Myocilin binding to Hep II domain of fibronectin inhibits cell spreading and incorporation of paxillin into focal adhesions.

Myocilin, a novel matricellular protein found in the human eye, can modify signaling events mediated by the Heparin II domain of fibronectin. Using myocilin produced in sf9 insect cells, myocilin inhibited spreading of cycloheximide-treated human skin fibroblasts plated on substrates co-coated with myocilin and either fibronectin or its Heparin II domain. Cell spreading could be rescued by adding back either substrate adsorbed or soluble Heparin II domains. Myocilin did not inhibit cell attachment to fibronectin even in the presence of a 2400 M excess of myocilin. Myocilin impaired focal adhesion formation and specifically blocked the incorporation of paxillin, but not vinculin, into focal adhesions. The Heparin II domain mediated the incorporation of paxillin into focal adhesions, since paxillin was not assembled into focal adhesions unless the Heparin II domain was present. The effect of myocilin on focal adhesions could be overcome by treating cells with either phorbol 12-myristate (PMA) or oleoyl-L-alpha-lysophosphatidic acid (LPA). Myocilin bound to the fibroblast cell surface, but its binding could not be competed with excess fibronectin, suggesting that myocilin does not compete for cell surface binding sites of fibronectin. Myocilin therefore appears to specifically block functions mediated by the Heparin II domain possibly through direct interactions with it.

Heparin II domain of fibronectin uses alpha4beta1 integrin to control focal adhesion and stress fiber formation, independent of syndecan-4.

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 alpha5beta1 and alpha4beta1 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 (alpha5beta1 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 alpha4beta1 integrin because soluble anti-alpha4 integrin antibodies inhibited Hep II domain-mediated cell spreading and soluble vascular cell adhesion molecule-1 (alpha4beta1 ligand)-induced cell spreading. This is the first demonstration of the Hep II domain mediating cell spreading and stress fiber formation through alpha4beta1 integrin. This novel pathway demonstrates a cooperative, rather than antagonistic, role between alpha5beta1 and alpha4beta1 integrins and suggests that interactions between the Hep II domain and alpha4beta1 integrin could modulate the strength of cytoskeleton-mediated processes in the trabecular meshwork of the human eye.

Effect of heparin II domain of fibronectin on aqueous outflow in cultured anterior segments of human eyes.

PURPOSE: To determine whether an integrin/syndecan-binding domain of fibronectin, called the heparin II (Hep II) domain, affects outflow facility in the human eye. METHODS: Anterior segments of human eyes were placed in perfusion organ culture. One eye of each pair received the Hep II domain, and the fellow eye received DMEM or a heat-denatured Hep II domain. The Hep II domain was produced as a recombinant glutathione S-transferase (GST)-fusion protein. Microscopic changes were assessed. RESULTS: Outflow facility in anterior segments treated with Hep II domain increased by 93% compared with that in anterior segments treated with DMEM. In contrast, facility in anterior segments treated with the heat-denatured Hep II domain showed very little change. Outflow facility remained high during Hep II domain perfusion and returned to baseline after removal of the protein. Electron microscopy revealed disruptions in the endothelial lining of Schlemm's canal in anterior segments fixed during maximum effect and in anterior segments after facility had returned to baseline. Scattered disruptions of canal cells were noted in control anterior segments. Trabecular cells in other regions looked normal. Major changes in the extracellular matrix of the juxtacanalicular tissue were not observed. Repeated doses of the Hep II domain administered after facility returned to baseline increased facility in two of three anterior segments. CONCLUSIONS: The Hep II domain of fibronectin increases outflow facility in the human anterior segment. This suggests that fibronectin-mediated interactions may have a role in modulating aqueous hydrodynamics. Such interactions may represent avenues of novel therapeutic interventions for glaucoma.

Alternative splicing of the IIICS domain in fibronectin governs the role of the heparin II domain in fibrillogenesis and cell spreading.

The Heparin (Hep) II-binding domain of fibronectin regulates the formation of focal adhesions and actin stress fibers and hence plays an important role in cell spreading, migration, and fibronectin fibrillogenesis. Using human skin fibroblast cultures, we demonstrate that alternative splicing of the neighboring IIICS domain may regulate the activities of the Hep II domain in cell spreading and fibronectin fibrillogenesis. Recombinant Hep II domains, adjacent to either the IIICS domain or the H89 splice variant that contains the amino-terminal sequence of the IIICS domain, blocked fibronectin fibrillogenesis and required sulfated proteoglycans to mediate cell spreading. If the Hep II domain was adjacent to either the H0 or H95 splice variants, which both lack the amino terminus of the IIICS domain, fibrillogenesis was not inhibited and cell spreading was independent of a sulfated proteoglycan-mediated mechanism. The effect of the splice variants on the Hep II domain could be mimicked using a Hep II domain that contained only 6 amino acids from the III(15) repeat or 10 amino acids from the IIICS domain suggesting that sequences proximal to the III(14) repeat determined the role of the Hep II domain in these processes. We propose that alternative splicing of the IIICS domain modulates interactions between heparan sulfate proteoglycans and the Hep II domain and that this serves as a mechanism to control the biological activities of fibronectin.