Heparin II Domain of Fibronectin Uses α4β1 Integrin to Control Focal Adhesion and Stress Fiber Formation, Independent of Syndecan-4

Abstract

Co-signaling events between integrins and cell surface proteoglycans play a critical role in the organization of the cytoskeleton and adhesion forces of cells. These processes, which appear to be responsible for maintaining intraocular pressure in the human eye, involve a novel cooperative co-signaling pathway between ␣5␤1 and ␣4␤1 integrins and are independent of heparan sulfate proteoglycans. Human trabecular meshwork cells isolated from the eye were plated on type III 7-10 repeats of fibronectin (␣5␤1 ligand) in the absence or presence of the heparin (Hep) II domain of fibronectin. In the absence of the Hep II domain, cells had a bipolar morphology with few focal adhesions and stress fibers. The addition of the Hep II domain increased cell spreading and the numbers of focal adhesions and stress fibers. Cell spreading and stress fiber formation were not mediated by heparan sulfate proteoglycans because treatment with chlorate, heparinase, or soluble heparin did not prevent Hep II domain-mediated cell spreading. Cell spreading and stress fiber formation were mediated by ␣4␤1 integrin because soluble anti-␣4 integrin antibodies inhibited Hep II domain-mediated cell spreading and soluble vascular cell adhesion molecule-1 (␣4␤1 ligand)-induced cell spreading. This is the first demonstration of the Hep II domain mediating cell spreading and stress fiber formation through ␣4␤1 integrin. This novel pathway demonstrates a cooperative, rather than antagonistic, role between ␣5␤1 and ␣4␤1 integrins and suggests that interactions between the Hep II domain and ␣4␤1 integrin could modulate the strength of cytoskeletonmediated processes in the trabecular meshwork of the human eye.Adhesive interactions between cells and extracellular matrix molecules form highly specific yet very diverse signaling conduits that regulate a wide variety of biological processes associated with cell morphology, proliferation, differentiation, migration, and survival (1-5). These adhesive events are mediated mainly by the integrin family of receptors and lead to the formation of dynamic multi-molecular structures called focal adhesions, focal complexes, and fibrillar adhesions (6, 7).The formation of focal contacts often depends on co-signaling events between integrins and cell surface proteoglycans. In fibroblasts and A375-SM melanoma cells plated on fibronectin, cooperative signaling between ␣5␤1 integrin and syndecan-4, a cell surface heparan sulfate proteoglycan (HSPG), 1 is needed to promote the formation of focal adhesions and stress fibers (8, 9). If fibronectin-null fibroblasts are plated on anti-␤1 integrin antibodies or the RGD cell binding domain of fibronectin, cells bind but do not assemble the actin cytoskeleton. However, if soluble antibody directed against the ectodomain of syndecan-4 is added, the cells will assemble focal adhesions and stress fibers (10). A similar scenario has been documented for ␣4␤1 integrin and cell surface chondroitin sulfate proteoglycans, supporting the idea that co-engagement of integrins and proteogylca...

Mutations in FN1 cause glomerulopathy with fibronectin deposits

“…HMEC plated on the 120-kDa fragment were able to attach, however, they showed few actin stress fibers, indicating they were not completely spread. If the III 12-14 wt was added to the media, the percent of stress fiberpositive cells was enhanced 3-fold (19), whereas this increase did not occur when either mutants were added at the same concentration (P Ͻ 0.01 vs. wild type; Fig. 3D).…”

“…But further progression to the formation of actin stress-fibers and focal contacts requires binding of Hep-II to cell-surface proteoglycans and integrins (18)(19)(20). We therefore hypothesized that lower heparin affinity and cell binding of the III [12][13][14] W1925R and III 12-14 L1974R resulted in impaired capability to trigger stress-fiber formation and cell spreading.…”

“…We therefore hypothesized that lower heparin affinity and cell binding of the III [12][13][14] W1925R and III 12-14 L1974R resulted in impaired capability to trigger stress-fiber formation and cell spreading. This possibility was tested in vitro in HMEC plated on a layer of 120-kDa fragments of FN containing the RGD domain but lacking the Hep-II domain, in the presence of the III [12][13][14] recombinants (19) (Fig. 3D).…”

Extracellular matrix in the trabecular meshwork

“…They are involved in focal adhesions (particularly syndecan-4), stress fiber formation (particularly syndecan-2), cytoskeletal organization and other cellular processes. (Couchman and Woods 1999;Couchman et al 2001;Santas et al 2002;Couchman 2003;Yoneda and Couchman 2003;FFilla et al 2004;Peterson et al 2005;Filla et al 2006) Syndecan-4 also appears to localize with the verticies of CLANs and syndecan-2 localizes with podosomes in the TM (Aga, et. al.…”

“…(Vittal et al 2005) This introduces two additional α4β1 integrin binding sites and has been shown to affect fibronectin's Hep II domain interactions with cells. (Santas et al 2002;Peterson et al 2005) TNF and IL-1 have modest effects and TGFβ and dexamethasone have stronger effects of the EIIIA and EIIIB splice isoforms, changing availability of integrin α9 sites (Keller et. al.…”