The Region Encoded by the Alternatively Spliced Exon IIIA in Mesenchymal Fibronectin Appears Essential for Chondrogenesis at the Level of Cellular Condensation

Gehris, Amy L.; Stringa, E.; Spina, Joseph; Desmond, Mary E.; Tuan, Rocky S.; Bennett, Vickie D.

doi:10.1006/dbio.1997.8693

Cited by 66 publications

(57 citation statements)

References 59 publications

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“…Blocking mAbs to EIIIA inhibited the condensation events that occur during chondrogenesis in chick embryos (50). Moreover, treatment with the EIIIA segment alone facilitated cartilage catabolism and markedly induced expression of matrix metalloproteinase in chondrocytes and synovial cells in an interleukin-1-dependent manner (32).…”

Section: Discussionmentioning

confidence: 99%

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Identification of the Peptide Sequences within the EIIIA (EDA) Segment of Fibronectin That Mediate Integrin α9β1-dependent Cellular Activities

Shinde¹,

Bystroff

Wang

et al. 2008

Journal of Biological Chemistry

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We have now performed site-directed mutagenesis within the EIIIA segment and carried out cell adhesion assays on these mutant EIIIAs. We find that the Asp 41 and Gly 42 residues within the C-C loop of EIIIA are necessary for integrin ␣9␤1 binding. Synthetic peptides based on the predicted important amino acid sequence from the C-C loop encode sufficient information to completely inhibit ␣9␤1-mediated cell adhesion. We also report that EIIIA promotes filopodial formation in ␣9␤1-expressing cells accompanied by Cdc42 activation. Our data provide a cellular activity for the EIIIA segment, evidence for conformational lability, and peptide sequences for probing EIIIA functions in vitro and in vivo.Guidance of cell function during adult tissue repair, the tumor microenvironment, and embryogenesis is highly orchestrated and involves the concerted action of extracellular matrix (ECM), 3 growth factors, and mechanical forces. Although it is now clear that the ECM serves both structural and instructional roles, the mechanisms that regulate the presentation of this "instruction set" to cells remain unclear. Potential mechanisms include alternative splicing to enhance the sequence complexity for an ECM gene product at a needed site. In addition, once expressed, ECM proteins can harbor cryptic sites that are exposed by specific proteolysis or by conformation changes induced by mechanical forces (3-6). The fibronectins (FNs) comprise a family of ECM proteins in which all three potential mechanisms have been implicated in its function.FNs are high molecular weight, multifunctional adhesive glycoproteins present in the ECM, connective tissues, basement membrane, and various body fluids. They provide excellent substrates for cell adhesion and spreading, thereby promoting cell migration during embryonic development, wound healing, and tumor progression and interact with other ECM proteins and cellular ligands, such as glycosaminoglycans, collagen, fibrin, and integrins (7). FNs are disulfide-bonded dimers of two closely related subunits, each consisting of three types of homologous repeating modules termed types I, II, and III (8). FN molecules have multiple isoforms generated from a single gene by alternative splicing of combinations of three exons: extra domain A (EDA/EIIIA), extra domain B (EDB/EIIIB), and connecting segment III (V). Both EIIIA and EIIIB exons are type III repeating units (7). Plasma fibronectin (pFN), produced by hepatocytes and abundant in plasma, lacks both the EIIIA and EIIIB domains. However, cellular FNs, produced by fibroblasts and epithelial and other cell types, are insoluble, and incorporated into the pericellular matrix, they contain the EIIIA and EIIIB segments in various combinations (9).

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Section: Discussionmentioning

confidence: 99%

“…Two of these mAbs, DH1, and IST-9 were reported to block function (30,31,50). Reduction in binding of mAbs IST-9, 3E2, and DH1 was also observed when Asp 41 and Gly 42 were mutagenized to Ala (2).…”

Section: Discussionmentioning

confidence: 99%

Identification of the Peptide Sequences within the EIIIA (EDA) Segment of Fibronectin That Mediate Integrin α9β1-dependent Cellular Activities

Shinde¹,

Bystroff

Wang

et al. 2008

Journal of Biological Chemistry

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“…Liver lipocytes and skin fibroblasts differentiate into myofibroblasts when adhering to FNs that include the EIIIA segment (10, 22). One monoclonal antibody (IST-9) to the EIIIA segment has been shown to inhibit myofibroblast differentiation, whereas another (DH1) blocks chondrogenesis during chick development (10,22,23). Moreover, the expression of MMP-9 is regulated by the EIIIA segment in chondrocytes and myelomonocytic cells potentially through toll-like receptors (24, 25).…”

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confidence: 99%

The EIIIA Segment of Fibronectin Is a Ligand for Integrins α9β1 and α4β1Providing a Novel Mechanism for Regulating Cell Adhesion by Alternative Splicing

Liao¹,

Gotwals²,

Koteliansky³

et al. 2002

Journal of Biological Chemistry

201

163

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Although it has been clear for many years that fibronectin (FN) 1 is alternatively spliced, the functions of, and receptors for, two alternatively spliced segments termed EIIIA (or ED-A) and EIIIB (or ED-B) segments have remained elusive. More is known about a non-homologous IIICS repeat encoding the CS-1 segment, which is a cell adhesive site and ligand for integrin ␣ 4 ␤ 1 (1). Both the EIIIA and EIIIB segments are homologous FN type III repeats and are prominently expressed during embryogenesis; homozygous mutations in FN are embryonic lethal (2-7). During wound healing (5, 8), lung, liver, and kidney fibrosis (9 -11), vascular intimal proliferation (12, 13), vascular hypertension (14), and cardiac transplantation (15), the expression of FNs containing the EIIIA and EIIIB domains is significantly increased. A ϳ170-kDa species of EIIIA-containing FNs is found in synovial fluid from patients with rheumatoid arthritis but not osteoarthritis (16). The EIIIB segment has been postulated to have a role in angiogenesis (17). The EIIIA segment has been observed to regulate cell adhesion and proliferation (18 -21). Liver lipocytes and skin fibroblasts differentiate into myofibroblasts when adhering to FNs that include the EIIIA segment (10, 22). One monoclonal antibody (IST-9) to the EIIIA segment has been shown to inhibit myofibroblast differentiation, whereas another (DH1) blocks chondrogenesis during chick development (10,22,23). Moreover, the expression of MMP-9 is regulated by the EIIIA segment in chondrocytes and myelomonocytic cells potentially through toll-like receptors (24, 25).We recently reported detailed epitope maps for functionblocking monoclonal antibodies that bind to the C-CЈ loop of the EIIIA segment (26). The FN type III (FN-III) repeats, of which the EIIIA segment is one, exhibit high structural homology (27-31) despite only 20 -40% identity in amino acid sequence (32). The canonical FN type III repeat is a conserved ␤-sandwich conformation consisting of two ␤ sheets comprising four strands (G, F, C, CЈ) and three strands (A, B, and E) (27). Epitope mapping of the EIIIA segment reveals that functionblocking mAbs interact with the loop between the C and CЈ ␤-strands and the adjacent Ile 43 and His 44 residues are critical to the epitope (26). Given that these monoclonal antibodies blocked EIIIA function we reasoned that the peptide comprising the C-CЈ loop region (EDGIHEL) could encode a sequence that bound cell surface receptors, possibly integrins.The integrins are a family of heterodimeric transmembrane receptors that mediate cell-extracellular matrix and cell-cell interactions (33). One integrin, ␣ 9 ␤ 1 , binds to a peptide sequence within the B-C loop of tenascin-C (34). This sequence (AEIDGIEL) is similar to the EDGIHEL sequence that we identified in the EIIIA segment (26). The ␣ 9 subunit binds unrelated sequences in other ligands including the vascular cell adhesion molecule-1 (VCAM-1) (35), osteopontin (36), the propolypeptide of von Willebrand factor (pp-vWF) (37), tissue transglutamina...

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“…The EDA and EDB type III repeats are expressed in very low levels in the adult, but are upregulated at various times during development, wound healing and tumor progression (Coito et al, 1997;Ffrench-Constant and Hynes, 1989;George et al, 2000;Jarnagin et al, 1998;Peters et al, 1986;Scarpino et al, 1999). One of these repeats, the EDA domain which is found between the 11th and 12th type III repeats, has been shown to affect several cellular processes including adhesion (Manabe et al, 1997), differentiation (Gehris et al, 1997) and gene expression (Saito et al, 1999). Recently, the α4β1 and α9β1 integrins have been identified as receptors for the EDA segment (Liao et al, 2002).…”

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confidence: 99%

Stimulation of extracellular matrix remodeling by the first type III repeat in fibronectin

Klein

Zheng

Ambesi

et al. 2003

Journal of Cell Science

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The fibronectin matrix contains cryptic sites which are thought to modulate cellular biological responses. One of these sites, located in fibronectin's first type III repeat (III1c), influences signaling pathways that are relevant to cytoskeletal organization and cell cycle progression. The purpose of this study was to identify possible mechanisms responsible for the effects of III1c on cell behavior. Recombinant peptides representing various type III repeats of fibronectin were compared for their effects on fibronectin matrix organization and activation of intracellular signaling pathways. III1c and III13 but not III11c or III10 bound to monolayers of human skin fibroblasts in a dose- and time-dependent manner and were localized to the extracellular matrix. Binding of III13, but not III1c, to matrix was sensitive to heparitinase, suggesting that the association of III1c with the matrix was not dependent on heparan sulfate proteoglycans. Quantitative and morphological assessment indicated that, in contrast to previously published reports, the binding of III1c to cell layers did not result in the loss or disruption of matrix fibronectin. Binding of III1c but not III13 to the extracellular matrix did result in the loss of a conformationally sensitive epitope present within the EDA type III module of cellular fibronectin. III1c-induced loss of the EDA epitope did not require the presence of cells, occurred within 1 hour and was associated with the activation of p38 mitogen-activated protein kinase (MAPK) followed by the formation of filopodia. Maximal phosphorylation of p38 MAPK occurred within 1 hour, whereas cytoskeletal changes did not appear until 12 hours later. These findings are consistent with a model in which the binding of III1c to the extracellular matrix results in a conformational remodeling of the fibronectin matrix, which has both short- and long-term effects on cell physiology.

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The Region Encoded by the Alternatively Spliced Exon IIIA in Mesenchymal Fibronectin Appears Essential for Chondrogenesis at the Level of Cellular Condensation

Cited by 66 publications

References 59 publications

Identification of the Peptide Sequences within the EIIIA (EDA) Segment of Fibronectin That Mediate Integrin α9β1-dependent Cellular Activities

Identification of the Peptide Sequences within the EIIIA (EDA) Segment of Fibronectin That Mediate Integrin α9β1-dependent Cellular Activities

The EIIIA Segment of Fibronectin Is a Ligand for Integrins α9β1 and α4β1Providing a Novel Mechanism for Regulating Cell Adhesion by Alternative Splicing

Stimulation of extracellular matrix remodeling by the first type III repeat in fibronectin

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