Gene Structure and Properties of TIGR, an Olfactomedin-related Glycoprotein Cloned from Glucocorticoid-induced Trabecular Meshwork Cells

Abstract

Expression of the trabecular meshwork inducible glucocorticoid response (TIGR) gene progressively increases from barely detectable levels to greater than 2% of total cellular mRNA over 10 days exposure of trabecular meshwork (TM) cells to dexamethasone. Cycloheximide blocked most of the TIGR mRNA induction, suggesting a requirement for ongoing protein synthesis. The genomic structure of TIGR (ϳ20 kilobases) consists of 3 exons, and a 5-kilobase promoter region that contains 13 predicted hormone response elements, including several glucocorticoid regulatory elements, and other potentially important regulatory motifs. TIGR cDNA encodes an olfactomedin-related glycoprotein of 504 amino acids with motifs for N-and O-linked glycosylation, glycosaminoglycan initiation, hyaluronic acid binding, and leucine zippers. Recombinant TIGR (rTIGR) showed oligomerization and specific binding to TM cells. Anti-rTIGR antibody detected multiple translational/post-translational forms of TIGR produced by the cells (including secreted 66 kDa/55 kDa glycoproteins/proteins in the media and 55 kDa cellular proteins), whereas Northern blot showed a single mRNA species. The findings suggest potential mechanisms by which TIGR could obstruct the aqueous humor fluid flow and participate in the pathogenesis of glaucoma.The trabecular meshwork inducible glucocorticoid response (TIGR) 1 protein, which has significant homology in its C-terminal domain with olfactomedins, was initially cloned in our laboratories as a candidate gene for glaucoma using differential library screening in a trabecular meshwork cell culture model (1, 2). Mutations were recently found in this gene that co-segregated with both juvenile and adult forms of the disease (3).Glaucoma is a major cause of blindness, with its most prevalent form thought to involve the specialized endothelial cells lining the outflow pathway of the eye, termed the trabecular meshwork (TM) (4, 5). The synthesis and/or degradation of a variety of extracellular molecules in the meshwork are thought to be regulated by the TM cells, and alterations in the type or amount of connective tissue elements have been postulated to explain the increased outflow resistance seen in glaucoma cases (6). However, an understanding of the biochemical changes that actually contribute to this process has remained elusive.Previously, we described a highly expressed protein and related glycoprotein (55 and 66 kDa, respectively) found in the media of TM cell culture, but not in other cell types examined, after a prolonged exposure to dexamethasone (DEX) (2). We used this observation to define a cell culture model for "steroidinduced glaucoma" and elevated intraocular pressure due to corticosteroids. The extracellular induced proteins appeared as reasonable candidates for being involved in steroid glaucoma since the time course and dose response of their induction mimicked the intraocular pressure elevation and increased outflow resistance seen in patients receiving glucocorticoid (GC) therapy (7,8).Coincident with our res...

Rho GTPase and cAMP/Protein Kinase A Signaling Mediates Myocilin-induced Alterations in Cultured Human Trabecular Meshwork Cells

“…Multiple mutations have been identified in a number of families (8,9). Myocilin was initially identified as a 57-/55-kDa protein secreted into the medium of TM cultures after induction with glucocorticoids, such as dexamethasone (10). The myocilin mRNA and protein are present in a variety of ocular and nonocular tissues, including the retina and the TM (11).…”

The effect of temperature on gene silencing by siRNAs: Implications for silencing in the anterior chamber of the eye

“…Myocilin was determined to be up-regulated when dexamethasone was added to cultures of human trabecular meshwork cells Nguyen et al, 1998;O'Brien et al, 1999;Polansky et al, 1997;Shepard et al, 2001). Expression of myocilin under normal conditions is particularly high in the trabecular meshwork (Karali et al, 2000).…”

Mutations including the promoter region of myocilin/TIGR gene

“…1 -3 MYOC/TIGR was originally discovered as a protein specifically induced in the human trabecular meshwork (HTM) cells by prolonged dexamethasone treatment 4 It was later reported to be present in the ciliary body 5 and in the connecting cilium of the photoreceptor cells. 6 The genomic organization of the MYOC gene is relatively simple.…”

“…7 The myocilin gene encodes a secretory protein of 504 amino acids. 4,5 The deduced amino-acid sequence shows significant homologies with N-terminal region. Homology with myosin is relatively low (25 -29%).…”

“…10 -14 It has been suggested that accumulation of myocilin in the extracellular matrix might contribute to the obstruction of the outflow pathway and result in an elevation of intraocular pressure (IOP). 4,15,16 However, the MYOC/TIGR gene might be involved in the pathogenesis of glaucoma in patients without mutations in the coding region. 7 In the present study, we have characterized five mutations in the promoter region of this gene.…”