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PubMed2006

The dependence of fibrillar adhesions in human fibroblasts on substratum chemistry.

Little is known about the influence of substratum properties and composition on the ability of cells to translocate alpha5beta1 integrins and to form fibrillar adhesion. We have examined the impact of self-assembled monolayers (SAMs) bearing different functional end groups (amines (NH2) or carboxylic acids (COOH)) on the presence of fibrillar adhesions in human fibroblasts attached to fibronectin-coated SAMs. Most of the fibroblasts incubated in serum-free medium for 2 h on COOH showed segregation of focal contact components (alphav integrin subunits, phosphotyrosine proteins) and fibrillar adhesions (alpha5 integrin subunits, tensin) while the majority of cells plated on NH(2) did not. Analysis of fibronectin fibril formation confirmed also that human fibroblasts plated on COOH formed matrix fibrils significantly better. The surface-associated alpha5 to alphav integrin ratio was smaller in cells on COOH than on NH2 due to a decreased alpha5 integrin binding. In addition, human fibroblasts migrated more readily on COOH in comparison to NH2 which points to a different binding strength of integrins to the substratum. Overall, the results indicate that the molecular composition of substrata has a strong influence on FN matrix formation by promoting or inhibiting segregation of focal and fibrillar adhesions.

Cell Adhesion↗
PubMedDate not supplied

Clinicopathological and immunohistochemical analysis of malignant features in mucinous cystic tumors of the pancreas.

BACKGROUND/AIMS: To investigate the malignancy of mucinous cystic tumors (MCTs) of the pancreas, we examined clinicopathological features and immunohistochemical findings of MCT. METHODOLOGY: We analyzed the expression of p53 protein, proliferating cell nuclear antigen, alpha6-integrin subunit, alpha5beta1-integrin, and interleukin-1 receptor type I in tumor specimens from eight patients with MCT. RESULTS: The tumors were classified as mucinous cyst adenoma (n=6) or mucinous cyst adenocarcinoma (n=2). The actuarial five-year survival rate was 83.3%. All in eight MCTs had 'ovarian-type' stroma in the cyst wall. The alpha6-integrin subunit and p53 protein were expressed in adenocarcinoma tissues of MCTs, and in two adenomas the alpha6-integrin subunit and p53 protein were also co-expressed. CONCLUSIONS: Our present results indicate that coexpression of the alpha6-integrin subunit and p53 protein should be appreciated as an indicator of malignancy in MCTs.

Adult↗
PubMed2000

A nonpeptide integrin antagonist can inhibit epithelial cell ingestion of Streptococcus pyogenes by blocking formation of integrin alpha 5beta 1-fibronectin-M1 protein complexes.

Streptococcus pyogenes can be efficiently internalized by a variety of human epithelial cells. beta-lactam antibiotics, commonly used to treat S. pyogenes infections, do not readily permeate mammalian cells. There is growing evidence that the ability of streptococci to enter host cells contributes to the frequent failure of antibiotics to eradicate the organism from infected individuals. Recent studies have suggested that host cell entry requires the formation of a complex of a bacterial fibronectin (Fn) binding protein (e.g., M1 protein or protein F1/SfbI), human Fn, and the epithelial cell Fn receptor, integrin alpha5beta1. We report here that a low molecular weight, nonpeptide antagonist of integrin alpha5beta1, SJ755, can inhibit internalization of streptococci by primary human tonsillar epithelial cells and immortalized human epithelial (A549) cells, thus increasing the extent of bacterial killing by antibiotics. SJ755 blocked Fn binding by human tonsillar epithelial and A549 cells, suggesting that integrin alpha5beta1 is the major Fn receptor expressed by both cell types. SJ755 did not affect Fn binding by purified M1 protein or M1(+) bacteria. Purified M1 protein failed to associate with integrin alpha5beta1 unless the integrin had been prebound by Fn. Also, SJ755 blocked formation of alpha5beta1-Fn-M1 complexes in vitro. These results support the previous proposal that Fn functions as a molecular bridge between M1 protein and integrin alpha5beta1. Furthermore, these results suggest that integrin antagonists may enhance the efficacy of antibiotics in treatment of S. pyogenes infections.

Adhesins, Bacterial↗
PubMed1998

Cross-talk between insulin receptor and integrin alpha5 beta1 signaling pathways.

The ligation and clustering of cell surface alphabeta heterodimeric integrins enhances cell adhesion and initiates signaling pathways that regulate such processes as cell spreading, migration, differentiation, proliferation and apoptosis. Here we show that insulin treatment of Chinese hamster ovary cells expressing insulin receptors (CHO-T) markedly promotes cell adhesion onto a fibronectin matrix, but not onto bovine serum albumin or poly-lysine. Incubation of cells with a GRGDSP peptide that specifically binds integrins (but not the nonspecific GRADSP peptide) abolishes this insulin effect, as does the potent phosphoinositide 3-kinase (PI 3-kinase) inhibitor wortmannin. Moreover, a specific blocking monoclonal anti-alpha5beta1 integrin antibody, PB-1, blocks insulin-stimulated cell adhesion onto fibronectin. Conversely, activating alpha5beta1 integrins on CHO-T cells by adherence onto fibronectin markedly potentiates the action of insulin to enhance insulin receptor and insulin receptor substrate (IRS)-1 tyrosine phosphorylation. Activation of alpha5beta1 integrin also markedly potentiates the recruitment of p85-associated PI 3-kinase activity to IRS-1 in response to submaximal levels of insulin in CHO-T cells. These data indicate that insulin potently activates integrin alpha5beta1 mediated CHO-T cell adhesion, while integrin alpha5beta1 signaling in turn enhances insulin receptor kinase activity and formation of complexes containing IRS-1 and PI 3-kinase. These findings raise the hypothesis that insulin receptor and alpha5beta1 integrin signaling act synergistically to enhance cell adhesion.

Androstadienes↗
PubMed1997

Defining the topology of integrin alpha5beta1-fibronectin interactions using inhibitory anti-alpha5 and anti-beta1 monoclonal antibodies. Evidence that the synergy sequence of fibronectin is recognized by the amino-terminal repeats of the alpha5 subunit.

The high affinity interaction of integrin alpha5beta1 with the central cell binding domain (CCBD) of fibronectin requires both the Arg-Gly-Asp (RGD) sequence (in the 10th type III repeat) and a second site (in the adjacent 9th type III repeat) which synergizes with RGD. We have attempted to map the fibronectin binding interface on alpha5beta1 using monoclonal antibodies (mAbs) that inhibit ligand recognition. The binding of two anti-alpha5 mAbs (P1D6 and JBS5) to alpha5beta1 was strongly inhibited by a tryptic CCBD fragment of fibronectin (containing both synergy sequence and RGD) but not by GRGDS peptide. Using recombinant wild type and mutated fragments of the CCBD, we show that the synergy region of the 9th type III repeat is involved in blocking the binding of P1D6 and JBS5 to alpha5beta1. In contrast, binding of the anti-beta1 mAb P4C10 to alpha5beta1 was inhibited to a similar extent by GRGDS peptide, the tryptic CCBD fragment, or recombinant proteins lacking the synergy region, indicating that the RGD sequence is involved in blocking P4C10 binding. P1D6 inhibited the interaction of a wild type CCBD fragment with alpha5beta1 but had no effect on the binding of a mutant fragment that lacked the synergy region. The epitopes of P1D6 and JBS5 mapped to the NH2-terminal repeats of the alpha5 subunit. Our results indicate that the synergy region is recognized primarily by the alpha5 subunit (in particular by its NH2-terminal repeats) but that the beta1 subunit plays the major role in binding of the RGD sequence. These findings provide new insights into the mechanisms, specificity, and topology of integrin-ligand interactions.

Animals↗
PubMed1997

Modulation of cell-adhesive activity of fibronectin by the alternatively spliced EDA segment.

Fibronectin (FN) has a complex pattern of alternative splicing at the mRNA level. One of the alternatively spliced segments, EDA, is prominently expressed during biological processes involving substantial cell migration and proliferation, such as embryonic development, malignant transformation, and wound healing. To examine the function of the EDA segment, we overexpressed recombinant FN isoforms with or without EDA in CHO cells and compared their cell-adhesive activities using purified proteins. EDA+ FN was significantly more potent than EDA- FN in promoting cell spreading and cell migration, irrespective of the presence or absence of a second alternatively spliced segment, EDB. The cell spreading activity of EDA+ FN was not affected by antibodies recognizing the EDA segment but was abolished by antibodies against integrin alpha5 and beta1 subunits and by Gly-Arg-Gly-Asp-Ser-Pro peptide, indicating that the EDA segment enhanced the cell-adhesive activity of FN by potentiating the interaction of FN with integrin alpha5beta1. In support of this conclusion, purified integrin alpha5beta1 bound more avidly to EDA+ FN than to EDA- FN. Augmentation of integrin binding by the EDA segment was, however, observed only in the context of the intact FN molecule, since the difference in integrin-binding activity between EDA+ FN and EDA- FN was abolished after limited proteolysis with thermolysin. Consistent with this observation, binding of integrin alpha5beta1 to a recombinant FN fragment, consisting of the central cell-binding domain and the adjacent heparin-binding domain Hep2, was not affected by insertion of the EDA segment. Since the insertion of an extra type III module such as EDA into an array of repeated type III modules is expected to rotate the polypeptide up to 180 degrees at the position of the insertion, the conformation of the FN molecule may be globally altered upon insertion of the EDA segment, resulting in an increased exposure of the RGD motif in III10 module and/or local unfolding of the module. Our results suggest that alternative splicing at the EDA exon is a novel mechanism for up-regulating integrin-binding affinity of FN operating when enhanced migration and proliferation of cells are required.

Alternative Splicing↗
PubMed1996

Activation-dependent alpha5beta1 integrin-mediated adhesion to fibronectin decreases proliferation of chronic myelogenous leukemia progenitors and K562 cells.

Chronic myelogenous leukemia (CML) is a malignant disease of the hematopoietic stem cell characterized by abnormal circulation and proliferation of malignant progenitors. In contrast to their normal counterparts, CML progenitors adhere poorly to bone marrow stroma or fibronectin (FN). Aside from anchoring progenitors in the marrow microenvironment, beta1 integrin-dependent adhesion of normal progenitors is also associated with inhibition of their proliferation. As the beta1 integrin expression on CML progenitors is normal, we hypothesized that decreased integrin affinity may underlie the abnormal adhesive and proliferative characteristics of CML progenitors. We examined the effect of affinity modulation by the activating antibody 8A2 on the adhesion and proliferation of CML progenitors and the CML cell line, K562. 8A2 induced alpha5Beta1-dependent adhesion of Philadelphia chromosome-positive (Ph+) CD34+/HLA-DR+ cells and K562 cells to FN. Increased adhesion was 8A2- and FN concentration-dependent, time-dependent, and energy-dependent. Further, 8A2-induced adhesion to FN significantly inhibited the proliferation of malignant CML progenitors as well as K562 cells independent of cell differentiation, necrosis, or apoptosis. These studies demonstrate that affinity modulation of the alpha5Beta1 integrin on CML progenitors and K562 cells by 8A2 results in increased adhesion to FN with subsequent decreased proliferation, suggesting that decreased beta1 integrin affinity contributes to the abnormal circulation and proliferation of malignant progenitors in CML.

Antibodies, Monoclonal↗
PubMed2002

Inhibition of endothelial cell survival and angiogenesis by protein kinase A.

Receptors for the provisional ECM are important regulators of angiogenesis. One of these receptors, integrin alpha5beta1, plays a critical role in tumor- and growth factor-induced angiogenesis, because antagonists of this integrin potently inhibit angiogenesis and tumor growth. Here we show that the integrin alpha5beta1 promotes endothelial cell survival during angiogenesis in vivo by suppressing the activity of protein kinase A (PKA). Antagonists of integrin alpha5beta1 activate PKA, which then leads to the activation of caspase-8 and induction of apoptosis. Direct activation of PKA by cAMP or by expression of the PKA catalytic subunit also induces endothelial cell apoptosis, resulting in angiogenesis inhibition in vivo. Our studies indicate that ligation of integrin alpha5beta1 during angiogenesis suppresses an apoptotic program that is dependent on PKA. These studies also indicate that induction of endothelial cell apoptosis in vivo by genetic or pharmacological activation of PKA may be a useful strategy to inhibit angiogenesis.

Angiogenesis Inhibitors↗
PubMed2004

Alpha5beta1, alphaVbeta3 and the platelet-associated integrin alphaIIbbeta3 coordinately regulate adhesion and migration of differentiating mouse trophoblast cells.

During blastocyst implantation, interaction between integrins on the apical surface of the trophoblast and extracellular matrix (ECM) in the endometrium anchors the embryo to the uterine wall. Strong adhesion of the blastocyst to fibronectin (FN) requires integrin signaling initiated by exogenous fibronectin. However, it is not known how integrin signaling enhances blastocyst adhesion. We present new evidence that the integrin, alphaIIbbeta3, plays a key role in trophoblast adhesion to fibronectin during mouse peri-implantation development. Trafficking of alphaIIb to the apical surface of the trophoblast increased dramatically after blastocysts were exposed to fibronectin, whereas other fibronectin-binding integrins, alpha5beta1 and alphaVbeta3, were resident at the apical surface before ligand exposure. Functional comparisons among the three integrins revealed that ligation of alpha5beta1 most efficiently strengthened blastocyst fibronectin-binding activity, while subsequent trophoblast cell migration was dependent primarily on the beta3-class integrins. In vivo, alphaIIb was highly expressed by invasive trophoblast cells in the ectoplacental cone and trophoblast giant cells of the parietal yolk sac. These data demonstrate that trafficking of alphaIIb regulates adhesion between trophoblast cells and fibronectin as invasion of the endometrium commences.

Animals↗
PubMed2001

Urokinase receptor and fibronectin regulate the ERK(MAPK) to p38(MAPK) activity ratios that determine carcinoma cell proliferation or dormancy in vivo.

We discovered that a shift between the state of tumorigenicity and dormancy in human carcinoma (HEp3) is attained through regulation of the balance between two classical mitogen-activated protein kinase (MAPK)-signaling pathways, the mitogenic extracellular regulated kinase (ERK) and the apoptotic/growth suppressive stress-activated protein kinase 2 (p38(MAPK)), and that urokinase plasminogen activator receptor (uPAR) is an important regulator of these events. This is a novel function for uPAR whereby, when expressed at high level, it enters into frequent, activating interactions with the alpha5beta1-integrin, which facilitates the formation of insoluble fibronectin (FN) fibrils. Activation of alpha5beta1-integrin by uPAR generates persistently high level of active ERK necessary for tumor growth in vivo. Our results show that ERK activation is generated through a convergence of two pathways: a positive signal through uPAR-activated alpha5beta1, which activates ERK, and a signal generated by the presence of FN fibrils that suppresses p38 activity. When fibrils are removed or their assembly is blocked, p38 activity increases. Low uPAR derivatives of HEp3 cells, which are growth arrested (dormant) in vivo, have a high p38/ERK activity ratio, but in spite of a similar level of alpha5beta1-integrin, they do not assemble FN fibrils. However, when p38 activity is inhibited by pharmacological (SB203580) or genetic (dominant negative-p38) approaches, their ERK becomes activated, uPAR is overexpressed, alpha5beta1-integrins are activated, and dormancy is interrupted. Restoration of these properties in dormant cells can be mimicked by a direct re-expression of uPAR through transfection with a uPAR-coding plasmid. We conclude that overexpression of uPAR and its interaction with the integrin are responsible for generating two feedback loops; one increases the ERK activity that feeds back by increasing the expression of uPAR. The second loop, through the presence of FN fibrils, suppresses p38 activity, further increasing ERK activity. Together these results indicate that uPAR and its interaction with the integrin should be considered important targets for induction of tumor dormancy.

Cell Division↗
PubMed2003

Ganglioside GM3 inhibits matrix metalloproteinase-9 activation and disrupts its association with integrin.

Gangliosides GM3 and GT1b both inhibit epithelial cell adhesion and migration on fibronectin. GT1b binds to integrin alpha5beta1 and blocks the integrin-fibronectin interaction; GM3 does not interact with integrin, and its effect is poorly understood. We evaluated the effects of endogenous modulation of GM3 expression on epithelial cell motility on several matrices and the mechanism of these effects. Endogenous accumulation of GM3 decreased cell migration on fibronectin, types I, IV, and VII collagen matrices; depletion of GM3 dramatically increased cell migration, regardless of matrix. GM3 overexpression and depletion in vitro correlated inversely with the expression and activity of matrix metalloproteinase-9; consistently, the cell migration stimulated by GM3 depletion is reversed by inhibition of matrix metalloproteinase-9 activity. Accumulation and depletion of GM3 in epithelial cells grown on fibronectin also correlated inversely with epidermal growth factor receptor and mitogen activated protein kinase phosphorylation and with Jun expression. Ganglioside depletion facilitated the co-immunoprecipitation of matrix metal-loproteinase-9 and integrin alpha5beta1, while endogenous accumulation of GM3, but not GT1b, blocked the co-immunoprecipitation. These data suggest modulation of epidermal growth factor receptor signaling and dissociation of integrin/matrix metalloproteinase-9 as mechanisms for the GM3-induced effects on matrix metalloproteinase-9 function.

Cell Line↗
PubMed1999

Fibronectin regulates assembly of actin filaments and focal contacts in cultured cells via the heparin-binding site in repeat III13.

Fibroblasts, when plated on the extracellular matrix protein fibronectin (FN), rapidly spread and form an organized actin cytoskeleton. This process is known to involve both the central alpha5beta1 integrin-binding and the C-terminal heparin-binding regions of FN. We found that within the heparin-binding region, the information necessary for inducing organization of stress fibers and focal contacts was located in a 29-amino acid segment of FN type III module 13 (III13). We did not find a cytoskeleton-organizing role for repeat III14, which had previously been implicated in this process. Within III13, the same five basic amino acids known to be most important for heparin binding were also necessary for actin organization. A substrate of III13 alone was only weakly adhesive but strongly induced formation of filopodia and lamellipodia. Stress fiber formation required a combination of III13 and III7-11 (which contains the integrin alpha5beta1 recognition site), either as a single fusion protein or as separate polypeptides, and the relative amounts of the two binding sites appeared to determine whether stress fibers or filopodia and lamellipodia were the predominant actin structures formed. We propose that a balance of signals from III13 and from integrins regulates the type of actin structures assembled by the cell.

Actins↗
PubMed2005

GSK3 and PKB/Akt are associated with integrin-mediated regulation of PTHrP, IL-6 and IL-8 expression in FG pancreatic cancer cells.

We have demonstrated recently that PTHrP is upregulated in pancreatic adenocarcinoma and that the ECM exerts regulatory control, at least in part, over PTHrP expression. In our present study, we examined the potential signaling interactions between these 2 pathways. Our results demonstrate that, under serum-free conditions, adhesion of FG pancreatic adenocarcinoma cells on Fn is mediated by the alpha5beta1 integrin, whereas adhesion to Type I collagen is mediated by the alpha2beta1 integrin. alpha5beta1 integrin-mediated adhesion to Fn results in a phenotype that includes a reduction in cell proliferation, increased E-cadherin localization in cell-cell contacts, increased beta-catenin localization throughout the cell, inhibition of haptokinetic cell migration, and increased expression of PTHrP, IL-6 and IL-8 relative to alpha2beta1 integrin-mediated adhesion on Type I collagen. A phosphoprotein immunoblotting screen of FG pancreatic cancer cells grown on either Fn or Type I collagen indicates that GSK3 and PKB/Akt are differentially phosphorylated on these 2 substrates. These results implicate GSK3 and PKB/Akt in the integrin-mediated regulation of PTHrP, IL-6 and IL-8 in pancreatic cancer.

Adenocarcinoma↗
PubMed1998

A key function for alphav containing integrins in mesodermal cell migration during Pleurodeles waltl gastrulation.

During cleavage of Pleurodeles waltl amphibian embryos, inner cells of the blastocoel roof (presumptive ectodermal and mesodermal cells) organize a fibrillar extracellular matrix (ECM) containing fibronectin on their basal surface by a beta1-integrin-dependent process. This matrix is used as a migratory substrate by mesodermal cells during gastrulation. While alpha5beta1 integrin is expressed on both ectodermal and mesodermal cell surface, we have shown previously that alphav containing integrins are essentially restricted to the surface of mesodermal cells (Alfandari, D., Whittaker, C. A., DeSimone, D. W., and Darribère, T., Dev. Biol. 170, 249-261, 1995). To investigate the function of alphav integrins during gastrulation, we have generated a function blocking antibody directed against the extracellular domain of the Pleurodeles integrin alphav subunit. The antibody did not prevent fibronectin fibril formation, whereas an antibody against the alpha5beta1 integrin did. When injected into the blastocoel, the antibody against integrin alphav subunit perturbed gastrulation and further development in a stage-dependent manner. Developmental defects were correlated to an abnormal positioning of the mesoderm layer. In vitro, the antibody blocked spreading of mesodermal cell to fibronectin or blastocoel roof ECM but not their attachment. In contrast, the antibody directed against the alpha5beta1 integrin inhibited both cell attachment and spreading to the same substrates. We propose that the alpha5beta1 integrin is required for fibronectin assembly into fibrils and mesodermal cell attachment to the blastocoel roof ECM, while the alphav containing integrins are necessary for cell spreading, and possibly migration, on this complex network.

Animals↗
PubMed2004

Surface chemistry modulates focal adhesion composition and signaling through changes in integrin binding.

Biomaterial surface properties influence protein adsorption and elicit diverse cellular responses in biomedical and biotechnological applications. However, the molecular mechanisms directing cellular activities remain poorly understood. Using a model system with well-defined chemistries (CH3, OH, COOH, NH2) and a fixed density of the single adhesive ligand fibronectin, we investigated the effects of surface chemistry on focal adhesion assembly and signaling. Surface chemistry strongly modulated integrin binding and specificity--alpha5beta1 integrin binding affinity followed the pattern OH>NH2=COOH>CH3, while integrin alphaVbeta3 displayed the relationship COOH>NH2>>OH=CH3. Immunostaining and biochemical analyses revealed that surface chemistry modulates the structure and molecular composition of cell-matrix adhesions as well as focal adhesion kinase (FAK) signaling. The neutral hydrophilic OH functionality supported the highest levels of recruitment of talin, alpha-actinin, paxillin, and tyrosine-phosphorylated proteins to adhesive structures. The positively charged NH2 and negatively charged COOH surfaces exhibited intermediate levels of recruitment of focal adhesion components, while the hydrophobic CH3 substrate displayed the lowest levels. These patterns in focal adhesion assembly correlated well with integrin alpha5beta1 binding. Phosphorylation of specific tyrosine residues in FAK also showed differential sensitivity to surface chemistry. Finally, surface chemistry-dependent differences in adhesive interactions modulated osteoblastic differentiation. These differences in focal adhesion assembly and signaling provide a potential mechanism for the diverse cellular responses elicited by different material properties.

3T3 Cells↗
PubMed2005

Fibronectin promotes brain capillary endothelial cell survival and proliferation through alpha5beta1 and alphavbeta3 integrins via MAP kinase signalling.

We showed previously that blood vessel maturation in the CNS is associated with a developmental switch in brain capillary endothelial cells (BCEC), from fibronectin signalling during angiogenesis to laminin signalling in the adult. To investigate the functional significance of this switch, we have examined the response of BCEC to different extracellular matrix (ECM) proteins. This showed that BCEC proliferation was significantly promoted by fibronectin (28.2 +/- 4.0%) and by vitronectin (14.8 +/- 2.1%) compared with uncoated glass (7.2 +/- 0.7%), while BCEC survival was significantly promoted by fibronectin (1130 +/- 131 cells), vitronectin (830 +/- 63 cells), collagen IV (703 +/- 77 cells) and laminin (680 +/- 34 cells) compared with the uncoated glass (367 +/- 48 cells). Biochemical studies showed that BCEC express a limited repertoire of integrins, including the beta1 integrins, alpha3beta1, alpha5beta1 and alpha6beta1, and the alphavbeta3 integrin. Function-blocking studies showed that the response to fibronectin was mediated equally by the alpha5beta1 and alphavbeta3 integrins. Analysis of signalling pathways revealed that fibronectin stimulated activation of the p44/p42 MAP kinase signalling pathway and pharmacological inhibitors of this pathway blocked BCEC proliferation on fibronectin. Taken together, these findings show that fibronectin exerts a strong angiogenic influence on endothelial cells (EC) in the CNS, and that this is mediated through the alpha5beta1 and alphavbeta3 integrins via MAP kinase signalling. In addition to a fundamental role in development, these findings may also have implications in pathological conditions of the CNS where fibronectin is re-expressed.

Animals↗
PubMed1998

Activation of distinct alpha5beta1-mediated signaling pathways by fibronectin's cell adhesion and matrix assembly domains.

The interaction of cells with fibronectin generates a series of complex signaling events that serve to regulate several aspects of cell behavior, including growth, differentiation, adhesion, and motility. The formation of a fibronectin matrix is a dynamic, cell-mediated process that involves both ligation of the alpha5beta1 integrin with the Arg-Gly-Asp (RGD) sequence in fibronectin and binding of the amino terminus of fibronectin to cell surface receptors, termed "matrix assembly sites," which mediate the assembly of soluble fibronectin into insoluble fibrils. Our data demonstrate that the amino-terminal type I repeats of fibronectin bind to the alpha5beta1 integrin and support cell adhesion. Furthermore, the amino terminus of fibronectin modulates actin assembly, focal contact formation, tyrosine kinase activity, and cell migration. Amino-terminal fibronectin fragments and RGD peptides were able to cross-compete for binding to the alpha5beta1 integrin, suggesting that these two domains of fibronectin cannot bind to the alpha5beta1 integrin simultaneously. Cell adhesion to the amino-terminal domain of fibronectin was enhanced by cytochalasin D, suggesting that the ligand specificity of the alpha5beta1 integrin is regulated by the cytoskeleton. These data suggest a new paradigm for integrin-mediated signaling, where distinct regions within one ligand can modulate outside-in signaling through the same integrin.

Amino Acid Sequence↗
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↗