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 α5ß1/αIIbß3 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-ß1-mediated cell signalling. We demonstrate that weakened α5ß1-mediated traction forces on FNsyn cause reduced TGF-ß1 release from its latent complex.

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 α5ß1 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 α5ß1 and αv-class integrins solely recognize the FN-RGD motif and that αv-class, but not α5ß1, 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.

Genetic abrogation of the fibronectin-α5ß1 integrin interaction in articular cartilage aggravates osteoarthritis in mice.

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 α5ß1 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 α5ß1-FN binding influences cartilage morphogenesis in vivo and whether non-ligated α5ß1 protects or aggravates the course of osteoarthritis in mice. We engineered mice (Col2a-Cre;Fn1RGE/fl), whose chondrocytes express an α5ß1 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 Col2a-Cre;Fn1RGE/fl mice was normal, while the exposure to high mechanical load induced osteoarthritis characterized by proteoglycan and collagen type II loss. In the Col2a-Cre;Fn1RGE/fl 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-α5ß1 adhesion as ECM sensor in circumstances of articular cartilage regeneration.

The fibronectin synergy site re-enforces cell adhesion and mediates a crosstalk between integrin classes.

Fibronectin (FN), a major extracellular matrix component, enables integrin-mediated cell adhesion via binding of α5ß1, αIIbß3 and αv-class integrins to an RGD-motif. An additional linkage for α5 and αIIb is the synergy site located in close proximity to the RGD motif. We report that mice with a dysfunctional FN-synergy motif (Fn1syn/syn) suffer from surprisingly mild platelet adhesion and bleeding defects due to delayed thrombus formation after vessel injury. Additional loss of ß3 integrins dramatically aggravates the bleedings and severely compromises smooth muscle cell coverage of the vasculature leading to embryonic lethality. Cell-based studies revealed that the synergy site is dispensable for the initial contact of α5ß1 with the RGD, but essential to re-enforce the binding of α5ß1/αIIbß3 to FN. Our findings demonstrate a critical role for the FN synergy site when external forces exceed a certain threshold or when αvß3 integrin levels decrease below a critical level.

Tribolium castaneum immune defense genes are differentially expressed in response to Bacillus thuringiensis toxins sharing common receptor molecules and exhibiting disparate toxicity.

In Tribolium castaneum larvae we have demonstrated by RNA interference knockdown that the Bacillus thuringiensis Cry3Ba toxin receptors Cadherin-like and Sodium solute symporter proteins are also functional receptors of the less active Cry3Aa toxin. Differences in susceptibility to B. thuringiensis infection might not only rely on toxin-receptor interaction but also on host defense mechanisms. We compared the expression of the immune related genes encoding Apolipophorin-III and two antimicrobial peptides, Defensin3 and Defensin2 after B. thuringiensis challenge. All three genes were up-regulated following Cry3Ba spore-crystal intoxication whereas only Defensins gene expression was induced upon Cry3Aa spore-crystal treatment, evidencing a possible association between host immune response and larval susceptibility to B. thuringiensis. We assessed the antimicrobial activity spectra of T. castaneum defensins peptide fragments and found that a peptide fragment of Defensin3 was effective against the human microbial pathogens, Escherichia coli, Staphylococcus aureus and Candida albicans, being S. aureus the most susceptible one.