Site-specific Phosphorylation of Kindlin-3 Protein Regulates Its Capacity to Control Cellular Responses Mediated by Integrin αIIbβ3

The contributions of integrins to cellular responses depend upon their activation, which is regulated by binding of proteins to their cytoplasmic tails. Kindlins are integrin cytoplasmic tail binding partners and are essential for optimal integrin activation, and kindlin-3 fulfills this role in hematopoietic cells. Here, we used human platelets and human erythroleukemia (HEL) cells, which express integrin α_IIbβ₃, to investigate whether phosphorylation of kindlin-3 regulates integrin activation. When HEL cells were stimulated with thrombopoietin or phorbol 12-myristate 13-acetate (PMA), α_IIbβ₃ became activated as evidenced by binding of an activation-specific monoclonal antibody and soluble fibrinogen, adherence and spreading on fibrinogen, colocalization of β₃ integrin and kindlin-3 in focal adhesions, and enhanced β₃ integrin-kindlin-3 association in immunoprecipitates. Kindlin-3 knockdown impaired adhesion and spreading on fibrinogen. Stimulation of HEL cells with agonists significantly increased kindlin-3 phosphorylation as detected by mass spectrometric sequencing. Thr⁴⁸² or Ser⁴⁸⁴ was identified as a phosphorylation site, which resides in a sequence not conserved in kindlin-1 or kindlin-2. These same residues were phosphorylated in kindlin-3 when platelets were stimulated with thrombin. When expressed in HEL cells, T482A/S484A kindlin-3 decreased soluble ligand binding and cell spreading on fibrinogen compared with wild-type kindlin-3. A membrane-permeable peptide containing residues 476–485 of kindlin-3 was introduced into HEL cells and platelets; adhesion and spreading of both cell types were blunted compared with a scrambled control peptide. These data identify a role of kindlin-3 phosphorylation in integrin β₃ activation and provide a basis for functional differences between kindlin-3 and the two other kindlin paralogs.     

A Role of Kindlin-3 in Integrin αMβ2 Outside-In Signaling and the Syk-Vav1-Rac1/Cdc42 Signaling Axis

Integrins mediate cell-cell and cell-extracellular matrix attachments. Integrins are signaling receptors because their cytoplasmic tails are docking sites for cytoskeletal and signaling proteins. Kindlins are a family of band 4.1-ezrin-radixin-moesin-containing intracellular proteins. Apart from regulating integrin ligand-binding affinity, recent evidence suggests that kindlins are involved in integrin outside-in signaling. Kindlin-3 is expressed in platelets, hematopoietic cells and endothelial cells. In humans, loss of kindlin-3 expression accounts for the rare autosomal disease leukocyte adhesion deficiency (LAD) type III that is characterized by bleeding disorders and defective recruitment of leukocytes into sites of infection. Studies have shown that the loss of kindlin-3 expression leads to poor ligand-binding properties of β1, β2 and β3 integrin subfamilies. The leukocyte-restricted β2 integrin subfamily comprises four members, namely αLβ2, αMβ2, αXβ2 and αDβ2. Integrin αMβ2 mediates leukocyte adhesion, phagocytosis, degranulation and it is involved in the maintenance of immune tolerance. Here we provide further evidence that kindlin-3 is required for integrin αMβ2-mediated cell adhesion and spreading using transfected K562 cells that expressed endogenous kindlin-3 but not β2 integrins. K562 stable cell line expressing si-RNA targeting kindlin-3, but not control-si-RNA, and transfected with constitutively activated integrin αMβ2N329S adhered and spread poorly on iC3b. We also show that kindlin-3 is required for the integrin αMβ2-Syk-Vav1 signaling axis that regulates Rac1 and Cdc42 activities. These findings reinforce a role for kindlin-3 in integrin outside-in signaling.     

A Role for the αvβ3 Integrin in the Transmigration of Monocytes

The β2 integrins and intercellular adhesion molecule-1 (ICAM-1) are important for monocyte migration through inflammatory endothelium. Here we demonstrate that the integrin αvβ3 is also a key player in this process. In an in vitro transendothelial migration assay, monocytes lacking β3 integrins revealed weak migratory ability, whereas monocytes expressing β3 integrins engaged in stronger migration. This migration could be partially blocked by antibodies against the integrin chains αL, β2, αv, or IAP, a protein functionally associated with αvβ3 integrin. Transfection of β3 integrin chain cDNA into monocytes lacking β3 integrins resulted in expression of the αvβ3 integrin and conferred on these cells an enhanced ability to transmigrate through cell monolayers expressing ICAM-1. These monocytes also engaged in αLβ2-dependent locomotion on recombinant ICAM-1 which was enhanced by αvβ3 integrin occupancy. Antibodies against IAP were able to revert this αvβ3 integrin-dependent cell locomotion to control levels. Finally, adhesion assays revealed that occupancy of αvβ3 integrin could decrease monocyte binding to ICAM-1.In conclusion, we show that αvβ3 integrin modulates αLβ2 integrin-dependent monocyte adhesion to and migration on ICAM-1. This could represent a novel mechanism to promote monocyte motility on vascular ICAM-1 and initiate subsequent transendothelial migration.     

B-Raf Regulation of Integrin α4β1-mediated Resistance to Shear Stress through Changes in Cell Spreading and Cytoskeletal Association in T Cells

The regulation of integrin-mediated adhesion is of vital importance to adaptive and innate immunity. Integrins are versatile proteins and mediate T cell migration and trafficking by binding to extracellular matrix or other cells as well as initiating intracellular signaling cascades promoting survival or activation. The MAPK pathway is known to be downstream from integrins and to regulate survival, differentiation, and motility. However, secondary roles for canonical MAPK pathway members are being discovered. We show that chemical inhibition of RAF by sorafenib or shRNA-mediated knockdown of B-Raf reduces T cell resistance to shear stress to α4β1 integrin ligands vascular cell adhesion molecule 1 (VCAM-1) and fibronectin, whereas inhibition of MEK/ERK by U0126 had no effect. Microscopy showed that RAF inhibition leads to significant inhibition of T cell spreading on VCAM-1. The association of α4β1 integrin with the actin cytoskeleton was shown to be dependent on B-Raf activity or expression, whereas α4β1 integrin affinity for soluble VCAM-1 was not. These effects were shown to be specific for α4β1 integrin and not other integrins, such as α5β1 or LFA-1, or a variety of membrane proteins. We demonstrate a novel role for B-Raf in the selective regulation of α4β1 integrin-mediated adhesion.     

Gln-362 of Angiopoietin-2 Mediates Migration of Tumor and Endothelial Cells through Association with α5β1 Integrin

Angiopoietin-2 (Ang-2) not only regulates angiogenesis by binding to its well known receptor Tie2 on endothelial cells but also controls sprouting of Tie2-negative angiogenic endothelial cells and invasion of Tie2-negative non-endothelial cells by binding to integrins. However, the molecular mechanism of the Ang-2/integrin association has been unclear. In this study, we found that the Gln-362 residue of Ang-2 was essential for binding to α5β1 integrin. A Q362E Ang-2 mutant, which still bound to Tie2, failed to associate with α5β1 integrin and was unable to activate the integrin downstream signaling of focal adhesion kinase. In addition, unlike wild-type Ang-2, the Q362E Ang-2 mutant was defective in mediating invasion of Tie2-negative glioma or Tie2-positive endothelial cells. Furthermore, the tailpiece domain of the α5 subunit in α5β1 integrin was critical for binding to Ang-2. Taken together, these results provide a novel insight into the mechanism of integrin regulation by Ang-2, which contributes to tumor invasion and endothelial cell migration in a Tie2-independent manner.     

Variation in One Residue Associated with the Metal Ion-Dependent Adhesion Site Regulates αIIbβ3 Integrin Ligand Binding Affinity

The Asp of the RGD motif of the ligand coordinates with the β I domain metal ion dependent adhesion site (MIDAS) divalent cation, emphasizing the importance of the MIDAS in ligand binding. There appears to be two distinct groups of integrins that differ in their ligand binding affinity and adhesion ability. These differences may be due to a specific residue associated with the MIDAS, particularly the β3 residue Ala²⁵² and corresponding Ala in the β1 integrin compared to the analogous Asp residue in the β2 and β7 integrins. Interestingly, mutations in the adjacent to MIDAS (ADMIDAS) of integrins α4β7 and αLβ2 increased the binding and adhesion abilities compared to the wild-type, while the same mutations in the α2β1, α5β1, αVβ3, and αIIbβ3 integrins demonstrated decreased ligand binding and adhesion. We introduced a mutation in the αIIbβ3 to convert this MIDAS associated Ala²⁵² to Asp. By combination of this mutant with mutations of one or two ADMIDAS residues, we studied the effects of this residue on ligand binding and adhesion. Then, we performed molecular dynamics simulations on the wild-type and mutant αIIbβ3 integrin β I domains, and investigated the dynamics of metal ion binding sites in different integrin-RGD complexes. We found that the tendency of calculated binding free energies was in excellent agreement with the experimental results, suggesting that the variation in this MIDAS associated residue accounts for the differences in ligand binding and adhesion among different integrins, and it accounts for the conflicting results of ADMIDAS mutations within different integrins. This study sheds more light on the role of the MIDAS associated residue pertaining to ligand binding and adhesion and suggests that this residue may play a pivotal role in integrin-mediated cell rolling and firm adhesion.     

Discoidin Domain Receptors Promote α1β1- and α2β1-Integrin Mediated Cell Adhesion to Collagen by Enhancing Integrin Activation

The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that bind to and are activated by collagens. Similar to collagen-binding β1 integrins, the DDRs bind to specific motifs within the collagen triple helix. However, these two types of collagen receptors recognize distinct collagen sequences. While GVMGFO (O is hydroxyproline) functions as a major DDR binding motif in fibrillar collagens, integrins bind to sequences containing Gxx’GEx”. The DDRs are thought to regulate cell adhesion, but their roles have hitherto only been studied indirectly. In this study we used synthetic triple-helical collagen-derived peptides that incorporate either the DDR-selective GVMGFO motif or integrin-selective motifs, such as GxOGER and GLOGEN, in order to selectively target either type of receptor and resolve their contributions to cell adhesion. Our data using HEK293 cells show that while cell adhesion to collagen I was completely inhibited by anti-integrin blocking antibodies, the DDRs could mediate cell attachment to the GVMGFO motif in an integrin-independent manner. Cell binding to GVMGFO was independent of DDR receptor signalling and occurred with limited cell spreading, indicating that the DDRs do not mediate firm adhesion. However, blocking the interaction of DDR-expressing cells with collagen I via the GVMGFO site diminished cell adhesion, suggesting that the DDRs positively modulate integrin-mediated cell adhesion. Indeed, overexpression of the DDRs or activation of the DDRs by the GVMGFO ligand promoted α1β1 and α2β1 integrin-mediated cell adhesion to medium- and low-affinity integrin ligands without regulating the cell surface expression levels of α1β1 or α2β1. Our data thus demonstrate an adhesion-promoting role of the DDRs, whereby overexpression and/or activation of the DDRs leads to enhanced integrin-mediated cell adhesion as a result of higher integrin activation state.     

C-terminal COOH of Integrin β1 Is Necessary for β1 Association with the Kindlin-2 Adapter Protein

Protein-protein interactions are driving forces in cellular processes. As a prime example, transmembrane integrins link extracellular matrix and intracellular proteins, resulting in bidirectional signaling that regulates cell migration, proliferation, differentiation, and survival. Here we provide the first evidence that interaction between the integrin β1 cytoplasmic tail and kindlin-2, a member of a family of adapters implicated in human disease pathogenesis, is mainly governed by the β1 C-terminal carboxylate moiety and is required for laterality organ development in zebrafish. Affinity measurements indicate that this unusual protein-protein interaction mode is coordinated by a putative carboxylate-binding motif in the kindlin-2 FERM subdomain F3. Contrary to the C terminus of proteins that engage PDZ domains, the C-terminal three residues of β1, per se, do not contribute to kindlin-2 binding or to laterality organ development. Thus, by employing zebrafish as an in situ physiological tool to correlate protein structure and function, we have discovered an unexpected association chemistry between an integrin and a key adapter involved in integrin signaling.     

Regulation of Integrin β1 Recycling to Lipid Rafts by Rab1a to Promote Cell Migration

Rab1a is a member of the Rab family of small GTPases with a well characterized function in the regulation of vesicle trafficking from the endoplasmic reticulum to the Golgi apparatus and within Golgi compartments. The integrin family heterodimeric transmembrane proteins serve as major receptors for extracellular matrix proteins, which play essential roles in cell adhesion and migration. Although effects on intracellular trafficking of integrins or other key cargos by Rab1a could influence cell migration, the regulatory mechanisms linking Rab1a to cell migration are not well understood. Here, we report identification of Rab1a as a novel regulator of cell migration using an unbiased RNAi screen targeting GTPases. Inhibition of Rab1a reduced integrin-mediated cell adhesion and spreading on fibronectins, reduced integrin β1 localization to lipid rafts, and decreased recycling of integrin β1 to the plasma membrane. Analysis of Rab1a effector molecules showed that p115 mediated Rab1a regulation of integrin recycling and lipid raft localization in cell migration. Taken together, these results suggest a novel function for Rab1a in the regulation of cell migration through controlling integrin β1 recycling and localization to lipid rafts via a specific downstream effector pathway.     

Modulation of Integrin Activity is Vital for Morphogenesis

Cells can vary their adhesive properties by modulating the affinity of integrin receptors. The activation and inactivation of integrins by inside-out mechanisms acting on the cytoplasmic domains of the integrin subunits has been demonstrated in platelets, lymphocytes, and keratinocytes. We show that in the embryo, normal morphogenesis requires the α subunit cytoplasmic domain to control integrin adhesion at the right times and places. PS2 integrin (α_PS2β_PS) adhesion is normally restricted to the muscle termini, where it is required for attaching the muscles to the ends of other muscles and to specialized epidermal cells. Replacing the wild-type α_PS2 with mutant forms containing cytoplasmic domain deletions results in the rescue of the majority of defects associated with the absence of the α_PS2 subunit, however, the mutant PS2 integrins are excessively active. Muscles containing these mutant integrins make extra muscle attachments at aberrant positions on the muscle surface, disrupting the muscle pattern and causing embryonic lethality. A gain- of-function phenotype is not observed in the visceral mesoderm, showing that regulation of integrin activity is tissue-specific. These results suggest that the α_PS2 subunit cytoplasmic domain is required for inside-out regulation of integrin affinity, as has been seen with the integrin α_IIbβ₃.     

Clustering of Syndecan-4 and Integrin β1 by Laminin α3 Chain–derived Peptide Promotes Keratinocyte Migration

Syndecans function as receptors for extracellular matrix (ECM) with integrins in cell spreading. However, the molecular mechanism of their specific involvement in cell migration or in wound healing has not been elucidated yet. Here, we report that a synthetic peptide, PEP75, which contains the syndecan-binding sequence of the laminin α3LG4 module, induces keratinocyte migration in in vitro and in vivo. Soluble PEP75 induced the clustering of syndecan-4 and conformation-modified integrin β1 colocalized with syndecan-4 in soluble PEP75-induced clusters. Treatment of cells in solution with PEP75 resulted in the exposure of the P4G11 antibody epitope of integrin β1 in immunostaining as well as in flow cytometry and augmented integrin β1–dependent cell adhesion to ECM. Pulldown assays demonstrated that PEP75 bound to syndecan-4, but not to integrin β1. A siRNA study revealed a role for syndecan-4 in PEP75-induced up-regulation of P4G11 antibody binding and migration of HaCaT cells. We conclude that binding of soluble PEP75 to syndecan-4 induces the coupling of integrin β1, which is associated with integrin β1-conformational changes and activation, and leads to keratinocyte migration. To activate integrin function through syndecans could be a novel therapeutic approach for chronic wound.     

Kindlins: essential regulators of integrin signalling and cell-matrix adhesion

Integrin-mediated cell-ECM (extracellular matrix) adhesion is a fundamental process that controls cell behaviour. For correct cell-ECM adhesion, both the ligand-binding affinity and the spatial organization of integrins must be precisely controlled; how integrins are regulated, however, is not completely understood. Kindlins constitute a family of evolutionarily conserved cytoplasmic components of cell-ECM adhesions that bind to beta-integrin cytoplasmic tails directly and cooperate with talin in integrin activation. In addition, kindlins interact with many components of cell-ECM adhesions--such as migfilin and integrin-linked kinase--to promote cytoskeletal reorganization. Loss of kindlins causes severe defects in integrin signalling, cell-ECM adhesion and cytoskeletal organization, resulting in early embryonic lethality (kindlin-2), postnatal lethality (kindlin-3) and Kindler syndrome (kindlin-1). It is therefore clear that kindlins, together with several other integrin-proximal proteins, are essential for integrin signalling and cell-ECM adhesion regulation.     

The syndecan-1 ectodomain regulates alphavbeta3 integrin activity in human mammary carcinoma cells

The α_vβ₃ integrin participates in cell morphogenesis, growth factor signaling, and cell survival. Activation of the integrin is central to these processes and is influenced by specific ECM components, which engage both integrins and syndecans. This paper demonstrates that the α_vβ₃ integrin and syndecan-1 (S1) are functionally coupled. The integrin is dependent on the syndecan to become activated and to mediate signals required for MDA-MB-231 and MDA-MB-435 human mammary carcinoma cell spreading on vitronectin or S1-specific antibody. Coupling of the syndecan to α_vβ₃ requires the S1 ectodomain (ED), as ectopic expression of glycosylphosphatidylinositol-linked S1ED enhances α_vβ₃ recognition of vitronectin; and treatments that target this domain, including competition with recombinant S1ED protein or anti-S1ED antibodies, mutation of the S1ED, or down-regulation of S1 expression by small-interfering RNAs, disrupt α_vβ₃-dependent cell spreading and migration. Thus, S1 is likely to be a critical regulator of many cellular behaviors that depend on activated α_vβ₃ integrins.     

Kindlin-2 Tyrosine Phosphorylation and Interaction with Src Serve as a Regulatable Switch in the Integrin Outside-in Signaling Circuit

Integrin-mediated cell-extracellular matrix (ECM) adhesion is critical for control of intracellular signaling; however, the mechanisms underlying this “outside-in” signaling are incompletely understood. Here we show that depletion of kindlin-2 impairs integrin outside-in signaling. Kindlin-2 is tyrosine-phosphorylated upon cell-ECM adhesion. Furthermore, kindlin-2 binds Src in a cell-ECM adhesion-regulatable fashion. At the molecular level, the kindlin-2·Src interaction is mediated by the kindlin-2 F0 and the Src SH2 and SH3 domains. Src activation increases kindlin-2 tyrosine phosphorylation and the kindlin-2·Src interaction. Conversely, inhibition of Src reduces kindlin-2 tyrosine phosphorylation and diminishes the kindlin-2·Src interaction. Finally, disruption of the kindlin-2·Src interaction, unlike depletion of kindlin-2, impairs neither cell-ECM adhesion nor cell-ECM adhesion-induced focal adhesion kinase Tyr-397 phosphorylation. However, it markedly inhibits cell-ECM adhesion-induced paxillin tyrosine phosphorylation, cell migration, and proliferation. These results suggest that kindlin-2 tyrosine phosphorylation and interaction with Src serve as a regulatable switch downstream of focal adhesion kinase in the integrin outside-in signaling circuit, relaying signals from cell-ECM adhesion to paxillin that control cell migration and proliferation.     

Integrin and Cadherin Synergy Regulates Contact Inhibition of Migration and Motile Activity

Integrin receptors play a central role in cell migration through their roles as adhesive receptors for both other cells and extracellular matrix components. In this study, we demonstrate that integrin and cadherin receptors coordinately regulate contact-mediated inhibition of cell migration. In addition to promoting proliferation (Sastry, S., M. Lakonishok, D. Thomas, J. Muschler, and A. Horwitz. 1996. J. Cell Biol. 133:169–184), ectopic expression of the α5 integrin in cultures of primary quail myoblasts promotes a striking contact-mediated inhibition of cell migration. Myoblasts ectopically expressing α5 integrin (α5 myoblasts) move normally when not in contact, but upon contact, they show inhibition of migration and motile activity (i.e., extension and retraction of membrane protrusions). As a consequence, these cells tend to grow in aggregates and do not migrate to close a wound. This phenotype is also seen with ectopic expression of β1 integrin, paxillin, or activated FAK (CD2 FAK) and therefore appears to result from enhanced integrin-mediated signaling. The contact inhibition observed in the α5 myoblasts is mediated by N-cadherin, whose expression is upregulated more than fivefold. Perturbation studies using low calcium conditions, antibody inhibition, and ectopic expression of wild-type and mutant N-cadherins all implicate N-cadherin in the contact inhibition of migration. Ectopic expression of N-cadherin also produces cells that show inhibited migration upon contact; however, they do not show suppressed motile activity, suggesting that integrins and cadherins coordinately regulate motile activity. These observations have potential importance to normal and pathologic processes during embryonic development and tumor metastasis.     

Gα12 Inhibits α2β1 Integrin–mediated Madin-Darby Canine Kidney Cell Attachment and Migration on Collagen-I and Blocks Tubulogenesis

Regulation of epithelial cell attachment and migration are essential for normal development and maintenance of numerous tissues. G proteins and integrins are critical signaling proteins regulating these processes, yet in polarized cells little is known about the interaction of these pathways. Herein, we demonstrate that Gα12 inhibits interaction of MDCK cells with collagen-I, the major ligand for α2β1 integrin. Activating Gα12 (QL point mutation or stimulating endogenous Gα12 with thrombin) inhibited focal adhesions and lamellipodia formation and led to impaired cell migration. Consistent with Gα12-regulated attachment to collagen-I, Gα12-silenced MDCK cells revealed a more adherent phenotype. Inhibiting Rho kinase completely restored normal attachment in Gα12-activated cells, and there was partial recovery with inhibition of Src and protein phosphatase pathways. Gα12 activation led to decreased phosphorylation of focal adhesion kinase and paxillin with displacement of α2 integrin from the focal adhesion protein complex. Using the MDCK cell 3D-tubulogenesis assay, activated Gα12 inhibited tubulogenesis and led to the formation of cyst-like structures. Furthermore, Gα12-silenced MDCK cells were resistant to thrombin-stimulated cyst development. Taken together, these studies provide direct evidence for Gα12–integrin regulation of epithelial cell spreading and migration necessary for normal tubulogenesis.     

Calcium and Calmodulin-dependent Serine/Threonine Protein Kinase Type II (CaMKII)-mediated Intramolecular Opening of Integrin Cytoplasmic Domain-associated Protein-1 (ICAP-1α) Negatively Regulates β1 Integrins

Focal adhesion turnover during cell migration is an integrated cyclic process requiring tight regulation of integrin function. Interaction of integrin with its ligand depends on its activation state, which is regulated by the direct recruitment of proteins onto the β integrin chain cytoplasmic domain. We previously reported that ICAP-1α, a specific cytoplasmic partner of β1A integrins, limits both talin and kindlin interaction with β1 integrin, thereby restraining focal adhesion assembly. Here we provide evidence that the calcium and calmodulin-dependent serine/threonine protein kinase type II (CaMKII) is an important regulator of ICAP-1α for controlling focal adhesion dynamics. CaMKII directly phosphorylates ICAP-1α and disrupts an intramolecular interaction between the N- and the C-terminal domains of ICAP-1α, unmasking the PTB domain, thereby permitting ICAP-1α binding onto the β1 integrin tail. ICAP-1α direct interaction with the β1 integrin tail and the modulation of β1 integrin affinity state are required for down-regulating focal adhesion assembly. Our results point to a molecular mechanism for the phosphorylation-dependent control of ICAP-1α function by CaMKII, allowing the dynamic control of β1 integrin activation and cell adhesion.     

Integrin Cross Talk: Activation of Lymphocyte Function-associated Antigen-1 on Human T Cells Alters α4β1- and α5β1-mediated Function

A regulated order of adhesion events directs leukocytes from the vascular compartment into injured tissues in response to inflammatory stimuli. We show that on human T cells, the interaction of the β2 integrin leucocyte function–associated antigen-1 (LFA-1) with its ligand intercellular adhesion molecule-1 (ICAM-1) will decrease adhesion mediated by α4β1 and, to a lesser extent, α5β1. Similar inhibition is also seen when T cells are exposed to mAb 24, which stabilizes LFA-1 in an active state after triggering integrin function through divalent cation Mg²⁺, PdBu, or T cell receptor/ CD3 complex (TCR/CD3) cross-linking. Such cross talk decreases α4β1 integrin–mediated binding of T cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In contrast, ligand occupancy or prolonged activation of β1 integrin has no effect on LFA-1 adhesion to ICAM-1. We also show that T cell migration across fibronectin, unlike adhesion, is mediated solely by α5β1, and is increased when the α4β1-mediated component of fibronectin adhesion is decreased either by cross talk or the use of α4-blocking mAb. The ability of mAb 24 Fab′ fragments to induce cross talk without cross-linking LFA-1 suggests signal transduction through the active integrin. These data provide the first direct evidence for cross talk between LFA-1 and β1 integrins on T cells. Together, these findings imply that activation of LFA-1 on the extravasating T cell will decrease the binding to VCAM-1 while enhancing the subsequent migration on fibronectin. This sequence of events provides a further level of complexity to the coordination of T cell integrins, whose sequential but overlapping roles are essential for transmigration.     

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.