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.     

Embryonic fibronectins are up-regulated following peripheral nerve injury in rats

Fibronectin mRNAs that include the alternatively spliced exons EIIIA, EIIIB, and V are prevalent during embryogenesis, and EIIIA and EIIIB reappear during wound healing. Using ribonuclease protection analyses, we found an up-regulation of V120 (containing the α₄β₁ integrin binding site), as well as EIIIA, and EIIIB in fibronectin mRNAs in the crush-injured adult rat sciatic nerve. In situ hybridization using splice variant-specific probes revealed that cells within endoneurial tubes of the injured nerve synthesize these embryonic forms of fibronectin. Our results suggest that embryonic fibronectins synthesized within the nerve contribute to the permissiveness of the peripheral nervous system to axon regrowth and a mechanism by which alternative splicing of the V region in fibronectin mRNA could enhance nervous system regeneration. © 1995 John Wiley & Sons, Inc.     

Expression of variant fibronectins in wound healing: cellular source and biological activity of the EIIIA segment in rat hepatic fibrogenesis

We have examined the cell-specific expression of two fibronectin isoforms, EIIIA and EIIIB, during experimental hepatic fibrosis induced by ligation of the biliary duct. AT the mRNA level, EIIIA and EIIIB were undetectable in normal liver but expressed early injury, preceding fibrosis. The cellular sources of these changes were determined by fractionating the liver at various time points after bile duct ligation into its constituent cell populations and extracting RNA from the fresh isolates. EIIIA-containing fibronectin mRNA was undetectable in normal sinusoidal endothelial cells but increased rapidly within 12 h of injury. By contrast, the EIIIB form was restricted to hepatic lipocytes (Ito or fat-storing cells) and appeared only after a lag of 12-24 h: it was minimal in sinusoidal endothelial cells. Both forms were minimal in hepatocytes. At the protein level, EIIIA-containing fibronectin was markedly increased within two days of injury and exhibited a sinusoidal distribution. Secretion of this form by endothelial cells was confirmed in primary culture. Matrices deposited in situ by endothelial cells from injured liver accelerated the conversion ("activation") of normal lipocytes to myofibroblast-like cells, and pretreatment of matrices with monoclonal antibody to the EIIIA segment blocked this response. Finally, recombinant fibronectin peptide containing the EIIIA segment was stimulatory to lipocytes in culture. We conclude that expression of EIIIA fibronectin by sinusoidal endothelial cells is a critical early event in the liver's response to injury and that the EIIIA segment is biologically active, mediating the conversion of lipocytes to myofibroblasts.     

Amphibian Pleurodeles waltl Fibronectin: cDNA Cloning and Developmental Expression of Spliced Variants

Partial cDNA clones encoding approximately the carboxy terminal half of Pleurodeles fibronectin (FN) were isolated. They account for 4.7 Kbp of the 3' region of the FN mRNA. The cDNA nucleotide sequence comprises all three alternatively spliced segments designated EIIIA, EIIIB and V-segment, respectively. All three segments are included in FN mRNA synthesized during early embryogenesis whereas, from the tailbud stage onward the V-region was partially excluded. The isolation of Pleurodeles cDNA clones including the three different spliced EIIIA, EIIIB and V segment raises new possibilities for the study of the precise role of specific regions of FN in early amphibian development.     

Spatial expression of the alternatively spliced EIIIB and EIIIA segments of fibronectin in the early chicken embryo

Using domain-specific antibodies, we have analyzed the tissue distribution of fibronectins (FNs) containing the alternatively spliced EIIIB and EIIIA segments relative to total FN in early chicken embryos. The results show a selective loss of EIIIA+ FN staining in the notochordal sheath and in cartilaginous structures between 4.5 and 7.0 days of development. In other regions, EIIIB+ and EIIIA+ FNs are extensively codistributed in and around mesoderm-derived structures (somites, notochord, heart, and blood vessels), in basal laminae of endoderm and ectoderm-derived structures, as well as within the vicinity of neural crest formation and migration. We also noted that EIIIA staining overlaps with spatial patterns of distribution that have previously been described for the alpha4 integrin subunit, a component of the EIIIA receptor alpha4beta1.     

Amphibian Pleurodeles waltl Fibronectin: cDNA Cloning and Developmental Expression of Spliced Variants

Partial cDNA clones encoding approximately the carboxy terminal half of Pleurodeles fibronectin (FN) were isolated. They account for 4.7 Kbp of the 3′ region of the FN mRNA. The cDNA nucleotide sequence comprises all three alternatively spliced segments designated EIIIA, EIIIB and V-segment, respectively. All three segments are included in FN mRNA synthesized during early embryogenesis whereas, from the tailbud stage onward the V-region was partially excluded. The isolation of Pleurodeles cDNA clones including the three different spliced EIIIA, EIIIB and V segment raises new possibilities for the study of the precise role of specific regions of FN in early amphibian development.     

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.     

Alternative splicing during chondrogenesis: cis and trans factors involved in splicing of fibronectin exon EIIIA

Primary chicken mesenchymal cells from limb buds and vertebral chondrocytes have been used to study the changes that occur in alternative mRNA splicing of fibronectin exon EIIIA during chondrogenesis. The mesenchymal cell phenotype (exon EIIIA included) and chondrocyte phenotype (exon EIIIA excluded) were preserved in culture. Both primary cell types were transfected with an EIIIA minigene and alternative splicing was monitored by S1 protection assay. Differential cell-specific splicing of the reporter was observed. The roles of two regulatory elements, an exon splicing enhancer (ESE) and an exon splicing silencer (ESS) were examined. Both elements were required for EIIIA inclusion into mRNA in mesenchymal cells. Gel mobility shift assays revealed that both chondrocyte- and mesenchymal cell-derived nuclear extracts contained exon EIIIA binding factors, but the RNA binding factors present in the two cell types appeared to be distinct. The ESE and ESS appeared to cooperate in the formation of both cell type-specific complexes. These results suggest a model in which inhibitory factors enriched in chondrocytes compete with positive factors enriched in mesenchymal cells for binding to exon EIIIA, determining whether the exon is included.     

Alternative splicing during chondrogenesis: cis and trans factors involved in splicing of fibronectin exon EIIIA

Primary chicken mesenchymal cells from limb buds and vertebral chondrocytes have been used to study the changes that occur in alternative mRNA splicing of fibronectin exon EIIIA during chondrogenesis. The mesenchymal cell phenotype (exon EIIIA included) and chondrocyte phenotype (exon EIIIA excluded) were preserved in culture. Both primary cell types were transfected with an EIIIA minigene and alternative splicing was monitored by S1 protection assay. Differential cell‐specific splicing of the reporter was observed. The roles of two regulatory elements, an exon splicing enhancer (ESE) and an exon splicing silencer (ESS) were examined. Both elements were required for EIIIA inclusion into mRNA in mesenchymal cells. Gel mobility shift assays revealed that both chondrocyte‐ and mesenchymal cell‐derived nuclear extracts contained exon EIIIA binding factors, but the RNA binding factors present in the two cell types appeared to be distinct. The ESE and ESS appeared to cooperate in the formation of both cell type‐specific complexes. These results suggest a model in which inhibitory factors enriched in chondrocytes compete with positive factors enriched in mesenchymal cells for binding to exon EIIIA, determining whether the exon is included. J. Cell. Biochem. 76:341–351, 1999. © 1999 Wiley‐Liss, Inc.     

Alternative splicing of fibronectin mRNAs in chondrosarcoma cells: role of far upstream intron sequences

The fibronectin (FN) gene encodes multiple mRNAs through the process of alternative splicing, and production of certain isoforms is characteristic of a given cell type. Chondrocytes produce FNs that completely lack alternative exon EIIIA, and loss of inclusion of the exon is tightly linked to chondrogenic condensation of mesenchymal cells. The inclusion of a second exon, EIIIB, is high in embryonic cartilage, but declines with age. Multiple exons are omitted to produce the (V + C)-form that is highly specific for cartilage and chondrocytes. A rat chondrosarcoma cell line, RCS, was identified that preserves key features of the cartilage-specific splicing phenotype. RCS cells, which exclude exon EIIIA, and HeLa cells, which include exon EIIIA similar to mesenchymal cells, were used to assess the contribution of intron sequences flanking exon EIIIA to splicing regulation. Deletion of most of the intron downstream of the exon had little effect on splicing in either cell type. However, deletions within upstream intron 32-A reduced inclusion of the alternative exon in both cell types. The sequences involved lie more than 200 nucleotides away from the exon, but could not be localized to a single region by deletion mapping. These intronic sequences contribute to the efficiency of exon EIIIA recognition, but not to cell-type specific regulation. The normally inhibitory factor polypyrimidine tract binding protein promotes exon EIIIA inclusion in a manner that is partially dependent on the regulatory sequences within intron 32-A.     

Alternative splicing of fibronectin: three variants, three functions.

Fibronectin (FN) is a multi-functional extracellular matrix protein required for cell adhesion and migration, blood clotting, wound healing, and oncogenic transformation. The functional complexity is paralleled by structural diversity in that multiple forms of FN are generated by cell type-specific alternative splicing. In the rat, up to 12 different combinations of the three alternatively spliced segments (EIIIA, EIIIB, and the V region) are produced. What effects do these segments have on FN function? Recently, progress has been made in the identification of specific activities for the three Variants of the V region, V120, V95, and V0. FN-mediated cell adhesion, FN synthesis and secretion, and incorporation into blood clots are differentially affected by these isoforms. These results suggest that cellular behavior is modulated by environmental cues provided by different types and proportions of alternatively spliced FN variants.     

Cellular fibronectin is induced by epidermal growth factor, but not by dexamethasone or cyclic AMP in rat liver epithelial cells

Epidermal growth factor (EGF) induces fibronectin (FN) and FN mRNA in rat liver epithelial cells, under conditions where the factor also induces the cells to migrate. Newly synthesized protein is secreted into the medium and deposited as substratum-bound extracellular matrix. The levels of mRNA and the amount of protein synthesized are not influenced by cyclic AMP or dexamethasone, factors that have been found to modulate FN expression in other cells. However, the cells are sensitive to the factors, suggesting a cell-specific regulation. The EGF-induced RNA contains the sequences EIIIA and EIIIB characteristic of cellular fibronectin.     

Alternative splicing during chondrogenesis: modulation of fibronectin exon EIIIA splicing by SR proteins

The alternative exon EIIIA of the fibronectin gene is included in mRNAs produced in undifferentiated mesenchymal cells but excluded from differentiated chondrocytes. As members of the SR protein family of splicing factors have been demonstrated to be involved in the alternative splicing of other mRNAs, the role of SR proteins in chondrogenesis-associated EIIIA splicing was investigated. SR proteins interacted with chick exon EIIIA sequences that are required for exon inclusion in a gel mobility shift assay. Addition of SR proteins to in vitro splicing reactions increased the rate and extent of exon EIIIA inclusion. Co-transfection studies employing cDNAs encoding individual SR proteins revealed that SRp20 decreased mRNA accumulation in HeLa cells, which make A+ mRNA, apparently by interfering with pre-mRNA splicing. Co-transfection studies also demonstrated that SRp40 increased exon EIIIA inclusion in chondrocytes, but not in HeLa cells, suggesting the importance of cellular context for SR protein activity. Immunoblot analysis did not reveal a relative depletion of SRp40 in chondrocytic cells. Possible mechanisms for regulation of EIIIA splicing in particular, and chondrogenesis associated splicing in general, are discussed.