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

Abstract

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...

Mammary Epithelial-Mesenchymal Interaction Regulates Fibronectin Alternative Splicing via Phosphatidylinositol 3-Kinase

“…This motif has been shown to promote cell adhesion by interacting with ␣ 4 ␤ 1 and ␣ 4 ␤ 7 integrins (26). Therefore, this newly synthesized FN (EDIϩ, IIICS-120) would have an enhanced potential for binding to a specific subset of integrins associated with cell adhesion, proliferation, migration, and matrix metalloproteinase induction (4,6,(27)(28)(29). Conversely, the EDIIϩ FN isoform was not detected in RNA from untreated cells, and none of the described stimuli altered this splicing pattern (Fig.…”

“…FN is the ligand for at least a dozen members of the integrin receptor family. EDI itself binds to ␣ 4 ␤ 1 and ␣ 9 ␤ 1 (29), and IIICS contains binding sites for ␣ 4 ␤ 1 and ␣ 4 ␤ 7 (47). Recently, a novel glycosaminoglycan-binding site has been identified within the FN IIICS, showing that cooperation between cell surface proteoglycans and integrins is important for mediating the adhesion of cells to FN (48).…”

Combinatorial activation of FAK and AKT by transforming growth factor-β1 confers an anoikis-resistant phenotype to myofibroblasts

“…FAK is also an important regulator of cell survival signalling [13]. Apoptosis induced by loss of adhesion or adhesion-mediated signalling (termed "anoikis") is associated with deactivation of FAK [14,15]. A role of FAKmediated signals in mesenchymal cell survival has been demonstrated in studies showing that fibroblasts in contractile gels are protected from apoptosis by activating antibodies to α5β1 integrins or through constitutive activation of FAK [16].…”

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

“…Moreover, adhesion of cells expressing either ␣9␤1 or ␣4␤1 was reduced when cultured on two of the shorter deletion mutants with intact C-CЈ loop of EIIIA (EIIIA-(24 -66) and EIIIA- (30 -57)). These data suggested that the optimal ligand binding site for ␣9␤1 and ␣4␤1 in the EIIIA segment could require both the C-CЈ loop and additional flanking sequences (1). In addition, mAbs IST-9 or 3E2 inhibited ␣9-and ␣4-mediated cell adhesion to the EIIIA segment, suggesting that the ligand binding site includes the C-CЈ loop (1).…”

“…Cell Culture-SW480, two human colon carcinoma cell lines, stably transfected with either integrin ␣9 or vector alone, were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 1% penicillin and streptomycin, and 2% Geneticin (Invitrogen) as described previously (1,47). Cells were incubated in a humidified incubator (Thermo Electron Corp., Waltham, MA) at 37°C in 5% CO 2 .…”