Extra Domain A Research Articles

Regulated splicing of the fibronectin EDA exon is essential for proper skin wound healing and normal lifespan.

Fibronectins (FNs) are multifunctional high molecular weight glycoproteins present in the blood plasma and in the ECMs of tissues. The FN primary transcript undergoes alternative splicing in three regions generating up to 20 main different variants in humans. However, the precise role of the FN isoforms is poorly understood. One of the alternatively spliced exons is the extra domain A (EDA) or extra type III homology that is regulated spatially and temporally during development and aging. To study its in vivo function, we generated mice devoid of EDA exon-regulated splicing. Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP–mediated deletion of the exon. Homozygous mouse strains with complete exclusion or inclusion of the EDA exon were viable and developed normally, indicating that the alternative splicing at the EDA exon is not necessary during embryonic development. Conversely, mice without the EDA exon in the FN protein displayed abnormal skin wound healing, whereas mice having constitutive inclusion of the EDA exon showed a major decrease in the FN levels in all tissues. Moreover, both mutant mouse strains have a significantly shorter lifespan than the control mice, suggesting that EDA splicing regulation is necessary for efficient long-term maintenance of biological functions.

Inhibition of cryogelation by the novel synthetic peptide (Gly–Arg–Lys–Lys–Thr): recognition site of extra domain A containing fibronectin for heparin in cryogelation

Cryogel is a physical gel formed by the heterophilic aggregation of extra domain A (EDA) containing fibronectin [EDA(+)FN], plasma fibronectin (pFN), fibrinogen (Fbg) and heparin (Hep) in the blood of rheumatoid arthritis (RA) patients. In cryogelation EDA(+)FN cross-links to form an interaggregate of cryogel with Hep. In the present study, we determined the recognition structure of Hep for EDA(+)FN by using oligo- and desulfonated-Hep. The affinity constant (KA) (1.2×10 8 per M) of oligo-Hep for EDA(+)FN did not change with a decrease in number-average molecular weight (4.9×10 4→6.0×10 3). The KA-value of desulfonated-Hep for EDA(+)FN decreased from 3.2×10 8 to 1.0×10 7 per M with a decrease in the sulfonation ratio (7.0→4.3%). We also determined the recognition structure of EDA(+)FN for Hep by an inhibition experiment on the heparin binding domain II (HepII) in EDA(+)FN with the synthetic peptides, Arg–Arg–Ala–Arg (RRAR), Asp–Gln–Ala–Arg (DNAR), Ile–Lys–Tyr–Glu–Lys (IKYEK), and Gly–Arg–Lys–Lys–Try (GRKKT). The GRKKT sequence clearly inhibited bonding between EDA(+)FN and Heps containing oligo- and desulfonated-Hep. The amount of cryogel formed in the RA-patient model plasma corresponded to the EDA(+)FN concentration in cryogel (36.7%) normalized by the EDA(+)FN concentration in plasma. When GRKKT was added to plasma, the EDA(+)FN concentration fell to 10.5%. These results demonstrated that inhibition of cryogelation in plasma could progress to a novel treatment for RA.

Promoter Architecture Modulates CFTR Exon 9 Skipping

Using hybrid minigene experiments, we have investigated the role of the promoter architecture on the regulation of two alternative spliced exons, cystic fibrosis transmembrane regulator (CFTR) exon 9 and fibronectin extra domain-A (EDB). A specific alternative splicing pattern corresponded to each analyzed promoter. Promoter-dependent sensitivity to cotransfected regulatory splicing factor SF2/ASF was observed only for the CFTR exon 9, whereas that of the EDB was refractory to promoter-mediated regulation. Deletion in the CFTR minigene of the downstream intronic splicing silencer element binding SF2/ASF abolished the specific promoter-mediated response to this splicing factor. A systematic analysis of the regulatory cis-acting elements showed that in the presence of suboptimal splice sites or by deletion of exonic enhancer elements the promoter-dependent sensitivity to splicing factor-mediated inhibition was lost. However, the basal regulatory effect of each promoter was preserved. The complex relationships between the promoter-dependent sensitivity to SF2 modulated by the exon 9 definition suggest a kinetic model of promoter-dependent alternative splicing regulation that possibly involves differential RNA polymerase II elongation.

Radial scars of the breast and breast carcinomas have similar alterations in expression of factors involved in vascular stroma formation

We recently reported that radial scars are an independent histologic risk factor for breast cancer. The reason for this association is not known. Given the importance of stromal–epithelial interactions in the pathogenesis of breast cancer, we studied radial scars for the expression of a number of factors known to be involved in the formation of vascular stroma in breast cancer. In situ hybridization was performed on formalin-fixed paraffin sections using 35S-labeled riboprobes for collagen type 1, total fibronectin, extra domain A (ED-A)+ fibronectin, thrombospondin 1, vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF), and one of its endothelial receptors, kinase insert domain–containing receptor (KDR) (vascular endothelial growth factor receptor [VEGFR-2]). Expression levels in radial scars (9 cases) were compared with those in normal breast tissue (15 cases) and infiltrating ductal breast carcinoma (4 cases). Factor VIII–related antigen immunostaining was used to define the distribution of microvessels in radial scars, carcinoma, and normal breast tissue. Compared with normal breast tissue, the radial scars showed focally increased numbers of blood vessels and focally increased expression of messenger RNA (mRNA) for collagen type 1, total fibronectin, ED-A+ fibronectin, thrombospondin 1, VPF/VEGF, and KDR. This pattern of mRNA overexpression was similar to that seen in the 4 invasive cancers. We conclude that there are similarities between radial scars and invasive breast cancers with regard to the level of mRNA expression for several factors involved in the formation of vascular stroma. These results suggest that a similar disturbance in stromal–epithelial interactions is present in both lesions. HUM PATHOL 33:29-38. Copyright © 2002 by W.B. Saunders Company

The Extra Domain A of Fibronectin Activates Toll-like Receptor 4

Cellular fibronectin, which contains an alternatively spliced exon encoding type III repeat extra domain A (EDA), is produced in response to tissue injury. Fragments of fibronectin have been implicated in physiological and pathological processes, especially tissue remodeling associated with inflammation. Because EDA-containing fibronectin fragments produce cellular responses similar to those provoked by bacterial lipopolysaccharide (LPS), we examined the ability of recombinant EDA to activate Toll-like receptor 4 (TLR4), the signaling receptor stimulated by LPS. We found that recombinant EDA, but not other recombinant fibronectin domains, activates human TLR4 expressed in a cell type (HEK 293 cells) that normally lacks this Toll-like receptor. EDA stimulation of TLR4 was dependent upon co-expression of MD-2, a TLR4 accessory protein. Unlike LPS, the activity of EDA was heat-sensitive and persisted in the presence of the LPS-binding antibiotic polymyxin B and a potent LPS antagonist, E5564, which completely suppressed LPS activation of TLR4. These observations provided a mechanism by which EDA-containing fibronectin fragments promote expression of genes involved in the inflammatory response.

The Fibronectin Extra Domain A Activates Matrix Metalloproteinase Gene Expression by an Interleukin-1-dependent Mechanism

The extra domain-A (EDA), present in fibronectin (FN) molecules arising from alternatively spliced transcripts, appears only during specific biological and pathogenic processes. However, its function is poorly understood. To define the physiologic role of this domain in joint connective tissue, the biological effects on rabbit cartilage explants, chondrocytes, and synovial cells were studied. A recombinant EDA protein (rEDA) increased proteoglycan release (3. 6-fold) in cartilage explant cultures and markedly induced production of matrix metalloproteinase (MMP)-1 in chondrocytes. In addition, rEDA induced MMP-1, MMP-3, and MMP-9 in synovial cells. These effects were elicited only by rEDA, while its neighboring type III repeats, III(11) or III(12), scarcely had any such effects. Interestingly, reorganization of F-actin stress fibers accompanied MMP-1 expression in synovial cells treated with rEDA, suggesting alteration of cellular phenotype. Subsequent Northern blotting revealed expression of pro-inflammatory cytokines, including interleukin (IL)-1alpha and IL-1beta, was induced by rEDA prior to MMP-1 expression. Delayed MMP-1 expression suggests that rEDA-induced IL-1s promote MMP-1 expression in an autocrine manner. This hypothesis is supported by the reduction of EDA-induced MMP-1 production by IL-1 receptor antagonist. The effect of EDA on MMP-1 production was reduced by connection with an adjacent type III repeat on either the NH(2) or COOH side of EDA and was abolished by connection on both sides of EDA, suggesting that exposure of either the NH(2) or COOH terminus of EDA domain by proteolytic cleavage releases the inducing activity. In agreement with these results, full-length cellular FN did not induce MMP-1 production. Furthermore, a 160-kDa EDA-positive FN fragment, which was purified from human placental tissue and corresponds to the region from NH(2) terminus through the EDA, induced MMP-1 production. Taken together, these results suggest that the EDA in FN fragments triggers alterations of cell physiology and plays a role in matrix degradation in joint connective tissue.

Cellular Fibronectin is Induced in Ultraviolet-Exposed Human Skin and Induces IL-10 Production by Monocytes/Macrophages

CD11b+ monocytic/macrophagic cells that infiltrate human skin after in vivo ultraviolet exposure potently produce interleukin-10. We hypothesized that binding of monocyte beta1 integrins to ultraviolet-induced extracellular matrix ligands, such as fibronectin, after entry of blood monocytes into the dermis, is involved in the modulation of immunoregulatory monocytic cytokines. Immunostaining of human skin and reverse transcriptase-polymerase chain reaction studies revealed that the embryonic isoform of cellular fibronectin, in which the extra domain A (EDA) segment is spliced in (EDA+ cellular fibronectin), and confers enhanced binding to beta1 integrins, is newly induced and is associated with infiltrating CD11b+ cells post in vivo ultraviolet exposure. We then tested the effect of fibronectin on resting purified peripheral monocytes in vitro. We found that monocyte interleukin-10, but not interleukin-12, was significantly induced in a concentration-dependent manner by in vitro binding to cellular fibronectin (n = 6), but not plasma fibronectin. Tumor necrosis factor-alpha was also induced in a concentration-dependent manner, but to a lesser extent. Monoclonal antibodies to beta1 integrins beta-subunit (CD29) also strongly induced tumor necrosis factor-alpha and interleukin-10 production, but not interleukin-12. Neutralization of tumor necrosis factor-alpha reduced by 54% the interleukin-10 production that was induced by monocytes binding to cellular fibronectin, indicating that interleukin-10 induction is at least in part dependent upon concomitant autocrine tumor necrosis factor-alpha release. In conclusion, ultraviolet skin injury results in increased production and deposition of EDA+ cellular fibronectin in the papillary dermis, which may be one of the key signals capable of inducing interleukin-10 but not interleukin-12 in monocytes that infiltrate micromilieu of human skin after ultraviolet exposure.

The distribution of laminins and fibronectins is modulated during extravillous trophoblastic cell differentiation and decidual cell response to invasion in the human placenta.

We studied the distribution of laminin (Ln) alpha1-alpha3, beta1-beta3, and gamma1 chains, and of the extradomain-A (EDA) and EDB and the oncofetal epitope of fibronectin (Onc-Fn) in extravillous trophoblastic cells and decidua in the human placenta by immunohistochemistry. We found that the transition from villous to extravillous trophoblast was accompanied by emergence of immunoreactivity for EDA-, EDB-, and Onc-Fn among the cells. Furthermore, whereas the villous trophoblastic basement membrane (BM) contains Ln alpha1, alpha2, beta1, beta2, and gamma1 chains, immunoreactivity for Ln alpha1, beta1, and gamma1, but not for Ln alpha2 and beta2 chains, was detected in association with extravillous trophoblastic cells. Interestingly, although immunoreactivity for the Ln alpha1, alpha2, beta1, beta2, and gamma1 chains was detected in all decidual cell BMs, EDB-Fn and Onc-Fn were detected only in decidua that had been invaded by the trophoblast. In summary, our results describe distinct changes in the distribution of Ln and Fn isoforms during the differentiation of villous trophoblast into extravillous trophoblastic cells. Furthermore, EDB- and Onc-Fn are preferentially found in decidua that has been invaded by the trophoblast, indicating that the deposition of these Fn isoforms reflects a decidual cell response to invasion.

Tenascin and fibronectin isoforms in human renal cell carcinomas, renal cell carcinoma cell lines and xenografts in nude mice

We studied the expression of tenascin (Tn) and isoforms of fibronectin (Fn) in human renal cell carcinomas (RCC) and oncocytomas, in RCC cell lines and in their s.c. implanted xenografts in nude mice. In well-differentiated RCCs and oncocytomas extra-domain A (EDA)-Fn and Tn immunoreactivities were confined to the basement membranes and blood vessels, while in less-differentiated RCCs they were also widely seen in the stroma, correlating with the morphological differentiation of the tumor. Expression of EDB-Fn and oncofetal (onc)-Fn was very scarce in most of the RCCs and oncocytomas. Western blotting results demonstrated the predominance of the M(r) 190,000 Tn subunit in most RCCs. Among the 4 RCC cell lines, 3 showed Tn in the extracellular matrix. As xenografts, they formed moderately or poorly differentiated tumors, with abundant Tn. Three of the RCC cell lines also showed secretion of EDA-Fn and 2 of them secretion of onc-Fn and EDB-Fn into the culture medium, while in xenografts there was a strong expression of all Fn isoforms. In xenografts, the expression of Tn closely recapitulated that seen in clinical tumors and in the cell lines in vitro, while the expression of Fn isoforms in cultured cells and their xenografts was highly discordant.

Transforming growth factor-beta regulates the expression of fibronectin and tenascin in BEAS 2B human bronchial epithelial cells.

We used monoclonal antibodies to study expression and extracellular matrix (ECM) incorporation of tenascin (Tn) and isoforms of fibronectin (Fn) in BEAS 2B immortalized human bronchial epithelial cells and the regulation of their synthesis by transforming growth factor (TGF)-beta 1 and -beta 2. In immunofluorescence microscopy, the control cells appeared negative for Tn. Extradomain A (EDA)-Fn was mainly seen in association with ECM fibers and, in a few cells, in an intracellular location. Immunoreactivity for oncofetal (onc)-Fn and extradomain B (EDB)-Fn was only seen in a few cells. In TGF-beta 1- and -beta 2-treated cells, a greatly enhanced immunostaining for Tn and three isoforms of Fn was seen both as to the number of positive cells and to the amount of immunoreactive material around them. In Western blotting of the untreated cells, EDA-Fn and onc-Fn were detected in the cell-free ECM and in the culture medium, whereas EDB-Fn was not detectable. An enhanced secretion and deposition of both EDA-Fn and onc-Fn and also secretion of EDB-Fn was seen upon treatment with TGF-beta s. In TGF-beta-treated cells, Tn was found exclusively in the ECM and not in the culture medium as shown by Western blotting of cell-free ECM and culture medium, respectively. Accentuation of tenascin staining in TGF-beta-treated cells was due to a greatly enhanced production of M(r) 280,000 and M(r) 190,000 isoforms of Tn.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular fibronectin in serum and plasma: a potential new tumour marker?

The concentration of cellular fibronectin (cFN) containing the extra domain A (EDA) was measured in 479 plasma and 300 serum samples from healthy blood donors by a competitive enzyme immunoassay (EIA). Serum and plasma samples contained low concentrations of EDAcFN. The mean concentration of EDAcFN was higher in plasma (2.46 mg l-1) than in serum (0.30 mg l-1). No significant differences between sexes or age groups were found. The EDAcFN concentrations were also measured in 120 patients with various malignancies. The mean values in serum were 4.28 mg l-1, 2.01 mg l-1 and 5.18 mg l-1 in patients with digestive tract malignancies, breast cancer and a group of miscellaneous cancers respectively. In plasma, the corresponding values were 12.26 mg l-1, 4.38 mg l-1 and 11.12 mg l-1 respectively. The serum EDAcFN concentration was higher than the 97.5th percentile level of healthy blood donors in 86% of patients with digestive tract and in 76% with miscellaneous malignancies. In patients with breast cancer 60% had elevated levels of EDAcFN. The corresponding figures for plasma samples in patients with digestive tract and miscellaneous malignancies were 79% and 71% respectively. In patients with breast cancer only 30% had elevated plasma levels of EDAcFN. The mean values in serum and plasma of 20 patients with benign diseases were below the cut-off levels. Consistent with the EIA results, Western blotting revealed increased amounts of EDAcFN in blood samples from cancer patients. Pregnancy did not affect the EDAcFN concentration. The mean values in 20 pregnant women were below the cut-off levels.

Competitive enzyme immunoassay for quantification of the cellular form of fibronectin (EDAcFN) in blood samples

A competitive enzyme immunoassay based on the monoclonal antibody DH1 was developed for the quantification of extradomain A (EDA)-containing isoform of cellular fibronectin (cFN). The average EDAcFN concentration in normal plasma was 2.46 μg/ml and in serum 0.68 μg/ml. Similar results were obtained by gelatin-Sepharose binding and immunoblotting. Studies on the effect of storage of plasma and serum as frozen samples indicated that the amount of EDAcFN decreased rapidly when stored at −20°C. Storage at −70°C resulted in less of a decrease in the concentration of EDAcFN. The method permits the determination of EDAcFN in blood samples from diseases showing increased amounts of the protein at the tissue level.

The alternative splicing of fibronectin pre-mRNA is altered during aging and in response to growth factors

The reverse transcription-polymerase chain reaction was used to examine alternative splicing at each of the three fibronectin exons known to undergo alternative splicing, i.e. extra domain A (ED-A), extra domain B (ED-B), and type III connecting sequence (IIICS). Ratios of fibronectin mRNAs with or without a given exon were determined in several rat tissues and human cell lines during aging in vivo and cellular senescence in vitro. We demonstrate that statistically significant shifts in the alternative splicing of fibronectin occur during aging in vivo and in vitro. Since all three alternatively spliced exons are spliced out at a higher frequency in aging tissues and cells, the fibronectin protein produced by old cells should be slightly smaller than that obtained from young cells. The reverse transcription-polymerase chain reaction demonstrates tissue-specific patterns of alternative splicing in several tissues. Whereas fibronectin mRNAs from adult rat tissues were found to range from 0 to 25% ED-A+ and from 0 to 10% ED-B+, fibronectin mRNAs from cultured cell lines were found to be approximately 50-60% ED-A+ and 15-25% ED-B+. We observed similarity in splicing of fibronectin RNA by the different cultured cell lines obtained from many tissues and attribute this observation to the effect of growth factors. We demonstrate that serum deprivation; placement of cells into primary culture; and growth factors such as transforming growth factor beta 1, retinoic acid, and 1,25-dihydroxyvitamin D3 can all change the alternative splicing of fibronectin pre-mRNA in the ED-A, ED-B, and type III connecting sequence exons. Possible mechanisms for the regulation of the alternative splicing of fibronectin RNA by growth factors are discussed.

Expression of fibronectin variants in vascular and visceral smooth muscle cells in development

Monoclonal antibodies recognizing extra domain A (ED-A) and extra domain B (ED-B) fibronectin (FN) sequences were used to characterize FN variants expressed in human vascular smooth muscle cells (SMC) during fetal and postnatal development and to compare spectrum of FN variants produced by vascular and visceral SMC. In 8- to 12-week-old fetuses both ED-A-containing FN (A-FN) and ED-B-containing FN (B-FN) were found in all smooth muscles studied—aorta, esophagus, stomach, and jejunum. By 20–25 weeks of gestation relative amounts of both A-FN and B-FN were reduced significantly in the aortic media (fivefold for A-FN and twofold for B-FN), while in visceral SMC only B-FN content was decreased. All the adult visceral smooth muscles examined contained A-FN rather than B-FN. Therefore, the cells from adult aortic media appear to be the only SMC so far known to produce FN that contains neither ED-A nor ED-B. Moreover, the data obtained show that, unlike other cells, medial SMC are embedded in vivo in the extracellular matrix that contains FN lacking both ED-A and ED-B. SMC from the minor intimal thickenings in the human child aorta as well as those from the atherosclerotic plaques produce A-FN rather than B-FN. We conclude that (1) vascular SMC change the spectrum of produced FN variants at least twice—during prenatal development between 12 and 20 weeks of gestation, and during the postnatal period, when they are recruited into the intimal cell population; (2) the production of FN variants in visceral SMC is also developmentally regulated; (3) all visceral SMC unlike the cells from adult aortic media produce A-FN; (4) the presence of ED-A and ED-B sequences in the FN molecule is not necessary for the extracellular matrix assembly in vivo.

Expression of extra domain A fibronectin sequence in vascular smooth muscle cells is phenotype dependent.

Different fibronectin (FN) variants arise from the single gene transcript alternatively spliced in a tissue-specific manner (Hynes, R. O. 1985. Annu. Rev. Cell Biol. 1:67-90; Owens, R. J., A. R. Kornblihtt, and F. E. Baralle. 1986. Oxf. Surv. Eurcaryotic Genes. 3:141-160). We used mAb IST-9, specific for extra domain A (ED-A) FN sequence, and cDNA probe to ED-A exon to determine whether ED-A is present in FN synthesized by vascular smooth muscle cells (SMCs) and, if so, whether expression of ED-A is SMC phenotype dependent. ED-A-containing FN (A-FN) was not revealed in tunica media of human arteries and normal rat aorta by immunofluorescence and immunoblotting techniques. A cDNA probe to ED-A exon did not hybridize with RNA isolated from human aortic media. A positive reaction with IST-9 was observed in (a) diffuse intimal thickening and atherosclerotic plaque from human arteries; (b) experimentally induced intimal thickening in rat aorta; and (c) cultured vascular SMCs. A-FN mRNA was present in the RNA preparation from human aortic intima as judged by hybridization with cDNA probe to ED-A. On the other hand, an mAb interacting with an epitope common for all FN variants revealed FN in both intima and media of human arteries and in the normal rat aorta. A cDNA probe to a sequence shared by all FN variants hybridized with RNA from both intima and media of human aorta, though the level of expression was higher in intima. The data suggest that ED-A exon is omitted during splicing of the FN mRNA precursor in medial SMCs while the expression of A-FN is characteristic of "modulated" SMCs--those of intimal thickenings, of atherosclerotic lesions, and growing in culture.

Human placental fibronectin: Demonstration of structural differences between the A and B chains in the extra domain-A region

Fibronectins are a class of cell adhesion proteins produced from a single gene. Soluble plasma fibronectin plays a role in wound healing and the insoluble cellular fibronectin form anchors cells to the substrata. The proteins possess multiple macromolecular binding domains including collagen, fibrin, and heparin. Alternative RNA splicing in at least three regions (ED-A, ED-B, and III CS) is responsible for this fibronectin polymorphism. We have been studying this polymorphism at the protein level in placental fibronectin, a poorly soluble form of cellular fibronectin. Cathepsin D-digested placental fibronectin applied to a heparin-agarose column and eluted with a NaCl stepwise gradient (0.1, 0.3, 0.5 m) gave two polypeptides (80–100 and 65 kDa) in the 0.3 m NaCl peak. Immunoblots with monoclonal antibodies IST-2 (specific for the carboxy-terminal heparin-binding domain) and IST-9 (specific for the ED-A portion of fibronectin) suggest that both peptides contain the carboxy-terminal heparin-binding (Hep-2) domain, but that only the larger fragment possesses the ED-A segment. The two peptides were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electrotransferred to Polybrene-coated polyvinyl difluoride membranes, and characterized by microsequence analysis. This analysis confirmed that both fragments start with the same amino acid sequence, 17 amino acids before the start of ED-A. These results demonstrate that placental fibronectin is a heterodimer, structurally distinct from plasma fibronectin due to the presence of a unique domain modification that is not seen in the plasma form.

Dibutyryl cyclic AMP decreases expression of ED-A fibronectin by canine chondrocytes

The presence of Extra Domain A (ED-A) in fibronectins from cartilage and from the culture medium of chondrocytes cultured under various conditions was determined with monoclonal antibody IST-9. Less than 2% of the fibronectin extracted from cartilage was fibronectin which contained the ED-A sequence (ED-A fibronectin). Chondrocytes placed in primary culture began to synthesize increased amounts of ED-A fibronectin (6% to 36% of the total fibronectin). Expression of ED-A fibronectin was selectively reduced when the chondrocytes were cultured in the presence of 0.5 mM dibutyryl cyclic AMP.