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1 From the Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, Wisconsin; 2 Cellular Pharmacology Laboratory, Department of Ophthalmology, University of California, San Francisco, California; and 3 Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, Wisconsin.
PURPOSE. To determine whether trabecular meshwork–inducible glucocorticoid response/myocilin (TIGR/MYOC) protein associates with the extracellular matrix (ECM) of human trabecular meshwork (HTM) cells.
METHODS. The extracellular localization of TIGR/MYOC was examined by immunofluorescence microscopy in HTM cultures treated with and without dexamethasone and ascorbate and in a transformed HTM cell line, TM-1, transiently transfected with TIGR/MYOC cDNA. Antibodies to TIGR/MYOC, fibronectin, laminin, type IV collagen, or thrombospondin were used to determine the extracellular localization of TIGR/MYOC. Solid phase binding assays using 125I-recombinant TIGR/MYOC and types I and IV collagens, fibronectin, and laminin were done to examine the association of TIGR/MYOC with these proteins and to identify a specific TIGR/MYOC binding site within fibronectin. The domains of fibronectin tested were the fibrin/collagen binding domain, the RGD domain, and the Heparin II (Hep II) domain.
RESULTS. TIGR/MYOC colocalized with fibronectin, laminin, and type IV collagen, but not thrombospondin in both dexamethasone and dexamethasone/ascorbate-treated HTM cultures and in TM-1 cultures transfected with TIGR/MYOC cDNA. In solid phase binding assays, 125I-TIGR/MYOC bound fibronectin but not laminin or type IV collagen. Binding to fibronectin could be competed with excess TIGR/MYOC or fibronectin. Specific binding was found for the Hep II domain of fibronectin.
CONCLUSIONS. TIGR/MYOC can associate with components of the ECM via interactions with the Hep II domain of fibronectin. The interactions with the Hep II domain of fibronectin could alter cell–matrix interactions in the TM and provides an interesting lead to explore the role(s) of TIGR/MYOC in both steroid-induced and primary open angle glaucoma.
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