The Novel α4B Murine α4 Integrin Protein Splicing Variant Inhibits α4 Protein-dependent Cell Adhesion

Integrins affect the motility of multiple cell types to control cell survival, growth, or differentiation, which are mediated by cell-cell and cell-extracellular matrix interactions. We reported previously that the α9 integrin splicing variant, SFα9, promotes WT α9 integrin-dependent adhesion. In this study, we introduced a new murine α4 integrin splicing variant, α4B, which has a novel short cytoplasmic tail. In inflamed tissues, the expression of α4B, as well as WT α4 integrin, was up-regulated. Cells expressing α4B specifically bound to VCAM-1 but not other α4 integrin ligands, such as fibronectin CS1 or osteopontin. The binding of cells expressing WT α4 integrin to α4 integrin ligands is inhibited by coexpression of α4B. Knockdown of α4B in metastatic melanoma cell lines results in a significant increase in lung metastasis. Expression levels of WT α4 integrin are unaltered by α4B, with α4B acting as a regulatory subunit for WT α4 integrin by a dominant-negative effect or inhibiting α4 integrin activation.     

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.     

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.     

p21-activated Kinase 4 Phosphorylation of Integrin β5 Ser-759 and Ser-762 Regulates Cell Migration

Modulation of integrin αvβ5 regulates vascular permeability, angiogenesis, and tumor dissemination. In addition, we previously found a role for p21-activated kinase 4 (PAK4) in selective regulation of integrin αvβ5-mediated cell motility (Zhang, H., Li, Z., Viklund, E. K., and Strömblad, S. (2002) J. Cell Biol. 158, 1287–1297). This report focuses on the molecular mechanisms of this regulation. We here identified a unique PAK4-binding membrane-proximal integrin β5-SERS-motif involved in controlling cell attachment and migration. We also mapped the integrin β5-binding site within PAK4. We found that PAK4 binding to integrin β5 was not sufficient to promote cell migration, but that PAK4 kinase activity was required for PAK4 promotion of cell motility. Importantly, PAK4 specifically phosphorylated the integrin β5 subunit at Ser-759 and Ser-762 within the β5-SERS-motif. Point mutation of these two serine residues abolished the PAK4-induced cell migration, indicating a functional role for these phosphorylations in migration. Our results may give important leads to the functional regulation of integrin αvβ5, with implications for vascular permeability, angiogenesis, and cancer dissemination.     

Differentiation of Human Skeletal Muscle Stem Cells into Odontoblasts Is Dependent on Induction of α1 Integrin Expression

Skeletal muscle stem cells represent an abundant source of autologous cells with potential for regenerative medicine that can be directed to differentiate into multiple lineages including osteoblasts and adipocytes. In the current study, we found that α7 integrin-positive human skeletal muscle stem cells (α7⁺hSMSCs) could differentiate into the odontoblast lineage under specific inductive conditions in response to bone morphogenetic protein-4 (BMP-4). Cell aggregates of FACS-harvested α7⁺hSMSCs were treated in suspension with retinoic acid followed by culture on a gelatin scaffold in the presence of BMP-4. Following this protocol, α7⁺hSMSCs were induced to down-regulate myogenic genes (MYOD and α7 integrin) and up-regulate odontogenic markers including dentin sialophosphoprotein, matrix metalloproteinase-20 (enamelysin), dentin sialoprotein, and alkaline phosphatase but not osteoblastic genes (osteopontin and osteocalcin). Following retinoic acid and gelatin scaffold/BMP-4 treatment, there was a coordinated switch in the integrin expression profile that paralleled odontoblastic differentiation where α1β1 integrin was strongly up-regulated with the attenuation of muscle-specific α7β1 integrin expression. Interestingly, using siRNA knockdown strategies revealed that the differentiation-related expression of the α1 integrin receptor positively regulates the expression of the odontoblastic markers dentin sialophosphoprotein and matrix metalloproteinase-20. These results strongly suggest that the differentiation of α7⁺hSMSCs along the odontogenic lineage is dependent on the concurrent expression of α1 integrin.     

A Highlights from MBoC Selection: CD47 plays a critical role in T-cell recruitment by regulation of LFA-1 and VLA-4 integrin adhesive functions

CD47 plays an important but incompletely understood role in the innate and adaptive immune responses. CD47, also called integrin-associated protein, has been demonstrated to associate in cis with β1 and β3 integrins. Here we test the hypothesis that CD47 regulates adhesive functions of T-cell α4β1 (VLA-4) and αLβ2 (LFA-1) in in vivo and in vitro models of inflammation. Intravital microscopy studies reveal that CD47^−/− Th1 cells exhibit reduced interactions with wild-type (WT) inflamed cremaster muscle microvessels. Similarly, murine CD47^−/− Th1 cells, as compared with WT, showed defects in adhesion and transmigration across tumor necrosis factor-α (TNF-α)–activated murine endothelium and in adhesion to immobilized intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion protein 1 (VCAM-1) under flow conditions. Human Jurkat T-cells lacking CD47 also showed reduced adhesion to TNF-α–activated endothelium and ICAM-1 and VCAM-1. In cis interactions between Jurkat T-cell β2 integrins and CD47 were detected by fluorescence lifetime imaging microscopy. Unexpectedly, Jurkat CD47 null cells exhibited a striking defect in β1 and β2 integrin activation in response to Mn²⁺ or Mg²⁺/ethylene glycol tetraacetic acid treatment. Our results demonstrate that CD47 associates with β2 integrins and is necessary to induce high-affinity conformations of LFA-1 and VLA-4 that recognize their endothelial cell ligands and support leukocyte adhesion and transendothelial migration.     

Cytoplasmic Domain Interactions of Syndecan-1 and Syndecan-4 with α6β4 Integrin Mediate Human Epidermal Growth Factor Receptor (HER1 and HER2)-dependent Motility and Survival

Epithelial cells are highly dependent during wound healing and tumorigenesis on the α6β4 integrin and its association with receptor tyrosine kinases. Previous work showed that phosphorylation of the β4 subunit upon matrix engagement depends on the matrix receptor syndecan (Sdc)-1 engaging the cytoplasmic domain of the β4 integrin and coupling of the integrin to human epidermal growth factor receptor-2 (HER2). In this study, HER2-dependent migration activated by matrix engagement is compared with migration stimulated by EGF. We find that whereas HER2-dependent migration depends on Sdc1, EGF-dependent migration depends on a complex consisting of human epidermal growth factor receptor-1 (HER1, commonly known as EGFR), α6β4, and Sdc4. The two syndecans recognize distinct sites at the extreme C terminus of the β4 integrin cytoplasmic domain. The binding motif in Sdc1 is QEEXYX, composed in part by its syndecan-specific variable (V) region and in part by the second conserved (C2) region that it shares with other syndecans. A cell-penetrating peptide containing this sequence competes for HER2-dependent epithelial migration and carcinoma survival, although it is without effect on the EGFR-stimulated mechanism. β4 mutants bearing mutations specific for Sdc1 and Sdc4 recognition act as dominant negative mutants to block cell spreading or cell migration that depends on HER2 or EGFR, respectively. The interaction of the α6β4 integrin with the syndecans appears critical for it to be utilized as a signaling platform; migration depends on α3β1 integrin binding to laminin 332 (LN332; also known as laminin 5), whereas antibodies that block α6β4 binding are without effect. These findings indicate that specific syndecan family members are likely to have key roles in α6β4 integrin activation by receptor tyrosine kinases.     

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.     

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.     

A Novel CD44-binding Peptide from the Pro-Matrix Metalloproteinase-9 Hemopexin Domain Impairs Adhesion and Migration of Chronic Lymphocytic Leukemia (CLL) Cells

(pro)MMP-9 binds to CLL cells through the PEX9 domain and contributes to CLL progression. To biochemically characterize this interaction and identify potential therapeutic targets, we prepared GST-PEX9 forms containing structural blades B1B2 or B3B4. We recently described a sequence in blade B4 (P3 sequence) that bound α4β1 integrin and partially impaired cell adhesion and migration. We have now studied the possible contribution of the B1B2 region to cell interaction with PEX9. CLL cells bound to GST-B1B2 and CD44 was the primary receptor. GST-B1B2 inhibited CLL cell migration as effectively as GST-B3B4. Overlapping synthetic peptides spanning the B1B2 region identified the sequence FDAIAEIGNQLYLFKDGKYW, present in B1 and contained in peptide P6, as the most effective site. P6 inhibited cell adhesion to PEX9 in a dose-dependent manner and with an IC₅₀ value of 90 μm. P6 also inhibited cell adhesion to hyaluronan but had no effect on adhesion to VCAM-1 (α4β1 integrin ligand), confirming its specific interaction with CD44. Spatial localization analyses mapped P6 to the central cavity of PEX9, in close proximity to the previously identified P3 sequence. Both P6 and P3 equally impaired cell adhesion to (pro)MMP-9. Moreover, P6 synergistically cooperated with P3, resulting in complete inhibition of CLL cell binding to PEX9, chemotaxis, and transendothelial migration. Thus, P6 is a novel sequence in PEX9 involved in cell-PEX9/(pro)MMP-9 binding by interacting with CD44. Targeting both sites, P6 and P3, should efficiently prevent (pro)MMP-9 binding to CLL cells and its pathological consequences.     

Matrix Metalloproteinase-2 Cleavage of the β1 Integrin Ectodomain Facilitates Colon Cancer Cell Motility

Cancer cell invasion is a key element in metastasis that requires integrins for adhesion/de-adhesion, as well as matrix metalloproteinases (MMPs) for focalized proteolysis. Herein we show that MMP-2 is up-regulated in resected colorectal tumors and degrades β1 integrins with the release of fragments containing the β1 I-domain. The β1 cleavage pattern is similar to that produced by digestion of α5β1 and α2β1 with MMP-2. Two such fragments, at 25 and 75 kDa, were identified after immunoprecipitation, with monoclonal antibody BD610468 reacting with the NH₂-terminal I-like ectodomain followed by SDS-PAGE and microsequencing using electrospray (ISI-Q-TOF-Micromass) spectrometry. Cleavage of the β1 integrin can be abolished by inhibition of MMP-2 activity; it can be induced by up-regulation of MMP-2 expression, as exemplified by HT29 colon cancer cells transfected with pCMV6-XL5-MMP-2. Co-immunoprecipitation studies of colon cancer cells showed that the β1 integrin subunit is associated with MMP-2. The MMP-2-mediated shedding of the I-like domain from β1 integrins resulted in decreased adhesion of colon cancer cells to collagen and fibronectin, thus abolishing their receptivity. Furthermore, such cells showed enhanced motility as evaluated by a “wound healing-like” assay and time-lapse microscopy, indicating their increased invasiveness. Altogether, our data demonstrate that MMP-2 amplifies the motility of colon cancer cells, not only by digesting the extracellular matrix components in the vicinity of cancer cells but also by inactivating their major β1 integrin receptors.     

The Integrin β1 Subunit Regulates Paracellular Permeability of Kidney Proximal Tubule Cells

Epithelial cells lining the gastrointestinal tract and kidney have different abilities to facilitate paracellular and transcellular transport of water and solutes. In the kidney, the proximal tubule allows both transcellular and paracellular transport, while the collecting duct primarily facilitates transcellular transport. The claudins and E-cadherin are major structural and functional components regulating paracellular transport. In this study we present the novel finding that the transmembrane matrix receptors, integrins, play a role in regulating paracellular transport of renal proximal tubule cells. Deleting the integrin β1 subunit in these cells converts them from a “loose” epithelium, characterized by low expression of E-cadherin and claudin-7 and high expression of claudin-2, to a “tight” epithelium with increased E-cadherin and claudin-7 expression and decreased claudin-2 expression. This effect is mediated by the integrin β1 cytoplasmic tail and does not entail β1 heterodimerization with an α-subunit or its localization to the cell surface. In addition, we demonstrate that deleting the β1 subunit in the proximal tubule of the kidney results in a major urine-concentrating defect. Thus, the integrin β1 tail plays a key role in regulating the composition and function of tight and adherens junctions that define paracellular transport properties of terminally differentiated renal proximal tubule epithelial cells.     

How Natalizumab Binds and Antagonizes α4 Integrins

Natalizumab antibody to α₄-integrins is used in therapy of multiple sclerosis and Crohn''s disease. A crystal structure of the Fab bound to an α₄ integrin β-propeller and thigh domain fragment shows that natalizumab recognizes human-mouse differences on the circumference of the β-propeller domain. The epitope is adjacent to but outside of a ligand-binding groove formed at the interface with the β-subunit βI domain and shows no difference in structure when bound to Fab. Competition between Fab and the ligand vascular cell adhesion molecule (VCAM) for binding to cell surface α₄β₁ shows noncompetitive antagonism. In agreement, VCAM docking models suggest that binding of domain 1 of VCAM to α₄-integrins is unimpeded by the Fab, and that bound Fab requires a change in orientation between domains 1 and 2 of VCAM for binding to α₄β₁. Mapping of species-specific differences onto α₄β₁ and α₄β₇ shows that their ligand-binding sites are highly conserved. Skewing away from these conserved regions of the epitopes recognized by current therapeutic function-blocking antibodies has resulted in previously unanticipated mechanisms of action.     

Integrin α2β1 Is a Receptor for the Cartilage Matrix Protein Chondroadherin

Chondroadherin (the 36-kD protein) is a leucine-rich, cartilage matrix protein known to mediate adhesion of isolated chondrocytes. In the present study we investigated cell surface proteins involved in the interaction of cells with chondroadherin in cell adhesion and by affinity purification. Adhesion of bovine articular chondrocytes to chondroadherin-coated dishes was dependent on Mg²⁺ or Mn²⁺ but not Ca²⁺. Adhesion was partially inhibited by an antibody recognizing β1 integrin subunit. Chondroadherin-binding proteins from chondrocyte lysates were affinity purified on chondroadherin-Sepharose. The β1 integrin antibody immunoprecipitated two proteins with molecular mass ~110 and 140 kD (nonreduced) from the EDTA-eluted material. These results indicate that a β1 integrin on chondrocytes interacts with chondroadherin. To identify the α integrin subunit(s) involved in interaction of cells with the protein, we affinity purified chondroadherin-binding membrane proteins from human fibroblasts. Immunoprecipitation of the EDTA-eluted material from the affinity column identified α2β1 as a chondroadherin-binding integrin. These results are in agreement with cell adhesion experiments where antibodies against the integrin subunit α2 partially inhibited adhesion of human fibroblast and human chondrocytes to chondroadherin. Since α2β1 also is a receptor for collagen type II, we tested the ability of different antibodies against the α2 subunit to inhibit adhesion of T47D cells to collagen type II and chondroadherin. The results suggested that adhesion to collagen type II and chondroadherin involves similar or nearby sites on the α2β1 integrin. Although α2β1 is a receptor for both collagen type II and chondroadherin, only adhesion of cells to collagen type II was found to mediate spreading.     

Proinflammatory Secreted Phospholipase A2 Type IIA (sPLA-IIA) Induces Integrin Activation through Direct Binding to a Newly Identified Binding Site (Site 2) in Integrins αvβ3, α4β1, and α5β1

Integrins are activated by signaling from inside the cell (inside-out signaling) through global conformational changes of integrins. We recently discovered that fractalkine activates integrins in the absence of CX3CR1 through the direct binding of fractalkine to a ligand-binding site in the integrin headpiece (site 2) that is distinct from the classical RGD-binding site (site 1). We propose that fractalkine binding to the newly identified site 2 induces activation of site 1 though conformational changes (in an allosteric mechanism). We reasoned that site 2-mediated activation of integrins is not limited to fractalkine. Human secreted phospholipase A2 type IIA (sPLA2-IIA), a proinflammatory protein, binds to integrins αvβ3 and α4β1 (site 1), and this interaction initiates a signaling pathway that leads to cell proliferation and inflammation. Human sPLA2-IIA does not bind to M-type receptor very well. Here we describe that sPLA2-IIA directly activated purified soluble integrin αvβ3 and transmembrane αvβ3 on the cell surface. This activation did not require catalytic activity or M-type receptor. Docking simulation predicted that sPLA2-IIA binds to site 2 in the closed-headpiece of αvβ3. A peptide from site 2 of integrin β1 specifically bound to sPLA2-IIA and suppressed sPLA2-IIA-induced integrin activation. This suggests that sPLA2-IIA activates αvβ3 through binding to site 2. sPLA2-IIA also activated integrins α4β1 and α5β1 in a site 2-mediated manner. We recently identified small compounds that bind to sPLA2-IIA and suppress integrin-sPLA2-IIA interaction (e.g. compound 21 (Cmpd21)). Cmpd21 effectively suppressed sPLA2-IIA-induced integrin activation. These results define a novel mechanism of proinflammatory action of sPLA2-IIA through integrin activation.     

Tenascin-C-derived Peptide TNIIIA2 Highly Enhances Cell Survival and Platelet-derived Growth Factor (PDGF)-dependent Cell Proliferation through Potentiated and Sustained Activation of Integrin α5β1

Tenascin-C is an adhesion modulatory matrix protein that is highly expressed in tumors; however, its biochemical activity involved in tumorigenesis is not fully understood. On the other hand, increasing evidence indicates the importance of integrin α5β1 in cancer development. We previously demonstrated that tenascin-C harbors a functional site that can be released as a proadhesive peptide such as TNIIIA2. Peptide TNIIIA2 is capable of inducing activation of β1-integrins including α5β1 via syndecan-4. In this study the proadhesive effect of TNIIIA2 was characterized by potentiated and sustained activation of integrin α5β1. Based on this effect, TNIIIA2 rendered nontransformed fibroblasts (NIH3T3) resistant to serum deprivation-elicited anoikis through activation of the Akt/Bcl-2 pathway. Moreover, TNIIIA2 hyperstimulated PDGF-dependent proliferation of NIH3T3 by activating integrin α5β1. Tenascin-C, a parental protein of TNIIIA2, also stimulated PDGF-dependent proliferation, which was blocked by a matrix metalloproteinase-2/9 inhibitor and an anti-TNIIIA2 function-blocking antibody, suggesting proteolytic exposure of the proadhesive effect of TNIIIA2. Mechanistic analyses revealed that TNIIIA2 induced a lateral association of PDGF receptor β with the molecular complex of activated integrin α5β1 and syndecan-4 in the membrane microdomains enriched with cholesterol/caveolin-1, resulting in prolonged activation of PDGF receptor β and the subsequent Ras/mitogen-activated protein kinase pathway in a PDGF-dependent manner. Of note, TNIIIA2 induced continuous proliferation in NIH3T3 in an integrin α5β1-dependent manner even after they formed a confluent monolayer. Thus, it was proposed that tenascin-C might be involved in deregulated cell growth through potentiated and sustained activation of integrin α5β1 after exposure of the proadhesive effect of TNIIIA2.     

β1 Integrin-Mediated Adhesion Signalling Is Essential for Epidermal Progenitor Cell Expansion

Background

There is a major discrepancy between the in vitro and in vivo results regarding the role of β1 integrins in the maintenance of epidermal stem/progenitor cells. Studies of mice with skin-specific ablation of β1 integrins suggested that epidermis can form and be maintained in their absence, while in vitro data have shown a fundamental role for these adhesion receptors in stem/progenitor cell expansion and differentiation.

Methodology/Principal Findings

To elucidate this discrepancy we generated hypomorphic mice expressing reduced β1 integrin levels on keratinocytes that developed similar, but less severe defects than mice with β1-deficient keratinocytes. Surprisingly we found that upon aging these abnormalities attenuated due to a rapid expansion of cells, which escaped or compensated for the down-regulation of β1 integrin expression. A similar phenomenon was observed in aged mice with a complete, skin-specific ablation of the β1 integrin gene, where cells that escaped Cre-mediated recombination repopulated the mutant skin in a very short time period. The expansion of β1 integrin expressing keratinocytes was even further accelerated in situations of increased keratinocyte proliferation such as wound healing.

Conclusions/Significance

These data demonstrate that expression of β1 integrins is critically important for the expansion of epidermal progenitor cells to maintain epidermal homeostasis.     

The integrins

The integrins are a superfamily of cell adhesion receptors that bind to extracellular matrix ligands, cell-surface ligands, and soluble ligands. They are transmembrane αβ heterodimers and at least 18 α and eight β subunits are known in humans, generating 24 heterodimers. Members of this family have been found in mammals, chicken and zebrafish, as well as lower eukaryotes, including sponges, the nematode Caenorhabditis elegans (two α and one β subunits, generating two integrins) and the fruitfly Drosophila melanogaster (five α and one β, generating five integrins). The α and β subunits have distinct domain structures, with extracellular domains from each subunit contributing to the ligand-binding site of the heterodimer. The sequence arginine-glycine-aspartic acid (RGD) was identified as a general integrin-binding motif, but individual integrins are also specific for particular protein ligands. Immunologically important integrin ligands are the intercellular adhesion molecules (ICAMs), immunoglobulin superfamily members present on inflamed endothelium and antigen-presenting cells. On ligand binding, integrins transduce signals into the cell interior; they can also receive intracellular signals that regulate their ligand-binding affinity. Here we provide a brief overview that concentrates mostly on the organization, structure and function of mammalian integrins, which have been more extensively studied than integrins in other organisms.     

Syndecan-1 and Syndecan-4 Capture Epidermal Growth Factor Receptor Family Members and the α3β1 Integrin Via Binding Sites in Their Ectodomains: NOVEL SYNSTATINS PREVENT KINASE CAPTURE AND INHIBIT α6β4-INTEGRIN-DEPENDENT EPITHELIAL CELL MOTILITY*

The α6β4 integrin is known to associate with receptor tyrosine kinases when engaged in epithelial wound healing and in carcinoma invasion and survival. Prior work has shown that HER2 associates with α6β4 integrin and syndecan-1 (Sdc1), in which Sdc1 engages the cytoplasmic domain of the β4 integrin subunit allowing HER2-dependent motility and carcinoma cell survival. In contrast, EGFR associates with Sdc4 and the α6β4 integrin, and EGFR-dependent motility depends on cytoplasmic engagement of β4 integrin with Sdc4. However, how HER2 and EGFR assimilate into a complex with the syndecans and integrin, and why kinase capture is syndecan-specific has remained unknown. In the present study, we demonstrate that HER2 is captured via a site, comprised of amino acids 210–240, in the extracellular domain of human Sdc1, and EGFR is captured via an extracellular site comprised of amino acids 87–131 in human Sdc4. Binding assays using purified recombinant proteins demonstrate that the interaction between the EGFR family members and the syndecans is direct. The α3β1 integrin, which is responsible for the motility of the cells, is captured at these sites as well. Peptides based on the interaction motifs in Sdc1 and Sdc4, called synstatins (SSTN_210–240 and SSTN_87–131) competitively displace the receptor tyrosine kinase and α3β1 integrin from the syndecan with an IC₅₀ of 100–300 nm. The syndecans remain anchored to the α6β4 integrin via its cytoplasmic domain, but the activation of cell motility is disrupted. These novel SSTN peptides are potential therapeutics for carcinomas that depend on these HER2- and EGFR-coupled mechanisms for their invasion and survival.