Glucocorticoids are primary stress hormones essential for life that are released by the adrenal cortex and function to maintain homeostasis. Glucocorticoids act on nearly all tissues and cells to regulate many diverse physiological processes including intermediary metabolism, immune function, skeletal growth, development, reproduction, cognition, and apoptosis.1,2 Because of their powerful anti-inflammatory and immunosuppressive actions, synthetic glucocorticoids are one of the most widely prescribed therapeutic agents in the world today for the treatment of acute and chronic inflammatory diseases, organ transplant rejection, and cancers of the lymphoid system.3 Over the last half-century, glucocorticoids have been a mainstay for the treatment of many ocular conditions including uveitis, keratitis, scleritis, conjunctivitis, macular edema, and postoperative management of cornea transplantation and cataract extraction.4
The therapeutic benefit of glucocorticoids, however, is limited by severe side effects that accompany chronic administration of these drugs.3,5 In the eye, long term topical or systemic glucocorticoid exposure frequently induces an increase in intraocular pressure (IOP) that is a major risk factor for the development of glaucoma, the leading cause of irreversible blindness in the world.6,7 The glucocorticoid-mediated elevation in IOP is due to increased resistance to aqueous humor outflow resulting from excessive extracellular matrix material deposition and aggregation within the trabecular meshwork.8
Identifying the genes responsible for ocular hypertension has been the subject of intense study. Myocilin, a secreted glycoprotein whose precise function is unknown, has received considerable attention as one of the major culprits of this pathology because its expression in trabecular meshwork cells is strongly induced by glucocorticoids and it has been genetically linked to primary glaucoma.9-11 Both the physiological and pharmacological actions of glucocorticoids are mediated by the glucocorticoid receptor (GR), a member of the nuclear receptor superfamily of intracellular proteins that function as liganddependent transcription factors.12 In this review, we discuss the GR signal transduction pathway in the context of secondary glaucoma, with particular emphasis on how the GR signaling profile can be modulated by the cellular complement of receptor isoforms and by the nature of the bound glucocorticoid.
GR SIGNAL TRANSDUCTION PATHWAY
GR is a modular protein comprised of three major
domains: an amino terminal transactivation domain
(NTD), a central DNA binding domain (DBD), and a carboxyl
terminal ligand-binding domain (LBD) (Figure
1A).13 In the absence of hormone, GR is found predominantly
in the cytoplasm of cells in a complex with various
chaperone proteins that maintain the receptor in a conformation
that is transcriptionally inactive but that binds ligand with high affinity (Figure 1B). Upon binding glucocorticoids,
the receptor undergoes a conformational
change resulting in the dissociation of the chaperone
proteins and the translocation of the receptor into the
nucleus where it regulates up to 20% of the human
genome.14 The ligand-bound receptor induces or represses
the expression of target genes by binding directly as a
homodimer to specific sequences of DNA termed glucocorticoid
responsive elements (GREs). Alternatively, GR
can regulate gene expression apart from direct DNA
binding by interacting with other promoter-bound transcription
factors and modulating their activity. For some
target genes, GR both directly binds DNA and interacts
with neighboring transcription factors to achieve its
effects on transcription. The latter two modes of action
account for GR-mediated repression of the proinflammatory
transcription factors AP-1 and NF-κΒ that leads to
the major anti-inflammatory and immunosuppressive
actions of glucocorticoids.15 Once associated with chromatin,
GR undergoes further conformational changes
that result in the coordinated recruitment of various
coactivators and corepressors necessary for chromatin
remodeling and the transcriptional response.16 The transcriptional
activation functions AF1 and AF2, found in
the receptor NTD and LBD, respectively, are important
for interacting with these coregulators as well as the basal
transcription machinery. Finally, the activity of GR can be
regulated at each step in the pathway by post-translational
modifications such as phosphorylation, ubiquitination,
sumoylation, and acetylation (Figure 1A).17,18
MULTIPLE GR ISOFORMS
Since the cloning of the human GR gene in 1985,19 the
prevailing assumption has been that a single receptor isoform
is responsible for the diverse actions of glucocorticoids.
Recent studies, however, have challenged this simple
“one gene-one receptor” paradigm by revealing an
unexpected array of GR subtypes with distinct expression,
functional, and gene regulatory profiles that arise by
alternative processing of the GR gene.20 Consequently,
the cellular response to glucocorticoids will ultimately be
determined by the composite action of the various GR
isoforms within the cell.
Alternative splicing produces two receptor isoforms, GRα and GRβ, which differ at their carboxyl termini.21 In contrast to the classic hormone-binding GRα isoform, the GRβ splice variant does not bind glucocorticoids, resides constitutively in the nucleus of cells, and has been shown to function as a dominant negative inhibitor of GRα. Elevated expression of GRβ has been associated with glucocorticoid resistance in a variety of inflammatory diseases including asthma, rheumatoid arthritis, and ulcerative consequent enhanced glucocorticoid responsiveness, might underlie the increased susceptibility to steroid-induced ocular hypertension observed in 30% to 40% of the normal population (steroid responders) and 90% of patients with primary glaucoma.28,31 Of interest will be to determine if persons harboring the A3669G allele resulting in elevated GRβ levels are less at risk of glucocorticoid-mediated rises in IOP.
Alternative translation initiation of the GRα mRNA was recently shown to produce an additional 8 receptor subtypes with progressively shorter NTDs: GRα-A, GRα-B, GRα-C1, GRα-C2, GRα-C3, GRα-D1, GRα-D2, and GRα- D3 (Figure 2).32 A similar set of translational isoforms would also be expected from the GRβ mRNA. The GRα translational isoforms are widely distributed but their relative levels vary both between and within tissues. In addition, the isoforms exhibit a similar capacity to bind glucocorticoids as well as GREs, but they differ in their subcellular distribution as the GR&alphA;-D isoforms reside predominantly in the nucleus of cells independent of glucocorticoid treatment. Marked differences have also been observed in the ability of the translational isoforms to regulate gene expression.32,33 The GRα-C isoforms are the most transcriptionally active and the GRα-D isoforms are the most deficient, and these effects have been attributed to subtype-specific differences in cofactor recruitment due to their altered NTDs. Whole genome microarray analysis on cells selectively expressing the individual isoforms has demonstrated that each receptor subtype regulates a common and unique set of genes in response to glucocorticoid treatment. Remarkably, over 90% of the genes were selectively regulated by different GRα subtypes. Moreover, the isoform specific differences in gene expression give rise to distinct functional outcomes on glucocorticoid-induced apoptosis. 33 The GRα translational isoforms are expressed in human trabecular meshwork cells, with GRα-C and GRα-D being more abundant than GRα-A and GRα-B.34 Whether the GRα isoform profile differs between glaucomatous and normal trabecular meshwork cells, and whether changes in the cellular composition of these subtypes underlie the differential susceptibility of individuals to steroidinduced rises in IOP has not yet been determined. Intriguingly, age is a known risk factor for secondary glaucoma, and the relative levels of the GRα translational isoforms in the brain were recently shown to change during the aging process.35
TRADITIONAL AND SELECTIVE GR AGONISTS
The cellular response to glucocorticoids will depend not
only on the GR isoform composition but also on the
nature of the glucocorticoid that binds and activates the
receptor. This was suggested early on by studies showing
that glucocorticoid analogs with different chemical structures
generate unique patters of GR nuclear distribution
and mobility.36,37 Indeed, microarray analyses of human trabecular
meshwork cells treated with structurally different
but similarly potent glucocorticoids used in the clinic (dexamethasone,
triamcinolone acetonide, and fluocinolone
acetonide) revealed subsets of genes both commonly and
uniquely regulated by these steroids.34,38Myocilin and
another glaucoma-associated gene angiopoietin-like 7 were
among the top regulated genes induced by each ligand.
Since mutations in all known glaucoma genes only account
for a small percentage of patients with the disease, identifying
genes commonly regulated by these three clinically relevant
steroids may lead to the discovery of new candidate
glaucoma genes. In addition, genes uniquely regulated by
these different glucocorticoids may give rise to steroid-specific
differences in the onset, rate, magnitude, and reversibility
of the rise in IOP. The distinct genetic signature of these
glucocorticoids suggests that their binding confers unique
conformations on the receptors that lead to differences in
DNA binding, cofactor recruitment, and/or chromatin
remodeling at target genes.39
Intense efforts have been made over the last decade to develop novel GR ligands with an improved therapeutic index. These molecules, termed dissociated or selective GR agonists (SEGRA), retain the negative regulation of gene expression that accounts for the anti-inflammatory actions of glucocorticoids but have lost (at least partially) the positive regulation that appears to contribute to some of their adverse effects.40,41 A number of promising SEGRAs have been described in the literature that strongly inhibit proinflammatory transcription factors and exhibit an improved safety profile in preclinical testing. For example, the non-steroidal SEGRA, BOL-303242-X, functions as an anti-inflammatory agent in a variety of human ocular cells with an efficacy and potency similar to that of dexamethasone and triamcinolone acetonide.42 In contrast, this receptor ligand was less effective than classical glucocorticoids at stimulating transcription of reporter and endogenous genes and inducing side effects such as thymocytes apoptosis, hyperglycemia, growth inhibition, and skin atrophy.43 Moreover, compared to dexamethasone and prednisolone acetate, BOL-303242-X was significantly compromised in its ability to induce the expression of myocilin mRNA and protein in monkey trabecular meshwork cells.44 As the SEGRAs progress down the clinical development path, future studies evaluating their gene regulatory profile on a genome-wide scale in human trabecular meshwork cells and their potential to induce ocular hypertension in vivo should be forthcoming.
CONCLUSION
Glucocorticoids continue to play a major role as therapeutic
agents in ocular disease. Both the cellular complement
of GR isoforms and the nature of the interacting ligand
are two principal factors dictating the glucocorticoid
responsiveness of cells such as the trabecular meshwork.
Understanding the factors that regulate the expression
and activity of GR are essential not only for understanding
the predisposition of certain individuals to develop glucocorticoid-
induced ocular hypertension and secondary
glaucoma but also for shedding light on the etiology of
primary glaucoma. Moreover, as the molecular details of
the GR signal transduction pathway are elucidated, the
design and development of new glucocorticoids with
improved safety profiles will be facilitated.
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