Patterns of alternative splicing of fibronectin pre-mRNA in human adult and fetal tissues

Alternative splicing of fibronectin pre-mRNA at two distinct regions, termed ED-A and IIICS, was investigated with human adult and fetal tissues by the nuclease S1 protection assay. A clear tissue specificity was observed in the splicing pattern at the ED-A region. More ED-A+ than ED-A- mRNAs were identified in lung, whereas ED-A- mRNAs were predominantly expressed in liver. Endometrium contained nearly equal amounts of ED-A+ and ED-A- mRNAs. The splicing pattern at the ED-A region was also different between adult and fetal liver but not between adult and fetal lung. Tissue type specific splicing was also observed at the IIICS region. Although the mRNA species containing the complete IIICS sequence comprised 40-65% of the total fibronectin mRNAs irrespective of tissue types, expression of the mRNA species lacking a part or all of the IIICS sequence was more pronounced in adult liver than in other tissues including fetal liver. These results strongly suggest that the alternative splicing of fibronectin pre-mRNA in vivo is regulated in a tissue type specific manner at both the ED-A and IIICS regions and that it is developmentally regulated in liver but not in lung. On the basis of these and other observations reported previously, a possibility that a part of the fibronectins synthesized and secreted by hepatocytes is deposited in the tissue matrix is discussed.     

The type III-9 repeat of human fibronectin is encoded by a single exon which is not alternatively spliced.

Type III homologies of human fibronectin are generally encoded by two exons, with the exception of the ED-A and ED-B repeats which are encoded by a single exon undergoing alternative splicing. We report that also the type III-9 homology is encoded by a single exon. Further more, RT-PCR analysis, performed on mRNA purified from fetal and adult tissues and from normal and tumor-derived cell types, showed that the III-9 region is not undergoing alternative splicing in all samples tested.     

Cell type specific trans-acting factors are involved in alternative splicing of human fibronectin pre-mRNA.

ED-A and ED-B are facultative type III homologies of fibronectin, encoded by alternatively spliced exons, described in man and in rat. A hybrid alpha-globin-fibronectin minigene containing the ED-B region from the human gene has been transfected in human cell lines derived from various tissues, in order to study the processing of the generated precursor RNA in the different cell environments. In most tested lines the pre-RNA is alternatively spliced and produces two mature RNAs, with and without the ED-B exon, in different ratios that closely resemble the corresponding endogenous fibronectin RNAs. In a hepatoma cell line, Hep 3B, only one RNA is produced, in which the ED-B exon is absent; the same pattern of splicing is observed in liver. The data show that all the information required to produce accurate and regulated alternative splicing of the ED-B exon is contained in the fragment used and cell specific factors are necessary for the pre-RNA to be differentially spliced in the various cell lines. In contrast, expression in Hep 3B of a similar gene containing the ED-A area failed to reproduce the liver specific splicing pattern. Therefore regulation of ED-A processing is likely to involve different mechanisms to those responsible for control of ED-B splicing.     

Sequence analysis and in vivo expression show that alternative splicing of ED-B and ED-A regions of the human fibronectin gene are independent events.

The structure of two alternatively spliced regions. ED-A and ED-B, of human fibronectin gene, was determined, in order to show whether any similarity was present between the two. Although some interesting features are present in each, no obvious common structure or sequence homology was found. Functional analysis of the alternative splicing events was carried out by transient expression in Hela cells. A hybrid gene was constructed by inserting the ED-B region into the third exon of the human alpha 1-globin gene. The transfected hybrid gene is expressed and produces, in Hela cells, two alternatively spliced RNAs, showing a pattern very similar to that observed for the endogenous fibronectin gene in fibroblasts. Cotransfection of this gene with a similar gene containing the ED-A region, shows that no interference is present between the two alternative splicing processes.     

Preparation of Phage Antibodies to the ED-A Domain of Human Fibronectin

The fibronectin (FN) isoform containing the alternative spliced ED-A domain is much more expressed in fetal, tumoral, and regenerating tissues than in normal adult tissues. The ED-A containing FN is up-regulated by numerous cytokines, such as TGF-β, and, although in normal adult liver the ED-A domain is undetectable, in regenerating rat liver the expression of ED-A is increased and mediates the conversion of fat storing cells to myofibroblasts. Here we describe the selection from a phage display library and the characterization of human antibody fragments directed against the ED-A sequence of FN. As they can be easily radiolabeled with³²P, these antibodies are very highly sensitive reagents for the determination of ED-A levels in tissues and biological fluids; in fact, use of these scFv induced a more than 10-fold increase in sensitivity with respect to the murine monoclonal IST-9. The possibility of preparing a range of human engineered antibodies should facilitate the development of antibody reagents with suitable pharmacokinetics, valency, functional affinity, and effector functions and that could be useful for clinical purposes.     

Monoclonal antibodies in the analysis of fibronectin isoforms generated by alternative splicing of mRNA precursors in normal and transformed human cells

Recent results showing that a single fibronectin gene can give rise to several different mRNAs by alternative splicing have offered an explanation for fibronectin polymorphism. Here we report on monoclonal antibodies that show specificity for a fibronectin segment (ED) that can be included or omitted from the molecule depending on the pattern of splicing of the mRNA precursors. Using these monoclonals, we have quantitatively analyzed the expression of the ED sequence in human fibronectin from different sources. The results demonstrated that, at the protein level, the ED segment is not expressed in plasma fibronectin and that, in fibronectin from the tissue culture medium of tumor-derived or simian virus-40-transformed human cells, the percentage of fibronectin molecules containing the ED segment is about 10 times higher than in fibronectin from normal human fibroblasts. These results suggest that in malignant cells the mechanisms that regulate the splicing of mRNA precursors are altered.     

Changes in extra cellular matrix remodelling and re-expression of fibronectin and tenascin-C splicing variants in human myocardial tissue of the right atrial auricle: implications for a targeted therapy of cardiovascular diseases using human SIP format antibodies

Cardiovascular diseases are accompanied by changes in the extracellular matrix (ECM) including the re-expression of fibronectin and tenascin-C splicing variants. Using human recombinant small immunoprotein (SIP) format antibodies, a molecular targeting of these proteins is of therapeutic interest. Tissue samples of the right atrial auricle from patients with coronary artery disease and valvular heart disease were analysed by PCR based ECM gene expression profiling. Moreover, the re-expression of fibronectin and tenascin-C splicing variants was investigated by immunofluoerescence labelling. We demonstrated changes in ECM gene expression depending on histological damage or underlying cardiac disease. An increased expression of fibronectin and tenascin-C mRNA in association to histological damage and in valvular heart disease compared to coronary artery disease could be shown. There was a distinct re-expression of ED-A containing fibronectin and A1 domain containing tenascin-C detectable with human recombinant SIP format antibodies in diseased myocardium. ED-A containing fibronectin showed a clear vessel positivity. For A1 domain containing tenascin-C, there was a particular positivity in areas of interstitial and perivascular fibrosis. Right atrial myocardial tissue is a valuable model to investigate cardiac ECM remodelling. Human recombinant SIP format antibodies usable for an antibody-mediated targeted delivery of drugs might offer completely new therapeutic options in cardiac diseases.     

Polarized regulation of fibronectin secretion and alternative splicing by transforming growth factor

Fibronectin is a multifunctional protein that is synthesized in several different forms that result from alternative splicing of mRNA. Although expression of splicing variants appears to be both developmentally regulated and tissue-specific, the functional significance of these isoforms is largely unknown. We found that cultured airway epithelial cells vectorially secrete two distinct species of fibronectin, one which contains the alternatively spliced EIIIA region (EIIIA+) and one in which the EIIIA segment is spliced out (EIIIA-). Fibronectin containing the EIIIA region is preferentially secreted apically. Although both apical and basal stimulation with transforming growth factor beta 1 increased fibronectin synthesis, the secretory response differed depending on which surface was being stimulated. Apical secretion of fibronectin and expression of EIIIA+ fibronectin mRNA increased only after apical stimulation. These data demonstrate a novel mechanism for the polarized regulation of targeted secretion and alternative splicing of fibronectin and suggest that the EIIIA segment may act as a targeting signal for the vectorial secretion of fibronectin.     

Mammary epithelial-mesenchymal interaction regulates fibronectin alternative splicing via phosphatidylinositol 3-kinase

The way alternative splicing is regulated within tissues is not understood. A relevant model of this process is provided by fibronectin, an important extracellular matrix protein that plays a key role in cell adhesion and migration and contains three alternatively spliced regions known as EDI, EDII, and IIICS. We used a cell culture system to simulate mammary epithelial-stromal communication, a process that is crucial for patterning and function of the mammary gland, and studied the effects of extracellular signals on the regulation of fibronectin pre-mRNA alternative splicing. We found that soluble factors from a mammary mesenchymal cell-conditioned medium, as well as the growth factors HGF/SF (hepatocyte growth factor/scatter factor), KGF (keratinocyte growth factor), and aFGF (acidic fibroblast growth factor), stimulate EDI and IIICS but not EDII inclusion into fibronectin mRNA in the mammary epithelial cell line SCp2, favoring fibronectin isoforms associated with proliferation, migration, and tissue remodeling. We explored the signaling pathways involved in this regulation and found that the mammary mesenchymal cell-conditioned medium and HGF/SF act through a phosphatidylinositol 3-kinase-dependent cascade to alter fibronectin alternative splicing. This splicing regulation is independent from promoter structure and de novo protein synthesis but does require two exonic elements within EDI. These results shed light on how extracellular stimuli are converted into changes in splicing patterns.     

Multiple sites of alternative splicing of the rat fibronectin gene transcript

We describe analyses of the structure and expression of the rat fibronectin gene with particular attention to the 40-kb stretch from the center of the gene which encodes 17 type-III repeating units. Each repeat is precisely separated from its neighbors by introns and most are encoded by pairs of exons. Three repeats are encoded precisely by single exons and two of these (EIIIA and EIIIB) are alternatively spliced in a cell type-specific fashion. A third site of alternative splicing (EIIIB) reported here is similar in expression to the previously described EIIIA segment. Both are excluded from mRNA in liver cells and are, therefore, absent from plasma fibronectin. These two alternative splices, plus a third one (V) reported previously, can occur in all possible combinations giving 12 fibronectin mRNAs from a single gene. These splicing variations account for most but not all of the known fibronectin subunit variants. We report investigations designed to detect other regions of alternative splicing. We also show that the pattern of alternative splicing is somewhat altered on oncogenic transformation.