Modulation of α5β1 and αVβ3 integrins on the cell surface during mitosis

One of the hallmarks of cells undergoing mitotic division is their rounded morphology and reduced adhesion to the substratum. We have studied and compared the attachment of interphase and mitotic cells to substrata coated with fibronectin and vitronectin. We have found that adhesion of mitotic cells, as compared to interphase cells, is significantly reduced to fibronectin, but is higher to vitronectin. These results correlate well with the expression of α5β1 and αVβ3 integrins, the respective receptors for fibronectin and vitronectin, on the cell surface. Mitotic cells show higher levels of αVβ3 and very low levels of α5β1 proteins on the cell surface as compared to interphase cells. This difference in the levels of these integrins also reflects in the total amounts of fibronectin and vitronectin present on the cell surface of these cells. We have further shown, by flow cytometry, that binding of vitronectin, or the synthetic peptide-GRGDSP-, causes an increase in the intracellular levels of Ca²⁻ in mitotic cells, but no change is seen in the interphase cells. Binding of fibronectin to either of these cells fails to elicit any response. One interesting feature of our results is that the levels of total, i.e., cytoplasmic plus membrane bound, α5β1 and αVβ3 integrins of mitotic and interphase cells remain the same, thus implying an alteration in the distribution of integrin chains between the plasma membrane and the cytoplasm during the conversion of interphase cells into the mitotic phase. © 1996 Wiley-Liss, Inc.     

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.     

INTEGRIN SYNTHESIS AND UTILIZATION IN CULTURED HUMAN OSTEOBLASTS

This study describes the adhesion of human osteoblasts, culturedin vitro, to proteins of the extracellular matrix, the biosynthesis of integrins, their topography and organization in focal contacts. The adhesion of osteoblasts to laminin, type I collagen, vitronectin and fibronectin was 77–100%, in 2h and at 55nm substrata concentration, and it was accompained by spreading of the cells. Adhesion to fibronectin (FN), laminin (LN) and type I collagen (COL) was inhibited by antibodies to the β1 integrin and antibodies to the α5 chain affected adhesion only to fibronectin. Using a panel of polyclonal antibodies against α2, α3, α5, αv, β1 andβ3 integrins we detected synthesis of α3β1, α5β1, αvβ3, and an αvβ1-like dimer by immunoprecipitation of metabolically labelled cell lysates. Studies of immunolocalization demonstrated the presence of the same integrins identified in lysates, plus α4, α1 and β5 subunits. In cells adhering in the presence of serum we showed organization of β3 and αv integrins in focal contacts. In cells adhering to fibronectin α5 and β1 integrins were localized in focal contacts. In cells spread on laminin or type I collagen none of the integrins investigated was localized in focal contacts.     

Cell adhesion and integrin expression are modulated by oxidative stress in EA.hy 926 cells

The effects of oxidative stress on integrin-mediated cell adhesion to the extracellular matrix (ECM) and related apoptosis were investigated using the EA.hy926 endothelial cells treated (or not) with two oxidants: the hypoxanthine/xanthine oxidase system (HX/XO) or the tert-butyl hydroperoxide (t-BHP) which both increased cell apoptosis. Cell adhesion onto vitronectin (Vn) and fibronectin (Fn) was increased at low concentrations of HX/XO (up to 5 mU/ml) or t-BHP (up to 125 μM) and prevented ROS-induced apoptosis. Flow cytometry analysis of integrin expression showed that the expression of integrin αv and α5 subunits was, respectively, increased and decreased. Cell adhesion inhibition experiments using function-blocking monoclonal antibodies against integrin subunits indicated that αvβ1 and αvβ3 integrins were involved in adhesion of cells to Vn, and αvβ3 integrin played a major role in oxidant-treated cells. For adhesion to Fn, α5β1 and αvβ1 integrins were required for oxidant-treated cells. Taken together, the results suggest that reactive oxygen species (ROS) produced either by HX/XO or t-BHP could affect expression and/or activation of specific integrins in the interaction of EA.hy926 cells with ECM.     

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.     

N-cadherin cell-cell adhesion complexes are regulated by fibronectin matrix assembly

Fibronectin is a principal component of the extracellular matrix. Soluble fibronectin molecules are assembled into the extracellular matrix as insoluble, fibrillar strands via a cell-dependent process. In turn, the interaction of cells with the extracellular matrix form of fibronectin stimulates cell functions critical for tissue repair. Cross-talk between cell-cell and cell-extracellular matrix adhesion complexes is essential for the organization of cells into complex, functional tissue during embryonic development and tissue remodeling. Here, we demonstrate that fibronectin matrix assembly affects the organization, composition, and function of N-cadherin-based adherens junctions. Using fibronectin-null mouse embryonic myofibroblasts, we identified a novel quaternary complex composed of N-cadherin, β-catenin, tensin, and actin that exists in the absence of a fibronectin matrix. In the absence of fibronectin, homophilic N-cadherin ligation recruited both tensin and α5β1 integrins into nascent cell-cell adhesions. Initiation of fibronectin matrix assembly disrupted the association of tensin and actin with N-cadherin, released α5β1 integrins and tensin from cell-cell contacts, stimulated N-cadherin reorganization into thin cellular protrusions, and decreased N-cadherin adhesion. Fibronectin matrix assembly has been shown to recruit α5β1 integrins and tensin into fibrillar adhesions. Taken together, these studies suggest that tensin serves as a common cytoskeletal link for integrin- and cadherin-based adhesions and that the translocation of α5β1 integrins from cell-cell contacts into fibrillar adhesions during fibronectin matrix assembly is a novel mechanism by which cell-cell and cell-matrix adhesions are coordinated.     

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.     

Selective integrin subunit reduction disrupts fibronectin extracellular matrix deposition and fibrillin 1 gene expression

Integrins are transmembrane receptors that can specifically bind extracellular matrix (ECM) proteins. Assembly of the ECM protein fibronectin into fibrils has been shown to be a cell-mediated process that requires integrins. Like fibronectin, fibrillin 1 is an ECM glycoprotein that can assemble into fibrils, but the role of integrins in fibril formation is not understood. To investigate the role of integrins in fibrillin 1 ECM deposition, cells that normally produce and assemble fibrillin 1 fibers in vitro were stably transfected with plasmid constructs encoding short interfering RNAs that target specific integrin subunits. Cells that were deficient in α2- and β3-integrin subunits produced and deposited fibronectin normally, but cells that were deficient for α5 and αV were unable to elaborate a fibronectin matrix, although they continued to produce and secrete the protein. Surprisingly, the cells that were unable to elaborate a fibronectin matrix also lost fibrillin 1 gene expression.     

Integrins and chondrocyte–matrix interactions in articular cartilage

The integrin family of cell adhesion receptors plays a major role in mediating interactions between cells and the extracellular matrix. Normal adult articular chondrocytes express α1β1, α3β1, α5β1, α10β1, αVβ1, αVβ3, and αVβ5 integrins, while chondrocytes from osteoarthritic tissue also express α2β1, α4β1, α6β1. These integrins bind a host of cartilage extracellular matrix (ECM) proteins, most notably fibronectin and collagen types II and VI, which provide signals that regulate cell proliferation, survival, differentiation, and matrix remodeling. By initiating signals in response to mechanical forces, chondrocyte integrins also serve as mechanotransducers. When the cartilage matrix is damaged in osteoarthritis, fragments of fibronectin are generated that signal through the α5β1 integrin to activate a pro-inflammatory and pro-catabolic response which, if left unchecked, could contribute to progressive matrix degradation. The cell signaling pathways activated in response to excessive mechanical signals and to fibronectin fragments are being unraveled and may represent useful therapeutic targets for slowing or stopping progressive matrix destruction in arthritis.     

Identification of the heparin-binding determinants within fibronectin repeat III1: role in cell spreading and growth

Fibronectins are high molecular mass glycoproteins that circulate as soluble molecules in the blood, and are also found in an insoluble, multimeric form in extracellular matrices throughout the body. Soluble fibronectins are polymerized into insoluble extracellular matrix (ECM) fibrils via a cell-dependent process. Recent studies indicate that the interaction of cells with the ECM form of fibronectin promotes actin organization and cell contractility, increases cell growth and migration, and enhances the tensile strength of artificial tissue constructs; ligation of integrins alone is insufficient to trigger these responses. Evidence suggests that the effect of ECM fibronectin on cell function is mediated in part by a matricryptic heparin-binding site within the first III1 repeat (FNIII1). In this study, we localized the heparin-binding activity of FNIII1 to a cluster of basic amino acids, Arg613, Trp614, Arg615, and Lys617. Site-directed mutagenesis of a recombinant fibronectin construct engineered to mimic the ECM form of fibronectin demonstrates that these residues are also critical for stimulating cell spreading and increasing cell proliferation. Cell proliferation has been tightly correlated with cell area. Using integrin- and heparin-binding fibronectin mutants, we found a positive correlation between cell spreading and growth when cells were submaximally spread on ECM protein-coated surfaces at the time of treatment. However, cells maximally spread on vitronectin or fibronectin still responded to the fibronectin matrix mimetic with an increase in growth, indicating that an absolute change in cell area is not required for the increase in cell proliferation induced by the matricryptic site of FNIII1.     

Syndecan-1 regulates cell migration and fibronectin fibril assembly

Corneal scarring is a major cause of blindness worldwide and can result from the deposition of abnormal amounts of collagen fibers lacking the correct size and spacing required to produce a clear cornea. Collagen fiber formation requires a preformed fibronectin (FN) matrix. We demonstrate that the loss of syndecan1 (sdc1) in corneal stromal cells (CSC) impacts cell migration rates, the sizes and composition of focal and fibrillar adhesions, the activation of integrins, and the assembly of fibronectin into fibrils. Integrin and fibronectin expression are not altered on sdc1-null CSCs. Cell adhesion, spreading, and migration studies using low compared to high concentrations of FN and collagen I (CNI) or vitronectin (VN) with and without activation of integrins by manganese chloride show that the impact of sdc1 depletion on integrin activation varies depending on the integrin-mediated activity evaluated. Differences in FN fibrillogenesis and migration in sdc1-null CSCs are reversed by addition of manganese chloride but cell spreading differences remain. To determine if our findings on sdc1 were specific to the cornea, we compared the phenotypes of sdc1-null dermal fibroblasts with those of CSCs. We found that without sdc1, both cell types migrate faster; however, cell-type-specific differences in FN expression and its assembly into fibrils exist between these two cell types. Together, our data demonstrate that sdc1 functions to regulate integrin activity in multiple cell types. Loss of sdc1-mediated integrin function results in cell-type specific differences in matrix assembly. A better understanding of how different cell types regulate FN fibril formation via syndecans and integrins will lead to better treatments for scarring and fibrosis.     

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.     

Dermatopontin interacts with fibronectin, promotes fibronectin fibril formation, and enhances cell adhesion

We report that dermatopontin (DP), an abundant dermal extracellular matrix protein, is found in the fibrin clot and in the wound fluid, which comprise the provisional matrix at the initial stage of wound healing. DP was also found in the serum but at a lower concentration than that in wound fluid. DP co-localized with both fibrin and fibronectin on fibrin fibers and interacted with both proteins. Both normal fibroblast and HT1080 cell adhesion to the fibrin-fibronectin matrix were dose-dependently enhanced by DP, and the adhesion was mediated by α5β1 integrin. The cytoskeleton was more organized in the cells that adhered to the fibrin-fibronectin-DP complex. When incubated with DP, fibronectin formed an insoluble complex of fibronectin fibrils as visualized by electron microscopy. The interacting sites of fibronectin with DP were the first, thirteenth, and fourteenth type III repeats (III(1), III(13), and III(14)), with III(13) and III(14) assumed to be the major sites. The interaction between III(2-3) and III(12-14) was inhibited by DP, whereas the interaction between I(1-5) and III(12-14) was specifically and strongly enhanced by DP. Because the interaction between III(2-3) and III(12-14) is involved in forming a globular conformation of fibronectin, and that between I(1-5) and III(12-14) is required for forming fibronectin fibrils, DP promotes fibronectin fibril formation probably by changing the fibronectin conformation. These results suggest that DP has an accelerating role in fibroblast cell adhesion to the provisional matrix in the initial stage of wound healing.     

The extracellular matrix and cytokines regulate microglial integrin expression and activation

Microglia are the primary immune effector cells resident within the CNS, whose activation into migratory, phagocytic cells is associated with increased expression of cell adhesion molecules of the integrin family. To determine which specific factors are important regulators of microglial activation and integrin expression, we have examined the influence of individual cytokines and extracellular matrix (ECM) substrates by quantifying cell surface expression of MHC and individual integrins by flow cytometry. We found that the proinflammatory cytokines TNF and IFN-alpha promoted microglial activation, as assessed by amoeboid morphology and increased expression of MHC class I, and also increased expression of the alpha(4)beta(1) and Mac-1 integrins. In contrast, TGF-beta1 had the opposite effect and was dominant over the other cytokines. Furthermore, the ECM substrates fibronectin and vitronectin, but not laminin, also promoted microglial activation and increased expression of the alpha(4)beta(1), alpha(5)beta(1) and Mac-1 integrins, but significantly, the influence of fibronectin and vitronectin was not diminished by TGF-beta1. Taken together, this work suggests that, in addition to cytokines, the ECM represents an important regulatory influence on microglial activity. Specifically, it implies that increases in the local availability of fibronectin or vitronectin, as a result of blood-brain barrier breakdown or increased expression in different pathological states of the CNS, could induce microglial activation and increased expression of integrins.     

Molecular interactions between fibronectin and integrins during mouse blastocyst outgrowth

To investigate the mechanism of trophoblast adhesion to fibronectin, we cultured blastocysts in serum-free medium on proteolytic fibronectin fragments containing its major functional domains, and localized fibronectin-binding integrins in outgrowing trophoblast cells by immunofluorescent staining. Outgrowth comparable to that obtained with intact fibronectin was observed using a 120 kD chymotryptic fragment containing the central cell-binding domain (FN-120) and the Arg-Gly-Asp (RGD) recognition sequence. A 40 kD COOH-terminal chymotryptic fragment of fibronectin containing both a heparin-binding region and an alternate (non-RGD) cell-binding site was inactive in supporting trophoblast adhesion. Three synthetic peptides derived from the heparin-binding domain, including the CS1 alternate cell-binding site, were also unable to promote trophoblast cell adhesion. A 75 kD recombinant protein, ProNectin F, containing 13 copies of the cell recognition epitope of fibronectin, Val-Thr-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ser, vigorously supported blastocyst outgrowth. Blastocyst outgrowth was not significantly different when surfaces were precoated with cellular fibronectin, which contains an alternatively spliced type III repeat and is the form actually encountered in vivo. Several putative fibronectin receptors were localized in trophoblast outgrowths by immunofluorescent labeling. Antibodies reactive with integrin subunits α3, α5, αllb, αv, β1 and β3, but not α4, all bound to trophoblast cells. Antibodies raised against either the β1 or β3 integrin subunits significantly inhibited fibronectin-mediated outgrowth. These findings demonstrate the key role of the central cell-binding domain of fibronectin in trophoblast adhesion, and suggest four RGD-binding integrins, α3β1, α5β1, αllbβ3, and αvβ3, that could mediate trophoblast adhesion in vitro and may play an important role during implantation. © 1995 Wiley-Liss, Inc.     

Coordinated Regulation of Fibronectin Fibril Assembly and Actin Stress Fiber Formation

Assembly of a fibronectin (FN) matrix is a multistep process which influences a number of cellular functions including intracellular cytoskeletal organization and signaling responses. We have previously reported on a recombinant FN (recFN), FNΔIII_1–7, which differs from native FN in its rate of fibril formation. To determine the intracellular consequences of a delay in assembly, we compared the distribution of cytoskeletal proteins during the formation of native and recFN matrices by immunofluorescence at various time points. CHOα5 cell cytoskeleton was reorganized in response to both native and recFN matrix formation. Assembly of native FN induced a rapid reorganization of actin into stress fibers and colocalization of α5131 integrin, focal adhesion kinase (FAK), vinculin, and paxillin to regions of cell-matrix contact. α5β1 integrins and FAK are also clustered upon binding of FNΔIII_1–7 to cells but actin reorganization and focal adhesion formation are delayed and appear to be dependent on the formation of FNΔIII_1–7 fibrils. These results suggest that the structural framework of the matrix plays an important role in the ability of FN to initiate intracellular responses.     

Role of integrins in angiotensin II-induced proliferation of vascular smooth muscle cells

Angiotensin II (AII) binds to G protein-coupled receptor AT(1) and stimulates extracellular signal-regulated kinase (ERK), leading to vascular smooth muscle cells (VSMC) proliferation. Proliferation of mammalian cells is tightly regulated by adhesion to the extracellular matrix, which occurs via integrins. To study cross-talk between G protein-coupled receptor- and integrin-induced signaling, we hypothesized that integrins are involved in AII-induced proliferation of VSMC. Using Oligo GEArray and quantitative RT-PCR, we established that messages for α(1)-, α(5)-, α(V)-, and β(1)-integrins are predominant in VSMC. VSMC were cultured on plastic dishes or on plates coated with either extracellular matrix or poly-d-lysine (which promotes electrostatic cell attachment independent of integrins). AII significantly induced proliferation in VSMC grown on collagen I or fibronectin, and this effect was blocked by the ERK inhibitor PD-98059, suggesting that AII-induced proliferation requires ERK activity. VSMC grown on collagen I or on fibronectin demonstrated approximately three- and approximately sixfold increases in ERK phosphorylation after stimulation with 100 nM AII, respectively, whereas VSMC grown on poly-d-lysine demonstrated no significant ERK activation, supporting the importance of integrin-mediated adhesion. AII-induced ERK activation was reduced by >65% by synthetic peptides containing an RGD (arginine-glycine-aspartic acid) sequence that inhibit α(5)β(1)-integrin, and by ～60% by the KTS (lysine-threonine-serine)-containing peptides specific for integrin-α(1)β(1). Furthermore, neutralizing antibody against β(1)-integrin and silencing of α(1), α(5), and β(1) expression by transfecting VSMC with short interfering RNAs resulted in decreased AII-induced ERK activation. This work demonstrates roles for specific integrins (most likely α(5)β(1) and α(1)β(1)) in AII-induced proliferation of VSMC.     

Hyaluronan Controls the Deposition of Fibronectin and Collagen and Modulates TGF-β1 Induction of Lung Myofibroblasts

The contribution of hyaluronan-dependent pericellular matrix to TGF-β1-driven induction and maintenance of myofibroblasts is not understood. Hyaluronan is an extracellular matrix (ECM) glycosaminoglycan important in cell adhesion, proliferation and migration, and is implicated in myofibroblast formation and maintenance. Reduced turnover of hyaluronan has been linked to differentiation of myofibroblasts and potentiation of lung fibrosis. Fibronectin is a fibril forming adhesive glycoprotein that is also upregulated following induction with TGF-β1. Although they are known to bind each other, the interplay between hyaluronan and fibronectin in the pericellular matrix during myofibroblast induction and matrix assembly is not clear. This study addresses the role of hyaluronan and its interaction with fibrillar matrix components during myofibroblast formation. Hyaluronan and fibronectin were increased and co-localized in the ECM following myofibroblast induction by TGF-β1. Inhibition of hyaluronan synthesis in TGF-β1-induced lung myofibroblasts over a 4 day period with 4-methyl umbelliferone (4-MU) further enhanced myofibroblast morphology, caused increased deposition of fibronectin and type I collagen in the ECM, and increased expression of alpha-smooth muscle actin and hyaluronan synthase 2 (HAS2) mRNA. Hyaluronan oligosaccharides or hyaluronidase treatment, which more effectively disrupted the pericellular matrix, had similar effects. CD44 and β1 integrins co-localized in the cell membrane and along some stress fibers. However, CD44 and hyaluronan were specifically excluded from focal adhesions, and associated primarily with cortical actin. Time-lapse imaging of the immediate effects of hyaluronidase digestion showed that hyaluronan matrix primarily mediates attachment of membrane and cortical actin between focal contacts, suggesting that surface adhesion through hyaluronan and CD44 is distinct from focal adhesion through β1 integrins and fibronectin. Fluorescein-labeled hyaluronan bound regularly along fibronectin fibers and co-localized more with β1 integrin and less with CD44. Therefore, the hyaluronan matrix can interfere with the assembly of fibrillar ECM components, and this interplay regulates the degree of myofibroblast formation. These data also suggest that adhesion through hyaluronan matrix impacts cytoskeletal organization, and is potentially part of a clutch mechanism that regulates stick and slip of myofibroblasts by affecting the adhesion to and organization of fibronectin and collagen.     

Cyclic strain induces reorganization of integrin α5β1 and α2β1 in human umbilical vein endothelial cells

Cyclic strain has been shown to modulate endothelial cell (EC) morphology, proliferation, and function. We have recently reported that the focal adhesion proteins focal adhesion kinase (pp125^FAK) and paxillin, are tyrosine phosphorylated in EC exposed to strain and these events regulate the morphological change and migration induced by cyclic strain. Integrins are also localized on focal adhesion sites and have been reported to induce tyrosine phosphorylation of pp125^FAK under a variety of stimuli. To study the involvement of different integrins in signaling induced by cyclic strain, we first observed the redistribution of α and β integrins in EC subjected to 4 h cyclic strain. Human umbilical vein endothelial cells (HUVEC) seeded on either fibronectin or collagen surfaces were subjected to 10% average strain at a frequency 60 cycles/min. Confocal microscopy revealed that β₁ integrin reorganized in a linear pattern parallel with the long axis of the elongated cells creating a fusion of focal adhesion plaques in EC plated on either fibronectin (a ligand for α₅β₁) or collagen (a ligand for α₂β₁) coated plates after 4 h exposure to cyclic strain. β₃ integrin, which is a vitronectin receptor, did not redistribute in EC exposed to cyclic strain. Cyclic strain also led to a reorganization of α₅ and α₂ integrins in a linear pattern in HUVEC seeded on fibronectin or collagen, respectively. The expression of integrins α₅, α₂, and β₁ did not change even after 24 h exposure to strain when assessed by immunoprecipitation of these integrins. Cyclic strain-induced tyrosine phosphorylation of pp125^FAK occurred concomitant with the reorganization of β₁ integrin. We concluded that α₅β₁ and α₂β₁ integrins play an important role in transducing mechanical stimuli into intracellular signals. J. Cell. Biochem. 64:505–513. © 1997 Wiley-Liss, Inc.