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PubMed2003

Glucose regulates monocyte adhesion through endothelial production of interleukin-8.

We have shown that glucose increases monocyte adhesion to human aortic endothelial cells (HAECs) in vitro.1 In the present study, we examined mechanisms by which glucose stimulates monocyte:endothelial interactions. HAECs cultured for 7 days in 25 mmol/L glucose had a 2-fold elevation in interleukin-8 (IL-8) secretion over control cells cultured in 5.5 mmol/L glucose (P<0.001). Use of a neutralizing antibody to IL-8 prevented glucose-mediated monocyte adhesion. Both glucose and IL-8 activated beta1 integrin on the HAEC surface, suggesting that both activate the alpha5beta1 integrin complex on the endothelial surface. The alpha5beta1 integrin complex is important for anchoring connecting segment-1 fibronectin on the HAEC surface for monocyte adhesion. Analysis of the human IL-8 promoter revealed binding sites for NF-kappaB and AP-1 as well as several aligned carbohydrate response elements (also known as E-boxes). Glucose dramatically stimulated IL-8 promoter activity. Using mutated IL-8 promoter constructs and EMSA, we found that the AP-1 element and the glucose-response element were responsible for much of the glucose-mediated activation of IL-8 transcription. Interestingly, inhibition of reactive oxygen species (ROS) production through use of pharmacological uncouplers of the mitochondrial electron transport chain significantly reduced glucose-mediated induction of IL-8 expression. These data indicate that glucose regulates monocyte:endothelial interactions through stimulation of IL-8 and ROS production and activation of the alpha5beta1 integrin complex on HAECs.

Animals↗
PubMed1997

Mesodermal defects and cranial neural crest apoptosis in alpha5 integrin-null embryos.

Alpha5beta1 integrin is a cell surface receptor that mediates cell-extracellular matrix adhesions by interacting with fibronectin. Alpha5 subunit-deficient mice die early in gestation and display mesodermal defects; most notably, embryos have a truncated posterior and fail to produce posterior somites. In this study, we report on the in vivo effects of the alpha5-null mutation on cell proliferation and survival, and on mesodermal development. We found no significant differences in the numbers of apoptotic cells or in cell proliferation in the mesoderm of alpha5-null embryos compared to wild-type controls. These results suggest that changes in overall cell death or cell proliferation rates are unlikely to be responsible for the mesodermal deficits seen in the alpha5-null embryos. No increases in cell death were seen in alpha5-null embryonic yolk sac, amnion and allantois compared with wild-type, indicating that the mutant phenotype is not due to changes in apoptosis rates in these extraembryonic tissues. Increased numbers of dying cells were, however, seen in migrating cranial neural crest cells of the hyoid arch and in endodermal cells surrounding the omphalomesenteric artery in alpha5-null embryos, indicating that these subpopulations of cells are dependent on alpha5 integrin function for their survival. Mesodermal markers mox-1, Notch-1, Brachyury (T) and Sonic hedgehog (Shh) were expressed in the mutant embryos in a regionally appropriate fashion. Both T and Shh, however, showed discontinuous expression in the notochords of alpha5-null embryos due to (1) degeneration of the notochordal tissue structure, and (2) non-maintenance of gene expression. Consistent with the disorganization of notochordal signals in the alpha5-null embryos, reduced Pax-1 expression and misexpression of Pax-3 were observed. Anteriorly expressed HoxB genes were expressed normally in the alpha5-null embryos. However, expression of the posteriormost HoxB gene, Hoxb-9, was reduced in alpha5-null embryos. These results suggest that alpha5beta1-fibronectin interactions are not essential for the initial commitment of mesodermal cells, but are crucial for maintenance of mesodermal derivatives during postgastrulation stages and also for the survival of some neural crest cells.

Animals↗
PubMed1996

Interaction between the insulin-like growth factor family and the integrin receptor family in tissue repair processes. Evidence in a rabbit ear dermal ulcer model.

We have determined previously that IGF-I is dependent on the presence of IGF binding protein-1 (IGFBP-1) to act as a wound healing agent. We sought to determine the mechanism whereby IGFBP-1 is able to enhance IGF-I bioactivity. As IGFBP-1 binds both the alpha5beta1 integrin as well as IGF-I in vitro, we asked which of the following interactions were important: (a) the ability of IGFBP-1 to interact with an integrin receptor, and/or (b) the binding of IGF-I by IGFBP-1. We used an IGF-1 analogue (des(1-3)IGF-I) with a > 100-fold reduction in affinity for IGFBP-1 as well as an IGFBP-1 mutant (WGD-IGFBP-1) which does not associate with the alpha5beta1 integrin to selectively abrogate each of these interactions. We also tested the ability of IGFBP-2, a related binding protein which has an arginine-glycine-aspartate sequence but does not associate with integrin family members, to enhance IGF-I bioactivity. Full-thickness dermal wounds were created on rabbit ears; various combinations of native IGF-I, native IGFBP-1, native IGFBP-2, and their respective analogues/mutants were applied to each wound. Wounds were harvested 7 d later for analysis. Only native IGF-I in combination with native IGFBP-1 was effective as a wound healing agent, enhancing reepithelialization and granulation tissue deposition by 64+/-5 and 83+/-12% over controls (P = 0.008 and 0.016, respectively). The same doses of IGF-I/WGD-IGFBP-1, des(1-3)IGF-I/IGFBP-1, and IGF-I/IGFBP-2 were ineffective. We propose that IGF-I physically interacts with IGFBP-1 and that IGFBP-1 also binds to an integrin receptor, most likely the alpha5beta1 integrin. This interaction is unique to IGFBP-1 as the closely related IGFBP-2 had no effect, a finding consistent with its inability to bind to integrin receptors. Our results suggest that activation of both the IGF-I receptor and the alpha5beta1 integrin is required for IGF-I to stimulate wound healing.

Animals↗
PubMed2006

IGFBP-1 inhibits EGF mitogenic activity in cultured endometrial stromal cells.

The properties of the insulin-like growth factor-binding proteins (IGFBP-1 to 6) are not limited to modulation of IGF actions. IGFBP-1, which shares an Arg-Gly-Asp (RGD) motif in its C-terminal domain, modulates cell motility by binding to integrin alpha5beta1. The cross-talks between integrins and growth factor receptor signalling pathways are extensively documented, particularly in the case of the epidermal growth factor receptor (EGFR). However, whether IGFBP-1 can modulate growth factor signalling through its interaction with integrin alpha5beta1 has not yet been studied. As EGF is involved in the decidualisation of endometrial stromal cells (ESCs) and as decidualised ESCs are a source of IGFBP-1, we investigated if IGFBP-1 can modulate EGF effects on ESCs. RGD- and IGF-independent inhibition of EGF mitogenic activity and EGFR signalling by IGFBP-1 were demonstrated in ESC primary cultures, A431, cells and in mouse fibroblasts lacking IGF receptors.

Animals↗
PubMed2006

Role of integrin cross-regulation in parvovirus B19 targeting.

Most viral vectors used for gene therapy lack the ability to target a defined cell population. Parvovirus B19 has a restricted tropism for human erythroid progenitor cells and uses activated alpha5beta1 integrins as coreceptors for entry [Weigel-Kelley, K.A., Yoder, M.C., and Srivastava, A. (2003). Blood 102, 3927-3933]. In this study we examined the role of coexpressed integrins in alpha5beta1 integrin coreceptor function. Antibody-mediated cross-linking of beta1, beta2, and beta3 integrins and the integrin-associated protein (IAP) increased parvovirus B19 entry into nontarget K562 cells. Functional silencing of one integrin group, however, reduced the virus uptake- promoting function of a subsequently activated integrin group, indicating that the three integrins did not operate in isolation but through shared signaling pathways. This was further corroborated by direct competition between simultaneously clustered beta2 and beta1 integrins that could be overcome by stabilizing clustered beta1 integrins in a high-affinity conformation. In contrast, parvovirus B19 entry into primary erythroid progenitor cells was characterized by strong clustering-induced beta1 integrin coreceptor activity that was not abolished by subsequent beta2 and beta3 integrin activation and was, in fact, substantially increased in the presence of preclustered beta2 and beta3 integrins. Thus, integrin function is regulated in a cell type-specific manner through coexpressed integrins and preferential parvovirus B19 entry into erythroid progenitor cells is promoted by a robust beta1 integrin response that is enhanced through stable preclustering of coexpressed integrins. These results have implications for other viral vectors that use integrins as receptors/coreceptors and for gene therapy of hematopoietic progenitor cells using parvovirus B19 vectors.

Antibodies, Viral↗
PubMed1998

Transforming growth factor-beta and platelet derived growth factor regulation of fibrillar fibronectin matrix formation by synovial fibroblasts.

OBJECTIVE: To investigate factors regulating fibronectin fibrillar matrix formation by synovial fibroblasts. METHODS: Basal and cytokine stimulated extracellular matrix (ECM) fibronectin produced by synovial fibroblasts was identified by immunofluorescence and Western blot. Alternative mRNA splicing of fibronectin was studied by reverse transcription polymerase chain reaction. The integrin receptor responsible for supporting fibronectin fibrillar matrix was identified by blocking antibodies and receptor levels studied by Western blot. RESULTS: Transforming growth factor-beta (TGF-beta) or platelet derived growth factor (PDGF), but not interleukin 1 or exogenous fibronectin, induced ECM fibronectin. ECM fibronectin was blocked by the addition of antibody to the alpha5beta1 integrin. Cytokines did not significantly change alternative mRNA splicing of fibronectin or levels of alpha5beta1 integrin expression. CONCLUSION: Synovial cell production of a fibrillar fibronectin matrix is induced by growth factors, including TGF-beta and PDGF. This induction is mediated by the alpha5beta1 integrin. Since fibrillar fibronectin formation was not strongly dependent on increased fibronectin or alpha5beta1 integrin levels, this effect may be mediated by growth factor induced changes in receptor affinity.

Alternative Splicing↗
PubMed1998

Collagen type I modulates the platelet-derived growth factor (PDGF) regulation of the growth and expression of beta1 integrins by rat mesangial cells.

Mesangial cell (MC) proliferation and the deposition of collagen type I (collagen I) are the major pathological features in many types of glomerulonephritis (GN). Recent work suggested that beta-integrins play a critical role in the cell proliferation and extracellular matrix (ECM) remodeling observed in tissue repair after injury. To examine the involvement of beta-integrins in MC proliferation in association with the interaction of MCs with pathological collagen I, we investigated the effect of a prominent mitogen, platelet-derived growth factor-BB (PDGF-BB) on the growth and expression of beta-integrins by MCs cultured on plastic or in a three-dimensional collagen I gel. Immunoprecipitation using 35S-metabolic labeling, flow cytometry and a 3H-thymidine-uptake analysis demonstrated that PDGF-BB stimulated the cell mitogenicity and the expression of alpha5beta1 integrin (a fibronectin receptor), but not alpha1beta1 integrin (a collagen and laminin receptor) of MCs on plastic, in a dose-dependent manner. In contrast, MCs in the collagen I gels showed no significant changes in mitogenicity or alpha1beta1 and alpha5beta1 integrin expression, but increased alpha1beta1 integrin-mediated gel contraction was observed after PDGF-BB stimulation. Thus, the parallel up-regulation of MC-mitogenicity and alpha5beta1 integrin expression by PDGF-BB suggested that alpha5beta1 integrin is an important ECM receptor involved in the proliferative phenotype of MC. A spatial interaction between MCs and pathological collagen I in GN may influence the PDGF regulation of the MC phenotype regarding the cell growth and the expression of beta1 integrins.

Animals↗
PubMed2003

ERK(MAPK) activity as a determinant of tumor growth and dormancy; regulation by p38(SAPK).

After dissemination from a primary tumor, cancer cells may resume growth, leading to overt metastasis, or enter a state of protracted dormancy. However, mechanisms that determine their fate, or markers that predict it, are mostly unavailable. We previously showed that in HEp3 human head and neck carcinoma, the extracellular signal-regulated kinase (ERK)(MAPK)/p38(SAPK) activity ratio predicts whether the cells will proliferate or enter a state of dormancy in vivo. The proliferative balance of high ERK/p38 ratio was induced by high urokinase (uPA) receptor (uPAR) expression, which activated alpha5beta1-integrin and epidermal growth factor receptor. This signaling pathway was additionally enhanced by uPA binding to uPAR and fibronectin binding to alpha5beta1-integrin. We tested whether the ERK/p38 balance is predictive of in vivo behavior in other cancer cell types and whether altering the balance will shift their phenotype between proliferation and dormancy. ERK and p38 activities were determined using either phospho-specific monoclonal antibodies or a trans-reporting system where GAL4-Elk and GAL4-CHOP trans-activation of luciferase gene served as reporters for ERK and p38 activities, respectively. We show that in breast, prostate, melanoma, and fibrosarcoma cell lines, the level of active phospho-ERK and the ERK/p38 activity ratio predict for the in vivo behavior in approximately 90% of the cell lines tested. Modulation of ERK/p38 activity ratio by multiple pharmacological and genetic interventions confirms that high ERK/p38 ratio favors tumor growth, whereas high p38/ERK ratio induces tumor growth arrest (dormancy) in vivo and that ERK is negatively regulated by p38. A melanoma cell line appeared to have developed an escape mechanism to avoid the growth inhibitory effect of high p38 activity. Mechanistic analysis implicated high uPAR expression and its interaction with and activation of alpha5beta1-integrin as determinants of the in vivo growth promoting high ERK/p38 ratio in several cell lines. The small GTPase, Cdc42, was implicated in activation of p38 and growth arrest. These results suggest that even cells that originate in advanced cancers retain a degree of dependence on surface receptors and matrix for their proliferative signals in vivo and provide a therapeutic opportunity to change their phenotype from tumorigenic to dormant.

Animals↗
PubMed2006

Immunohistochemical assessment of fibronectin and tenascin and their integrin receptors alpha5beta1 and alpha9beta1 in gastric and colorectal cancers with lymph node and liver metastases.

The aim of the current study was to assess immunohistochemically and compare the level of expression of tenascin (TN) and fibronectin (FN) and their integrin receptors alpha9beta1 and alpha5beta1 in the primary colorectal and gastric tumors, and in corresponding lymph node and liver metastases from 53 patients. We detected similar high deposition of the studied ECM proteins and their receptors in the stroma of primary tumors and in liver metastases and a lower deposition in lymph node metastases. Cytoplasmic immune reaction for FN and TN was also seen in the tumor cells. A pronounced co-localization of immune deposits for FN and TN and their receptors was found in the stroma of the center and the invasion front (IF) (p<0.0001). A significant decrease of FN immune signal was observed in the IF in primary tumors and liver metastases (p<0.0001). The levels of immunolabeling of FN and TN correlated with the differentiation grade of primary tumors (p<0.0001). In conclusion, we may say that there is heterogeneous deposition of TN, FN and their integrin receptors in the different areas of primary colorectal and gastric tumors and of their metastases. These findings imply that the studied proteins may be involved in cell processes such as growth, adhesion, migration and apoptosis.

Adult↗
PubMed1997

Decrease in fibronectin occurs coincident with the increased expression of its integrin receptor alpha5beta1 in stress-deprived ligaments.

Stress deprivation secondary to immobilization leads to atrophic changes in periarticular soft tissues. The changes in ligaments include a disorganization of collagen and cellular ultrastructure with varied biochemical alterations resulting in a functionally weaker tissue. This study tests the hypothesis that alterations in fibronectin (Fn) and the expression of its integrin receptor alpha5beta1 in ligament fibroblasts accompany the extracellular matrix remodeling which occurs in stress-deprived knee ligaments. The left knees of eighteen New Zealand white rabbits were surgically immobilized in acute flexion. Fibroblasts within three nine week and three twelve week stress-deprived anterior cruciate ligaments (ACLs) and medial collateral ligaments (MCLs) demonstrated markedly increased immunostaining for the beta1 and alpha5 integrin subunits, as compared to fibroblasts in the contralateral unoperated control ligaments. The effects of stress deprivation on the concentration of Fn was measured by competitive ELISA on the remaining twelve rabbits. Decreases in Fn of 54.0 percent and 63.7 percent occurred in the ACL after nine and twelve weeks of stress deprivation when compared to contralateral controls. The MCL had less of a decrease, losing 37.7 percent and 41.7 percent at nine and twelve weeks, respectively. These results suggest an important role for the Fn-specific integrin receptor alpha5beta1 in remodeling stress-deprived periarticular ligamentous tissue, and the importance of maintaining normal stresses on periarticular ligaments to prevent the degradation of extracellular matrix components such as Fn.

Animals↗
PubMed2006

Significance of tenascin-C, fibronectin, laminin, collagen IV, alpha5beta1 and alpha9beta1 integrins and fibrotic capsule formation around liver metastases originating from cancers of the digestive tract.

The formation of a fibrotic capsule around liver metastases may functionally act as a barrier to local invasion. However, the prognostic significance of exstracellular matrix (ECM) and of some integrins' deposition around liver metastases remains unclear. An immunohistochemical investigation was carried out on 55 patients with synchronous liver metastases from colorectal and gastric cancers. Encapsulated metastases were detected in 60% of the cases. The 'non-capsular' cases showed clear immunostaining for tenascin-C, fibronectin, collagen IV, laminin, alphaSMA and integrins. On opposite, most of the cases with 'capsule' were negative for the studied ECM proteins and the two integrins. The patients with 'capsular' pattern had significantly longer median survival after the surgery compared to those with non-encapsulated metastases. The presence of tenascin, fibronectin, fibronectin receptor and laminin, as well as the strong immune signal for alphaSMA and collagen type IV in the sinusoids attached to the liver metastases was associated with a worse prognosis. The cells, forming ECM in the sinusoids attached to metastases in the 'non-capsular' pattern were alphaSMA-positive myofibroblasts. It was shown ultrastructurally that they were HSCs. The results indicate that fibrotic capsule formation is associated with longer survival after surgery. The appearance of tenascin-C and of its receptor at the periphery of liver metastases could be used as a sign of invasiveness.

Adenocarcinoma↗
PubMed2004

A correlation between membrane cholesterol level, cell adhesion and tumourigenicity of polyoma virus transformed cells.

We have investigated the implications of the rise in membrane cholesterol levels on several in vitro and in vivo properties of polyoma virus transformed rat fibroblasts (PyF), with a special emphasis on alpha5beta1 integrin functions. We show that increased membrane cholesterol causes the PyF cells to change their shape and become more bipolar in appearance. These cells also show significantly higher adhesion to the cell-binding domain of fibronectin, increased localization of alpha5beta1 integrin and talin molecules in focal adhesions and a more robust actin cytoskeleton organization. PyF cells with increased membrane cholesterol show reduced growth in vitro and tumours caused by these cells in nude mice are slow growing. These changes in the growth properties of PyF cells are reversible when the cholesterol levels of PyF cells become normal. Our results suggest that changes in membrane cholesterol levels influence the growth and morphological properties of transformed cells, which can be exploited in controlling the growth of tumours in vivo.

Actins↗
PubMed2003

Fibronectin fragment activation of proline-rich tyrosine kinase PYK2 mediates integrin signals regulating collagenase-3 expression by human chondrocytes through a protein kinase C-dependent pathway.

Fibronectin fragments (FN-f), including the 110-kDa fragment that binds the alpha5beta1 integrin, stimulate collagenase-3 (MMP-13) production and cartilage destruction. In the present study, treatment of chondrocytes with the 110-kDa FN-f or an activating antibody to the alpha5beta1 integrin was found to increase tyrosine autophosphorylation (Tyr-402) of the proline-rich tyrosine kinase-2 (PYK2) without significant change in autophosphorylation (Tyr-397) of focal adhesion kinase (FAK). The tyrosine kinase inhibitor tyrphostin A9, shown previously to block a PYK2-dependent pathway, blocked the FN-f-stimulated increase in MMP-13, whereas tyrphostin A25 did not. FN-f-stimulated PYK2 phosphorylation and MMP-13 production was also blocked by reducing intracellular calcium levels. Adenovirally mediated overexpression of wild type but not mutant PYK2 resulted in increased MMP-13 production. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate stimulated PYK2 phosphorylation and MMP-13 production. MMP-13 expression stimulated by either phorbol 12-myristate 13-acetate or FN-f was blocked by PKC inhibitors including the PKCdelta inhibitor rottlerin. Furthermore, PKCdelta translocation from cytosol to membrane was noted within 5 min of stimulation with FN-f. Immortalized human chondrocytes, transiently transfected with MMP-13 promoter-luciferase reporter constructs, showed increased promoter activity after FN-f treatment that was inhibited by co-transfection with either of two dominant negative mutants of PYK2 (Y402F and K457A). No inhibition was seen after cotransfection with wild type PYK2, a dominant negative of FAK (FRNK) or empty vector plasmid. FN-f-stimulated MMP-13 promoter activity was also inhibited by chemical inhibitors of ERK, JNK, and p38 mitogen-activated protein (MAP) kinases or by co-transfection of dominant negative MAP kinase mutant constructs. These studies have identified a novel pathway for the MAP kinase regulation of MMP-13 production which involves FN-f stimulation of the alpha5beta1 integrin and activation of the nonreceptor tyrosine kinase PYK2 by PKC, most likely PKCdelta

Cells, Cultured↗
PubMed1999

Different integrins mediate cell spreading, haptotaxis and lateral migration of HaCaT keratinocytes on fibronectin.

Collaborative role of various fibronectin-binding integrins (alpha5beta1, alphavbeta1 and alphavbeta6) as mediators of cell adhesion and migration on fibronectin was studied using cultured HaCaT keratinocytes. This cell line spontaneously expressed all three fibronectin-binding integrins. In addition, the expression of alphavbeta6 integrin was strongly and specifically upregulated by transforming growth factor-beta1 (TGFbeta1) whereas the amount of other integrins remained practically unchanged on the cell surface. Adhesion, spreading and motility of HaCaT keratinocytes on fibronectin were promoted by TGFbeta1. Based on antibody blocking experiments, both untreated and TGFbeta1-treated HaCaT cells used alphavbeta6 integrin as their main fibronectin receptor for cell spreading. In contrast to TGFbeta1-treated cells, the untreated cells also needed alpha5beta1 integrin for maximal cell spreading on fibronectin. Combinations of antibodies blocking both of these receptors totally prevented spreading of both untreated and TGFbeta1-treated cells. Haptotactic motility of individual HaCaT cells through fibronectin-coated membranes was again mainly dependent on alphavbeta6 integrin, while alphavbeta1 and alpha5beta1 integrins played a lesser role both in untreated and TGFbeta1-treated HaCaT cells. However, unlike haptotaxis, lateral migration of HaCaT cell sheet was mainly mediated by beta1 integrins, and alphavbeta6 integrin showed a minor role. The migration process appeared to involve a number of beta1 integrins that could adaptively replace each other when blocking antibodies were present. Thus, keratinocytes appear to use different fibronectin receptors for different functions, such as cell spreading, haptotaxis and lateral migration. The cells can also adapt to a situation where one receptor is unfunctional by switching to another receptor of the same ligand.

Antigens, Neoplasm↗
PubMed2003

Endostatin associates with lipid rafts and induces reorganization of the actin cytoskeleton via down-regulation of RhoA activity.

Endostatin, the C-terminal fragment of collagen XVIII, is a potent inhibitor of angiogenesis. Observations that endostatin inhibits endothelial cell migration and induces disassembly of the actin cytoskeleton provide putative cellular mechanisms for this effect. To understand the mechanisms of endostatin-induced intracellular signaling, we analyzed the association of recombinant endostatin with endothelial cell lipid rafts and the roles of its heparin- and integrin-binding properties in this interaction. We observed that a fraction of cell surface-bound endostatin partitioned in low density membrane raft fractions together with caveolin-1. Heparinase treatment of cells prevented the recruitment of endostatin to the lipid rafts but did not affect the association of endostatin with the non-raft fraction, whereas preincubation of endostatin with soluble alpha5beta1 integrin prevented the association of endostatin with the endothelial cell membrane. Endostatin treatment induced recruitment of alpha5beta1 integrin into the raft fraction via a heparan sulfate proteoglycan-dependent mechanism. Subsequently, through alpha5beta1 integrin, heparan sulfate, and lipid raft-mediated interactions, endostatin induced Src-dependent activation of p190RhoGAP with concomitant decrease in RhoA activity and disassembly of actin stress fibers and focal adhesions. These observations provide a cell biological mechanism, which plausibly explains the anti-angiogenic mechanisms of endostatin in vivo.

Actins↗
PubMed1999

Differences in phosphatase modulation of alpha4beta1 and alpha5beta1 integrin-mediated adhesion and migration of B16F1 cells.

It is well established that a biphasic relationship exists between the adhesive strength of beta1 integrins and their ability to mediate cell movement. Thus, cell movement increases progressively with adhesive strength, but beyond a certain point of optimal interaction, cell movement is reduced with further increases in adhesive function. The interplay between the various kinase and phosphatase activities provides the balance in beta1 integrin-mediated cell adhesion and migration. In the present study, the significance of protein tyrosine phosphatases (PTP) and ser/thr protein phosphatases (PP) in alpha4beta1 and alpha5beta1 integrin-mediated mouse melanoma B16F1 cell anchorage and migration on fibronectin was characterized using phosphatase inhibitors. At low fibronectin concentration, alpha5beta1 functioned as the predominant receptor for cell movement; a role for alpha4beta1 in B16F1 cell migration increased progressively with fibronectin concentration. Treatment of B16F1 cells with PTP inhibitors, sodium orthovanadate (Na3VO4) and phenylarsine oxide (PAO), or PP-1/2A inhibitor, okadaic acid (OA), abolished cell movement. Inhibition of cell movement by PAO and OA was associated by a reduction in the adhesive strength of alpha4beta1 and alpha5beta1. In contrast, treatment of B16F1 cells with Na3VO4 resulted in selective stimulation of the adhesive function of alpha5beta1, but not alpha4beta1. Therefore, our results demonstrate that (i) both PTP and PP-1/2A have roles in cell movement, (ii) modulation of cell movement by PTP and PP-1/2A may involve either a stimulation or reduction of beta1 integrin adhesive strength, and (iii) distinct phosphatase-mediated signaling pathways for differential regulation of the various beta1 integrins exist.

Animals↗
PubMed2004

Integrin-mediated action of insulin-like growth factor binding protein-2 in tumor cells.

The neoplastic production of the insulin-like growth factor binding protein (IGFBP)-2 often correlates with tumor malignancy and aggressiveness. Since IGFBP-2 contains an RGD motif in its C-terminus, it was hypothesized that this protein may act independently of IGF on tumor cells through integrins. To investigate this, integrin binding, intracellular signaling and the impact of IGFBP-2 on cell adhesion and proliferation were examined in two tumor cell lines. In tracer displacement studies, up to 30% of the added (125)I-hIGFBP-2 specifically bound to the cells. Bound (125)I-hIGFBP-2 was reversibly displaced by IGFBP-2, IGFBP-1 and RGD-(Gly-Arg-Asp)-containing peptides, but not by IGFBP-3, -4, -5, -6 and RGE-(Gly-Arg-Glu)-containing peptides. Blocking with antibodies directed against different integrins and with fibronectin demonstrated that IGFBP-2 cell surface binding is specific for alpha5beta1-integrin. Incubation of IGFBP-2 with equimolar quantities of IGF-I and IGF-II annihilated RGD-specific binding. IGFBP-2 binding at the cell surface led to dephosphorylation of the focal adhesion-kinase (FAK) of up to 37% (P<0.01), and of the p42/44 MAP-kinases of up to 40% (P<0.01). In addition, IGFBP-2 promoted de-adhesion of the cells dose-dependently by up to 30% (P<0.05), and reduced proliferation by 24% (P<0.01). Since one of the cell lines used does not express a functional IGF-I receptor, these data demonstrate that IGFBP-2 can act in an IGF-independent manner, at least in part by an interaction with alpha5beta1-integrin.

Cell Adhesion↗
PubMed2001

Proteolysis of integrin alpha5 and beta1 subunits involved in retinoic acid-induced apoptosis in human hepatoma Hep3B cells.

Our previous report demonstrated that all-trans-retinoic acid (ATRA) induces detachment and death under serum starvation in several human tumor cell lines. In this study, we examined the influence of cell-extracellular matrix interaction on the ability of ATRA to induce apoptosis. Plating of human hepatoma Hep3B cells onto poly-hydroxyethylmethacrylate-coated plates in the absence of serum resulted in the acceleration of ATRA-induced apoptosis. In contrast, ATRA-induced apoptosis was significantly suppressed by plating cells onto Matrigel-coated plates but not suppressed by culturing onto collagen-, laminin-, vitronectin-, or fibronectin-coated plates. Exogenously added soluble collagen, laminin, fibronectin, vitronectin or Matrigel failed to suppress ATRA-induced apoptosis. Results from the adhesion assay indicated that the cell attachment to fibronectin was significantly inhibited by ATRA. Treatment with perturbing antibody against integrin alpha5 or beta1 subunits resulted in promotion of ATRA-induced apoptosis. Moreover, the proteolytic cleavage of alpha5beta1 integrin and focal adhesion kinase (FAK) proteins is linked to the early phase of the ATRA-induced apoptotic process. Furthermore, ATRA-induced detachment, death, and cleavage of alpha5beta1 integrin and FAK were drastically suppressed by plating cells onto Matrigel-coated plates. These findings provide evidence that abrogation of cell adhesion, through proteolysis of alpha5beta1 integrin and FAK, is closely linked to ATRA-induced apoptosis in Hep3B cells.

Antineoplastic Agents↗