New insights into form and function of fibronectin splice variants

Abstract

The extracellular matrix (ECM) is a highly dynamic structure that not only provides a physical framework for cells within connective tissues, but also imparts instructive signals for development, tissue homeostasis and basic cell functions through its composition and ability to exert mechanical forces. The ECM of tissues is composed of, in addition to proteoglycans and hyaluronic acid, a number of proteins, most of which are generated after alternative splicing of their pre-mRNA. However, the precise function of these protein isoforms is still obscure in most cases. Fibronectin (FN), one of the main components of the ECM, is also one of the best-known examples of a family of proteins generated by alternative splicing, having at least 20 different isoforms in humans. Over the last few years, considerable progress on elucidating the functions of the alternatively spliced FN isoforms has been achieved with the essential development of key engineered mouse strains. Here we summarize the phenotypes of the mouse strains having targeted mutations in the FN gene, which may lead to novel insights linking function of alternatively spliced isoforms of fibronectin to human pathologies.

Figure 1. Fibronectin primary structure

The scheme shows a representation of a fibronectin dimer and its interactions (Panel A). The different types of homologies (twelve Type I, two Type II and fifteen Type III) are represented. Numbering of Type III homologies excludes EDA and EDB domains. Type I, II and III domains are constituted of 40, 60 and 90 aa, respectively. Constitutive (RGD) and alternatively spliced (LDV), synergy (PHSRN) and EDA (EDGIHEL) cell-binding sites are indicated, together with their integrin receptor partners. EDA and EDB splicing is similar in all species (either total inclusion or exclusion), while that of the IIICS region is species-specific (five variants in humans, three in rodents, and two in chickens). Type III homologies are organized in seven anti-parallel β strands (Spatial and planar representations are shown in Panels B and C, respectively).

Figure 2. Mechanisms of EDA and EDB alternative splicing

Panel A: EDA exon splicing is positively regulated by the splicing factor SF2/ASF, which recognizes the exonic splicing enhancer (ESE) displayed in a loop region of stem-loop structure of the FN pre-mRNA. Skipping of the EDA exon is favored by hnRNP A1. Panel B: The splicing factor SRp40 promotes EDB exon inclusion by binding to a purine-rich sequence (ESE) within the EDB exon itself, or by recognizing the intronic splicing enhancers (ISE) located in the adjacent downstream intron. The presence of weak splice sites favors skipping of the EDA and EDB exons.

Figure 3. Unrooted phylogenetic diagram showing the relationships of the EDA and EDB domains among vertebrates

The tree was constructed by comparing the amino acid sequence of the EDA and EDB domains with the ClustalW2 software (http://www.ch.embnet.org/software/ClustalW.html) by the neighbour-joining method [137]. The dnd file generated was plotted using the TreeViewPPC package. The abbreviations are described in Legend to Figures 3 and 4.

Figure 4. Role of EDA cFN in lung fibrosis

Fibroblast differentiation into myofibroblasts occurs only on the presence of EDA cFN that could be either produced and secreted by the fibroblast (EDA^wt/wt fibroblast, Panel A) or already present in the ECM, since EDA^−/− fibroblasts differentiate into myofibroblasts only when plated on a EDA cFN (Panels A and B). EDA cFN secreted by the cells or already present in the ECM activates latent TGF β (Panels A and B) and fibroblast differentiation proceeds, leading to lung fibrosis. In the absence of EDA cFN (produced either by the EDA^−/− cells or by the ECM prepared from EDA^−/− fibroblasts, Panel C), the absence of latent TGF β activation limits differentiation into myofibroblasts preventing lung fibrosis.

Figure 5. Schemes of the possible conformational changes of FN by the inclusion of the extra domains EDA and EDB

In the “closed” or “compact” conformation of pFN the RGD loop and nearby synergy sites are not available for interaction by integrins (Panel A). The inclusion of the EDA domain in EDA cFN (Panel B) triggers a conformational change enhancing the exposition of the RGD loop and the synergy site, and the binding of integrin receptors (indicated). The model postulates that inclusion of the EDB domain may also enhance the exposition of the RGD loop and the synergy site (Panel C). Asterisks in pFN indicate the expected position of the EDB (left asterisks) and EDA (right asterisks) domains if inserted (Panel A).