In vitro localization of TIGR/MYOC in trabecular meshwork extracellular matrix and binding to fibronectin.
OBJECTIVE
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.
Filla
Mark S
MS
Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, Wisconsin 53706, USA.
Liu
Xuyang
X
Nguyen
Thai D
TD
Polansky
Jon R
JR
Brandt
Curtis R
CR
Kaufman
Paul L
PL
Peters
Donna M
DM
D000328:Adult; D001205:Ascorbic Acid / Q000494:pharmacology; D001665:Binding Sites; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D003598:Cytoskeletal Proteins; D003907:Dexamethasone / Q000494:pharmacology; D005109:Extracellular Matrix / Q000378:metabolism*; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism*; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007797:Laminin / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D014129:Trabecular Meshwork / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D014162:Transfection; D000099179:Myocilin
D001205:Ascorbic Acid; D003094:Collagen; D003598:Cytoskeletal Proteins; D003907:Dexamethasone; D005136:Eye Proteins; D005353:Fibronectins; D006023:Glycoproteins; D007797:Laminin; D000099179:Myocilin