Synergistic control of keratinocyte adhesion through muscarinic and nicotinic acetylcholine receptor subtypes

The biological mechanisms involved in initiating, coordinating, and ultimately terminating cell-cell adhesion in the stratified epithelium are not well understood at present. This study was designed to elucidate the roles of the muscarinic M3, the nicotinic alpha3, and the mixed muscarinic-nicotinic alpha9 acetylcholine receptors in physiologic control of keratinocyte adhesion. Both muscarinic and nicotinic antagonists caused keratinocyte detachment and reversibly increased the permeability of keratinocyte monolayers, indicative of the involvement of both muscarinic and nicotinic pathways in the cholinergic control of keratinocyte adhesion. Since phosphorylation of adhesion proteins plays an important role in rapid assembly and disassembly of intercellular junctions, we measured muscarinic and nicotinic effects on phosphorylation of keratinocyte adhesion molecules. The phosphorylation levels of E-cadherin, beta-catenin, and gamma-catenin increased following pharmacological blockage of muscarinic receptors. Long-term blocking of alpha3, alpha9, and M3 receptor signaling pathways with antisense oligonucleotides resulted in cell-cell detachment and changes in the expression levels of E-cadherin, beta-catenin, and gamma-catenin in cultured human keratinocytes. Simultaneous inhibition of several receptor subtypes with a mixture of antisense oligonucleotides produced intensified abnormalities with cell adhesion. Moreover, altered cell-cell adhesion was found in the stratified epithelium of alpha3, alpha9, and M3 receptor knockout mice. Keratinocytes from these mice exhibited abnormal expression of adhesion molecules at both the protein and the mRNA levels. Thus, our data indicate that the alpha3, alpha9, and M3 acetylcholine receptors play key roles in regulating in a synergistic mode keratinocyte adhesion, most probably by modulating cadherin and catenin levels and activities. These findings may aid in the development of novel methods useful for the treatment of skin adhesion diseases and tumor metastasis.     

Integrin alpha4beta1 involvement in stromal cell-derived factor-1alpha-promoted myeloma cell transendothelial migration and adhesion: role of cAMP and the actin cytoskeleton in adhesion

The chemokine stromal cell-derived factor-1alpha (SDF-1alpha) is expressed by bone marrow (BM) stromal cells and plays key roles in cell homing to and retention into the bone marrow. In multiple myeloma, blood-borne malignant plasma cells home to the BM and accumulate in contact with stromal cells, implicating myeloma cell migration across endothelium. Myeloma cells express the SDF-1alpha receptor CXCR4, as well as the integrin alpha4beta1, which mediates their attachment to BM stroma. We show here that SDF-1alpha promotes transendothelial migration of purified BM myeloma cells and myeloma-derived NCI-H929 cells, involving a transient upregulation of alpha4beta1-dependent cell adhesion to the endothelium. Characterization of intracellular signaling pathways involved in the modulation by SDF-1alpha of alpha4beta1-mediated myeloma cell adhesion revealed that intracellular cAMP amounts associated with the activation of protein kinase A play key roles in this modulation. Furthermore, a functional link between cAMP actions on the dynamics of actin cytoskeleton, RhoA activation, and alpha4beta1-dependent cell adhesion in response to SDF-1alpha has been found. The regulation of alpha4beta1-mediated myeloma cell adhesion by SDF-1alpha could play key roles during myeloma cell homing into and trafficking inside the BM, and characterization of the molecular events involved in SDF-1alpha-activated modulation of this adhesion will contribute to a better understanding of mechanisms participating in cell migration.     

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.     

Anti-β1 integrin antibody inhibits schwann cell meylination

Schwann cells (SCs) co-cultured with sensory neurons require ascorbate supplementation for basal lamina assembly and differentiation into myelinating cells. The ascorbate requirement can be bypased by adding a purifed basal lamina component, laminin, to SC/neuron cocultures. We have examined the role of laminin receptors, Namely, the β1 subfamily of integrins, in the process of myelination. We demonstrate by immunostaining or immunoprecipitation that undifferentiated SCs in contact with axons express large amounts of the β1 subunit in association with the α1 or α6 subunit. In co-cultures of myelinating SCs, α1β1 is no longer present, α6β1 is still present but at reduced levels, and α6β4 is expressed at much higher levels than in co-cultures of undifferentiated SCs. Immunogold labelling at the electron microscope level suggested that β1 integrins are randomly distributed on undifferentiated SCs, become localized to the SC surface contacting basal lamina in differentiating SCs before the onset of myelination, and are not detected on myelinating SCs. Fab fragments of β1 function-blocking antibody block both attachment of isolated SCs to laminin and formation of myelin sheaths by SCs co-cultured with neurons in ascorbate-supplemented medium. SCs unable to myelinate in the presence of the anti-β1 antibody assemble patchy basal lamina that is only loosely attached to the cell surface and in some cases appears to be detaching from the membrane. In contrast, an α1β1 function-blocking antibody only partially blocks attachment of isolated SCs to laminin but has no inhibitory effect on SC myelination. These results are consistent with the hypothesis that a member of the β1 subfamily of integrins other than α1β1 binds laminin present in basal lamina to the SC surface and transduces signals that are critical for initiation of SC differentiation into a myelinating cell. 1994 John Wiley & Sons, Inc.     

Aza-bicyclic amino acid carboxamides as alpha4beta1/alpha4beta7 integrin receptor antagonists

A series of N-carboxy, N-alkyl, and N-carboxamido azabicyclo[2.2.2]octane carboxamides were prepared and assayed for inhibition of alpha4beta1-VCAM-1 and alpha4beta7-MAdCAM-1 interactions. Potency and alpha4beta1/alpha4beta7 selectivity were sensitive to the substituent R1-R3 in the structures 6, 7, and 8. Several compounds demonstrated low nanomolar balanced alpha4beta1/alpha4beta7 in vitro activity. Two compounds were selected for in vivo leukocytosis studies and demonstrated increases in circulating lymphocytes up to 250% over control.     

Characterization of the oligosaccharide component of alpha3beta1 integrin from human bladder carcinoma cell line T24 and its role in adhesion and migration

Malignant transformation is highly associated with altered expression of cell surface N-linked oligosaccharides. These changes concern integrins, a family of cell surface glycoproteins involved in the attachment and migration of cells on various extracellular matrix proteins. The integrin alpha3beta1 is particularly interesting because of its role in migration and invasion of several types of metastatic tumours. In this study, alpha3beta1 from human bladder T24 carcinoma cells was purified and treated with peptide N-glycosidase F. Then the N-glycans of the alpha3 and beta1 subunits were characterized using matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). In alpha3beta1 integrin the presence of high-mannose, hybrid and predominantly complex type N-oligosaccharides was shown. Unlike to normal epithelium cells, in both subunits of alpha3beta1 integrin from cancer cells, the sialylated tetraantennary complex type glycan Hex7HexNAc6FucSia4 was present. In a direct ligand binding assay, desialylated alpha3beta1 integrin exhibited significantly higher fibronectin-binding capability than untreated integrin, providing evidence that sialic acids play a direct role in ligand-receptor interaction. Moreover, alpha3beta1 integrin was shown to take part in T24 cell migration on fibronectin: anti-alpha3 antibodies induced ca 30% inhibition of wound closure. Treatment of T24 cells with swainsonine reduced the rate of bladder carcinoma cell migration by 16%, indicating the role of beta1,6 branched complex type glycans in this process. Our data show that alpha3beta1 integrin function may be altered by glycosylation, that both subunits contribute to these changes, and that glycosylation may be considered a newly found mechanism in the regulation of integrin function.     

The muscle integrin binding protein (MIBP) interacts with alpha7beta1 integrin and regulates cell adhesion and laminin matrix deposition

Integrins are alphabeta transmembrane receptors that function in key cellular processes, including cell adhesion, differentiation, and extracellular matrix deposition through interactions with extracellular, membrane, and cytoplasmic proteins. We previously identified and cloned a muscle beta1 integrin cytoplasmic binding protein termed MIBP and found that the expression level of MIBP is critical in the decision-making process of terminal myogenic differentiation. We report here that MIBP interacts with the alpha7beta1 integrin but not the alpha5beta1 integrin in C2C12 myoblasts, suggesting an important role of integrin alpha chains in the regulation of the beta1-MIBP interaction. Furthermore, consistent with its selective binding activity toward the alpha7beta1 laminin receptor, we have found that overexpression of MIBP in C2C12 myoblasts resulted in a significant reduction of cell adhesion to laminin and inhibition of laminin matrix deposition. By contrast, neither cell adhesion to fibronectin nor fibronectin matrix deposition was significantly altered in cells overexpressing MIBP. Finally, we show that both the protein level and tyrosine phosphorylation of paxillin, a key signaling molecule involved in the cellular control of myogenic differentiation, are increased by MIBP. These results suggest that MIBP functions in the control of myogenic differentiation by regulating alpha7beta1 integrin-mediated cell interactions with laminin matrix and intracellular signaling through paxillin.     

Upmodulation of αvβ1 integrin expression on human tumor cells by human interleukin for DA cells/leukemia inhibitory factor and oncostatin M: Correlation with increased cell adhesion on fibronectin

Integrins belong to a large family of heterodimeric membrane glycoproteins which mediate cell-cell or cell-extracellular matrix interactions. These interactions could play a major role during the migration of tumor cells across the extracellular matrix and vascular endothelium and would thus appear to be requisite for the metastatic process. Pretreatment of the Foss human melanoma cell line with HILDA/LIF or OSM, two cytokines involved in acute-phase response, increased the expression of membrane αvβ1 1.5–2-fold. The same phenomenon was observed on the SK-N-SH human neuroblastoma cell line. αvβ1 upmodulation was concomitant with improved tumor cells attachment to the fibronectin matrix. This greater adhesion of tumor cells to fibronectin was inhibited by specific monoclonal antibodies against αv or β1 integrin subunits. Similar results were obtained after TNF-α treatment. Our findings demonstrate the ability of HILDA/LIF and OSM to modulate tumor cell capacity to adhere to the matrix component, suggesting a potential role for these cytokines in modulation of tumoral progression.     

Induction of mouse β integrin expression following transfection with human α4 chain

We report here an analysis of the expression and function of the α chain of human VLA-4 in stable mouse L cell transfectants and the requirement for the β chain in these processes. L cells were transfected with human α4 cDNA or α4 and human β1 cDNA. Unexpectedly, human α4 cDNA, when transfected alone, could induce de novo surface expression of host β7 and increased expression of host β1. Induction of mouse β7 and β1 surface expression was not due to de novo gene activation, but instead represented α4/β intracellular subunit association and transport to the cell surface. Transfection with human β1 prevented surface expression of mouse β integrins. Whereas human α4 and human β1 subunits associated very tightly in anti-α4 immunoprecipitates, human α4 and mouse β subunits were only partially associated. Furthermore, binding of human/mouse chimeric receptors to recombinant VCAM, a major ligand for α4β7 and α4β1, was very poor, whereas human α4/human β1 receptors bound strongly to VCAM. One α4 transfectant, which exhibited a tight human α4/mouse β1 association, could be induced, but only after PMA activation, to bind strongly to VCAM. These results indicate that α4 subunits have specific affinity for β7 and β1 integrins and require β subunits for surface expression as well as high affinity ligand binding activity. Our results indicate that a tight association between the α4 and β subunit appears to be critical for ligand binding, consistent with a direct as well as regulatory role for the β subunit in ligand binding. Furthermore, these studies demonstrate that expression of foreign recombinant proteins can alter host cell protein expression resulting in de novo surface protein expression. © 1996 Wiley-Liss, Inc.     

The integrin alpha(v)beta8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-beta1

Integrins, matrix metalloproteases (MMPs), and the cytokine TGF-beta have each been implicated in homeostatic cell behaviors such as cell growth and matrix remodeling. TGF-beta exists mainly in a latent state, and a major point of homeostatic control is the activation of TGF-beta. Because the latent domain of TGF-beta1 possesses an integrin binding motif (RGD), integrins have the potential to sequester latent TGF-beta (SLC) to the cell surface where TGF-beta activation could be locally controlled. Here, we show that SLC binds to alpha(v)beta8, an integrin expressed by normal epithelial and neuronal cells in vivo. This binding results in the membrane type 1 (MT1)-MMP-dependent release of active TGF-beta, which leads to autocrine and paracrine effects on cell growth and matrix production. These data elucidate a novel mechanism of cellular homeostasis achieved through the coordination of the activities of members of three major gene families involved in cell-matrix interactions.     

Cyclic AMP induces integrin-mediated cell adhesion through Epac and Rap1 upon stimulation of the beta 2-adrenergic receptor

cAMP controls many cellular processes mainly through the activation of protein kinase A (PKA). However, more recently PKA-independent pathways have been established through the exchange protein directly activated by cAMP (Epac), a guanine nucleotide exchange factor for the small GTPases Rap1 and Rap2. In this report, we show that cAMP can induce integrin-mediated cell adhesion through Epac and Rap1. Indeed, when Ovcar3 cells were treated with cAMP, cells adhered more rapidly to fibronectin. This cAMP effect was insensitive to the PKA inhibitor H-89. A similar increase was observed when the cells were transfected with Epac. Both the cAMP effect and the Epac effect on cell adhesion were abolished by the expression of Rap1-GTPase-activating protein, indicating the involvement of Rap1 in the signaling pathway. Importantly, a recently characterized cAMP analogue, 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate, which specifically activates Epac but not PKA, induced Rap-dependent cell adhesion. Finally, we demonstrate that external stimuli of cAMP signaling, i.e., isoproterenol, which activates the G alpha s-coupled beta 2-adrenergic receptor can induce integrin-mediated cell adhesion through the Epac-Rap1 pathway. From these results we conclude that cAMP mediates receptor-induced integrin-mediated cell adhesion to fibronectin through the Epac-Rap1 signaling pathway.     

Fibrillin-1 and alpha8 integrin are co-expressed in the glomerulus and interact to convey adhesion of mesangial cells

Fibrillin-1 is a microfibrillar extracellular matrix protein that was described to be a ligand for α8 integrin. α8 integrin is a matrix receptor specifically expressed in mesangial and smooth muscle cells of the kidney. In previous studies we detected glomerular expression of fibrillin-1. Moreover, fibrillin-1 promoted adhesion, migration, and proliferation of mesangial cells. We hypothesized that fibrillin-1 and α8 integrin might interact in the glomerulus, and thus, regulate mesangial cell properties. Our studies showed that fibrillin-1 and α8 integrin colocalize in the glomerular mesangium. Induction of experimental glomerulonephritis led to an increase of both fibrillin-1 and α8 integrin expression. In vitro studies revealed that mesangial cells deficient for α8 integrin adhere weaker to fibrillin-1 and migrate more easily on fibrillin-1 than wild-type mesangial cells. Baseline proliferation on fibrillin-1 is higher in α8 integrin-deficient mesangial cells, but the induction of proliferation is not different in α8 integrin-deficient and wild-type mesangial cells. We conclude that fibrillin-1 and α8 integrin interact, and thus, regulate mesangial cell adhesion and migration. The concomitant induction of both fibrillin-1 and α8 integrin in a self-limited model of glomerular injury points to a protective role of the interaction of fibrillin-1 with α8 integrin in the glomerulus resulting in reduced damage of the glomerular tuft as a consequence of firm adhesion of mesangial cells.     

Differentiation-inducing factor-1 induces cyclin D1 degradation through the phosphorylation of Thr286 in squamous cell carcinoma

Differentiation-inducing factors (DIFs) are morphogens which induce cell differentiation in Dictyostelium. We reported that DIF-1 and DIF-3 inhibit proliferation and induce differentiation in mammalian cells. In this study, we investigated the effect of DIF-1 on oral squamous cell carcinoma cell lines NA and SAS, well differentiated and poorly differentiated cell lines, respectively. Although DIF-1 did not induce the expression of cell differentiation makers in these cell lines, it inhibited the proliferation of NA and SAS in a dose-dependent manner by restricting the cell cycle in the G0/G1 phase. DIF-1 induced cyclin D1 degradation, but this effect was prevented by treatment with lithium chloride and SB216763, the inhibitors of glycogen synthase kinase-3beta (GSK-3beta). Depletion of endogenous GSK-3beta by RNA interference also attenuated the effect of DIF-1 on cyclin D1 degradation. Therefore, we investigated the effect of DIF-1 on GSK-3beta and found that DIF-1 dephosphorylated GSK-3beta on Ser9 and induced the nuclear translocation of GSK-3beta, suggesting that DIF-1 activated GSK-3beta. Then, we examined the effect of DIF-1 on cyclin D1 mutants (Thr286Ala, Thr288Ala, and Thr286/288Ala). We revealed that Thr286Ala and Thr286/288Ala mutants were highly resistant to DIF-1-induced degradation compared with wild-type cyclin D1, indicating that the phosphorylation of Thr286 was critical for cyclin D1 degradation induced by DIF-1. These results suggest that DIF-1 induces degradation of cyclin D1 through the GSK-3beta-mediated phosphorylation of Thr286.     

Leukotriene D4-induced adhesion of Caco-2 cells is mediated by prostaglandin E2 and upregulation of alpha2beta1-integrin

Cell-cell and extracellular matrix adhesions play important roles in the progression of cancer. We investigated the involvement of the inflammatory mediator leukotriene D4 (LTD4) in the regulation of cell-matrix adhesion of colon cancer (Caco-2) cells. We observed that LTD4 acted via its CysLT1 receptor in these cells to induce increased adhesion to collagen I. LTD4 also enhanced the activation and expression of alpha2beta1-integrins on the cell surface, which we found to be responsible for mediating the increased adhesion to collagen I. LTD4 simultaneously augmented expression of the prostaglandin-generating enzyme cyclooxygenase-2 (COX-2) and increased prostaglandin E2 (PGE2) production in Caco-2 cells. The adhesive capacity of the Caco-2 cells was reduced by specific inhibition of COX-2 and was subsequently restored by PGE2, but not by LTD4. A selective PGE2 receptor antagonist abolished the increased adhesion and the augmented alpha2beta1-integrin expression induced by both PGE2 and LTD4. Summarizing, the inflammatory mediator LTD4 regulates the adhesive properties and migration of the Caco-2 cell line by upregulating COX-2 and stimulating PGE2-induced expression of alpha2beta1-integrins. This suggests that inflammatory mediators such as LTD4 can be involved in the dissemination and survival of colon cancer cells.     

Vascular endothelial growth factor A (VEGF-A) induces endothelial and cancer cell migration through direct binding to integrin {alpha}9{beta}1: identification of a specific {alpha}9{beta}1 binding site

Integrin α9β1 mediates accelerated cell adhesion and migration through interactions with a number of diverse extracellular ligands. We have shown previously that it directly binds the vascular endothelial growth factors (VEGF) A, C, and D and contributes to VEGF-induced angiogenesis and lymphangiogenesis. Until now, the α9β1 binding site in VEGF has not been identified. Here, we report that the three-amino acid sequence, EYP, encoded by exon 3 of VEGF-A is essential for binding of VEGF to integrin α9β1 and induces adhesion and migration of endothelial and cancer cells. EYP is specific for α9β1 binding and neither requires nor activates VEGFR-2, the cognate receptor for VEGF-A. Following binding to EYP, integrin α9β1 transduces cell migration through direct activation of the integrin signaling intermediates Src and focal adhesion kinase. This interaction is biologically important because it mediates in vitro endothelial cell tube formation, wound healing, and cancer cell invasion. These novel findings identify EYP as a potential site for directed pharmacotherapy.     

Fibronectin and heparin binding domains of latent TGF-beta binding protein (LTBP)-4 mediate matrix targeting and cell adhesion

Latent transforming growth factor (TGF)-β binding proteins are extracellular matrix (ECM) proteins involved in the regulation of TGF-β sequestration and activation. In this study, we have identified binding domains in LTBP-4, which mediate matrix targeting and cell adhesion. LTBP-4 was found to possess heparin binding activity, especially in its N-terminal region. The C-terminal domain of LTBP-4 supported fibroblast adhesion, a property reduced by soluble heparin. In addition, we found that LTBP-4 binds directly to fibronectin (FN), which was indispensable for the matrix assembly of LTBP-4. The FN binding sites were also located in the N-terminal region. Interestingly, heparin was able to reduce the binding of LTBP-4 to FN. In fibroblast cultures, LTBP-4 colocalized first with FN and subsequently with fibrillin-1, pointing to a role for FN in the early assembly of LTBP-4. In FN −/− fibroblasts, LTBP-mediated ECM targeting was disturbed, resulting in increased TGF-β activity. These results revealed new molecular interactions which are evidently important for the ECM targeting, but which also are evidence of novel functions for LTBP-4 as an adhesion molecule.