Glucocorticoid (GC) therapy can lead to the development of glaucomatous ocular hypertension and secondary open-angle glaucoma that is clinically similar to primary open-angle glaucoma . The elevated intraocular pressure (IOP) is due to increased aqueous humor outflow resistance and is associated with morphological and biochemical changes in the trabecular meshwork (TM) . These changes are associated with increased deposition of extracellular matrix material in the outflow pathway , which may be due, in part, to an inhibition of TM phagocytosis , decreased extracellular activity of stromelysin and tissue plasminogen activator , alteration of the actin cytoskeleton  and formation of intercellular junctions . Most of the effects of GCs on TM cells and tissues are likely due to GC-mediated TM cell gene expression, including the induction of myocilin, serum amyloid A1, α A-crystallin, insulin growth factor binding protein 2, growth arrest-specific 1 and other genes [8–13]. It is currently unclear which genes or combinations of genes are modulated by GCs and ultimately lead to increased IOP.
The biological actions of glucocorticoids are mediated through the cytoplasmic glucocorticoid receptor (GR), which belongs to the nuclear receptor subfamily that includes receptors for mineralocorticoids, estrogen and thyroid hormones, retinoic acid, and vitamin D . Upon hormone binding, the activated ligand-bound receptor translocates into the nucleus and binds as a homodimer to glucocorticoid response elements within the promoter region of target genes. The GR can positively or negatively regulate gene expression, depending on the response element sequence and promoter context. The GR also modulates gene expression, independent of glucocorticoid response elements, by physically interacting with other transcription factors (e.g., activating protein AP-1 and nuclear factor NF-κB) .
GRα and GRβ are the two major splice variants of GR as a result of alternative splicing. The GRβ isoform acts as a natural dominant negative inhibitor of GRα-induced transactivation of glucocorticoid-responsive genes . Amino acid sequence analysis revealed that GRα and GRβ isoforms are identical from the amino terminus to amino acid 727 but diverge beyond this position, with GRα having an additional 50 amino acids and GRβ having an additional, non homologous, 15 amino acids. The expression of both GRα and GRβ was reported in cultured human TM cells [17, 18].
Recent evidence indicates that at least eight different GRα or GRβ N-terminal isoforms are generated from one single GR gene by alternative translation initiation . For GRα, receptor isoforms are designated GRα-A, -B, -C1, -C2, -C3, -D1, -D2 and -D3 . The GRα-A isoform is the full-length receptor containing amino acids 1-777. The other GRα isoforms have shortened N termini. The apparent molecular weights for GRα-A, -B, -C and -D are 94, 91, 82-84 and 53-56 KDa, respectively. All GRα isoforms are functional receptors and contain the identical intact ligand-binding domain that binds GCs. The GRα isoforms transcriptionally regulate common and unique sets of genes within the context of a single cell type . Furthermore, the tissue expression patterns of GRα translational isoforms have been determined in both rats and mice and the levels of the GRα isoforms differ widely among tissues . Thus, the unique transcriptional activities and distinct tissue-specific distribution patterns of GRα isoforms could provide a novel mechanism for tissue-specific glucocorticoid responses. Further studies are needed to confirm this in human tissues. For GRβ, receptor isoforms are designated GRβ-A, -B, -C1, -C2, -C3, -D1, -D2 and -D3 . The GRβ-A isoform is the full-length receptor containing amino acids 1-742 with an apparent molecular weight of 90 KDa. The other GRβ isoforms have shortened N termini. Although little is known about the transcriptional activity and tissue distribution of GRβ isoforms, Fruchter et al demonstrated that the potency of the dominant negative effect of GRβ on GRα-induced transactivation depends on both the type and the dose of the synthetic glucocorticoids in use .
Recently, Schaaf et al showed that the intranuclear distribution and mobility of the GR is highly dependent on the chemical structure of the glucocorticoid with which it is associated . Some ligands, especially high-affinity synthetic ligands like dexamethasone and triamcinolone acetonide, induce a highly punctate GC-GRα distribution organized in discrete domains of high receptor concentration. In contrast, other ligands, mainly naturally occurring low-affinity ligands like cortisone and cortexolone, induce a more homogeneous, although still not entirely random, distribution. More importantly, structure-function analysis revealed that the 9-fluoro and 17-hydroxy groups on the steroid significantly impact nuclear receptor distribution. The effects of GC-mediated GR nuclear distribution and mobility patterns on GR-dependent transcriptional activity are still unclear.
Microarray technology provides a comprehensive, rapid and efficient method for large scale profiling of gene expression changes in biological samples (e.g., treatment versus control, disease versus normal). The advantages of DNA microarray technology include the ability to analyze expression patterns of thousands of genes simultaneously. Other advantages include the ability to characterize relationships between genes and the changes in biological processes such as disease states, developmental stages and responses to drugs [22, 23]. More importantly, Canales et al recently showed that results generated from five different microarray platforms correlated exceedingly well with real-time quantitative polymerase chain reaction (RT-PCR), as well as other non-microarray-based approaches to determine transcript abundance .
In this study, using two primary human TM cell lines isolated from either a newborn or an adult donor, we examined the distribution of GRα and GRβ isoforms and determined global gene expression profiles after treatment with 1 μM of three potent GR agonists (dexamethasone, fluocinolone acetonide and triamcinolone acetonide). TM 86 and TM 93 expressed all known GRα and GRβ isoforms as determined by Western blot. The gene expression profiles and Ingenuity Pathway Analysis demonstrated that each of the three GCs regulated a common and unique subset of genes that is associated with a cell lineage-dependent specific signaling pathway.