The Fibronectin RGD and synergy sites function in integrin interaction

Fibronectin (FN) is a large glycoprotein component of the extracellular matrix (ECM). It presents two forms: soluble in plasma or insoluble (fibrillar) within the ECM surrounding the cells in tissues. FN is one of the most important proteins in the ECM, it directly mediates cell adhesion, and therefore is essential for several biological processes such as embryonic development or blood clotting. Its involvement in these processes relay in its interaction with the cellular receptors integrins. The major binding site for integrins in FN is the so-called RGD site located in the 10th FN type III repeat (FNIII10), which is recognized by a5b1, aIIbb3 and all the av-containing integrins. Additionally, the a5b1 and aIIbb3 integrins can also bind the synergy site (DRVPPSRN) in the 9th FN type III repeat (FNIII9). In this work, we aimed to understand the function of these two different FN binding sites in interaction with integrins and fibrillar ECMs formation. When FN fibrillogenesis is disabled either due to insufficient FN production or defective assembly can lead to organ or tissue dysfunction. Integrin interaction with the RGD motif in FNIII10 has been considered the backbone of FN fibrillogenesis. This work has explored new RGDindependent mechanisms for FN fibrillogenesis. The results from this work showed that despite lacking the RGD site, FNdRGD can be assembled in a disorganized fibrillar matrix. The FNdRGD fibrillogenesis takes place through a different binding site located in the 12-14th FN type III repeat (heparinII) through binding of receptors distinct of integrins, the syndecan family of heparan sulphate. Although the RGD sequence in the FNIII10 domain is the key binding site for integrins, the synergy site has been demonstrated in adhesion experiments to cooperate in the interaction of the RGD motif with a5b1 and aIIbb3 in vitro. With the aim of analysing the role of the synergy site in vivo, in this project we analysed a mouse strain in with mutations in the sequence DRVPPSRN. The mutation does not affect mouse development, however the mutant synergy mice (Fn1syn/syn) have prolonged haemorrhages upon vessel injury, indicating that platelet-FN interaction is altered. To further study the function of the synergy site in integrin binding, we developed a series of in vivo and in vitro. In this work we show that the synergy site is not essential for cell adhesion or FN fibrillogenesis, but it is important to strengthen the bond to FN under shear forces. The a5b1 adhesion to FN reinforcement allows regulation of integrin-FN downstream signalling, assembly of focal adhesions and reorganization of the cytoskeleton. Integrin-FN reinforcement through the synergy site also modulates cellular adaptation to different rigidities. Moreover, we could demonstrate that the function of the synergy site can be compensated by av integrins in mesenchymal cells and by fibrinogen, a plasmatic ECM protein which binds aIIbb3.Fibronectin (FN) is a large glycoprotein component of the extracellular matrix (ECM). It presents two forms: soluble in plasma or insoluble (fibrillar) within the ECM surrounding the cells in tissues. FN is one of the most important proteins in the ECM, it directly mediates cell adhesion, and therefore is essential for several biological processes such as embryonic development or blood clotting. Its involvement in these processes relay in its interaction with the cellular receptors integrins. The major binding site for integrins in FN is the so-called RGD site located in the 10th FN type III repeat (FNIII10), which is recognized by a5b1, aIIbb3 and all the av-containing integrins. Additionally, the a5b1 and aIIbb3 integrins can also bind the synergy site (DRVPPSRN) in the 9th FN type III repeat (FNIII9). In this work, we aimed to understand the function of these two different FN binding sites in interaction with integrins and fibrillar ECMs formation. When FN fibrillogenesis is disabled either due to insufficient FN production or defective assembly can lead to organ or tissue dysfunction. Integrin interaction with the RGD motif in FNIII10 has been considered the backbone of FN fibrillogenesis. This work has explored new RGDindependent mechanisms for FN fibrillogenesis. The results from this work showed that despite lacking the RGD site, FNdRGD can be assembled in a disorganized fibrillar matrix. The FNdRGD fibrillogenesis takes place through a different binding site located in the 12-14th FN type III repeat (heparinII) through binding of receptors distinct of integrins, the syndecan family of heparan sulphate. Although the RGD sequence in the FNIII10 domain is the key binding site for integrins, the synergy site has been demonstrated in adhesion experiments to cooperate in the interaction of the RGD motif with a5b1 and aIIbb3 in vitro. With the aim of analysing the role of the synergy site in vivo, in this project we analysed a mouse strain in with mutations in the sequence DRVPPSRN. The mutation does not affect mouse development, however the mutant synergy mice (Fn1syn/syn) have prolonged haemorrhages upon vessel injury, indicating that platelet-FN interaction is altered. To further study the function of the synergy site in integrin binding, we developed a series of in vivo and in vitro. In this work we show that the synergy site is not essential for cell adhesion or FN fibrillogenesis, but it is important to strengthen the bond to FN under shear forces. The a5b1 adhesion to FN reinforcement allows regulation of integrin-FN downstream signalling, assembly of focal adhesions and reorganization of the cytoskeleton. Integrin-FN reinforcement through the synergy site also modulates cellular adaptation to different rigidities. Moreover, we could demonstrate that the function of the synergy site can be compensated by av integrins in mesenchymal cells and by fibrinogen,

αv-Class integrin binding to fibronectin is solely mediated by RGD and unaffected by an RGE mutation

Fibronectin (FN) is an essential glycoprotein of the extracellular matrix; binds integrins, syndecans, collagens, and growth factors; and is assembled by cells into complex fibrillar networks. The RGD motif in FN facilitates cell binding- and fibrillogenesis through binding to α5β1 and αv-class integrins. However, whether RGD is the sole binding site for αv-class integrins is unclear. Most notably, substituting aspartate with glutamate (RGE) was shown to eliminate integrin binding in vitro, while mouse genetics revealed that FNRGE preserves αv-class integrin binding and fibrillogenesis. To address this conflict, we employed single-cell force spectroscopy, engineered cells, and RGD motif-deficient mice (Fn1ΔRGD/ΔRGD) to search for additional αv-class integrin-binding sites. Our results demonstrate that α5β1 and αv-class integrins solely recognize the FN-RGD motif and that αv-class, but not α5β1, integrins retain FN-RGE binding. Furthermore, Fn1ΔRGD/ΔRGD tissues and cells assemble abnormal and dysfunctional FNΔRGD fibrils in a syndecan-dependent manner. Our data highlight the central role of FN-RGD and the functionality of FN-RGE for αv-class integrins

The Role of the Fibronectin Synergy Site for Skin Wound Healing

Skin is constantly exposed to injuries that are repaired with different outcomes, either regeneration or scarring. Scars result from fibrotic processes modulated by cellular physical forces transmitted by integrins. Fibronectin (FN) is a major component in the provisional matrix assembled to repair skin wounds. FN enables cell adhesion binding of α5β1/αIIbβ3 and αv-class integrins to an RGD-motif. An additional linkage for α5/αIIb is the synergy site located in close proximity to the RGD motif. The mutation to impair the FN synergy region (Fn1syn/syn) demonstrated that its absence permits complete development. However, only with the additional engagement to the FN synergy site do cells efficiently resist physical forces. To test how the synergy site-mediated adhesion affects the course of wound healing fibrosis, we used a mouse model of skin injury and in-vitro migration studies with keratinocytes and fibroblasts on FNsyn. The loss of FN synergy site led to normal re-epithelialization caused by two opposing migratory defects of activated keratinocytes and, in the dermis, induced reduced fibrotic responses, with lower contents of myofibroblasts and FN deposition and diminished TGF-β1-mediated cell signalling. We demonstrate that weakened α5β1-mediated traction forces on FNsyn cause reduced TGF-β1 release from its latent complex

Genetic abrogation of the fibronectin-α5β1 integrin interaction in articular cartilage aggravates osteoarthritis in mice

<div><p>The balance between synthesis and degradation of the cartilage extracellular matrix is severely altered in osteoarthritis, where degradation predominates. One reason for this imbalance is believed to be due to the ligation of the α5β1 integrin, the classic fibronectin (FN) receptor, with soluble FN fragments instead of insoluble FN fibrils, which induces matrix metalloproteinase (MMP) expression. Our objective was to determine whether the lack of α5β1-FN binding influences cartilage morphogenesis <i>in vivo</i> and whether non-ligated α5β1 protects or aggravates the course of osteoarthritis in mice. We engineered mice (<i>Col2a-Cre;Fn1</i>^{<i>RGE/fl</i>}), whose chondrocytes express an α5β1 binding-deficient FN, by substituting the aspartic acid of the RGD cell-binding motif with a glutamic acid (FN-RGE). At an age of 5 months the knee joints were stressed either by forced exercise (moderate mechanical load) or by partially resecting the meniscus followed by forced exercise (high mechanical load). Sections of femoral articular knees were analysed by Safranin-O staining and by immunofluorescence to determine tissue morphology, extracellular matrix proteins and matrix metalloproteinase expression. The articular cartilage from untrained control and <i>Col2a-Cre;Fn1</i>^{<i>RGE/fl</i>} mice was normal, while the exposure to high mechanical load induced osteoarthritis characterized by proteoglycan and collagen type II loss. In the <i>Col2a-Cre;Fn1</i>^{<i>RGE/fl</i>} articular cartilage osteoarthritis progressed significantly faster than in wild type mice. Mechanistically, we observed increased expression of MMP-13 and MMP-3 metalloproteinases in FN-RGE expressing articular cartilage, which severely affected matrix remodelling. Our results underscore the critical role of FN-α5β1 adhesion as ECM sensor in circumstances of articular cartilage regeneration.</p></div

Immunofluorescence and quantification of MMP-3 and MMP-13 in femoral knee cartilage exposed to high load.

<p>A) Immunofluorescence staining for MMP-3 levels and quantification of pixel density per mm² in the articular cartilage of mice 10 days after exposure to normal activity (normal), moderate load (FEx) and high load (10 DAI). B) MMP-13 levels. Values represent mean ± SEM. Scale bars, 50 μm. Statistical significances: *p<0.05, **p<0.01 and ***p<0.001.</p

Morphology of articular cartilage in <i>Fn1</i>^<i>RGE/-</i> mice.

<p>A) Representative Safranin-O stained sections from wild type (<i>Fn1</i>^<i>wt/wt</i>) and mutant (<i>Fn1</i>^<i>RGE/-</i>) knee joint cartilage from 5-month-old male mice. Femoral condyle (fc) and tibial plateau (tp) are shown. B) High magnification showing the femoral cartilage. The dotted line indicates the tidemark that separates the non-calcified (superficial) from the calcified zone. C, D) Hematoxylin and immunoperoxidase staining to detect aggrecan, collagen type II and FN in the femoral condyle (C) and tibial (D) articular cartilage. Scale bars, 50 μm.</p