Extracellular Matrix Molecules Fibronectin Research Articles

Δ133p53 coordinates ECM-driven morphogenesis and gene expression in three-dimensional mammary epithelial acini.

Growing evidence indicates that p53 (encoded by TP53) has a crucial role in normal tissue development. The role of the canonical p53 (p53α) and its 12 isoforms in development and homeostasis of healthy tissue remains poorly understood. Here, we demonstrate that the Δ133p53 isoforms, the three short isoforms of p53, respond specifically to laminin-111 and play an important regulatory role in formation of mammary organoids in concert with p53α. We demonstrate that down-modulation of Δ133p53 isoforms leads to changes in gene expression of the extracellular matrix molecules fibronectin (FN), EDA+-FN, laminin α5 and laminin α3 in human breast epithelial cells. These changes resulted in increased actin stress fibers and enhanced migratory behavior of cells in two-dimensional culture. We found that α5β1-integrin coupled with the extracellularly deposited EDA+-FN activates the Akt signaling pathway in three-dimensional (3D) culture when Δ133p53 is dysregulated. Cells that do not express detectable Δ133p53 isoforms or express low levels of these isoforms failed to form polarized structures in 3D. These results uncover that Δ133p53 isoforms coordinate expression and deposition of organ-specific ECM molecules that are critical for maintenance of tissue architecture and function.

Platelet-Derived PDGFB Promotes Recruitment of Cancer-Associated Fibroblasts, Deposition of Extracellular Matrix and Tgfβ Signaling in the Tumor Microenvironment.

Simple SummaryThe aim of this study was to investigate the relative contribution of PDGFB derived specifically from platelets to the remodeling of the extracellular matrix (ECM) in tumors. Platelets are a major source of growth factors, which are released from the platelet granules upon activation. Platelets are continuously activated in the tumor microenvironment, due to their similarities to a wound. However, the role of platelet-derived factors in ECM remodeling has not been fully addressed. To this end, we made use of a mouse model with conditional deletion of PDGFB in platelets, which was crossbred to the RIP1-Tag2 model for pancreatic neuroendocrine carcinoma. The amount of tumor-associated PDGFB protein showed a 10-fold reduction in mice lacking PDGFB in platelets. Moreover, ECM deposition, the amount of cancer-associated fibroblasts and TGFβ signaling were all reduced in tumors from mice with platelet-specific deletion of PDGFB, demonstrating the significant contribution of a platelet-derived factor to ECM remodeling in tumors.Platelets constitute a major reservoir of platelet-derived growth factor B (PDGFB) and are continuously activated in the tumor microenvironment, exposing tumors to the plethora of growth factors contained in platelet granules. To address the specific role of platelet-derived PDGFB in the tumor microenvironment, we have created a mouse model with conditional knockout of PDGFB in platelets (pl-PDGFB KO). Lack of PDGFB in platelets resulted in 10-fold lower PDGFB concentration in the tumor microenvironment, fewer cancer-associated fibroblasts and reduced deposition of the extracellular matrix (ECM) molecules fibronectin and collagen I in the orthotopic RIP1-Tag2 model for pancreatic neuroendocrine cancer. Myosin light chain phosphorylation, promoting cell contraction and, consequently, the mechano-induced release of active transforming growth factor (TGF) β from extracellular compartments, was reduced in tumors from pl-PDGFB KO mice. In agreement, TGFβ signaling, measured as phosphorylated Smad2, was significantly hampered in tumors from mice lacking PDGFB in their platelets, providing a plausible explanation for the reduced deposition of extracellular matrix. These findings indicate a major contribution of platelet-derived PDGFB to a malignant transformation of the tumor microenvironment and address for the first time the role of PDGFB released specifically from platelets in the remodeling of the ECM in tumors.

Induced Pluripotent Stem Cells From Subjects With Primary Sclerosing Cholangitis Develop a Senescence Phenotype Following Biliary Differentiation.

Primary sclerosing cholangitis (PSC) is a chronic fibroinflammatory disease of the biliary tract characterized by cellular senescence and periportal fibrogenesis. Specific disease features that are cell intrinsic and either genetically or epigenetically mediated remain unclear due in part to a lack of appropriate, patient‐specific, in vitro models. Recently, our group developed systems to create induced pluripotent stem cell (iPSC)‐derived cholangiocytes (iDCs) and biliary epithelial organoids (cholangioids). We use these models to investigate whether PSC cholangiocytes are intrinsically predisposed to cellular senescence. Skin fibroblasts from healthy controls and subjects with PSC were reprogrammed to pluripotency, differentiated to cholangiocytes, and subsequently grown in three‐dimensional matrigel‐based culture to induce formation of cholangioids. RNA sequencing (RNA‐seq) on iDCs showed significant differences in gene expression patterns, including enrichment of pathways associated with cell cycle, senescence, and hepatic fibrosis, that correlate with PSC. These pathways also overlapped with RNA‐seq analysis on isolated cholangiocytes from subjects with PSC. Exome sequencing on the subjects with PSC revealed genetic variants of unknown significance in the genes identified in these pathways. Three‐dimensional culture revealed smaller size, lack of a central lumen, and increased cellular senescence in PSC‐derived cholangioids. Congruent with this, PSC‐derived iDCs showed increased secretion of the extracellular matrix molecule fibronectin as well as the inflammatory cytokines interleukin‐6, and chemokine (C‐C motif) ligand 2. Conditioned media (CM) from PSC‐derived iDCs more potently activated hepatic stellate cells compared to control CM. Conclusion: We demonstrated efficient generation of iDCs and cholangioids from patients with PSC that show disease‐specific features. PSC cholangiocytes are intrinsically predisposed to cellular senescence. These features are unmasked following biliary differentiation of pluripotent stem cells and have functional consequences in epithelial organoids.

Streptococcuspneumoniae surface protein PfbA is a versatile multidomain and multiligand-binding adhesin employing different binding mechanisms.

Streptococcuspneumoniae, one of the major human respiratory pathogens, uses its repertoire of surface proteins to adhere to the epithelium of the nasopharynx and lungs leading to colonization. PfbA is a conserved surface protein of S.pneumoniae and helps the bacterium to colonize the host by recognizing the extracellular matrix (ECM) molecule fibronectin, as well as blood proteins like plasminogen and human serum albumin. The crystal structure of rPfbA150-607 revealed it to possess a beta-helical region similar to those of carbohydrate-active enzymes as well as a C-terminal segment that resembles the fibronectin-binding regions of fibronectin-binding proteins. To get more insight into the putative carbohydrate-binding property of PfbA and its binding to various host molecules, we generated three different constructs of PfbA and characterized them by ELISA, isothermal titration calorimetry and bio-layer interferometry experiments. Importantly, the isothermal titration calorimetry experiments revealed that PfbA binds to different saccharides. Further, ELISA and bio-layer interferometry experiments identified that (a) apart from fibronectin and plasminogen, the beta helix of PfbA also binds to other ECM molecules (b) lysines are not responsible for PfbA's binding to plasminogen, (c) in comparison with native fibrinogen, deglycosylated-fibrinogen exhibits reduced binding affinity towards PfbA implying the importance of sugar molecule-PfbA interaction and (d) the C-terminal region of PfbA binds exclusively to the N-terminal F1 modules of fibronectin. Thus, the results of this study show PfbA to be a versatile multidomain and multiligand-binding protein employing different binding mechanisms. These results could be useful for structure-based designing of inhibitors against PfbA.

Expression of cytokine and extracellular matrix mRNAs in fetal hepatic stellate cells.

In mouse fetal liver, hepatoblasts, sinusoidal endothelial cells and macrophages (or erythroblastic islands) promote differentiation and proliferation of hematopoietic cells through cell-cell interactions and secretion of cytokines and extracellular matrix factors. Until now, we have had little knowledge of the hematopoietic cytokines or extracellular matrix mRNAs expressed in hepatic stellate cells. Using p75 neurotrophin receptor (p75NTR) to mark this cell population, we sorted 12.5, 14.5 and 16.5 dpc hepatic stellate cells and analyzed expression of cytokines and extracellular matrix mRNAs. Among cytokines, insulin-like growth factor 2 (Igf2) was highly expressed at all three stages analyzed. The extracellular matrix molecule fibronectin (Fn1) was highly expressed in 12.5 dpc cells, whereas vitronectin (Vtn) was highly expressed in 14.5 and 16.5 dpc hepatic stellate cells. Among liver cells, Igf2 was predominantly expressed in hepatoblast-like cells at all three stages examined, suggesting that hepatoblast-like cells are an essential part of the niche that maintains homeostasis of hematopoietic cells in embryonic mouse liver. Defining these expression patterns could facilitate our understanding of cross talk between cytokine and extracellular matrix molecules in hepatic stellate cells and benefit research in developmental hematopoiesis as well as the study of liver biology.

In vitro investigation of molecules involved in Lactobacillus gasseri SBT2055 adhesion to host intestinal tract components.

The adhesion ability of Lactobacillus gasseri SBT2055 was investigated invitro by searching for its adhesion molecules. Lactobacillus gasseri SBT2055 showed adherence to host components, including two commercially available mucins, Caco-2 epithelial-like cells and the extracellular matrix molecule fibronectin (Fn). Its adhesion rates to host components were generally higher than those of other Lactobacillus strains. We examined sortase-dependent proteins (SDPs) anchored by a sortase enzyme encoded by srtA1. The adhesion rates of an srtA1 disruptant were lower than those of Lact.gasseri SBT2055, and the relative adherences were as follows: two mucins, 43 and 40%; Caco-2, 66% and Fn, 28%. Seven additional gene disruptants were generated to determine the precise SDPs that contribute to adhesion to each component. The adhesion ability of Lact.gasseri SBT2055 was superior to those of other Lactobacillus strains. Additionally, four adhesion molecules were newly identified from candidate SDPs. Although the contribution of SDPs to adhesion has been reported using sortase gene disruptants, this is the first report to identify the precise SDPs that act as adhesion molecules. Our results will contribute to achieving better understanding of probiotic bacterial adherence.

Overexpression of fibronectin confers cell adhesion-mediated drug resistance (CAM-DR) against 5-FU in oral squamous cell carcinoma cells

The tumor-associated microenvironment has been shown to protect tumor cells from treatment, and the extracellular matrix (ECM) is known to affect drug resistance as a key regulator of the tumor microenvironment. However, little is known about cell adhesion-mediated drug resistance (CAM-DR) due to cell-ECM contact in patients with oral squamous cell carcinoma (OSCC). In the present study, we evaluated the ECM molecule fibronectin (FN) using DNA microarray data obtained from parental and 5-FU-resistant OSCC cell lines. We investigated the effects of cell adhesion to FN on 5-FU resistance in OSCC cells and examined the activation of FN receptor β1 integrin-mediated survival regulators such as ILK, Akt and NF-κB. In addition, we investigated whether FNIII14, a 22-mer peptide derived from FN that potently prevents β1 integrin-mediated adhesion to FN, could overcome CAM-DR against 5-FU in OSCC cells and examined the activation of survival regulators and apoptosis-related molecules. Consequently, we obtained the following results. FN was extracellularly overexpressed in the 5-FU-resistant cells compared with that observed in the 5-FU-sensitive cells. Cell adhesion to FN enhanced 5-FU resistance and activated integrin-mediated ILK/Akt/NF-κB survival signaling in the 5-FU-resistant OSCC cells. Furthermore, the inhibition of cell adhesion to FN by FNIII14 enhanced chemosensitivity to 5-FU and apoptosis by suppressing ILK/Akt/NF-κB signaling in the 5-FU-resistant cells. These novel findings demonstrate that FN is a potentially useful biomarker and therapeutic target for improving the treatment of OSCC, particularly in the setting of 5-FU resistance.

Fibronectin aggregation in multiple sclerosis lesions impairs remyelination

Remyelination following central nervous system demyelination is essential to prevent axon degeneration. However, remyelination ultimately fails in demyelinating diseases such as multiple sclerosis. This failure of remyelination is likely mediated by many factors, including changes in the extracellular signalling environment. Here, we examined the expression of the extracellular matrix molecule fibronectin on demyelinating injury and how this affects remyelination by oligodendrocytes progenitors. In toxin-induced lesions undergoing efficient remyelination, fibronectin expression was transiently increased within demyelinated areas and declined as remyelination proceeded. Fibronectin levels increased both by leakage from the blood circulation and by production from central nervous system resident cells. In chronically demyelinated multiple sclerosis lesions, fibronectin expression persisted in the form of aggregates, which may render fibronectin resistant to degradation. Aggregation of fibronectin was similarly observed at the relapse phase of chronic experimental autoimmune encephalitis, but not on toxin-induced demyelination, suggesting that fibronectin aggregation is mediated by inflammation-induced demyelination. Indeed, the inflammatory mediator lipopolysaccharide induced fibronectin aggregation by astrocytes. Most intriguingly, injection of astrocyte-derived fibronectin aggregates in toxin-induced demyelinated lesions inhibited oligodendrocyte differentiation and remyelination, and fibronectin aggregates are barely expressed in remyelinated multiple sclerosis lesions. Therefore, these findings suggest that fibronectin aggregates within multiple sclerosis lesions contribute to remyelination failure. Hence, the inhibitory signals induced by fibronectin aggregates or factors that affect fibronectin aggregation could be potential therapeutic targets for promoting remyelination.

Osteopontin and Fibronectin Levels Are Decreased in Vitreous of Autoimmune Uveitis and Retinal Expression of Both Proteins Indicates ECM Re-Modeling

Autoimmune uveitis is an intraocular inflammation that arises through autoreactive T-cells attacking the inner eye, eventually leading to blindness. However, the contributing molecular pathomechanisms within the affected tissues remain as yet elusive. The extracellular matrix (ECM) is a highly dynamic structure that varies tremendously and influences the encompassing tissue. In order to assess ECM re-modeling in autoimmune uveitis, we investigated the expression of ECM molecules fibronectin and osteopontin in vitreous and retina samples. This was carried out in the only spontaneous animal model for human autoimmue uveitis, namely equine recurrent uveitis (ERU) that resembles the human disease in clinical as well as in immunopathological aspects. ERU is a naturally occurring autoimmune disease in horses that develops frequently and has already proved its value to study disease-related pathomechanisms. Western blot analysis of fibronectin and osteopontin in healthy and uveitic vitreous revealed significant reduction of both proteins in uveitis. Immunohistochemical expression of fibronectin in healthy retinas was restricted to the inner limiting membrane abutting vimentin positive Müller cell endfeet, while in uveitic sections, a disintegration of the ILM was observed changing the fibronectin expression to a dispersed pattern extending toward the vitreous. Retinal expression of osteopontin in control tissue was found in a characteristic Müller cell pattern illustrated by co-localization with vimentin. In uveitic retinas, the immunoreactivity of osteopontin in gliotic Müller cells was almost absent. The ability of Müller cells to express fibronectin and osteopontin was additionally shown by immunocytochemistry of primary cultured equine Müller cells and the equine Müller cell line eqMC-7. In conclusion, severe ECM re-modeling in autoimmune uveitis reported here, might affect the adhesive function of fibronectin and thus the anchoring of Müller cell endfeet to the ILM. Furthermore, the absence of osteopontin in gliotic Müller cells might represent reduced neuroprotection, an osteopontin attribute that is intensively discussed.

Effect of cannabidiol on human gingival fibroblast extracellular matrix metabolism: MMP production and activity, and production of fibronectin and transforming growth factor β

Marijuana (Cannabis sativa) use may be associated with gingival enlargement, resembling that caused by phenytoin. Cannabidiol (CBD), a nonpsychotropic Cannabis derivative, is structurally similar to phenytoin. While there are many reports on effects of phenytoin on human gingival fibroblasts, there is no information on effects of Cannabis components on these cells. The objective of this study was to determine effects of CBD on human gingival fibroblast fibrogenic and matrix-degrading activities. Fibroblasts were incubated with CBD in serum-free medium for 1-6 d. The effect of CBD on cell viability was determined by measuring activity of a mitochondrial enzyme. The fibrogenic molecule transforming growth factor β and the extracellular matrix molecule fibronectin were measured by ELISA. Pro-MMP-1 and total MMP-2 were measured by ELISA. Activity of MMP-2 was determined via a colorimetric assay in which a detection enzyme is activated by active MMP-2. Data were analysed using ANOVA and Scheffe's F procedure for post hoc comparisons. Cannabidiol had little or no significant effect on cell viability. Low CBD concentrations increased transforming growth factor β production by as much as 40% (p < 0.001), while higher concentrations decreased it by as much as 40% (p < 0.0001). Cannabidiol increased fibronectin production by as much as approximately 100% (p < 0.001). Lower CBD concentrations increased MMP production, but the highest concentrations decreased production of both MMPs (p < 0.05) and decreased MMP-2 activity (p < 0.02). The data suggest that the CBD may promote fibrotic gingival enlargement by increasing gingival fibroblast production of transforming growth factor β and fibronectin, while decreasing MMP production and activity.

Fibronectin and tenascin-C: accomplices in vascular morphogenesis during development and tumor growth

In addition to soluble factors, the extracellular matrix (ECM) also plays a vital role in normal vasculogenesis and in the pathological angiogenesis of many disease states. Here we will review what is known about the role of the ECM molecules fibronectin and tenascin-C in the vasculature and highlight a potential collaborative interplay between these molecules in developmental and tumorigenic angiogenesis. We will address the evolution of these modular proteins, their cellular interactions and how they become assembled into an insoluble matrix that impacts the assembly of other ECM proteins and the bioavailability of pro-angiogenic factors. The role of fibronectin and tenascin-C networks in tumor angiogenesis and metastasis will be described. We will elaborate on lessons learned about their role in vessel function from the functional ablation or the ectopic expression of both molecules. We will also elaborate on potential mechanisms of how fibronectin and tenascin-C affect cell adhesion and signaling that are relevant to angiogenesis.

Fibronectin is a T H1-specific molecule in human subjects

T(H)1 cell-mediated immunity is essential for host defense against a variety of intracellular pathogens, such as mycobacteria, salmonella, and Leishmania species. A major T(H)1-mediated effector mechanism involves the IFN-gamma-induced killing of the pathogen by infected macrophages. The range of known T(H)1-specific effector molecules is limited, especially in human subjects. We sought to identify novel effector molecules that might be involved in T(H)1-mediated pathogen clearance. We performed microarray-based analysis of human T(H)1 and T(H)2 cells to identify T(H)1-specific molecules. These analyses identified the extracellular matrix molecule fibronectin as a highly expressed T(H)1-specific molecule. We examined the expression of fibronectin in a variety of human cell types by using real-time RT-PCR, ELISA, and Western blotting. We also studied the role of fibronectin in modulating monocyte phenotype using in vitro culture. We show that human T(H)1 cells constitutively express and secrete fibronectin after in vitro differentiation from naive precursors. Furthermore, we demonstrate that ex vivo human T(H)1 cells selectively express fibronectin when compared with T(H)2 cells. The predominant isoform of fibronectin expressed by T(H)1 cells contains additional domains of the protein responsible for alpha4beta1 integrin binding and activation of Toll-like receptor 4. We show that treatment of monocytes with T(H)1 cell-derived fibronectin induces expression of the proinflammatory cytokine IL-6 while inhibiting IL-10 expression. Because fibronectin also plays a major role in the attachment and opsonization of numerous intracellular pathogens, we propose that it might be a critical molecule produced by T(H)1 cells involved in pathogen eradication.

Adhering effect of Leptospira interrongan to major extracellular matrix molecules

To determine the adhering ability of Leptospira interrogans to extracellular matrix molecules (ECM) of host cells and its diversity. The infection models of L. interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 to J774A.1, L929 and Vero cells were established. Fontana silver impregnation method was applied to demonstrate the adhering effect of microbes to extracellular matrix (ECM) of the cells. ELISA methods were established to detect the adhering effects of L. interrogans to ECM molecules laminin (LN), fibronectin (FN), decorin (DEN) and collagen1, 2, 4 (COL1, 2, 4). A competitive inhibition test was performed to verify the results. L. interrogans strain 56601 adhered the ECMs of L929, J774A.1 and Vero cells with its one or two sites. L. interrogans strain 56601 adhered all six ECM molecules; the adhering effects to COL1, LN, COL4 were relatively stronger. The adhering effects were markedly decreased after the microbes were pre-incubated with corresponding ECM molecules. L. interrogans adheres to host cells through ECM molecules; LN, FN, DEN, COL1, COL2 and COL4 are the receptor molecules with different adhesion intensity.

Syndecan-1 supports integrin α2β1-mediated adhesion to collagen

Several different receptor molecules act in concert to regulate cell adhesion. Among these are cell-surface proteoglycans and integrins, which collaborate extensively in mediating binding of cells to extracellular matrix molecules fibronectin and vitronectin. However, very little is known about possible functional synergism between proteoglycans and integrins during adhesion of cells to collagen, although collagen is the most abundant protein in the human body. Here we show that cell-surface heparan sulphate proteoglycans (HSPGs) support integrin α2β1-mediated adhesion to collagen. Cells made devoid of HSPGs either by genetic means or by enzymatic digestions were unable to adhere to collagen via α2β1 integrin. HSPG-deficient cells also displayed impaired spreading and actin organization on collagen. Among different HSPG molecules syndecan-1 was found to play an important role in supporting α2β1 integrin-mediated adhesion. Using overexpression and knock-down experiments we demonstrated that syndecan-1, but not syndecan-2 or -4, enhanced binding of α2β1 to collagen. Moreover, syndecan-1 co-localized with α2β1 integrin and contributed to proper organization of cortical actin. Finally, crosstalk between syndecan-1 and α2β1 integrin was found to enhance the transcription of matrix metalloproteinase-1 in response to collagen binding. Our findings thus suggest that a previously unknown link between integrin α2β1 and syndecan-1 is important in regulating cell adhesion to collagen and in triggering integrin downstream signalling.

MMP-2 AND MMP-9 SECRETION BY RPE IS STIMULATED BY ANGIOGENIC MOLECULES FOUND IN CHOROIDAL NEOVASCULAR MEMBRANES

Matrix metalloproteinases (MMP)-2 and -9 play an important role in the pathogenesis of choroidal neovascularization (CNV). Retinal pigment epithelial cells (RPE) are an important source of MMPs in the outer retinal environment, however little is known about the local factors that modulate MMP secretion in these cells. The purpose of this study was to determine the effects of CNV involved growth factors and the extracellular matrix molecule fibronectin on MMP-2 and -9 secretion by cultured human RPE. MMP-2 and -9 secretion was studied using gelatin zymography, Western blot, and ELISA assay of RPE culture supernatants. The effects of stimulating the cells for 36 hours with vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bGFG), tumor necrosis factor-alpha (TNF-alpha), or fibronectin (FN), all angiogenic factors found in CNV membranes, was determined. Resting RPE cells secreted MMP-2 but not MMP-9. Stimulation with TNF-alpha induced secretion of MMP-9 and increased the secretion of MMP-2. MMP-2 secretion was also increased by stimulation with FN and VEGF, but not bFGF. The results indicated that the angiogenic molecules VEGF, FN, and TNF-alpha stimulate MMP-2 and -9 secretion from RPE and thus further promote CNV.

Epidermal Growth Factor-Induced Epithelio-Mesenchymal Transition in Human Breast Carcinoma Cells

PMC42-LA cells display an epithelial phenotype: the cells congregate into pavement epithelial sheets in which E-cadherin and β-catenin are localized at cell–cell borders. They abundantly express cytokeratins, although 5% to 10% of the cells also express the mesenchymal marker vimentin. Stimulation of PMC42-LA cells with epidermal growth factor (EGF) leads to epithelio-mesenchymal transition–like changes including up-regulation of vimentin and down-regulation of E-cadherin. Vimentin expression is seen in virtually all cells, and this increase is abrogated by treatment of cells with an EGF receptor antagonist. The expression of the mesenchyme-associated extracellular matrix molecules fibronectin and chondroitin sulfate proteoglycan also increase in the presence of EGF. PMC42-LA cells adhere rapidly to collagen I, collagen IV, and laminin-1 substrates and markedly more slowly to fibronectin and vitronectin. EGF increases the speed of cell adhesion to most of these extracellular matrix molecules without altering the order of adhesive preference. EGF also caused a time-dependent increase in the motility of PMC42-LA cells, commensurate with the degree of vimentin staining. The increase in motility was at least partly chemokinetic, because it was evident both with and without chemoattractive stimuli. Although E-cadherin staining at cell–cell junctions disappeared in response to EGF, β-catenin persisted at the cell periphery. Further analysis revealed that N-cadherin was present at the cell–cell junctions of untreated cells and that expression was increased after EGF treatment. N- and E-cadherin are not usually coexpressed in human carcinoma cell lines but can be coexpressed in embryonic tissues, and this may signify an epithelial cell population prone to epithelio-mesenchymal–like responses.

The Eighth FIII Domain of Human Fibronectin Promotes Integrin α5β1 Binding via Stabilization of the Ninth FIII Domain

Binding of the extracellular matrix molecule fibronectin to the integrin receptor alpha(5)beta(1) elicits downstream signaling pathways that modulate cell function. Fibronectin-alpha(5)beta(1) interaction occurs via the conserved RGD sequence in the tenth FIII (FIII10) domain of fibronectin. A synergistic site containing the sequence PHSRN in the adjacent FIII9 domain has also been identified. Here we investigate the function of the eighth FIII domain in integrin-mediated cell adhesion using a wide range of methods, including biochemical, biological, and biophysical assays of integrin binding, cell adhesion, and protein denaturation. Mutation of the FIII9 synergistic site (PHSRN to PHAAA) in FIII9-10 reduced the binding activity for integrin alpha(5)beta(1) to levels observed for FIII10 alone, but the corresponding mutant in FIII8-9-10 showed no loss of binding activity. Cell adhesion assays also demonstrated enhanced functional activity of constructs containing FIII8. Equilibrium chemical denaturation studies indicated that FIII8 confers conformational stability upon FIII9, but only if the exposed loops, PHSRN and VKNEED on FIII9 and FIII8, respectively, are intact. These results demonstrate that the loss of integrin binding activity, observed upon alteration of the PHSRN synergistic site of FIII9-10, results partly from a loss of conformational stability of FIII9. Our data suggest a mechanism for integrin alpha(5)beta(1)-fibronectin interaction, which in addition to the primary RGD binding event, involves a conformation-sensitive scanning by the integrin for accessible sites on the ligand, whereupon full activation of downstream signaling occurs.

α4 Integrin Is Expressed during Peripheral Nerve Regeneration and Enhances Neurite Outgrowth

We have shown previously that repair in the peripheral nervous system is associated with a reversion to an embryonic pattern of alternative splicing of the extracellular matrix molecule fibronectin. One of the consequent changes is a relative increase in the number of fibronectins expressing the binding site for alpha4 integrins. Here we show that alpha4 integrins are expressed on dorsal root ganglion neuron cell bodies and growth cones in the sciatic nerve during regeneration and that the interaction of alpha4 integrin with alternatively spliced isoforms of recombinant fibronectins containing the alpha4 binding site enhances neurite outgrowth in dorsal root ganglion neurons. The pheochromocytoma (PC12) neuronal cell line, which normally extends neurites poorly on fibronectin, does so efficiently when alpha4 is expressed in the cells. Experiments using chimeric integrins expressed in PC12 cells show that the alpha4 cytoplasmic domain is necessary and sufficient for this enhanced neurite outgrowth. In both dorsal root ganglion neurons and PC12 cells the alpha4 cytoplasmic domain is tightly linked to the intracellular adapter protein paxillin. These experiments suggest an important role for alpha4 integrin and paxillin in peripheral nerve regeneration and show how alternative splicing of fibronectin may provide a mechanism to enhance repair after injury.

Identification of a novel heparin-binding site in the alternatively spliced IIICS region of fibronectin: roles of integrins and proteoglycans in cell adhesion to fibronectin splice variants.

The extracellular matrix molecule fibronectin (FN) is a glycoprotein whose major functional property is to support cell adhesion. FN contains at least two distinct cell-binding domains: the central cell-binding domain and the HepII/IIICS region. The HepII region comprises type III repeats 12-14 and contains proteoglycan-binding sites, while the alternatively spliced IIICS segment possesses the major alpha4beta1 integrin-binding sites. Both cell surface proteoglycans and integrins are important for mediating the adhesion of cells to this region of FN. By comparing heparin binding to different recombinant splice variants of the HepII/IIICS region, evidence was obtained for the existence of a novel heparin-binding site in the centre of the IIICS. Site-directed mutagenesis of basic amino acid sequences in this region reduced heparin binding to recombinant HepII/IIICS proteins and, in conjunction with mutations in the HepII region, caused a synergistic loss of activity. Using the H/120 variant of FN, which contains type III repeats 12-15 and the full-length IIICS region, and the H/95 variant of FN, which contains type III repeats 12-15 but lacks the high affinity integrin-binding LDV sequence, the relative roles played by cell-surface proteoglycans and integrins in mediating cell adhesion have been investigated. This was achieved by studying the effects of anti-integrin antibodies and exogenous heparin on A375 melanoma cell attachment to the wild-type and three different mutants of H/120 and H/95 in which the potential proteoglycan-binding sites were partially or completely removed. A375 cell adhesion to H/120 and its mutants was found to involve the co-operative action of both integrin and cell-surface proteoglycan binding, although integrin made a dominant contribution. Anti-integrin antibodies and exogenous heparin were capable of inhibiting melanoma cell adhesion to H/95 and in this case adhesion was due primarily to cell-surface proteoglycan and not integrin binding.

Opposing views on tensegrity as a structural framework for understanding cell mechanics.

Donald E. Ingber: Important new theories in science often ignite heated debates. If they do not, they are probably of little significance. Thus a strong argument in support of the importance of the tensegrity model of cell and tissue architecture first proposed almost 20 years ago ([23][1], [24][2