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.     

Physiological evidence of integrin-antibody reactive proteins influencing the innate cellular immune responses of larval Galleria mellonella hemocytes

Larval Galleria melonella(L.)hemocytes form microaggregates in response to stimulation by Gram-positive bacteria Hemocyte adhesion to foreign materials is mediated by the CAMP/protein kinase A pathway and the B-subunit of cholera toxin using a cAMP-independent mechanism.Cholera toxin-induced microaggregation was inhibited by the integrin inhibitory RGDS peptide,implying integrins may be part of the mechanism.Based on the types of mammalian integrin-antibody reactive proteins affecting hemocyte adhesion and bacterial-induced responses ars,ory,Ai,and B3 subunits occred on both granular cell and plasmatocyte hemocyte subtypes.A fluorescent band representing the binding of rabbit as-integrin subunit antibodies occurred between adhering heterotypic hemocytes.The frequency of the bands was increased by cholera toxin.The as andβrabbit integrin subunit antibodies inhibited removal of Bacillus subtilis(Cohn)from the hemolymph in vivo,A as ir-specific synthetic peptide blocker similarly diminished hemocyte function whereas the 0v Bs-specific inhibitory peptide and the corresponding integrin subunit antibodies did not influence nonself hemocyte activities.Western blots revealed several proteins reacting with a given integrin-antibody subtype.Thus integrin-antibody reactive proteins(which may include integrins)with possible as and B epitopes modulate immediate hemocyte function.Confocal microscopy established plasmatocyte adhesion to and rosetting over substrata followved by granular cell microaggregate adhesion to plasmatocytes during early stage nodulation.     

α9β1 Integrin in melanoma cells can signal different adhesion states for migration and anchorage

Cell surface integrins are the primary receptors for cell migration on extracellular matrix, and exist in several activation states regulated in part by ectodomain conformation. The α9 integrin subunit, which pairs only with β1, has specific roles in the immune system and may regulate cell migration. Melanoma cells express abundant α9β1 integrin, and its role in cell migration was assessed. Ligands derived from Tenascin-C and ADAM12 supported α9β1 integrin-mediated cell attachment and GTP-Rac dependent migration, but not focal adhesion formation. Manganese ions induced α9β1 integrin- and Rho kinase-dependent focal adhesion and stress fibre formation, suggesting that the activation status of α9β1 integrin was altered. The effect of manganese ions in promoting focal adhesion formation was reproduced by β1 integrin activating antibody. The α9β1 integrin translocated to focal adhesions, where active β1 integrin was also detected by conformation-specific antibodies. Focal adhesion assembly was commensurate with reduced cell migration. Endogenous α9β1 integrin-mediated adhesion was sensitive to the PP1 chemical inhibitor and an inhibitor of endosomal vesicle recycling, but not inhibitors of protein kinase C or the small GTPase Rho. Our results demonstrated that although α9β1 integrin can induce and localise to focal adhesions in a high activation state, its intermediate activity state normally supports cell adhesion consistent with migration.     

Glucose-Induced Alteration of Integrin Expression and Function in Cultured Human Mesangial Cells

Alteration in mesangial volume, due to an increase of the matrix surrounding mesangial cells, is a hallmark indicator of nephropathy in diabetes. Mesangial cells may also play a significant role in the development of nephropathy. Therefore, we examined the effect of glucose on the expression of integrins by cultured human mesangial cells and their ability to interact with collagen IV, a major component of the mesangial matrix. Human mesangial cells were grown in 5 and 25 mM glucose and their integrin profile was examined by immunoprecipitation and flow cytometry in each experimental condition. The results indicate that when mesangial cells were grown in 25 mM glucose, the expression of integrin subunit α2, was increased, while the α1 subunit was considerably decreased, as compared to cells grown in 5 mM glucose. Additionally, mesangial cells were tested for their ability to adhere to collagen IV in a solid-phase assay in the presence of neutralizing antibodies to integrin subunits. The results of these experiments indicate that both α1 and α2 complexed to β1 (α2β1 and α1β1) are major mesangial cell receptors for adhesion to collagen IV both in 5 and 25 mM glucose. The two receptors act in concert to mediate adhesion of mesangial cells to type IV collagen. When cell surface expression of the α1 subunit in 25 mM glucose was reduced, the α2 subunit was involved in adhesion to a greater extent than it was in 5 mM glucose. Immunoperoxidase histochemical studies localized both α1 and α2 integrin subunits in the mesangium of normal adult kidneys, suggesting that in vivo interaction with collagen IV could involve both of these receptors. These observations suggest that glucose-induced alterations in integrin expression may modify the ability of mesangial cells to interact with collagen IV.     

Structural basis of integrin transmembrane activation

Integrins are cell adhesion receptors that transmit bidirectional signals across plasma membrane and are crucial for many biological functions. Recent structural studies of integrin transmembrane (TM) and cytoplasmic domains have shed light on their conformational changes during integrin activation. A structure of the resting state was solved based on Rosetta computational modeling and experimental data using intact integrins on mammalian cell surface. In this structure, the α_IIb GXXXG motif and their β₃ counterparts of the TM domains associate with ridge‐in‐groove packing, and the α_IIb GFFKR motif and the β₃ Lys‐716 in the cytoplasmic segments play a critical role in the α/β association. Comparing this structure with the NMR structures of the monomeric α_IIb and β₃ (represented as active conformations), the α subunit helix remains similar after dissociation whereas β subunit helix is tilted by embedding additional 5–6 residues into the lipid bilayer. These conformational changes are critical for integrin activation and signaling across the plasma membrane. We thus propose a new model of integrin TM activation in which the recent NMR structure of the α_IIbβ₃ TM/cytoplasmic complex represents an intermediate or transient state, and the electrostatic interaction in the cytoplasmic region is important for priming the initial α/β association, but not absolutely necessary for the resting state. J. Cell. Biochem. 109: 447–452, 2010. © 2009 Wiley‐Liss, Inc.     

Localization of Urokinase Type Plasminogen Activator to Focal Adhesions Requires Ligation of Vitronectin Integrin Receptors

Previous studies have shown that the adhesion protein, vitronectin, directs the localization of urokinase-type plasminogen activator (uPA) to areas of cell-substrate adhesion, where uPA is thought to regulate cell migration as well as pericellular proteolysis. In the present study, HT-1080 cell lines expressing either wild-type vitronectin or vitronectin containing a single amino-acid substitution in the integrin binding domain were used to assess whether ligation of the α_vβT₅ integrin was required for uPA localization to focal adhesions. The synthesis of wild-type vitronectin by HT-1080 cells adherent to either collagen or fibronectin resulted in the redistribution of both the α_vβT₅ integrin as well as uPA to focal adhesion structures. In contrast, cells synthesizing mutant vitronectin, containing the amino-acid substitution in the integrin binding domain, were unable to direct the redistribution of either α_vβT₅ or uPA to focal adhesions. Recombinant forms of wild-type and mutant vitronectin were prepared in a baculovirus system and compared for their ability to direct the redistribution of vitronectin integrin receptors as well as uPA on human skin fibroblasts. In the absence of vitronectin, fibroblast cells adherent to fibronectin assemble focal adhesions which contain the βT₁ integrin but do not contain uPA. Addition of recombinant wild-type, but not mutant, vitronectin to fibroblasts adherent to fibronectin resulted in the redistribution of α_vβT₃, α_vβT₅, and uPA into focal adhesions. However, when cells were plated directly onto antibodies directed against either the α_vβT₃ or α_vβT₅ integrins, uPA was not localized on the cell surface. These data indicate that ligation of vitronectin integrin receptors is necessary but not sufficient for the localization of uPA to areas of cell-matrix adhesion, and suggest that vitronectin may promote cell migration by recruiting vitronectin integrin receptors and components of the plasminogen activator system to areas of cell matrix contact.     

Altered glycosylation of α(s)β1 integrins from rat colon carcinoma cells decreases their interaction with fibronectin

Malignant cell transformation is generally accompanied by changes in their interactions with environing matrix proteins in a way to facilitate their migration and generate invasion. Our results show the binding of rat colon adenocarcinoma PROb cells to fibronectin strongly reduced when compared to normal rat intestine epithelial cells. This decrease was not due to the level of α(s)β1 integrins expressed at the surface of the cell line. However, β₁- and α_(s)-associated subunits appeared to be structurally altered as shown by immunoprecipitation followed by electrophoresis. Pulse chase experiments using ³⁵S methionine evidenced differences in the biosynthesis of β1- and α (s) associated integrins: normal epithelial IEC18 cells required 16 h for maximal biosynthesis of the completely mature β1 subunit, while PROb cells did it within 4-6 h. Studies using endoglycosidases O, H, D, and N glycanase confirmed that the molecular weight alterations were due to abnormal glycosylation and suggested that α(s)β1 integrins of PROb cells could bear both mature complex and immature high mannose types while IEC18 cells borne only mature complex type oligosaccharidic chains. Treatment of both cell types with castanospermine, an inhibitor of N-glycosylation, reduced the differences observed in their adhesion to the fibronectin without significantly affecting β1 receptors expression at the cell surface. These results strongly suggest a role of the glycosylation of β1 receptors in the adhesion of rat colon adenocarcinoma PROb cells to fibronectin substrata. © 1996 Wiley-Liss, Inc.     

Transfection of β4 Integrin Subunit into a Neoplastic Keratinocyte Line Fails to Restore Terminal Differentiation Capacity or Influence Proliferation

Loss of expression of specific integrins is a feature of poorly differentiated oral squamous cell carcinomas (SCCs) and cell lines derived from them. In order to test whether there is a direct link between reduced α₆β₄ integrin expression and abnormal keratinocyte growth and differentiation we ‘repaired' an SCC line. H376, by transfection of the β₄ integrin subunit. We analysed five independent β₄ transfectant clones and compared them with four empty vector control clones and with the parental cell line. Elevated cell surface expression of α₆β₄ was not correlated with changes in anchorage dependent or independent growth and was not sufficient to induce expression of the terminal differentiation marker, involucrin. Introduction of the β₄ integrin subunit did not have a major effect on cell adhesion to laminin 1 or 5 and did not result in formation of stable anchoring contacts. We conclude that loss of α₆β₄ is not directly responsible for the abnormal behaviour of the H376 cell line.     

Titanium with nanotopography induces osteoblast differentiation through regulation of integrin αV

Topographical modifications of titanium (Ti) at the nanoscale level generate surfaces that regulate several signaling pathways and cellular functions, which may affect the process of osseointegration. Here, we investigated the participation of integrin αV in the osteogenic capacity of Ti with nanotopography. Machined titanium discs (untreated) were submitted to treatment with H₂SO₄/H₂O₂ to produce the nanotopography (nanostructured). First, the greater osteogenic capacity of the nanotopography that increased osteoblast differentiation of mesenchymal stem cells compared with untreated topography was shown. Also, the nanostructured surface increased (regulation ≥ 1.9-fold) the gene expression of 6 integrins from a custom array plate utilized to evaluate the gene expression of 84 genes correlated with cell adhesion signaling pathway, including integrin αV, which is involved in osteoblast differentiation. By silencing integrin αV in MC3T3-E1 cells cultured on nanotopography, the impairment of osteoblast differentiation induced by this surface was observed. In conclusion, it was shown that nanotopography regulates the expression of several components of the cell adhesion signaling pathway and its higher osteogenic potential is, at least in part, due to its ability to upregulate the expression of integrin αV. Together with previous data that showed the participation of integrins α1, β1, and β3 in the nanotopography osseoinduction activity, we have uncovered the pivotal role of this family of membrane receptors in the osteogenic potential of this surface.     

Vesicle-associated membrane protein 2 mediates trafficking of α5β1 integrin to the plasma membrane

Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of α5β1 integrin. VAMP2 was present on vesicles containing endocytosed β1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface α5β1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of α5β1, without altering cell surface expression of α2β1 integrin or α3β1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of α5β1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.     

Single cell tracking assay reveals an opposite effect of selective small non-peptidic α5β1 or αvβ3/β5 integrin antagonists in U87MG glioma cells

BackgroundIntegrins are extracellular matrix receptors involved in several pathologies. Despite homologies between the RGD-binding α5β1 and αvβ3 integrins, selective small antagonists for each heterodimer have been proposed. Herein, we evaluated the effects of such small antagonists in a cellular context, the U87MG cell line, which express both integrins. The aim of the study was to determine if fibronectin-binding integrin antagonists are able to impact on cell adhesion and migration in relationships with their defined affinity and selectivity for α5β1 and αvβ3/β5 purified integrins.MethodsSmall antagonists were either selective for α5β1 integrin, for αvβ3/β5 integrin or non-selective. U87MG cell adhesion was evaluated on fibronectin or vitronectin. Migration assays included wound healing recovery and single cell tracking experiments. U87MG cells stably manipulated for the expression of α5 integrin subunit were used to explore the impact of α5β1 integrin in the biological assays.ResultsU87MG cell adhesion on fibronectin or vitronectin was respectively dependent on α5β1 or αvβ3/β5 integrin. Wound healing migration was dependent on both integrins. However U87MG single cell migration was highly dependent on α5β1 integrin and was inhibited selectively by α5β1 integrin antagonists but increased by αvβ3/β5 integrin antagonists.ConclusionsWe provide a rationale for testing new integrin ligands in a cell-based assay to characterize more directly their potential inhibitory effects on integrin cellular functions.General significanceOur data highlight a single cell tracking assay as a powerful cell-based test which may help to characterize true functional integrin antagonists that block α5β1 integrin-dependent cell migration.     

Effect of hypoxia on integrin-mediated adhesion of endothelial progenitor cells

Homing of endothelial progenitor cells (EPCs) is crucial for neoangiogenesis, which might be negatively affected by hypoxia. We investigated the influence of hypoxia on fibronectin binding integrins for migration and cell‐matrix‐adhesion. AMP‐activated kinase (AMPK) and integrin‐linked kinase (ILK) were examined as possible effectors of hypoxia.Human EPCs were expanded on fibronectin (FN) and integrin expression was profiled by flow cytometry. Cell‐matrix‐adhesion‐ and migration‐assays on FN were performed to examine the influence of hypoxia and AMPK‐activation. Regulation of AMPK and ILK was shown by Western blot analysis. We demonstrate the presence of integrin β₁, β₂ and α₅ on EPCs. Adhesion to FN is reduced by blocking β₁ and α₅ (49% and 2% of control, P < 0.05) whereas α₄‐blockade has no effect. Corresponding effects were shown for migration. Hypoxia and AMPK‐activation decrease adhesion on FN. Although total AMPK‐expression remains unchanged, phospho‐AMPK increases eightfold.The EPCs require α₅ for adhesion on FN. Hypoxia and AMPK‐activation decrease adhesion. As α₅ is the major adhesive factor for EPCs on FN, this suggests a link between AMPK and α₅‐integrins. We found novel evidence for a connection between hypoxia, AMPK‐activity and integrin activity. This might affect the fate of EPCs in ischaemic tissue.     

Negative Cooperativity between α3β1and α2β1Integrins in Human Mammary Carcinoma MDA MB 231 Cells

The α₃β₁integrin has been implicated as a receptor for several matrix components, including collagen, fibronectin, and laminins. The function of α₃β₁seems to be very versatile involving cell adhesion to or migration on ECM, establishment of cell–cell contacts in aggregates, as well as linkage to intracellular tyrosine phosphorylation cascades. Here we report a strong induction of attachment of α₃β₁integrin expressing human breast carcinoma cell line MDA MB 231 to matrix proteins by two α₃integrin subunit function-blocking monoclonal antibodies (P1B5 and ASC-1). In contrast, stimulation of adhesion to ECM by inhibitory α₃integrin-specific antibodies was not observed in the α₃β₁integrin-expressing nonmalignant human mammary epithelial cell line MCF-10A or the human breast carcinoma cell line MDA MB 468 that expressed relatively low amounts of α₃β₁integrin at the cell surface. This increase was specific for collagens and not observed on fibronectin or laminin. Physiological concentrations of bivalent cations were not required. MAb P1B5 did not induce homotypic aggregation of MDA MB 231 cells. The P1B5-induced increase in cell attachment to collagens could be prevented but not reduced below control levels by blocking mAb to the α₂integrin subunit. Function blocking anti-α₅integrin subunit mAb was without effect while anti-β₁-mAb completely abolished adhesion. Our data indicate that negative cooperativity between integrins results in transdominant inhibition of α₂β₁function by α₃β₁in human MDA MB 231 but not MDA MB 468 tumor cells or nonmalignant MCF-10A cells.     

Characterization of adhesion properties of the cardiomyocyte integrins and extracellular matrix proteins using atomic force microscopy

Integrins are transmembrane adhesion receptors that play important roles in the cardiovascular system by interacting with the extracellular matrix (ECM). However, direct quantitative measurements of the adhesion properties of the integrins on cardiomyocyte (CM) and their ECM ligands are lacking. In this study, we used atomic force microscopy (AFM) to quantify the adhesion force (peak force and mean force) and binding probability between CM integrins and three main heart tissue ECM proteins, ie, collagen (CN), fibronectin (FN), and laminin (LN). Functionalizing the AFM probes with ECM proteins, we found that the peak force (mean force) was 61.69 ± 5.5 pN (76.54 ± 4.0 pN), 39.26 ± 4.4 pN (59.84 ± 3.6 pN), and 108.31 ± 4.2 pN (129.63 ± 6.0 pN), respectively, for the bond of CN‐integrin, FN‐integrin, and LN‐integrin. The binding specificity between CM integrins and ECM proteins was verified by using monoclonal antibodies, where α₁₀‐ and α₁₁‐integrin bind to CN, α₃‐ and α₅‐integrin bind to FN, and α₃‐ and α₇‐integrin bind to LN. Furthermore, adhesion properties of CM integrins under physiologically high concentrations of extracellular Ca²⁺ and Mg²⁺ were tested. Additional Ca²⁺ reduced the adhesion mean force to 68.81 ± 4.0 pN, 49.84 ± 3.3 pN, and 119.21 ± 5.8 pN and binding probability to 0.31, 0.34, 0.40 for CN, FN, and LN, respectively, whereas Mg²⁺ caused very minor changes to adhesion properties of CM integrins. Thus, adhesion properties between adult murine CM integrins and its main ECM proteins were characterized, paving the way for an improved understanding of CM mechanobiology.     

The effect of differentiation inducers on the integrin expression of K562 erythroleukemia cells.

We studied the effects of differentiation-inducers on the integrin profile and adhesive properties of K562 leukemia cells. The fibronectin (Fn) receptor integrin, α₅β₁, was the only integrin expressed in suspension cultured K562 cells. When the cells were exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA) immunoreactivity for the β₁ integrin subunit was slightly enhanced. TPA exposure also induced the appearance of the α₂, α₃, α_v and β₃ integrin subunits, but the platelet integrin subunit α_IIb was not detected. On the other hand, hemin chloride-induced erythroid differentiation of K562 cells diminished the expression of the α₅β₁ integrin on the surface of the cells. Adhesion experiments with TPA-exposed K562 cells indicated that although the adherence to the extracellular matrix (ECM) proteins as a rule was low a few cells spread on these proteins. The present results specify the effects of differentiation inducers on the integrin profile of K562 cells and excludes the comprehension that TPA would induce expression of the platelet integrin α_IIb on their surface. Our results also show, that an increased expression of a certain integrin does not necessarily lead to a comparable adhesion ability on its ligand in vitro.     

The regulation of integrin function by divalent cations

Integrins are a family of α/β heterodimeric adhesion metalloprotein receptors and their functions are highly dependent on and regulated by different divalent cations. Recently advanced studies have revolutionized our perception of integrin metal ion-binding sites and their specific functions. Ligand binding to integrins is bridged by a divalent cation bound at the MIDAS motif on top of either α I domain in I domain-containing integrins or β I domain in α I domain-less integrins. The MIDAS motif in β I domain is flanked by ADMIDAS and SyMBS, the other two crucial metal ion binding sites playing pivotal roles in the regulation of integrin affinity and bidirectional signaling across the plasma membrane. The β-propeller domain of α subunit contains three or four β-hairpin loop-like Ca²⁺-binding motifs that have essential roles in integrin biogenesis. The function of another Ca²⁺-binding motif located at the genu of α subunit remains elusive. Here, we provide an overview of the integrin metal ion-binding sites and discuss their roles in the regulation of integrin functions.     

Stimulated single‐cell force spectroscopy to quantify cell adhesion receptor crosstalk

To control their attachment to substrates and other cells, cells regulate their adhesion receptors. One regulatory process is receptor crosstalk, where the binding of one type of cell adhesion molecule influences the activity of another type. To identify such crosstalk and gain insight into their mechanisms, we developed the stimulated single‐cell force spectroscopy assay. In this assay, the influence of a cells adhesion to one substrate on the strength of its adhesion to a second substrate is examined. The assay quantifies the adhesion of the cell and the contributions of specific adhesion receptors. This allows mechanisms by which the adhesion is regulated to be determined. Using the assay we identified crosstalk between collagen‐binding integrin α₁β₁ and fibronectin‐binding integrin α₅β₁ in HeLa cells. This crosstalk was unidirectional, from integrin α₁β₁ to integrin α₅β₁, and functioned by regulating the endocytosis of integrin α₅β₁. The single‐cell assay should be expandable for the screening and quantification of crosstalk between various cell adhesion molecules and other cell surface receptors.     

The roles of αV integrins in lens EMT and posterior capsular opacification

Posterior capsular opacification (PCO) is the major complication arising after cataract treatment. PCO occurs when the lens epithelial cells remaining following surgery (LCs) undergo a wound healing response producing a mixture of α‐smooth muscle actin (α‐SMA)‐expressing myofibroblasts and lens fibre cells, which impair vision. Prior investigations have proposed that integrins play a central role in PCO and we found that, in a mouse fibre cell removal model of cataract surgery, expression of α_V integrin and its interacting β‐subunits β1, β5, β6, β8 are up‐regulated concomitant with α‐SMA in LCs following surgery. To test the hypothesis that α_V integrins are functionally important in PCO pathogenesis, we created mice lacking the α_V integrin subunit in all lens cells. Adult lenses lacking α_V integrins are transparent and show no apparent morphological abnormalities when compared with control lenses. However, following surgical fibre cell removal, the LCs in control eyes increased cell proliferation, and up‐regulated the expression of α‐SMA, β1‐integrin, fibronectin, tenascin‐C and transforming growth factor beta (TGF‐β)–induced protein within 48 hrs, while LCs lacking α_V integrins exhibited much less cell proliferation and little to no up‐regulation of any of the fibrotic markers tested. This effect appears to result from the known roles of α_V integrins in latent TGF‐β activation as α_V integrin null lenses do not exhibit detectable SMAD‐3 phosphorylation after surgery, while this occurs robustly in control lenses, consistent with the known roles for TGF‐β in fibrotic PCO. These data suggest that therapeutics antagonizing α_V integrin function could be used to prevent fibrotic PCO following cataract surgery.     

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

Collaborative role of various fibronectin-binding integrins (α5β1, αvβ1 and αvβ6) as mediators of cell adhesion and migration on fibronectin was studied using cultured HaCaT keratinocytes. This cell line spontaneously expressed all three fibronectin-binding integrins. In addition, the expression of αvβ6 integrin was strongly and specifically upregulated by transforming growth factor-β1 (TGFβ1) whereas the amount of other integrins remained practically unchanged on the cell surface. Adhesion, spreading and motility of HaCaT keratinocytes on fibronectin were promoted by TGFβ1. Based on antibody blocking experiments, both untreated and TGFβ1-treated HaCaT cells used αvβ6 integrin as their main fibronectin receptor for cell spreading. In contrast to TGFβ1-treated cells, the untreated cells also needed α5β1 integrin for maximal cell spreading on fibronectin. Combinations of antibodies blocking both of these receptors totally prevented spreading of both untreated and TGFβ1-treated cells. Haptotactic motility of individual HaCaT cells through fibronectin-coated membranes was again mainly dependent on αvβ6 integrin, while αvβ1 and α5β1 integrins played a lesser role both in untreated and TGFβ1-treated HaCaT cells. However, unlike haptotaxis, lateral migration of HaCaT cell sheet was mainly mediated by β1 integrins, and αvβ6 integrin showed a minor role. The migration process appeared to involve a number of β1 integrins that could adaptively replace each other when blocking antibodies were present. Thus, keratinocytes appear to use different fibronectin receptors for different functions, such as cell spreading, haptotaxis and lateral migration. The cells can also adapt to a situation where one receptor is unfunctional by switching to another receptor of the same ligand.