagenase enzyme activity may affect the invasive potential of SiHa cells.

Cell Adhesion↗

PubMed2004

Integrin signaling in inflammatory and neuropathic pain in the rat.

Many painful conditions are associated with alterations in the extracellular matrix (ECM) of affected tissues. While several integrins, the receptors for ECM proteins, are present on sensory neurons that mediate pain, the possible role of these cell adhesion molecules in inflammatory or neuropathic pain has not been explored. We found that the intradermal injection of peptide fragments of domains of laminin and fibronectin important for adhesive signaling selectively inhibited the hyperalgesia caused by prostaglandin E2 (PGE2) and epinephrine (EPI), respectively. The block of EPI hyperalgesia was mimicked by other peptides containing the RGD integrin-binding sequence. Monoclonal antibodies (mAbs) against the alpha1 or alpha3 integrin subunits, which participate in laminin binding, selectively blocked PGE2 hyperalgesia, while a mAb against the alpha5 subunit, which participates in fibronectin binding, blocked only EPI-induced hyperalgesia. A mAb against the beta1 integrin subunit, common to receptors for both laminin and fibronectin, inhibited hyperalgesia caused by both agents, as did the knockdown of beta1 integrin expression by intrathecal injection of antisense oligodeoxynucleotides. The laminin peptide, but not the fibronectin peptides, also reversibly abolished the longer lasting inflammatory hyperalgesia induced by carrageenan. Finally, the neuropathic hyperalgesia caused by systemic administration of the cancer chemotherapy agent taxol was reversibly inhibited by antisense knockdown of beta1 integrin. These results strongly implicate specific integrins in the maintenance of inflammatory and neuropathic hyperalgesia.

Animals↗

PubMed2004

Relations of the type and branch of surface N-glycans to cell adhesion, migration and integrin expressions.

The relations between the structure of cell surface N-glycans to cell behaviors were studied in H7721 human hepatocarcinoma cell line, which predominantly expressed complex-type N-glycans on the surface. 1-Deoxymannojirimycin (DMJ) and swaisonine (SW), the specific inhibitor of Golgi alpha-mannosidase II or I, were selected to block the processing of N-glycans at the steps of high mannose and hybrid type respectively. All-trans retinoic acid (ATRA) and antisense cDNA of N-acetylglucosaminyltransferase-V (GnT-V) were used to suppress the expression of GnT-V and decreased the GlcNAc beta1,6-branching or tri-/tetra-antennary structure of surface N-glycans. The structural alterations of N-glycans were verified by sequential lectin affinity chromatography of [3H] mannose-labeled glycans isolated from the cell surface. The cell adhesions to fibronectin (Fn) and human umbilical vein epithelial cell (HUVEC), as well as cell migration (including chemotaxis and invasion) were selected as the parameters of cell behaviors. It was found that cell adhesion and migration were significantly decreased in SW and DMJ treated cells, suggesting that complex type N-glycan is critical for the above cell behaviors. ATRA and antisense GnTV enhanced cell adhesion to Fn but reduce cell adhesion to HUVEC and cell migration. These results reveal that cell surface complex-type N-glycans with GlcNAc beta1,6 branch are more effective than those without this branch in the cell adhesion to HUVEC and cell migration, but N-glycan without GlcNAc beta1,6-branch is the better one in mediating the cell adhesion to Fn. The integrin alpha5beta1 (receptor of Fn) on cell surface was unchanged by DMJ and SW. In contrast, ATRA up regulated alpha5, but not beta1, and antisense GnT-V decreased both alpha5 and beta1. This findings suggest that both the structure of N-glycan and the expression of integrin on cell surface are two of the important factors in the determination of cell adhesion to Fn, a complex biological process.

1-Deoxynojirimycin↗

PubMed1996

Induction of fibroblast gelatinase B expression by direct contact with cell lines derived from primary tumor but not from metastases.

During cancer progression, tumor cells interact with stromal cells. As a consequence, matrix metalloproteinases are produced that contribute to the degradation of the extracellular matrix. This study used coculture systems to investigate fibroblast interaction with three colon cancer cell lines isolated from a single patient. Cells from primary colorectal carcinoma, but not from corresponding liver or lymph node metastases, induced gelatinase B expression by fibroblasts of different tissue origin. Remarkably, direct cell-cell contact was required for this induction, which occurred at the pretranslational level (as revealed by Northern blot analysis) and was completely blocked by anti-beta1 integrin monoclonal antibody, but only partially blocked by anti-alpha5 or anti-alpha(v). Induction was also inhibited by cytochalasin D, staurosporine, or dexamethasone, suggesting the need, respectively, for an organized actin cytoskeleton, protein kinase C, and AP-1-driven gene transcription. Our data suggest that direct tumor-stromal cell contact is one inductive event involved in matrix metalloproteinase expression by stromal cells.

Animals↗

PubMed1998

Extracellular signal-regulated kinase and c-Jun NH2-terminal kinase activation by mechanical stretch is integrin-dependent and matrix-specific in rat cardiac fibroblasts.

Integrins, which connect the cytoskeleton to the extracellular matrix and mediate a variety of signaling cascades, may transduce mechanical stimuli into biochemical signals. We studied integrin- and matrix-dependent activation of extracellular signal-regulated kinase (ERK2), c-Jun NH2-terminal kinase (JNK1), and p38 in response to 4% static biaxial stretch in rat cardiac fibroblasts. ERK2 and JNK1, but not p38, were rapidly activated by stretch when the fibroblasts were allowed to synthesize their own matrices. When the cells were limited to specific matrix substrates, ERK2 and JNK1 were differentially activated: ERK2 was only activated when the cells were plated on fibronectin, while JNK1 was activated when the cells were plated on fibronectin, vitronectin, or laminin. Plating cells on collagen before stretching did not activate either kinase. Adhesion to all matrices was integrin-dependent because it could be blocked by inhibitors of specific integrins. ERK2 activation could be blocked with a combination of anti-alpha4 and -alpha5 antibodies and an arginine-glycine-aspartic acid (RGD) peptide, while the antibodies or peptide used separately failed to block ERK2 activation. This result suggests that at least two integrins, alpha4beta1 and an RGD-directed, non-alpha5beta1 integrin, activate ERK2 in response to mechanical stimulation. Activation of JNK1 could not be blocked with the inhibitors, suggesting that an RGD-independent integrin or integrins other than alpha4beta1 can activate JNK1 in cells adherent to fibronectin. This study demonstrates that integrins act as mechanotransducers, providing insight into potential mechanisms for in vivo responses to mechanical stimuli.

Animals↗

PubMed2002

Fibronectin fragments and blocking antibodies to alpha2beta1 and alpha5beta1 integrins stimulate mitogen-activated protein kinase signaling and increase collagenase 3 (matrix metalloproteinase 13) production by human articular chondrocytes.

OBJECTIVE: To determine if integrin-mediated signaling results in activation of chondrocyte mitogen-activated protein (MAP) kinases that lead to increased expression of matrix metalloproteinase 13 (MMP-13; collagenase 3), a potent mediator of cartilage matrix degradation. METHODS: Human articular chondrocytes isolated from normal ankle and knee cartilage obtained from tissue donors were cultured in monolayers. The cells were treated with a 120-kd fibronectin fragment (FN-f) that binds the alpha5beta1 integrin or with antibodies to specific integrin receptors. Activation of MAP kinases was determined by immunoblotting with phosphospecific antibodies. MMP production was measured by gelatin zymography, and MMP-13 production and activation were determined by immunoblotting and by a fluorogenic peptide assay. RESULTS: Human articular chondrocytes were found to respond to the 120-kd FN-f and to adhesion-blocking antibodies to the alpha2beta1 and alpha5beta1 integrins with increased phosphorylation of the extracellular signal-regulated kinase 1 (ERK1)/ERK2, c-Jun N-terminal kinase (JNK), and p38 MAP kinases. Intact FN and integrin-blocking antibodies to alpha1, alpha3, and alphaVbeta3 and a nonblocking alpha5 antibody had no effect. After MAP kinase activation, increased phosphorylation of c-Jun and the nuclear factor kappaB inhibitor was noted, followed by increased pro- and activated MMP-13 in the conditioned media. Inhibitors of mitogen-activated protein kinase kinase, p38, and JNK were each able to inhibit increased MMP-13 production, while the interleukin-1 receptor antagonist (IL-1Ra) protein did not. However, the IL-1Ra partially inhibited FN-f-induced activation of MMP-13. CONCLUSION: Integrin-mediated MAP kinase signaling stimulated by FN-f is associated with increased production and release of pro- and active MMP-13. Autocrine production of IL-1 appears to result in additional MMP-13 activation. These processes may play a key role in feedback loops responsible for progressive cartilage degradation in arthritis.

Antibodies↗

PubMed1998

Isolation and characterization of laminin-10/11 secreted by human lung carcinoma cells. laminin-10/11 mediates cell adhesion through integrin alpha3 beta1.

A panel of human tumor cell lines was screened for selective expression of laminin alpha5 chain, a newly identified laminin subunit comprising laminin-10 (alpha5 beta1 gamma1) and -11 (alpha5 beta2 gamma1). The lung adenocarcinoma cell line A549 was found to express the alpha5 chain at relatively high levels but no detectable amounts of other alpha chains. The laminin variants containing alpha5 chain were purified from the conditioned medium of A549 cells by immunoaffinity chromatography using the anti-laminin monoclonal antibody 4C7 which was shown recently to recognize the laminin alpha5 chain (Tiger, C.-F., Champliaud, M.-F., Pedrosa-Domellof, F., Thornell, L.-E., Ekblom, P., and Gullberg, D. (1997) J. Biol. Chem. 272, 28590-28595). The purified laminin variants consisted of three chains with molecular masses of 350, 220, and 210 kDa. The 350-kDa chain was specifically recognized by another anti-alpha5 chain monoclonal antibody capable of recognizing denatured alpha5 chain on immunoblots, whereas the 210-kDa chain was recognized by an anti-gamma1 chain antibody. The purified alpha5 chain-containing laminin variants (hereafter referred to as laminin-10/11) were highly active in mediating adhesion of A549 cells to the substratum with potency as high as that of laminin-5 and significantly higher than those of laminin-1, laminin-2/4, or fibronectin. Adhesion to substrata coated with laminin-10/11 was specifically inhibited by anti-integrin antibodies directed against the integrin alpha3 or beta1 subunit but not by those against alpha2 or alpha6 subunit, indicating that laminin-10/11 is specifically recognized by integrin alpha3 beta1. Given the wide distribution of laminin-10/11 in the basement membrane of various tissue types and dominant expression of integrin alpha3 beta1 in most epithelial cells, specific interaction of laminin-10/11 with integrin alpha3 beta1 may play an important role in in vivo regulation of proliferation and differentiation of epithelial cells through the basement membrane.

Cell Adhesion↗

PubMed2001

Integrin and FAK-mediated MAPK activation is required for cyclic strain mitogenic effects in Caco-2 cells.

Rhythmic strain stimulates Caco-2 proliferation. We asked whether mitogen-activated protein kinase (MAPK) activation mediates strain mitogenicity and characterized upstream signals regulating MAPK. Caco-2 cells were subjected to strain on collagen I-precoated membranes or antibodies to integrin subunits. Twenty-four hours of cyclic strain increased cell numbers compared with static conditions. MAPK-extracellular signal-regulated kinase (ERK) kinase inhibition (20 microM PD-98059) blocked strain mitogenicity. p38 Inhibition (10 microM SB-202190) did not. Strain rapidly and time-dependently activated focal adhesion kinase (FAK), paxillin, ERK1 and 2, and p38 on collagen. c-Jun NH(2)-terminal kinase (JNK)1 and 2 exhibited delayed activation. Similar activation occurred when Caco-2 cells were subjected to strain on a substrate of functional antibody to the alpha2-, alpha3-, alpha6-, or beta1-integrin subunits but not on a substrate of functional antibody to the alpha5-subunit. FAK inhibition by FAK397 transfection blocked ERK2 and JNK1 activation by in vitro kinase assays, but pharmacological protein kinase C inhibition did not block ERK1 or 2 activation by strain. Strain-induced ERK signals mediate strain's mitogenic effects and may require integrins and FAK activation.

Caco-2 Cells↗

PubMed1999

Expression of integrins of the beta1 family in thyroid cells from patients with Graves' disease in vivo and in vitro.

The expression of the beta1 family of integrins was determined in thyroid follicular cells from patients with Graves' disease (GD). Integrin expression was quantitated by flow fluorocytometry of single cell suspensions with antibodies against the common beta1 chain and the alpha1-alpha6 subunits. Results indicated that also in thyroid glands of GD, as previously observed in nodular goiters, two follicular cell populations with different patterns of beta1 integrin expression coexist (VLAalpha3beta1 and VLAalpha1,3,5,6beta1). The VLAalpha1,3,5,6beta1 thyrocyte population in GD was more abundant than in nodular goiters, ranging from 40 to 70% of the total follicular cells and the overall expression of the beta1 integrins was a two-fold higher. In thyrocytes from patients with GD cultured in vitro, alpha3 and alpha2 expression was regulated by cell-to-cell contact as previously described in normal thyroid cells, while the expression of alpha1, alpha5 and alpha6 was quickly lost during the culture. Our data suggest that the integrin profile of the VLAalpha1,3,5,6beta1 thyrocyte population in GD is induced by micro-environmental conditions rather than being the expression of a constitutive phenotype.

Cells, Cultured↗

PubMed1999

Real-time analysis of integrin-mediated chemotactic migration of T lymphocytes within 3-D extracellular matrix-like gels.

We have developed a novel 3-D gel reconstituted with major extracellular matrix (ECM) glycoproteins to follow the dynamics of migration of human T cells locomoting, in real-time, on gradients formed by representative chemoattractants: the C-C chemokine RANTES, and the pro-inflammatory cytokine IL-2. In the absence of chemoattractants, none of the T cells migrated directionally and the levels of random migration or cell polarization were low. However, major fractions of T cells placed in IL-2 and RANTES gradients in the gels polarized immediately after exposure to the chemoattractants. Shortly after polarization, 25% of the T cells migrated, in either a random or directional fashion, towards the sources of the chemoattractants; additional 5-10% of the cells remained polarized but stationary. The number of T cells migrating directionally towards RANTES or IL-2 peaked along with the formation of the chemotactic gradients. The directional migration of T cells was increased by a short pre-exposure to low doses of IL-2, which did not alter the level of expression of the beta1 integrins. The directional migration of T cells towards IL-2 and RANTES was mediated by IL-2R and pertussis toxin-sensitive receptors, respectively, and the directional, and to a lesser degree, the random locomotion of T cells induced by both chemoattractants required intact tyrosine kinase signaling and activities of the alpha4, alpha5, and, to a lesser degree, the alpha2 and alpha6 members the beta1 integrins. Our system enables the real-time tracking of individual locomoting lymphocytes and the analysis of their dynamic interactions with ECM components and cytokines.

Cell Movement↗

PubMed2000

Beta1 integrins play an essential role in adhesion and invasion of pancreatic carcinoma cells.

To investigate the role of beta1 integrins in pancreatic carcinoma invasion, we analyzed the relationship between the activity of beta1 integrins and the invasive ability of human pancreatic carcinoma cell lines. AsPC1, BxPC3, PANC1, SU8686, KP1NL, KP2, and H48N cells had high expression of beta1 and alpha6 subunits, and various levels of alpha2, alpha3, and alpha5 expression as determined by flow cytometry. Cell adhesion assay revealed that alpha2beta1, alpha5beta1, and alpha6beta1 integrins were the predominant adhesion receptors for collagen, fibronectin, and laminin, respectively. Beta1 integrins on different cell types showed a wide range of constitutive activity. Anti-beta1 monoclonal antibody (MAB) TS2/16 rapidly activated beta1 integrins, and thus TS2/16 requirement in cell adhesion represented the levels of constitutive activity of beta1 integrins. Notably, as the result of in vitro chemoinvasion assay, the levels of constitutive activity of beta1 integrins correlated with the invasive ability of pancreatic carcinoma cells. The inhibitory anti-beta1 MAB 13 completely blocked the invasion of these cell lines. Alternatively, the stimulatory anti-beta1 MAB TS2/16 strongly inhibited the invasion. These results show an essential role of beta1 integrins in invasion of pancreatic carcinoma cells and also suggest subtle regulatory mechanisms of cell invasion.

Antibodies, Monoclonal↗

PubMed1996

Stimulation of tyrosine phosphorylation after ligation of beta7 and beta1 integrins on human B cells.

B lymphocytes express several members of the integrin family of adhesion molecules that mediate cell-cell and cell-extracellular matrix interactions. In addition to beta1 integrins, predominantly alpha4 beta1, mature B cells also express alpha4 beta7, which is a receptor for vascular cell adhesion molecule-1 and fibronectin, and is also involved in the homing of B cells to mucosal sites through binding to a third ligand, mucosal address in cell adhesion molecule-1. Here we describe that crosslinking of alpha4 beta7 integrins on B cell lines and normal tonsillar B cells, induces tyrosine phosphorylation of multiple substrates of 105-130 kD, indicating that beta7 integrin plays a role as signaling molecule in B cells. This pattern of phosphorylated proteins was very similar to that induced following ligation of alpha4 beta1. Interestingly, ligation of alpha5 beta1 or alpha6 beta1 also stimulated the 105-125 kD group of phosphorylated proteins, whereas ligation of beta2 integrins did not. The focal adhesion tyrosine kinase p125FAK was identified as one of these substrates. Beta1 or beta7 mediated tyrosine phosphorylations were markedly decreased when the microfilament assembly was inhibited by cytochalasin B. These results suggest that intracellular signals initiated by different integrins in B cells may converge, to similar cytoskeleton-dependent tyrosine phosphorylated proteins.

Antibodies, Monoclonal↗