High affinity interaction of integrin alpha4beta1 (VLA-4) and vascular cell adhesion molecule 1 (VCAM-1) enhances migration of human melanoma cells across activated endothelial cell layers

The capacity of tumor cells to form metastatic foci correlates with their ability to interact with and migrate through endothelial cell layers. This process involves multiple adhesive interactions between tumor cells and the endothelium. Only little is known about the molecular nature of these interactions during extravasation of tumor cells. In human melanoma cells, the integrin alphavbeta3 is involved in transendothelial migration and its expression correlates with metastasis. However, many human melanoma cells do not express beta3 integrins. Therefore, it remained unclear how these cells undergo transendothelial migration. In this study we show that human melanoma cells with different metastatic potency, which do not express beta2 or beta3 integrins, express the VCAM-1 receptor alpha4beta1. VCAM-1 is up-regulated on activated endothelial cells and is known to promote transendothelial migration of leukocytes. Interestingly, despite comparable cell surface levels of alpha4beta1, only the highly metastatic melanoma cell lines MV3 and BLM, but not the low metastatic cell lines IF6 and 530, bind VCAM-1 with high affinity without further stimulation, and are therefore able to adhere to and migrate on isolated VCAM-1. Moreover, we demonstrate that function-blocking antibodies against the integrin alpha4beta1, as well as siRNA-mediated knock-down of the alpha4 subunit in these highly metastatic human melanoma cells reduce their transendothelial migration. These data imply that only high affinity interactions between the integrin alpha4beta1 on melanoma cells and VCAM-1 on activated endothelial cells may enhance the metastatic capacity of human beta2/beta3-negative melanoma cells.     

Transendothelial migration of human basophils

During allergic reactions, basophils migrate from the blood compartment to inflammatory sites, where they act as effector cells in concert with eosinophils. Because transendothelial migration (TEM) represents an essential step for extravasation of cells, for the first time we have studied basophil TEM using HUVEC. Treatment of HUVEC with IL-1beta significantly enhanced basophil TEM, which was further potentiated by the presence of a CCR3-specific ligand, eotaxin/CCL11. In addition to CCR3 ligands, MCP-1/CCL2 was also active on basophil TEM. Although stromal cell-derived factor-1/CXCL12, a CXCR4 ligand, failed to induce TEM in freshly isolated basophils, it caused strong TEM in 24-h cultured cells. IL-3 enhanced basophil TEM by increasing the chemokinetic response. Spontaneous TEM across activated HUVEC was inhibited by treatment of cells with anti-CD18 mAb, but not with anti-CD29 mAb, and also by treatment of HUVEC with anti-ICAM-1 mAb. Anti-VCAM-1 mAb alone failed to inhibit TEM, but showed an additive inhibitory effect in combination with anti-ICAM-1 mAb. In contrast, eotaxin- and IL-3-mediated TEM was significantly inhibited by anti-CD29 mAb as well as anti-CD18 mAb. These results indicate that beta2 integrins play the primary role in basophil TEM, but beta1 integrins are also involved, especially in TEM of cytokine/chemokine-stimulated basophils. In conclusion, the regulatory profile of basophil TEM is very similar to that reported for eosinophils. Our results thus support the previous argument for a close relationship between basophils and eosinophils and suggest that the in vivo kinetics of these two cell types are similar.     

Tumor Cell Extravasation Mediated by Leukocyte Adhesion is Shear Rate Dependent on IL-8 Signaling^*

To complete the metastatic journey, cancer cells have to disseminate through the circulation and extravasate to distal organs. However, the extravasation process, by which tumor cells leave a blood vessel and invade the surrounding tissue from the microcirculation, remains poorly understood at the molecular level. In this study, tumor cell adhesion to the endothelium (EC) and subsequent extravasation were investigated under various flow conditions. Results have shown polymorphonuclear neutrophils (PMNs) facilitate melanoma cell adhesion to the EC and subsequent extravasation by a shear-rate dependent mechanism. Melanoma cell-PMN interactions are mediated by the binding between intercellular adhesion molecule-1 (ICAM-1) on melanoma cells andb2integrins on PMNs. In addition, the fluid convection affects the extent of activation ofb2integrins on PMNs by endogenously secreted interleukin 8 (IL-8) within the tumor microenvironment. Results also indicate that shear rate affects the binding kinetics between PMNs and melanoma cells, which may contribute to the shear-rate dependence of melanoma extravasation in a shear flow when mediated by PMNs.     

Role of galectin-3 as an adhesion molecule for neutrophil extravasation during streptococcal pneumonia

Recruitment of neutrophils from blood vessels to sites of infection represents one of the most important elements of innate immunity. Movement of neutrophils across blood vessel walls to the site of infection first requires that the migrating cells firmly attach to the endothelial wall. Generally, neutrophil extravasation is mediated at least in part by two classes of adhesion molecules, beta(2) integrins and selectins. However, in the case of streptococcal pneumonia, recent studies have revealed that a significant proportion of neutrophil diapedesis is not mediated by the beta(2) integrin/selectin paradigm. Galectin-3 is a beta-galactoside-binding lectin implicated in inflammatory responses as well as in cell adhesion. Using an in vivo streptococcal pneumonia mouse model, we found that accumulation of galectin-3 in the alveolar space of streptococcus-infected lungs correlates closely with the onset of neutrophil extravasation. Furthermore, immunohistological analysis of infected lung tissue revealed the presence of galectin-3 in the lung tissue areas composed of epithelial and endothelial cell layers as well as of interstitial spaces. In vitro, galectin-3 was able to promote neutrophil adhesion to endothelial cells. Promotion of neutrophil adhesion by galectin-3 appeared to result from direct cross-linking of neutrophils to the endothelium and was dependent on galectin-3 oligomerization. Together, these results suggest that galectin-3 acts as an adhesion molecule that can mediate neutrophil adhesion to endothelial cells. However, accumulation of galectin-3 in lung was not observed during neutrophil emigration into alveoli induced by Escherichia coli infection, where the majority of neutrophil emigration is known to be beta(2) integrin dependent. Thus, based on our results, we propose that galectin-3 plays a role in beta(2) integrin-independent neutrophil extravasation, which occurs during alveolar infection with Streptococcus pneumoniae.     

Fibronectin receptor functions in embryonic cells deficient in alpha 5 beta 1 integrin can be replaced by alpha V integrins.

alpha 5 beta 1 integrin mediates cell adhesion to extracellular matrix by interacting with fibronectin (FN). Mouse lines carrying null mutations in genes encoding either the alpha 5 integrin subunit or FN have been generated previously. Both mutations are embryonic lethal with overlapping defects, but the defects of alpha 5-null embryos are less severe. Primary embryonic cells lacking alpha 5 beta 1 are able to adhere to FN, form focal contacts, migrate on FN, and assemble FN matrix. These results suggest the involvement of (an)other FN receptors(s). In this study, we examined functions of alpha 4 beta 1 and alpha V integrins in embryonic cells lacking alpha 5 beta 1. Our analysis of cells lacking both alpha 4 beta 1 and alpha 5 beta 1 showed that alpha 4 beta 1 is also not required for these FN-dependent functions. Using alpha V-specific blocking reagents, we showed that alpha V integrins are required for alpha 5-null cells, but not wild-type cells, to adhere and spread on FN. Our data also showed that, although the expression levels of alpha V integrins on the wild-type and alpha 5-null cells are similar, there is an increase in recruitment of alpha V integrins into focal contacts in alpha 5-null cells plated on FN, indicating that alpha V integrins can compensate functionally for the loss of alpha 5 beta 1 in focal contacts of alpha 5-null cells. Finally, our data suggested possible roles for alpha V integrins in replacing the role of alpha 5 beta 1 in FN matrix assembly in vitro and in FN-dependent embryonic functions in vivo.     

Collagen XVI harbors an integrin alpha1 beta1 recognition site in its C-terminal domains

Collagen XVI is integrated tissue-dependently into distinct fibrillar aggregates, such as D-banded cartilage fibrils and fibrillin-1-containing microfibrils. In skin, the distribution of collagen XVI overlaps that of the collagen-binding integrins alpha1 beta1 and alpha2 beta1. Basal layer keratinocytes express integrin alpha2 beta1, whereas integrin alpha1 beta1 occurs in smooth muscle cells surrounding blood vessels, in hair follicles, and on adipocytes. Cells bearing the integrins alpha1 beta1 and alpha2 beta1 attach and spread on recombinant collagen XVI. Furthermore, collagen XVI induces the recruitment of these integrins into focal adhesion plaques, a principal step in integrin signaling. Of potential physiological relevance, these integrin-collagen XVI interactions may connect cells with specialized fibrils, thus contributing to the organization of fibrillar and cellular components within connective tissues. In cell-free binding assays, collagen XVI is more avidly bound by alpha1 beta1 integrin than by alpha2 beta1 integrin. Both integrins interact with collagen XVI via the A domain of their alpha subunits. A tryptic collagen XVI fragment comprising the collagenous domains 1-3 is recognized by alpha1 beta1 integrin. Electron microscopy of complexes of alpha1 beta1 integrin with this tryptic collagen XVI fragment or with full-length collagen XVI revealed a unique alpha1 beta1 integrin-binding site within collagen XVI located close to its C-terminal end.     

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.     

The involvement of Gab1 and PI 3-kinase in beta1 integrin signaling in keratinocytes

The control of the stem cell compartment in epidermis is closely linked to the regulation of keratinocyte proliferation and differentiation. Beta1 integrins are expressed 2-fold higher by stem cells than transit-amplifying cells. Signaling from these beta1 integrins is critical for the regulation of the epidermal stem cell compartment. To clarify the functional relevance of this differential expression of beta1 integrins, we established HaCaT cells with high beta1 integrin expression by repeated flow cytometric sorting of this population from the parental cell line. In these obtained cells expressing beta1 integrins by 5-fold, MAPK activation was markedly increased. Regarding the upstream of MAPK, Gab1 phosphorylation was also higher with high beta1 integrin expression, while Shc phosphorylation was not altered. In addition, enhanced phosphatidylinositol 3-kinase activation was also observed. These observations suggest that Gab1 and phosphatidylinositol 3-kinase play pivotal roles in the beta1 integrin-mediated regulation of the epidermal stem cell compartment.     

Beta1 integrins are distributed in adhesion structures with fibronectin and caveolin and in coated pits.

Integrins are found in adhesion structures, which link the extracellular matrix to cytoskeletal proteins. Here, we attempt to further define the distribution of beta1 integrins in the context of their association with matrix proteins and other cell surface molecules relevant to the endocytic process. We find that beta1 integrins colocalize with fibronectin in fibrillar adhesion structures. A fraction of caveolin is also organized along these adhesion structures. The extracellular matrix protein laminin is not concentrated in these structures. The alpha4beta1 integrin exhibits a distinct distribution from other beta1 integrins after cells have adhered for 1 h to extracellular matrix proteins but is localized in adhesion structures after 24 h of adhesion. There are differences between the fibronectin receptors: alpha5beta1 integrins colocalize with adaptor protein-2 in coated pits, while alpha4beta1 integrins do not. This parallels our earlier observation that of the two laminin receptors, alpha1beta1 and alpha6beta1, only alpha1beta1 integrins colocalize with adaptor protein-2 in coated pits. Calcium chelation or inhibition of mitogen-activated protein kinase kinase, protein kinase C, or src did not affect localization of alpha1beta1 and alpha5beta1 integrins in coated pits. Likewise, the integrity of coated-pit structures or adhesion structures is not required for integrin and adaptor protein-2 colocalization. This suggests a robust and possibly constitutive interaction between these integrins and coated pits.     

Role of multiple beta1 integrins in cell adhesion to the disintegrin domains of ADAMs 2 and 3

ADAM disintegrin domains can support integrin-mediated cell adhesion. However, the profile of which integrins are employed for adhesion to a given disintegrin domain remains unclear. For example, we suggested that the disintegrin domains of mouse sperm ADAMs 2 and 3 can interact with the alpha6beta1 integrin on mouse eggs. Others concluded that these disintegrin domains interact instead with the alpha9beta1 integrin. To address these differing results, we first studied adhesion of mouse F9 embryonal carcinoma cells and human G361 melanoma cells to the disintegrin domains of mouse ADAMs 2 and 3. Both cell lines express alpha6beta1 and alpha9beta1 integrins at their surfaces. Antibodies to the alpha6 integrin subunit inhibited adhesion of both cell lines. An antibody that recognizes human alpha9 integrin inhibited adhesion of G361 cells. VLO5, a snake disintegrin that antagonizes alpha4beta1 and alpha9beta1 integrins, potently inhibited adhesion of both cell lines. We next explored expression of the alpha9 integrin subunit in mouse eggs. In contrast to our ability to detect alpha6beta1, we were unable to convincingly detect alpha9beta1 integrin on the surface of mouse eggs. Moreover, treatment of mouse eggs with 250 nm VLO5, which is 250 fold over its approximately IC(50) for inhibition of somatic cell adhesion, had minimal effect on sperm-egg binding or fusion. We did detect alpha9 integrin protein on epithelial cells of the oviduct. Additional studies showed that antibodies to the alpha6 and alpha7 integrins additively inhibited adhesion of mouse trophoblast stem cells and that an antibody to the alpha4 integrin inhibited adhesion of MOLT-3 cells to these disintegrin domains: Our data suggest that multiple integrins (on the same cell) can participate in adhesion to a given ADAM disintegrin domain and that interactions between ADAMs and integrins may be important for sperm transit through the oviduct.     

Expression and functional importance of collagen-binding integrins,alpha 1 beta 1 and alpha 2 beta 1, on virus-activated T cells

Adhesive interactions are crucial to cell migration into inflammatory sites. Using murine lymphocytic choriomeningitis virus as an Ag model system, we have investigated expression and function of collagen-binding integrins, alpha(1)beta(1) and alpha(2)beta(1), on activated and memory T cells. Using this system and MHC tetramers to define Ag-specific T cells, we demonstrate that contrary to being VLAs, expression of alpha(1)beta(1) and alpha(2)beta(1) can be rapidly induced on acutely activated T cells, that expression of alpha(1)beta(1) remains elevated on memory T cells, and that expression of alpha(1)beta(1) parallels that of viral-specific effector CD8(+) T cells (defined by tetramer and IFN-gamma staining). In an adoptive transfer model, mAb-mediated blockade of these integrins on activated effector and memory T cells inhibited Ag-specific delayed-type hypersensitivity responses; similar decreased responses were seen upon transfer of alpha(1)-deficient activated/memory T cells. Thus, expression of alpha(1)beta(1) and alpha(2)beta(1) integrins on activated T cells is directly functionally important for generation of inflammatory responses within tissues. Finally, the inhibitory effect of alpha(1)beta(1) blockade on the delayed-type hypersensitivity response could be bypassed by direct injection of Ag-specific T cells to inflammatory sites, demonstrating for the first time in vivo that collagen-binding integrins are involved in leukocyte migration into tissues.     

Integrin engagement mediates the human polymorphonuclear leukocyte response to a fungal pathogen-associated molecular pattern

Extravasation of leukocytes from peripheral blood is required for an effective inflammatory response at sites of tissue infection. Integrins help mediate extravasation and navigate the leukocyte to the infectious source. A novel role for integrins in regulating the effector response to a cell wall component of fungal pathogens is the subject of the current study. Although phagocytosis is useful for clearance of unicellular fungi, the immune response against large, noningestible hyphae is not well-understood. Fungal beta-glucan, a pathogen-associated molecular pattern, activates production of superoxide anion in leukocytes without the need for phagocytosis. To model polymorphonuclear leukocyte (PMN) recognition of fungi under conditions in which phagocytosis cannot occur, beta-glucan was covalently immobilized onto tissue culture plastic. Plasma membrane-associated respiratory burst was measured by reduction of ferricytochrome C. Results show that the human PMN oxidative burst response to immobilized beta-glucan is suppressed by addition of beta(1) integrin ligands to the beta-glucan matrix. Suppression was dose dependent and steric hindrance was ruled out. beta(1) integrin ligands did not affect respiratory burst to ingestible beta-glucan-containing particles, phorbol esters or live yeast hyphae. Furthermore, in the absence of matrix, Ab activation of VLA3 or VLA5, but not other beta(1) integrins, also prevented beta-glucan-induced respiratory burst. beta(1)-induced suppression was blocked and burst response restored by treating neutrophils with either the cell-binding fragment of soluble human Fn, cyclic RGD peptide, or Ab specific to VLA3 or VLA5. Together these findings extend the functional role of beta(1) integrins to include modulating PMN respiratory burst to a pathogen-associated molecular pattern.     

The integrin alpha9beta1 mediates adhesion to activated endothelial cells and transendothelial neutrophil migration through interaction with vascular cell adhesion molecule-1

The integrin alpha9beta1 has been shown to be widely expressed on smooth muscle and epithelial cells, and to mediate adhesion to the extracellular matrix proteins osteopontin and tenascin-C. We have found that the peptide sequence this integrin recognizes in tenascin-C is highly homologous to the sequence recognized by the closely related integrin alpha4beta1, in the inducible endothelial ligand, vascular cell adhesion mole-cule-1 (VCAM-1). We therefore sought to determine whether alpha9beta1 also recognizes VCAM-1, and whether any such interaction would be biologically significant. In this report, we demonstrate that alpha9beta1 mediates stable cell adhesion to recombinant VCAM-1 and to VCAM-1 induced on human umbilical vein endothelial cells by tumor necrosis factor-alpha. Furthermore, we show that alpha9beta1 is highly and selectively expressed on neutrophils and is critical for neutrophil migration on VCAM-1 and tenascin-C. Finally, alpha9beta1 and alpha4 integrins contribute to neutrophil chemotaxis across activated endothelial monolayers. These observations suggest a possible role for alpha9beta1/VCAM-1 interactions in extravasation of neutrophils at sites of acute inflammation.     

Adhesion of human monocytic THP-1 cells to endothelial cell adhesion molecules or extracellular matrix proteins via beta1 integrins regulates heparin binding epidermal growth factor-like growth factor (HB-EGF) expression

Adherence to endothelium and then to the extracellular matrix is a prerequisite for extravasation of monocytes into injured tissues. There, monocytes differentiate into macrophages and express heparin binding epidermal growth factor-like growth factor (HB-EGF), a key growth factor involved in normal wound healing. We investigated whether the interaction of human monocytic THP-1 cells with the endothelial cell adhesion molecules (vascular CAM-1, VCAM-1; intercellular adhesion molecule-1 ICAM-1 and endothelial-selectin, E-selectin), or the extracellular matrix (ECM) proteins (fibronectin, FN; laminin, LN and fibrinogen, FG) regulate HB-EGF expression. We have shown that adherence of THP-1 cells via VCAM-1, E-selectin or FN, which are all overexpressed at sites of inflammation, potentiates HB-EGF mRNA expression. In contrast, adhesion of THP-1 cells via ICAM-1 or FG, has no significant effect. Since THP-1 cells interact with ICAM-1 and FG through beta2 integrins, and with VCAM-1 and FN via beta1 integrins, regulation of HB-EGF expression appears to be specific to beta1 integrin ligation. In addition, we demonstrate that THP-1 binding to LN, through the beta1 integrin VLA-6, down regulates HB-EGF expression. Thus physiologically, transient destruction of LN and expression of VCAM-1, E-selectin and fibronectin at sites of inflammation, may locally induce HB-EGF overexpression.     

Cell adhesion to extracellular matrix regulates the life cycle of integrins.

The expression of alpha 5 beta 1 integrin on the surface of fibroblasts requires adhesion to substratum. We have examined the basis for this adhesion-dependent surface expression by comparing the life cycle of integrins in parallel cultures of adherent and nonadherent cells. Results of biosynthetic labeling experiments in NRK fibroblasts showed that the synthesis and biosynthetic processing of the beta 1 integrin subunit proceed in the absence of cell attachment; however, when examining the behavior of preexisting cell surface integrins, we observed that the alpha beta 1 integrins are internalized and degraded when adhesion to substratum is blocked. A kinetic analysis of integrin internalization in cycloheximide-treated NRK cells showed that each of the fibroblast integrins we examined (in both the beta 1 and beta 3 families) are lost from the cell surface after detachment from substratum. Thus, the default integrin life cycle in fibroblasts involves continuous synthesis, processing, transport to the cell surface, and internalization/degradation. Interestingly, studies with NIH-3T3 cells expressing alpha 1 beta 1 integrin showed that the loss of cell-surface alpha 5 beta 1 integrin is blocked by adhesion of cells to dishes coated with type IV collagen (a ligand for alpha 1 beta 1 integrin) as well as fibronectin. Similarly, adhesion of these cells to dishes coated with type IV collagen stabilizes the surface expression of alpha 5 beta 1 as well as alpha 1 beta 1 integrin. We propose that the adhesion of fibroblasts to extracellular matrix protein alters the integrin life cycle and permits retention of these proteins at the cell surface where they can play important roles in transmitting adhesion-dependent signals.     

Divergent effects of platelet-endothelial cell adhesion molecule-1 and beta 3 integrin blockade on leukocyte transmigration in vivo

The final stage in the migration of leukocytes to sites of inflammation involves movement of leukocytes through the endothelial cell layer and the perivascular basement membrane. Both platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) and the integrin alphavbeta3 have been implicated in this process, and in vitro studies have identified alphavbeta3 as a heterotypic ligand for PECAM-1. In the present study we have addressed the roles of these molecules by investigating and comparing the effects of PECAM-1 and alphavbeta3 blockade on leukocyte migration in vivo. For this purpose we have examined the effects of neutralizing Abs directed against PECAM-1 (domain 1-specific, mAb 37) and beta3 integrins (mAbs 7E3 and F11) on leukocyte responses in the mesenteric microcirculation of anesthetized rats using intravital microscopy. The anti-PECAM-1 mAb suppressed leukocyte extravasation, but not leukocyte rolling or firm adhesion, elicited by IL-1beta in a dose-dependent manner (e.g., 67% inhibition at 10 mg/kg 37 Fab), but had no effect on FMLP-induced leukocyte responses. Analysis by electron microscopy suggested that this suppression was due to an inhibition of neutrophil migration through the endothelial cell barrier. By contrast, both anti-beta3 integrin mAbs, 7E3 F(ab')2 (5 mg/kg) and F11 F(ab')2 (5 mg/kg), selectively reduced leukocyte extravasation induced by FMLP (38 and 46%, respectively), but neither mAb had an effect on IL-1beta-induced leukocyte responses. These findings indicate roles for both PECAM-1 and beta3 integrins in leukocyte extravasation, but do not support the concept that these molecules act as counter-receptors in mediating leukocyte transmigration.     

Expression of chicken integrin beta 1 subunit in rat PC12 cells

We transfected rat pheochromocytoma (PC12) cells with a cDNA encoding chicken integrin beta 1 subunit. The chicken integrin beta 1 subunit produced in stable transfectants associated with two major alpha subunits of rat integrins to form interspecific chimeric receptors. These receptors mediated cell spreading and initial neurite outgrowth on laminin as did corresponding endogenous integrins, although they were slightly less effective in inducing cell adhesion to laminin. These results indicate that chicken integrin beta 1 may functionally substitute for beta 1 subunit of rat integrins in PC12 cells. Apparently, the structure of the integrin beta 1 subunit is highly conserved in the evolution of these species.     

Hyposialylation of integrins stimulates the activity of myeloid fibronectin receptors

Despite numerous reports suggesting that beta(1) integrin receptors undergo differential glycosylation, the potential role of N-linked carbohydrates in modulating integrin function has been largely ignored. In the present study, we find that beta(1) integrins are differentially glycosylated during phorbol ester (PMA)-stimulated differentiation of myeloid cells along the monocyte/macrophage lineage. PMA treatment of two myeloid cell lines, U937 and THP-1, induces a down-regulation in expression of the ST6Gal I sialyltransferase. Correspondingly, the beta(1) integrin subunit becomes hyposialylated, suggesting that the beta(1) integrin is a substrate for this enzyme. The expression of hyposialylated beta(1) integrin isoforms is temporally correlated with enhanced binding of myeloid cells to fibronectin, and, importantly, fibronectin binding is inhibited when the Golgi disrupter, brefeldin A, is used to block the expression of the hyposialylated form. Consistent with the observation that cells with hyposialylated integrins are more adhesive to fibronectin, we demonstrate that the enzymatic removal of sialic acid residues from purified alpha(5)beta(1) integrins stimulates fibronectin binding by these integrins. These data support the hypothesis that unsialylated beta(1) integrins are more adhesive to fibronectin, although desialylation of alpha(5) subunits could also contribute to increased fibronectin binding. Collectively our results suggest a novel mechanism for regulation of the beta(1) integrin family of cell adhesion receptors.     

Matrix-specific suppression of integrin activation in shear stress signaling

Atherosclerotic plaque develops at sites of disturbed flow. We previously showed that flow activates endothelial cell integrins, which then bind to the subendothelial extracellular matrix (ECM), and, in cells on fibronectin or fibrinogen, trigger nuclear factor-kappaB activation. Additionally, fibronectin and fibrinogen are deposited into the subendothelial ECM at atherosclerosis-prone sites at early times. We now show that flow activates ECM-specific signals that establish patterns of integrin dominance. Flow induced alpha2beta1 activation in cells on collagen, but not on fibronectin or fibrinogen. Conversely, alpha5beta1 and alphavbeta3 are activated on fibronectin and fibrinogen, but not collagen. Failure of these integrins to be activated on nonpermissive ECM is because of active suppression by the integrins that are ligated. Protein kinase A is activated specifically on collagen and suppresses flow-induced alphavbeta3 activation. Alternatively, protein kinase Calpha is activated on fibronectin and mediates alpha2beta1 suppression. Thus, integrins actively cross-inhibit through specific kinase pathways. These mechanisms may determine cellular responses to complex extracellular matrices.     

Human ether-a-go-go-related gene 1 channels are physically linked to beta1 integrins and modulate adhesion-dependent signaling

Adhesive receptors of the integrin family are primarily involved in cell-extracellular matrix adhesion. Additionally, integrins trigger multiple signaling pathways that are involved in cell migration, proliferation, survival, and differentiation. We previously demonstrated that the activation of integrins containing the beta(1) subunit leads to a selective increase in potassium currents carried by the human ether-a-go-go-related gene (hERG) channels in neuroblastoma and leukemia cells; this current activation modulates adhesion-dependent differentiation in these cells. We hypothesized that the cross-talk between integrins and hERG channels could be traced back to the assembly of a macromolecular signaling complex comprising the two proteins. We tested this hypothesis in both SH-SY5Y neuroblastoma cells and in human embryonic kidney 293 cells stably transfected with hERG1 and, therefore, expressing only the full-length hERG1 protein on the plasma membrane. The beta(1) integrin and hERG1 coprecipitate in these cells and colocalize in both intracellular and surface membrane compartments. The two proteins also coprecipitate with caveolin-1, suggesting the localization of the complex in lipid rafts/caveolae. hERG1-transfected cells undergo an activation of hERG currents after beta(1) integrin-mediated adhesion to fibronectin; concomitant with this activation, the focal adhesion kinase associates with the hERG1 protein and becomes tyrosine phosphorylated. Using hERG1-specific inhibitors, we show that the tyrosine phosphorylation of focal adhesion kinase is strictly dependent on hERG channel activity. Similarly, the activity of the small GTPase Rac1 turned out to be dependent on hERG currents. On the whole, these data indicate that the hERG1 protein associates with beta(1) integrins and modulates adhesion receptor signaling.