PII: S1350-9462(98)00035-4

E€ects of Glucocorticoids on the Trabecular Meshwork:

Towards a Better Understanding of Glaucoma
Robert J. Wordinger* and Abbot F. Clark
Department of Anatomy and Cell Biology, University of North Texas, Health Science Center, Fort Worth,
Texas, USA and Glaucoma Research, Alcon Laboratories, Inc., Forth Worth, Texas, USA

CONTENTS

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 630
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 631
2. The association of glucocorticoids with primary open angle glaucoma . . . . . . . . . . . . . . . . . . . . . . . . . . . 631
3. Anatomy of the human trabecular meshwork . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 632
3.1. Schlemm's canal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 632
3.2. Juxtacanalicular tissue (JCT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633
3.3. Corneoscleral meshwork . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633
3.4. Uveal meshwork . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633
3.5. Trabeculum morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633
4. The e€ect of glucocorticoids on the trabecular meshwork . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 634
4.1. In vivo e€ects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 634
4.2. Perfusion cultured human eyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 635
4.3. In vitro e€ects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 635
4.3.1. Trabecular meshwork cells in culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 635
4.3.2. Dexamethasone alters trabecular meshwork cell morphology in vitro . . . . . . . . . . . . . . . . . . 637
4.3.3. Gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 637
4.3.4. Extracellular matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 637
4.3.5. Cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 638
4.3.6. Cell adhesion molecules. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 640
4.3.7. Inhibition of trabecular meshwork cell functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 640

4.3.8. Similarities between glucocorticoid-mediated and glaucomatous changes within the

trabecular meshwork . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 640
5. The glucocorticoid receptor complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 641
5.1. Structure of the glucocorticoid receptor (GRa) complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 641
5.2. Role of the heat shock proteins (Hsp) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 642
5.3. Glucocorticoid receptor activation and nuclear translocation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 643
5.4. Receptor phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 644
6. Mechanisms of glucocorticoid receptor action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 645
6.1. The type 1 mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 645

*Corresponding author. Department of Anatomy and Cell Biology, North Texas Eye Research Institute, University of North
Texas Health Science Center at Fort Worth, 3500 Camp Bowie Blvd., Fort Worth, TX 76107, USA. Tel.: 817-735-2045; Fax: 817-
735-2610; E-mail: rwording@hsc.unt.edu

Progress in Retinal and Eye Research Vol. 18, No. 5, pp. 629 to 667, 1999
# 1999 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
1350-9462/99/$ - see front matter
630 R. J. Wordinger and A. F. Clark

6.2. The type 2 mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 646

6.3. Glucocorticoid receptors and co-activators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 647
6.4. Glucocorticoid receptor association with an RNA-binding nuclear matrix protein . . . . . . . . . . . 647
6.5. Functional cooperation of the glucocorticoid receptor and other signaling pathways . . . . . . . . . 648
6.6. Glucocorticoid responsiveness unrelated to the glucocorticoid response element (GRE) . . . . . . 648
7. Non-genomic steroid e€ects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 649
8. The potential role of the glucocorticoid receptor complex in glaucoma . . . . . . . . . . . . . . . . . . . . . . . . . . 649
8.1. Glucocorticoid receptor expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 649
8.2. Intracellular hormone availability and 11b-hy-
droxysteroid dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 649
8.3. Hormone binding anity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 650
8.4. Hormone-induced conformational change and dissociation from the Hsp complex . . . . . . . . . . . 650
8.5. The GRb isoform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 651
8.6. Alternate isoform of the estrogen receptor (ERR2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 653
9. The potential role of nuclear transcription factors in glaucoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 653
9.1. Activating protein-1 (AP-1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 653
9.2. NF-kB/IkB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 654
10. The GLC1A/MYOC gene and glaucoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 655
11. Future research trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 657
11.1. Glucocorticoid responsiveness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 657
11.2. GR isoforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 658
11.3. Gene regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 658
11.4. Functional cooperation between signaling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 659
11.5. Trabecular meshwork cytoskeleton and extracellular matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 659
11.6. Steroid inducible GLC1A gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 659
11.7. Growth factors and growth factor receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 659
12. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660

AbstractÐGlucocorticoid e€ects on the human trabecular meshwork can be used as a model system in which to study
glaucomatous damage to the trabecular meshwork. One of the most important risk factors for glaucoma is an elevated
intraocular pressure. The administration of glucocorticoids also can cause elevated intraocular pressure in some individ-
uals. In addition, there is suggestive evidence linking glucocorticoids with the development of glaucoma. Glucocorticoids
cause multiple e€ects on the human trabecular meshwork including changes in extracellular matrix metabolism, organis-
ation of the cytoskeleton, and changes in gene expression and cell function. New discoveries on the molecular mechanisms
of glucocorticoid receptor action provide new opportunities to study the possible role of this receptor in the development
of glaucoma. For example, alternate spliced forms of the glucocorticoid receptor, glucocorticoid receptor response element
half-sites, numerous modulatory factors, and direct e€ects of nuclear transcription factors have been recently described.
Other recent information has shown that the new glaucoma gene (GLC1A/myocilin) is induced in the human trabecular
meshwork by glucocorticoids. Although the exact function of myocilin is currently unknown, it o€ers the opportunity to
dissect the molecular pathways regulating aqueous humor out¯ow. Future challenges include determining (1) which gluco-
corticoid e€ects in the human trabecular meshwork are responsible for elevated intraocular pressure; and (2) the signi®-
cance of these ®ndings to the development of glaucoma. # 1999 Elsevier Science Ltd. All rights reserved
 E€ects of glucocorticoids on the trabecular meshwork
1. INTRODUCTION
For over 30 years, there has been a suggested link
between glucocorticoids (GCs) and primary open
angle glaucoma (POAG). The suggestive evidence
for this link includes the following: (1) GC ad-
ministration can lead to the development of ocu-
lar hypertension and a secondary open angle
glaucoma in certain individuals. (2) Steroid
responsiveness (i.e., development of ocular hyper-
tension upon GC administration) appears to be
associated with POAG. (3) There are reports of
elevated levels of GCs and altered GC metab-
olism in some POAG patients. (4) Ocular hyper-
tension induced by glucocorticoids, as well as the
ocular hypertension associated with POAG, is
due to increased aqueous humor out¯ow resist-
ance at the trabecular meshwork (TM). In ad-
dition there have been a number of studies on the
e€ects of GCs on the TM as a model system to
better understand the pathogenic mechanisms
involved in ocular hypertension, GC-induced
glaucoma, for POAG. These studies are sugges-
tive that steroid responsiveness to glucocorticoids
may be a major risk factor for POAG. It is also
possible that glucocorticoids may play a role in
the pathogenesis of glaucoma in a subset of
POAG patients.
This review article will address the role gluco-
corticoids may play in POAG and will emphasize
the (a) association of glucocorticoids with POAG,
(b) in vivo and in vitro e€ects of glucocorticoids
on the trabecular meshwork, (c) glucocorticoid
receptor complex and its potential involvement in
POAG, (d) physiological signi®cance of nuclear
transcription factors in POAG, and (e) GLC1A/
MYOC gene and glaucoma. We conclude the
review by o€ering our perspectives on future
directions for research into the role glucocorti-
coids may play in POAG.
2. THE ASSOCIATION OF
GLUCOCORTICOIDS WITH PRIMARY
OPEN ANGLE GLAUCOMA
The administration of glucocorticoids can
cause ocular hypertension which is dependent on
631
the potency of the glucocorticoid, the GC dose
and duration of treatment, the route of adminis-
tration, as well as individual susceptibility (Clark,
1995a). Topical ocular administration of a potent
glucocorticoid for 4±6 weeks can produce an elev-
ated intraocular pressure in approximately 40%
of the general population. Three levels of respon-
siveness have been reported with 4±6% of the
GC-treated individuals developing quite signi®-
cant elevations in IOP. These individuals often
are referred to as ``steroid responders'' (Becker,
1965; Armaly, 1965). It has been reported that
steroid responders are more likely to develop
POAG compared to non-responders (Kitazawa
and Horie, 1981; Lewis et al., 1988). There also
have been several studies (Becker and Chevrette,
1966; Paterson, 1966; Davies, 1968; Bartlett et al.,
1993) reporting increased steroid responsiveness
in relatives of POAG patients and reports of ster-
oid responsiveness being hereditary (Becker, 1965;
Armaly, 1965). Thus, steroid responsiveness
appears to be an important risk factor in the
development of POAG. In addition, the elevated
IOP induced by GCs in steroid responders is due
to increased aqueous humor out¯ow resistance
and is associated with morphological changes in
the trabecular meshwork. These ®ndings are simi-
lar to what occurs in POAG.
Other reports also indicate, although do not
prove, that glucocorticoids are associated with
POAG. For example, it has been reported that
POAG patients have elevated levels of the en-
dogenous glucocorticoid, cortisol, in their blood
compared to age-matched subjects without
POAG (Ray et al., 1977; Rozsival et al., 1981;
Schwartz et al., 1987; Meredig et al., 1980;
McCarty and Schwartz, 1991). There also has
been one report of higher levels of cortisol in the
aqueous humor of POAG patients compared to
non-glaucomatous patients undergoing cataract
extraction (Rozsival et al., 1981). Several early
studies (Tassman, 1957; Weinstein, 1954) have
suggested that stress is a risk factor for the devel-
opment of POAG which may implicate stress hor-
mones, such as cortisol, as being associated with
the disease. Chronic, slightly higher levels of cor-
tisol may lead to slowly progressive changes in
the TM which could eventually lead to ocular
hypertension and glaucoma.
632 R. J. Wordinger and A. F. Clark

Additional studies have suggested that POAG

may be due to an ocular and/or systemic defect in
cortisol metabolism (Southern et al., 1983;
Weinstein et al., 1985, 1996). Changes in the ac-
tivities of two important cortisol metabolising
enzymes have been reported in the TM and/or
peripheral white blood cells of POAG patients.
These researchers have speculated that the altered
activities of these enzymes would lead to the ac-
cumulation of the cortisol metabolite dihydrocor-
tisol which they believe is involved in altering the
TM leading to elevated IOP. However, the same
researchers failed to ®nd di€erences in the con-
centrations of dihydrocortisol in the aqueous
humor between POAG patients and normals
(Weinstein et al., 1991).
These studies are suggestive that glucocorti-
coids are associated with POAG but the studies
are not conclusive. To date, there is no clear pic-
ture. It is possible that glucocorticoids are just
one of the many risk factors associated with
POAG, or that glucocorticoids may play a role in
the pathogenesis of glaucoma in only a subset of
POAG patients. Since elevated IOP induced by
glucocorticoids in steroid responders is due to
aqueous humor out¯ow resistance which is
re¯ected by changes in trabecular meshwork mor-
phology, we will address the e€ects of glucocorti-
coids on the trabecular meshwork and the
potential role(s) in POAG. We begin with a brief
introduction to the anatomy of the human trabe- Fig. 1. Transmission electron micrograph of the human
cular meshwork. trabecular meshwork. Aqueous humor exits the eye
through the trabecular meshwork which imparts resistance
to aqueous out¯ow. Morphological changes occur in the
trabecular meshwork of glaucomatous eyes. These
changes are associated with the development of elevated
intraocular pressure in many forms of glaucoma. (SC)
Canal of Schlemm, (IW) Inner wall of the canal of
3. ANATOMY OF THE HUMAN Schlemm, (JCT) juxtacanalicular tissue, (*) Trabecular
TRABECULAR MESHWORK beam, (arrowheads) Trabecular cell. Bar = 20 mm.

The anatomy of the human trabecular mesh-

work (Fig. 1) is a complex consisting of four
regions which are termed the inner wall of
Schlemm's canal, juxtacanalicular meshwork, cor-
nealscleral meshwork, and uveal meshwork. Each
of these regions has a distinct morphology and
will be considered separately. A detailed review of
the human trabecular meshwork morphology can
be obtained in Lutjen-Drecoll and Rohen (1996,
1994).
3.1. Schlemm's Canal
The canal of Schlemm is a complete circular
canal just posterior to the trabecular meshwork
into which aqueous humor ®lters. Schlemm's
canal is lined by a single layer of vascular endo-
thelial-like cells which contain gap junctions and
 E€ects of glucocorticoids on the trabecular meshwork
incomplete tight junctions. Aqueous humor
appears to pass through the inner wall of
Schlemm's canal by paracellular routes as well as
transcellular microchannels or pores. Aqueous
humor exits the canal of Schlemm via collector
channels where it subsequently enters the venous
circulation.
3.2. Juxtacanalicular Tissue (JCT)
The juxtacanilicular tissue (JCT) or cribriform
meshwork is morphologically distinct from other
regions of the trabecular meshwork. The inner
wall cells of Schlemm's canal attach to the JCT,
and together these structures serve as the last
region of the trabecular meshwork through which
aqueous humor must pass before entering
Schlemm's canal. The JCT and the inner wall of
Schlemm's canal are the major source of aqueous
humor out¯ow resistance (Maepea and Bill,
1989). Morphological and biochemical changes in
the JCT are associated with the development of
elevated intraocular pressure in POAG (Lutjen-
Drecoll and Rohen, 1996). The juxtacanalicular
meshwork consists of a layer of connective tissue
lined on either side by cells. The juxtacanilicular
meshwork forms the outermost part of the
human trabecular meshwork as well as contribut-
ing to the inner wall of Schlemm's canal. The
connective tissue component lacks descrete open-
ings and contains a network of irregularly
oriented ®brils and a number of elongated cells
arranged in layers. Extracellular matrix can be
seen between the layers of cells and type III col-
lagen has been shown to be present in this region.
The outer layer of the juxtacanalicular meshwork
comprises the inner wall of Schlemm's canal while
the inner layer is continuous with the corneoscl-
eral meshwork.
3.3. Corneoscleral Meshwork
The corneoscleral meshwork extends from the
scleral spur to the anterior wall of the scleral sul-
cus. The corneoscleral meshwork morphology
consists of equatorially oriented sheets of trabecu-
lae (e.g., trabecular beams) which are perforated
633
by elliptical openings. Aqueous humor ¯ows
through the openings and around the trabeculae
as it moves towards the juxtacanalicular mesh-
work. The openings get progressively smaller (50±
5 mm) closer towards the juxtacanalicular mesh-
work. The trabeculae are surrounded by a single
layer of ¯attened, trabecular meshwork cells
which reside on a distinct basal lamina. Each tra-
beculae contains a core of collagenous and elastic
®bers.
3.4. Uveal Meshwork
The uveal meshwork is immediately adjacent to
the aqueous humor in the anterior chamber and
consists of radially oriented strands of connective
tissue trabeculae. These connective tissue stands
extend from the iris root and ciliary body to the
peripheral cornea. The arrangement of the trabe-
culae within the uveal meshwork creates irregular
openings which contains aqueous humor and
which vary in size from 25±75 mm across.
3.5. Trabeculum Morphology
Each connective tissue trabeculum contains
several distinct regions which have been pre-
viously described. First there is an inner, ®brous
connective tissue core. Immunohistochemical stu-
dies indicate that collagen type I and III and elas-
tin are present within this core. The elastic ®bers
which are present may provide ¯exibility to the
trabeculum. Immediately outside the connective
tissue core is a region consisting of delicate ®la-
ments embedded in extracellular matrix. On the
surface of the trabecular beam is a noncontinuous
basement membrane. Finally, a layer of trabecu-
lar meshwork cells covers the trabeculum and
forms a continuous layer surrounding each beam.
634 R. J. Wordinger and A. F. Clark
4. THE EFFECT OF GLUCOCORTICOIDS
ON THE TRABECULAR MESHWORK
4.1. In Vivo E€ects
A number of investigators have examined the
e€ects of glucocorticoids on the trabecular mesh-
work in an attempt to better understand gluco-
corticoid induced glaucoma and primary open
angle glaucoma. Cortisol is the natural glucocor-
ticoid in man, and approximately 90±95% of cir-
culating cortisol is bound to the serum binding
proteins transcortin (cortisol binding globulin)
and albumin. For many of the synthetic glucocor-
ticoids, such as dexamethasone (DEX), there is
very little serum protein binding which, along
with an increased receptor anity and resistance
to metabolic inactivation, accounts for their
greater biological activity. Glucocorticoids not
complexed with serum binding proteins permeate
the cellular membranes and bind to soluble intra-
cellular glucocorticoid receptors (Section 5).
Human trabecular meshwork cells contain glu-
cocorticoid receptors (Weinreb et al., 1981;
Hernandez et al., 1983) and would therefore be
expected to respond to glucocorticoid adminis-
tration. The administration of potent glucocorti-
coids in man can generate a progressively
elevated intraocular pressure which is dependent
on glucocorticoid potency, pharmacokinetics,
duration of treatment, route of administration, as
well as di€erences in individual responsiveness.
Glucocorticoid-induced ocular hypertension is
due to increased aqueous humor out¯ow resist-
ance (Bernstein and Schwartz, 1962; Becker and
Mills, 1963; Armaly, 1963; Paterson, 1966) and is
associated with speci®c morphological changes in
the trabecular meshwork. Glucocorticoid-induced
ocular hypertension is usually reversible after ces-
sation of glucocorticoid administration (Becker
and Mills, 1963; Armaly, 1963). Morphological
examination of the trabecular meshwork of
patients with glucocorticoid-induced glaucoma
has shown an increased deposition of extracellu-
lar material in the trabecular beams and in the
juxtacanalicular tissue (cribriform region), a
decrease in intratrabecular spaces, as well as an
apparent ``activation'' of the trabecular mesh-
work cells (Rohen et al., 1973; Roll and Benedict,
1979; Toriyama, 1979; Rohen and Lutjen-
Drecoll, 1989; Johnson et al., 1997). Two charac-
teristic types of extracellular deposits seen in the
TM of steroid glaucoma patients are ®ngerprint-
like depositions of material in the uveal mesh-
work (Rohen et al., 1973; Johnson et al., 1997)
and ®ne ®brillar material in the JCT region
(Johnson et al., 1997). The ocular hypertension
caused by GC administration generally takes
weeks to months to occur, although there has
been one report of GC-induced ocular hyperten-
sion within hours of systemically administered
dexamethasone in POAG patients (Weinreb et al.,
1985).
In addition to man, glucocorticoid adminis-
tration leads to ocular hypertension in several
other animal species including rabbit, cat and
monkey. Topical ocular administration of a
potent glucocorticoid (such as dexamethasone or
betamethasone) causes ocular hypertension in
rabbits (Lorenzetti, 1970; Levine et al., 1974;
Ticho et al., 1979; Francois et al., 1984; Knepper
et al., 1978; Knepper et al., 1985; Bodor et al.,
1992) and similar to man, this IOP elevation is
due to a decrease in the aqueous out¯ow facility
(Lorenzetti, 1970). The glucocorticoid-induced
ocular hypertension in rabbits is associated with
morphological changes in the TM including
thickening of trabecular beams, activation of TM
cells, and increased deposition of extracellular
material (Ticho et al., 1979; Francois et al., 1984).
This GC-induced ocular hypertension is associ-
ated with biochemical changes in the trabecular
meshwork glycosaminoglycan composition with
decreased hyaluronic acid and an increased depo-
sition of chondroitin sulfate and GAGase resist-
ant material (Knepper et al., 1978). However,
there is one major drawback with the rabbit as a
model in which to study GC-induced ocular
hypertension. The rabbit is very sensitive to GCs
that can cause signi®cant systemic e€ects such as
loss in body weight, adrenal atrophy, and hepato-
megaly, which is seen even after topical GC ad-
ministration.
Topical ocular administration of dexametha-
sone to cats also induces ocular hypertension
(Zhan et al., 1992; Palkama et al., 1994) which is
due to a decrease in the out¯ow facility and is as-
sociated with increased deposition of extracellular
 E€ects of glucocorticoids on the trabecular meshwork
matrix material in the trabecular meshwork.
Finally, several groups have reported the gener-
ation of glucocorticoid-induced ocular hyperten-
sion in monkeys by topical ocular administration
(Clark et al., 1996) or by sub-Tenon's injection
(DeSantis et al., 1990) of dexamethasone. Five
out of eleven monkeys were found to be steroid
responders after topical ocular dexamethasone
administration. As in man, this IOP elevation was
reversible after stopping DEX treatment and was
reproducible after a second DEX challenge
(Clark et al., 1996).
4.2. Perfusion Cultured Human Eyes
It can be argued that topical administration of
glucocorticoids may not have a direct e€ect on
the trabecular meshwork. However, recent studies
have demonstrated that glucocorticoids do in fact
have a direct e€ect. The isolated, perfusion cul-
tured human eye model (Johnson and
Tschumper, 1987) has been used to develop dexa-
methasone-induced ocular hypertension. After 2
weeks in culture, 30% of the eyes have pressure
rises averaging 17 mmHg (Clark et al., 1995c). It
should be noted that the ``responder'' rate in
these perfusion cultured eyes is very similar to the
responder rate of the normal human population
to topical ocular dexamethasone administration
(Becker, 1965; Armaly and Becker, 1965). The
dexamethasone-induced ocular hypertension in
the perfusion cultured eyes was associated with a
thickening of trabecular beams, decreased inter-
trabecular spaces, and an increased deposition of
extracellular material in the JCT region. At least
a portion of the JCT material was shown to be
due to an altered distribution of the extracellular
matrix glycoprotein ®bronectin. In addition,
Johnson et al. (1990) have shown an increase in
GAGase resistant glycosaminoglycans in per-
fusion cultured human eyes treated for 21 days
with dexamethasone.
The perfusion cultured eye model also has been
used to study glucocorticoid regulation of phago-
cytosis and prostaglandin metabolism. For
example, glucocorticoids have been shown to
inhibit arachidonate metabolism in a number of
di€erent cells and tissues. Shaw et al. (1993) have
635
shown that PGF2a and PGE2 are the major ara-
chidonate metabolites in perfusion cultured
human eyes and that arachidonate metabolism is
signi®cantly inhibited by perfusion with dexa-
methasone. The dexamethasone-induced inhi-
bition of PGF2a production has been suggested to
contribute to the IOP elevating e€ects of gluco-
corticoids because the FP prostaglandins have
been shown to have ocular hypotensive properties
(Camras, 1996). Perfusion of human anterior seg-
ments with DEX for 3 weeks also alters trabecu-
lar meshwork function. Recently, Matsumoto and
Johnson (1997) reported that DEX signi®cantly
inhibited trabecular meshwork phagocytosis.
4.3. In Vitro E€ects
4.3.1. Trabecular meshwork cells in culture
A number of investigators have cultured
human trabecular meshwork cells in order to
directly study the e€ects of glucocorticoids on
TM cell structure and function. Cultured TM
cells grow as a monolayer, are round to partially
elongated in shape, and contain numerous pro-
cesses with overlapping borders (Fig. 2)
(Polansky et al., 1979; Alvarado et al., 1982;
Tripathi and Tripathi, 1982; Hernandez et al.,
1987; Wilson et al., 1993; Clark et al., 1994).
Cultured TM cells share many properties with
TM cells in situ; both (1) synthesize and secrete
very similar extracellular matrix molecules
(Hernandez et al., 1987; Yun et al., 1989;
Hernandez and Neufeld, 1989), (2) have identical
cytoskeletal elements including smooth muscle a-
actin (Clark et al., 1994; Ryder et al., 1988;
Weinreb and Ryder, 1990; Flugel et al., 1992), (3)
are phagocytic (Alvarado et al., 1982; Tripathi
and Tripathi, 1982; Barak et al., 1988; Rohen and
van der Zyphen, 1968; Grierson and Lee, 1973),
(4) express similar extracellular proteinases
(Alexander et al., 1991), and (5) have similar junc-
tional complexes (Raviola and Raviola, 1981;
McCartney et al., 1993; Bhatt et al., 1995). It is
therefore argued that cultured TM cells are an
appropriate model for studying many of the bio-
logical properties of the TM. The TM, like many
tissues, functions as a tissue matrix which
636 R. J. Wordinger and A. F. Clark

Fig. 2. Light micrograph of normal and DEX treated human trabecular meshwork cells in culture. (A) Control human
trabecular meshwork cells appear as partially elongated cells with overlapping cell processes. (B) DEX treatment for 14
days (0.1 mM) causes an increase in cell and nuclear size.
 E€ects of glucocorticoids on the trabecular meshwork
involves a dynamic interaction between the extra-
cellular matrix, the TM cell membrane, the cytos-
keleton, and the TM cell nucleus. Because of this
extensive connectivity, it is not unexpected that
glucocorticoids have been shown to in¯uence all
of these components of the TM.
4.3.2. Dexamethasone alters trabecular meshwork cell
morphology in vitro
Glucocorticoids appear to a€ect the mor-
phology of TM cells. Three weeks of treatment
with cortisol caused an increase in TM nuclear
size and DNA content which was most likely due
to endoreplication (Tripathi et al., 1989). Treating
cultured TM cells for several weeks with DEX
caused a signi®cant increase (50±100%) in aver-
age TM cell size in addition to increased nuclear
size (Fig. 2) (Wilson et al., 1993; Clark et al.,
1994; Matsumoto et al., 1998). Dexamethasone-
induced ultrastructural changes included prolifer-
ation and activation of the endoplasmic reticulum
and Golgi apparatus as well as increased depo-
sition of extracellular matrix material (Wilson et
al., 1993). There was also an apparent increase in
fusion vesicles which were often found arranged
in linear rows at the surface of the TM cell mem-
brane (McCartney et al., 1993). The apparent ac-
tivation of TM cells by glucocorticoids has been
reported in glucocorticoid-induced glaucoma in
man (Rohen et al., 1973; Rohen and Lutjen-
Drecoll, 1989) and DEX-induced ocular hyper-
tension in rabbits (Ticho et al., 1979; Francois et
al., 1984).
Glucocorticoids increase the size of cultured
TM cells (Wilson et al., 1993; Clark et al., 1994;
Tripathi et al., 1989). Although it has been shown
that this increase in cell size is associated with
glucocorticoid-induced changes in the TM cytos-
keleton (Wilson et al., 1993; Clark et al., 1994)
there may be additional explanations. Recently
Putney et al. (1997) have shown that glucocorti-
coids alter the expression of the TM Na/K/Cl co-
transporter which causes TM cell swelling. These
authors propose that this co-transporter may be
responsible for regulating trabecular out¯ow
(O'Donnell et al., 1995). Although DEX does
appear to alter the expression of the TM Na/K/
637
Cl cotransporter, its e€ects are transient and do
not appear to coincide with either the acute
(within hours) or more chronic (weeks) e€ects of
glucocorticoids on IOP. In addition, a separate
study questions the role of the Na/K/Cl co-trans-
porter in regulating IOP (Gabelt et al., 1997).
4.3.3. Gene expression
The physiological and pharmacological action
of steroids is mediated through the regulation of
transcriptional activity (Section 6). This appears
to be the major mechanism by which glucocorti-
coids alter TM cell function. The treatment of
cultured TM cells with a potent glucocorticoid,
such as dexamethasone, causes increased ex-
pression of speci®c genes and decreased ex-
pression of other genes which have been studied
using subtractive cDNA libraries (Nguyen et al.,
1991, 1998) or quantitative PCR techniques
(Nguyen et al., 1993). The altered expression of
TM gene products has also been examined using
polyacrylamide gel electrophoresis (PAGE) to
separate proteins made by the TM cells (Polansky
et al., 1991; Kawase et al., 1994; Clark et al.,
1998). Using high resolution 2-dimensional
PAGE of radiolabeled TM cell proteins, it
appears that glucocorticoid treatment a€ects the
expression of approximately 2±5% of the TM cell
proteins (Kawase et al., 1994).
4.3.4. Extracellular matrix
The treatment of cultured TM cells with gluco-
corticoids has a demonstrable e€ect on the extra-
cellular matrix. There have been con¯icting
reports on the e€ects of DEX on TM collagen
including increased TM collagen deposition (Yue,
1996) and decreased collagen synthesis (Hernadez
et al., 1985). However, this apparent discrepancy
may be due to the very di€erent methodologies
used in these two studies. GCs also a€ect the
composition of TM glycosaminoglycans (GAGs).
The TM of cultured human eyes perfused for 14±
21 days with DEX had a signi®cant increase in
the amount of GAGase resistant material
(Johnson et al., 1990). Cultured primate TM cells
638 R. J. Wordinger and A. F. Clark
exposed to DEX for 12 days had a signi®cant re-
duction in hyaluronan synthesis (Engelbrecht-
Schnur et al., 1997). Biochemical analysis of the
GAGs extracted from cultured TM cells treated
with DEX for 14 days also con®rms that GCs
alter the GAG pro®le by increasing chondroitin
sulfate and decreasing hyaluronate deposition
(Knepper and Clark, unpublished observations).
The treatment of TM cells with DEX for 17 days
increased the deposition of the ECM glyco-
proteins ®bronectin (Steely et al., 1992) and lami-
nin (Dickerson et al., 1998). The DEX-induced
increase in ®bronectin was progressive over time,
and there was some heterogeneity in DEX
responsiveness between the various human TM
cell lines tested (Steely et al., 1992). The e€ects of
GCs on the TM extracellular matrix may be due
to altered rates of protein synthesis, protein
degradation, or a combination of both. Recent
studies have shown that DEX treatment inhibits
the activity of several TM extracellular metallo-
proteinases which are involved in the turnover of
the ECM (Snyder et al., 1993; Samples et al.,
1993; Fig. 3, Clark et al., unpublished obser-
vations). The matrix metalloproteinases stromcly-
sin and gelatinase as well as tissue plasminogen
activator activities are inhibited within 2±3 days
of DEX exposure.
DEX treatment has been reported to increase
the secretion of elastin (Yun et al., 1989). Elastin
is the predominant component of elastic ®bers
Fig. 3. E€ect of interleukin-1 (IL-1) and dexamethasone
(DEX) on stromelysin (MMP-3) expression in cultured
human TM ceils. IL-1 signi®cantly upregulates MMP-3
expression. DEX treatment downregulates TM cell MMP-
3 expression in both control and IL-1 induced cultures
(Pang, Shade, and Clark, unpublished observations).
and has been localized in both normal and glau-
comatous human trabecular meshwork (Umihira
et al., 1994). Of particular interest is the report by
Umihira et al., 1994 which demonstrated that in
POAG there are marked changes in the distri-
bution and the quantity of elastin. When POAG
samples were compared to normals, there was an
increased amount of elastin immunoreactivity
along the inner canal endothelium which also
contained ®ne ®brillar-like material. It is therefore
signi®cant that Del Monaco et al. (1997) reported
the presence of three novel/putative glucocorti-
coid response elements (GREs) within the promo-
ter region of the human elastin gene. The GREs
were within the downstream half-site sequence
TGTTCC that has homology with the consensus
GRE. However, the upstream half-site showed no
homology. The authors showed that all three
GREs could bind the GR and they suggested that
these GREs would be active and could mediate
the glucocorticoid-induced up-regulation of the
human elastin promoter.
4.3.5. Cytoskeleton
In addition to their e€ects on the ECM, gluco-
corticoids have profound e€ects on the trabecular
meshwork cytoskeleton. The majority of f-actin
micro®laments in cultured human TM cells are
localized in actin ®lament bundles known as
stress ®bers (Ryder et al., 1988; Wilson et al.,
1993; Clark et al., 1994). Upon treatment with
glucocorticoids, the TM micro®laments reorgan-
ise into geodesic dome-like structures of cross-
linked actin networks (CLANs) (Wilson et al.,
1993; Clark et al., 1994) (Fig. 4). The GC-induced
formation of CLANs was found to be: (1) time
dependent progressing over the course of days to
weeks, (2) speci®c for glucocorticoids, (3) dose
dependent matching the anity of GCs for the
glucocorticoid receptor, (4) speci®c for TM cells,
and (5) reversible upon withdrawal of the GC
(Clark et al., 1994). This GC-induced micro®la-
ment reorganisation was associated with changes
in the expression of speci®c micro®lament pro-
teins. DEX-treatment of cultured TM cells caused
a 2±3X increase in the expression of the actin
binding proteins a-actinin and meta-vinculin. In
 E€ects of glucocorticoids on the trabecular meshwork 639

Fig. 4. Epi¯uorescent micrograph (Rhodamine/phalloidin labeled) of actin micro®laments in normal (A) and DEX-treated
(B) TM cells. Control cells (A) contain normal stress ®bers. In contrast, DEX treated cells (0.1 mM DEX for 14 days)
(B) have cross-linked actin networks (CLANs) (arrows) localized within the cells. CLANs are geodesic dome-like micro-
®lamentous structures.

contrast, the expression of total actin, smooth
muscle a-actin and tropomyosin remained
unchanged (Clark et al., 1997). Elevated levels of
CLANs have been reported in a number of TM
cell lines derived from glaucomatous donors
suggesting that these structures may play a role in
POAG (Clark, 1995a; Clark et al., 1997).
Dexamethasone treatment also induced changes
in the organisation of microtubules in TM cells.
A translocation of the microtubule organising
center as well as the development of microtubule
tangles was found in TM cells treated with DEX
for 14 days (McCartney et al., 1994). These GC-
mediated changes in the TM cytoskeleton may
have signi®cant e€ects on a variety of TM cell
functions. The cytoskeleton has been shown to
regulate cellular behavior including: attachment
to the extracellular matrix and adjacent cells, cell
shape, cell division, cell movement, phagocytosis,
protein synthesis, and protein secretion
(Bershadsky and Vasiliev, 1988; Bray and White,
1988). Trabecular meshwork cells treated with
640 R. J. Wordinger and A. F. Clark
DEX have been shown to be resistant to the in-
duction of cell retraction by ethacrymic acid and
EGTA which suggests that glucocorticoids stabil-
ise the TM cytoskeleton as well as cell-cell and
cell-matrix attachment (O'Brien et al., 1996).
4.3.6. Cell adhesion molecules
Cell adhesion molecules are involved in cell-cell
and cell-matrix interactions. The intracellular
cytoskeleton is linked to speci®c membrane ad-
hesion receptors which bind to other cells or to
the extracellular matrix. Because glucocorticoids
alter the TM cell cytoskeleton and the TM extra-
cellular matrix, it would be reasonable to expect
glucocorticoid-mediated changes in TM cell ad-
hesion molecules as well. Recent work has shown
that a2, a5, and av integrin expression is altered
in TM cells cultured with dexamethasone for 18
days (Dickerson et al., 1998). Even junctional
complexes between TM cells are a€ected by DEX
treatment. There was a realignment of gap junc-
tional complexes (McCartney et al., 1994) and an
apparent increased expression of the tight junc-
tion protein ZO-1 (Underwood et al., 1993) in
TM cells cultured with DEX for several weeks.
These GC-induced changes in TM cell adhesion
molecules and cell junctions may alter cell beha-
vior and/or alter TM tissue permeability.
4.3.7. Inhibition of trabecular meshwork cell functions
A number of TM cell functions are inhibited
upon GC treatment. Normal TM cells have been
demonstrated to produce certain arachidonate
metabolises such as PGE2 and PGF2a in cell cul-
ture, and DEX treatment signi®cantly inhibited
TM cell arachidonic acid metabolism (Weinreb et
al., 1983; Gerritsen et al., 1986) similar to that seen
in perfusion cultured human eyes (Shaw et al.,
1993). The role of TM cell arachidonic acid metab-
olism in the regulation of IOP and/or TM cell
function is currently not known. Dexamethasone
treatment of cultured TM cells for several weeks
signi®cantly inhibited phagocytic activity
(Polansky et al., 1991; Shirato et al., 1988).
Glucocorticoid-mediated inhibition of TM phago-
cytosis leading to the accumulation of extracellular
debris has been suggested as a cause of corticoster-
oid-induced ocular hypertension (Bill, 1975). In
addition, DEX has been shown to inhibit TM cell
migration (Clark et al., 1994) and proliferation
(Clark et al., 1994; Polansky et al., 1991).
Dexamethasone treatment also has been shown to
inhibit hydraulic ¯ow through TM cells cultured
on ®lters (Underwood et al., 1993) possibly due to
DEX-induced changes in cell size, altered TM
cytoskeletal organization, and/or in TM cell tight
junctional complexes. A summary of the multitude
of e€ects of glucocorticoids on cultured trabecular
meshwork cells is shown in Fig. 5 and Fig. 6.
4.3.8. Similarities between glucocorticoid-mediated and
glaucomatous changes within the trabecular meshwork
Many GC-induced changes in the trabecular
meshwork appear to be similar to glaucomatous
changes in the TM including e€ects on the ECM,
cytoskelton, and gene expression. Glucocorticoids
can change the composition of the TM ECM (see
Section 4.3.4). A similar increased deposition of
®bronectin (Babizhayev and Brodskaya, 1989), of
elastin (Umihira et al., 1994), and decreased hya-
luronan expression (Knepper et al., 1996a,b) have
been reported in the trabecular meshwork of
patients with primary open angle glaucoma.
Likewise, the cytoskeletal CLANs that are
induced by GCs in normal TM cells (Section
4.3.5) also are seen in glaucomatous TM cells cul-
tured in the absence of GCs (Clark et al., 1995b).
Trabecular meshwork cells exposed to GCs leads
to the induction of the GLC1A gene and
increased expression of the myocilin protein in
the TM (Section 4.3.3 and Section 10). A number
of mutations in the GLC1A gene can lead to the
development of juvenile and adult onset open
angle glaucoma. Despite many of these simi-
larities with glaucomatous changes to the TM,
there is still no proof that the GC-induced
changes to the ECM, cytoskelton or expression of
myocilin are responsible for increased out¯ow
resistance and elevated IOP.
 E€ects of glucocorticoids on the trabecular meshwork 641

Fig. 5. Composite schematic representation of previously reported DEX-induced changes within human trabecular mesh-
work cells. (1) Proliferation of the Golgi apparatus. (2) Enlarged and pleomorphic nuclei. (3) Increased numbers of geode-
sic dome-like cross-linked actin ®lament networks (CLANs). (4) Development of microtubule tangles. (5) Altered
expression and localization of junctional complexes. (6) Increased linear stacks of endoplasmic reticulum. (7) Altered ex-
pression of speci®c integrins. (8) Increased synthesis and secretion of amorphous extracellular material with ®brillar com-
ponents. (9) Increased numbers of electron lucent fusion vacuoles.

5. THE GLUCOCORTICOID RECEPTOR transcription factors (Evans, 1988; Mangelsdorf

COMPLEX
5.1. Structure of the Glucocorticoid Receptor (GRa
a)
Complex
Almost all known biological e€ects of glucocor-
ticoids are mediated via the glucocorticoid recep-
tor (GRa). In the absence of ligand, the GRa
exists as a large (>200 kDa) heterocomplex con-
taining both heat shock proteins (hsp) and at
least one immunophilin (Pratt and Toft, 1997)
(Fig. 7). GRa belongs to a superfamily of highly
conserved proteins including receptors for miner-
alocorticoids, androgens, estrogens, thyroid hor-
mone, retinoic acid, progestins, and vitamin D
(Mangelsdorf et al., 1995). Since these receptors
bind to and modulate speci®c gene promoters
they have also been termed ligand-dependent
et al., 1995).
The GRa and other members of the receptor
superfamily share certain characteristics. First,
the N-terminal region spans approximately the
®rst 439 amino acids of the receptor and contains
sequences involved in activation of target genes
(Giguere et al., 1986; Hollenberg et al., 1987;
Dahlman-Wright et al., 1994). Second, there are
two highly conserved ``zinc ®ngers'' located in the
central portion of the receptor which contains the
DNA-binding domain (DBD). This region, which
is about 65 amino acids in length, also contains
sequences which participate in receptor dimeriza-
tion (Tsai et al., 1988), nuclear translocation
(Picard and Yamamoto, 1987), and transactiva-
tion (Hollenberg et al., 1987; Lefstin et al., 1994).
Thirdly, the C-terminal region of the receptor
contains the hormone-binding domain (HBD) as
642 R. J. Wordinger and A. F. Clark
Fig. 6. Quantitative changes within trabecular meshwork
cells following DEX treatment. (A) DEX treatment
altered the expression of cytoskeleton (CLANs), ®bronec-
tin (FN), laminin (LM), integrins (alpha 5 and alpha 2
subunits), and myocilin. (B) DEX treatment signi®cantly
inhibited trabecular meshwork cell proliferation, phagocy-
tosis, and migration while increasing cell size.
well as speci®c sequences for binding to heat
shock protein (hsp) chaperones, nuclear translo-
cation and other sequences which apparently
``silence'' the GR in the absence of the ligand.
The main function of the heterocomplex appears
to be involved in facilitating three-dimensional
folding which maintains the HBD in a high-a-
nity conformation thus allowing it to bind ligand
(Pratt and Toft, 1997). The hormone binding
domain extends for about 250 amino acids.
Recently, a second isoform of the glucocorti-
coid receptor (GRb) has been ``rediscovered'' and
examined for possible physiological signi®cance
in human tissues. Originally, Hollenberg et al.
(1985) reported the primary structure and ex-
pression of the human GR cDNA. Encio and
Detera-Wadleigh (1991) later reported the geno-
mic structure. Previous reports have indicated
that alternative splicing of the glucocorticoid
receptor pre-mRNA produces a highly homolo-
gous mRNA and protein isoform termed gluco-
corticoid receptor beta (GRb) in comparison to
the original glucocorticoid receptor which is now
termed GRa (Hollenberg et al., 1985; Encio and
Detera-Wadleigh, 1991). The physiological signi®-
cance of GRb and its potential role in glaucoma
are discussed later in the review (Section 8.5).
5.2. Role of the Heat Shock Proteins (Hsp)
The core glucocorticoid receptor heterocomplex
is composed of one GR molecule, two molecules
of hsp 90, one molecule of hsp 70, and one mol-
ecule of the immunophilin FKBP52 also known
as hsp 56 (Hutchison et al., 1993; Pratt, 1993;
Czar et al., 1994a; Czar et al., 1994b) (Fig. 7). In
the absence of the glucocorticoid ligand, the
receptor heterocomplex may in fact undergo
cycles of dissociation/reassociation (Hu et al.,
1994; Hutchison et al., 1994). The heat shock pro-
teins act as chaperones and may themselves exist
as multiprotein complexes independent of GR as-
sociation (Pratt and Toft, 1997). Heat shock pro-
tein 90 is an abundant cytoplasmic protein that is
widely distributed in vertebrate cells and has been
reported to account for as much as 1±2% of all
cytosolic proteins (Pratt and Toft, 1997). Two
genes (hsp 90a and hsp 90b) are known to encode
for hsp 90 proteins in mammalian cells with hsp
90 a having 86% amino acid homology with hsp
90b (Hickey et al., 1989). Homodimers of hsp 90
(a/a and b/b) have been reported by Minami et
al., 1991 and Minami et al., 1994. The signi®cance
of the hsp 90 b isoform is not currently known. It
is clear however that the GR must be bound to
and directly interact with hsp 90 for the HBD of
the GR to be in a high-anity, steroid binding
con®guration. The dissociation of hsp 90 from
the GR results in the inability the GR to bind
GC (Bresnick et al., 1989).
Other heat shock proteins are also involved in
maintaining the stability of the GR-hsp 90 com-
plex. One of these is hsp 70 which has been
shown to be di€usely located in both the cyto-
 E€ects of glucocorticoids on the trabecular meshwork
plasm and nucleus. Members of the hsp 70 family
act as chaperones which bind in an ATP-depen-
dent manner and assist in protein folding and
protein translocation (Pratt and Toft, 1997).
Although hsp 70 is required for the assembly of
the GR-hsp 90 complex, there is no evidence that
it is directly involved in receptor action.
Proteins termed immunophilins (e.g., cyclophi-
lins) are highly conserved in mammalian cells and
bind immunosuppressant drugs such as cyclos-
porin (Schrieber, 1991). The protein FKBP52,
also known as hsp 56, is a member of the immu-
nophilin family and associates with the GR com-
plex. The exact function of hsp 56 is not clear,
but it is known to bind directly to hsp 90. In fact,
hsp 90 contains a common immuophilin binding
region (Pratt and Toft, 1997). One function for
hsp 56 may be involved with the tracking of the
GR from the cytoplasm to the nucleus. For
example, Czar et al. (1995) have shown that the
injection of an antibody against hsp 56 impedes
the DEX-mediated shift of the GR into the
nucleus. It is also known that hsp 56 is not
required for GR complex assembly or for proper
folding of the GR. However, as stressed by
Bamberger et al. (1996), it is not conclusively
known which of the hsp components actually
remain bound to the receptor during transloca-
tion.
Two other proteins may also be associated with
the GR-hsp heterocomplex. The protein p23 has
been shown to bind directly to hsp 90 via an ATP
driven mechanism (Johnson and Toft, 1995;
Johnson et al., 1996). The protein p60 is only pre-
sent in the receptor heterocomplex as a transient
participant during assembly (Pratt and Toft,
1997). Currently the exact role of p23 and p60 in
the action of the GR has not been established.
5.3. Glucocorticoid Receptor Activation and Nuclear
Translocation
Glucocorticoids cross the cell membrane and
interact with the cytoplasmic GR-hsp 90 hetero-
complex (Fig. 7). Studies indicate that the binding
of GC and subsequent GR activation depends on
the association of the GR with hsp 90 through
643
the HBD (Pratt, 1997). For example, geldanamy-
cin, a hsp 90 binding agent, inhibits GR function
(Bamberger et al., 1997) and impedes DEX-
dependent tracking of the GR from the cyto-
plasm to the nucleus (Czar et al., 1997). In re-
sponse to the presence of the GC, the GR is
transformed from a silent to an active transcrip-
tion factor. Although the exact mechanism is cur-
rently unknown, ligand binding to the GR
induces a conformational change in the GR
which allows the receptor to disassociate from the
hsp complex (Tsai and O'Malley, 1994;
Hutchison et al., 1993).
Following disassociation from the hsp complex,
the nuclear localization signals within the ligand-
binding domain are ``unmasked'' and transloca-
tion of the receptor takes place (Akner et al.,
1995). At the present time it has not been con-
clusively shown if any component of the hsp com-
plex remains bound to the receptor during
shuttling to the nucleus (Bamberger et al., 1996).
The cytoskeleton may be involved in the translo-
cation of the GR in some manner since hsp 90 is
thought to be associated with both microtubules
and micro®laments (Czar et al., 1996). In ad-
dition, the activated GR forms a dimer after dis-
sociation from the heterocomplex but prior to
interacting with discrete regions on the chromatin
(Fig. 7).
At one time the GR complex, in the absence of
the ligand, was considered to be static and to be
localized primarily in the cytoplasm of most cells.
Recent evidence indicates that this may not be
entirely correct. The GR complex may undergo
bi-directional shuttling between the cytoplasm
and the nucleus (Pratt, 1993; Madan and
DeFranco, 1993). In fact Bamberger et al. (1996)
have suggested that the exact composition of the
glucocorticoid receptor heterocomplex may deter-
mine the predominant direction of movement.
Further evidence for the cytoplasmic to nucleus
translocation of the activated glucocorticoid
receptor comes from experiments with green ¯u-
orescent protein (GFP) labeled GR (Carey et al.,
1996). The authors demonstrated that after trans-
fection into baby hamster kidney cells of the
GFP-GR complex, the addition of DEX caused a
rapid translocation (e.g. 5 min) of the chimera
protein from the cytosol into the nucleus.
644 R. J. Wordinger and A. F. Clark

Fig. 7. Schematic representation of the Type 1 (e.g. classic) glucocorticoid response within a target cell. The glucocorticoid
(GC) ligand passes through the cell membrane, binds to the cytoplasmic glucocorticoid receptor (GR) heterocomplex
causing liberation of the heat shock protein chaperones (90, 70, and 56). The activated GR-ligand complex is translocated
to the nucleus and forms a homodimer prior to binding to speci®c glucocorticoid response elements (GRE) located within
the promoter region of target genes. This interaction, coupled with the presence of speci®c transcription factors (TF),
leads to altered trancription of the target gene. The ultimate biological response is the result of the translation of a speci®c
protein.

5.4. Receptor Phosphorylation tors which contained mutations of hormone indu-

Lastly, the e€ect of phosphorylation on recep-
tor function is currently not known but may play
a crucial role in regulating receptor levels and
control receptor functions. Webster et al. (1997)
reported that the mouse GR phosphorylation sta-
tus in¯uences multiple functions of the receptor
protein. For example, the phosphorylation status
had a profound e€ect on the ability to transacti-
vate a promoter containing a glucocorticoid re-
sponse element (GRE) and on the stability of the
glucocorticoid receptor protein. However, Jewell
et al. (1995) created mouse glucocorticoid recep-
cible phosphorylation sites. These mutant GR
receptors still exhibited translocation properties
similar to the wild type GR receptors indicating
that the phosphorylation sites did not play a
major role in hormone inducible nuclear translo-
cation.
Krstic et al. (1997) have shown that the gluco-
corticoid receptor is a target for multiple kinases.
Mitogen-activated protein kinase (mapk) and
cyclin-dependent kinase (cdk) can phosphorylate
the rat GR at distinct sites. The kinases could
either positively or negatively regulate receptor
transcriptional activity. The ability to control
 E€ects of glucocorticoids on the trabecular meshwork
receptor transcription activity adds another ad-
ditional level of regulatory input which must be
considered.
6. MECHANISMS OF GLUCOCORTICOID
RECEPTOR ACTION
One of the most distinctive features of the GRa
is its ability to both up-regulate and down-regu-
late gene expression depending on the target gene
and experimental conditions. Bamberger et al.
(1996) have suggested that when the ligand-acti-
vated GRa is translocated to the nucleus it may
act via at least two possible mechanisms. The
type 1 mechanism is characterized by the GRa
interacting with speci®c DNA sequences and rep-
resents the ``classic model'' of GR action. The
type 2 mechanism involves GRa interacting with
other transcription factors such as AP-1 or NF-
kB in the absence of DNA binding. Both type 1
and type 2 mechanisms of glucocorticoid action
are understood to take place within the nucleus.
In addition to the type 1 and type 2 mechanisms,
other potential mechanisms of GR action have
been recently reported and are included in this
section.
6.1. The Type 1 Mechanism
This mechanism is considered the ``classical''
mechanism. In this mechanism, a GRa homodi-
mer binds to speci®c DNA sequences called glu-
cocorticoid response elements (GRE) in the
promoter region of glucocorticoid-responsive
genes (Fig. 7). The binding of the GRa homodi-
mer leads to activation of the target gene. This in-
teraction enhances transcription by RNA
polymerase II. It is also possible that the binding
of the GR to speci®c GREs rearranges the chro-
matin structure and allows other transcription
factors to bind which were unable to bind pre-
viously. The net e€ect of this mechanism is to
enhance transcription of a speci®c mRNA
(Fig. 7).
However, recent information also indicates that
ligand-activated GR may bind to negative gluco-
corticoid response elements (nGRE) which act to
645
cause an inhibition rather than enhancement of
transcription (Drouin et al., 1993). Although the
exact mechanism is not known, the interaction is
not identical to the enhancer mechanism since
three molecules of GR bind to the nGRE rather
than two (Fig. 8). In addition, some nGREs
appear to contain only half a palindromic GRE
thus potentially allowing only one GR moiety to
contact the DNA (Subramaniam et al., 1997).
Several examples of repression of gene transcrip-
tion by GC involving nGREs have been reported
including bovine prolactin (Subramaniam et al.,
1997), mouse gonadotropin-releasing hormone
(Chandran et al., 1996), human glycoprotein hor-
mone alpha subunit (Chatterjee et al., 1991), pro-
opiomelanocortin (Drouin et al., 1993), human
IL-1b (Zhang et al., 1997), human osteocalcin
(Morrison and Eisman, 1993), rat VIP receptor
(Pei, 1996), and human insulin (Goodman et al.,
1996). Negative control via GR binding directly
to DNA adds yet another level of complexity in
our understanding of how GCs function in cells
within the trabecular meshwork.
Several glucocorticoid-responsive genes do not
contain ``classical'' GRE palindromic sequences.
These sites are sometimes referred to as GRE
half-sites or single GRE sites (sGRE) containing
only nucleotide hexamers (e.g. TGTnCC). These
sites have been shown to bind activated glucocor-
ticoid receptors by a variety of methods including
DNase I footprinting, gel mobility shift assays,
protection from DNA methylation, and transfec-
tion of these regulatory sites linked to reporter
genes. There are often multiple sGRE sites in a
single gene. GRs bind less tightly to sGRE sites
compared to the classical palindromic GREs.
Glucocorticoid receptor modi®cation (e.g., level
of phosphorylation) may a€ect the anity of
GRs for the sGRE site. In contrast to a GR
homodimer binding to the palindromic GRE, it
appears that a single activated GR may bind a
sGRE in addition to other hormone-independent
transcription factors. In some cases, such as glu-
cocorticoid regulation of the rat a2a-globulin gene
(Chan et al., 1991), the steroid-induced transcrip-
tion of genes containing sGRE sites is delayed
compared to genes containing classical GREs.
The potential importance and relevance of GRE
half-sites in the TM and glaucoma will be dis-
646 R. J. Wordinger and A. F. Clark

cussed further in Section 9 (GLC1A/MYOC
Gene and Glaucoma).
6.2. The Type 2 Mechanism
The type 2 mechanism o€ers an additional way
for the GR to inhibit rather than activate gene ex-
pression. It is known that GCs can inhibit gene
activation in the absence of nGRE or other GC
binding sites within the promoter regions of tar-
get genes (Jonat et al., 1990; Northrop et al.,
1992; Vacca et al., 1992; Paliogianni et al., 1993).
Thus a di€erent activation sequence must be envi-
sioned when considering the type 2 mechanism.
Current information indicates that inhibition of
gene expression may occur via the inactivation of
other nuclear transcription factors such as acti-
vating protein-1 (AP-1). The AP-1 transcription
factor is composed of dimers of the proteins Jun
and Fos. When the AP-1 complex binds to
speci®c target sequences in the promoter regions
of target genes, gene expression is activated.
However, the association of the GR with AP-1
leads to inhibition of promoter activity in selected
genes, a process also termed transrepression. One
example of this type of inhibition is the transre-
pression of the IL-2 gene (Northrop et al., 1992;
Vacca et al., 1992; Paliogianni et al., 1993). The
potential role(s) of glucocorticoid-AP-1 inter-
actions in glaucoma are considered in Section 8.2.
The GR is also known to interact with other
transcription factors most notably the p65 subu-
nit of NF-kB (Ray and Prefontaine, 1994;
Caldenhoven et al., 1995; Scheinman et al.,
1995a). Not only does the GR physically interact
with the p65 subunit but it also suppresses NF-
kB activity by inducing the IkB inhibitory pro-
tein. The IkB protein traps NF-kB in inactive
cytoplasmic complexes and prevents activation of
speci®c genes (Auphan et al., 1995; Scheinmann
et al., 1995b). The net e€ect of the type 2 mechan-

Fig. 8. Schematic representation of glucocorticoid interaction with a negative glucocorticoid response element (nGRE).
Compared to the mechanism in Figure 7, note that following translocation to the nucleus, an activated GR-ligand trimer
forms and binds to the nGRE of speci®c genes. This binding blocks mRNA transcription and protein translation.
 E€ects of glucocorticoids on the trabecular meshwork
ism is the inhibition of expression of speci®c
genes.
What is the predominant mechanism of GR
mediated gene regulation: (1) activation of GR
binding to positive GREs, (2) repression of GR
binding to nGREs, or (3) transcriptional interfer-
ence by interaction by GR with other transcrip-
tion factors? Recent studies provide further
evidence for the GR's role in transcriptional inter-
ference by interactions of GR with transcriptional
factors independent of GREs. As discussed, the
GR binds to GREs as homodimers. GR dimeriza-
tion involves the D loop of the second zinc-®nger
in the DNA-binding domain of the GR, and a
speci®c mutation in this domain (e.g., A458T)
prevents GR dimerization and binding to GREs.
However, dimerization mutations in the GR
(GRdim) does not prevent suppresion of AP-1
regulated genes (Heck et al., 1994). Recent exper-
iments with homoallelic GRdim/GRdim ``knock-
in'' mice further support these ®ndings (Reichardt
et al., 1998). Unlike GRÿ/GRÿ knock-out mu-
tations which are lethal (Cole et al., 1995), the
knock-in GRdim/GRdim mice remained viable and
healthy within a normal laboratory environment.
These mice were unresposive to several GRE
mediated responses such as TAT (tyrosine amino-
transferase) induction in the liver as well as GC-
mediated killing of thymocytes and thymic invo-
lution. However, these GRdim/GRdim mice still
were able to suppress AP-1 regulated genes such
as collagenase. In addition to transcriptional
interference, it is possible that the GRdim mutants
can still partially regulate GRE containg genes by
non-cooperative binding to multipe GRE half
sites in some promoters (Karin, 1998). These stu-
dies suggest that GC regulation occurs predomi-
nately through mechanisms not requiring classic
GR/GRE gene transcription, at least in the non-
stressed state.
6.3. Glucocorticoid Receptors and Co-Activators
In order to achieve full transcriptional activity,
steroid receptors must associate with steroid
receptor co-activators upon binding to DNA
(Glass et al., 1997; Shibota et al., 1997; Hong et
647
al., 1997). Several co-activators have been ident-
i®ed and characterized which comprise a family
of proteins termed steroid receptor coactivators
(SRCs). Members of the SRC family include
SRC-1, SRC-2 (TIF2), GRIP1 and SRC-3
(Shibota et al., 1997). Members of the SRC
family can bind to the ligand-dependent transacti-
vation site of the ligand binding domain (AF-2)
on glucocorticoid receptors and signi®cantly
enhance the expression of glucocorticoid-depen-
dent target genes. Two of the better characterized
co-activators are SRC-1 (steroid receptor co-acti-
vator-1) and SRC-2 (TIF2) ((transcription inter-
mediary factor-2) or its murine ortholog GRIP1
(glucocorticoid receptor intermediary protein 1)).
Both SRC-1 and SRC-2 (TIF2) are co-activators
of other nuclear receptors in addition to glucocor-
ticoid receptors. These co-activators appear to
form complexes with the activated steroid recep-
tors which may further stabilize the transcrip-
tional machinery thus leading to enhanced
transcription of steroid-activated genes. It is quite
possible that some of the variable e€ects of gluco-
corticoids on the trabecular meshwork are due in
part to the levels of expression of these or other
co-activators. The role co-activators play in the
pathophysiology of the trabecular meshwork war-
rants further study.
6.4. Glucocorticoid Receptor Association with An RNA-
Binding Nuclear Matrix Protein
The structural organization of chromatin and
the integrity of the nucleus is maintained by the
nuclear matrix. Several studies suggest that the
nuclear matrix can interact with chromatin and
transcription factors. In fact DNA replication,
RNA processing, and gene transcription seem to
be connected to the nuclear matrix. In addition,
steroid hormone receptors have been localized to
the nuclear matrix. Thus it was with interest that
Eggert et al. (1997) recently reported that the GR
is associated with an RNA-binding protein in the
nuclear matrix. They demonstrated for the ®rst
time that GR is complexed with hnRNP U, a
matrix protein for which binding to RNA and to
sca€old attachment regions has been previously
648 R. J. Wordinger and A. F. Clark
described (Kiledjian and Dreyfuss, 1992; Romig
et al., 1992). The protein hnRNP U has been
reported to be an abundant nuclear protein and
belongs to a family of proteins known to be
involved in the processing and transport of
mRNA (Dreyfuss et al., 1993). The authors
suggested three possible functions for the associ-
ation of GR with the hnRNP U protein. These
included, (1) an e€ect on RNA processing or
RNA stability, (2) a mechanism for storage of
inactive GR molecules, and (3) GC-induced tran-
scription may indeed take place in the nuclear
matrix. Future studies will be needed to determine
which, if any, of these functions is physiologically
signi®cant and if there is a role in glaucoma.
6.5. Functional Cooperation of the Glucocorticoid
Receptor and Other Signaling Pathways
A recent report indicates that the GR can inter-
act with Stat 5. Stoecklin et al. (1997) used mam-
mary epithelial cells to study the molecular
mechanisms by which prolactin and glucocorti-
coids cooperate in the induction of speci®c milk
proteins. They established that the GR and Stat 5
form a molecular complex which induces tran-
scription of the b-casein gene. They report a
novel function for GR in that GR can act as a
coactivator of Stat 5 which is independent of
both the GR ligand and GR DNA binding
domains. However, the activation function 1
(AF-1) in the N-terminal domain of the GR is
indispensable for cooperation. This study raises
the possibility that there are other GR-Stat inter-
actions which occur. In fact, Stoecklin et al.
(1997) have suggested that in di€erent cells, cyto-
kine-speci®c interactions between Stat factors and
non-Stat transcription factors (e.g., GR) may lead
to di€erent phenotypic e€ects generated by indi-
vidual cytokines. These interactions may also be
altered in pathological conditions leading to a
lack of cooperation or a minimal cytokine re-
sponse for example within the trabecular mesh-
work.
Using di€erential display, Kemppainen and
Behrend (1998) have identi®ed a new Ras super-
family gene (Dexras1) which is induced by DEX
in mouse-derived corticotroph tumor cells (AtT-
20). Expression of the gene has also been ident-
i®ed in mouse heart, brain, liver, and kidney and
could be induced by DEX. Interestingly, the
deduced protein sequence shows regions of hom-
ology with members of the Ras superfamily of
small GTPases. While the function of this gene is
unknown, its wide distribution and rapid induc-
tion by DEX suggests the possibility of a role of
glucocorticoid action in a variety of tissues via
Dexras1. These ®ndings are of great interest
because Ras proteins act as molecular switches
which are active when GTP is bound and inactive
when GDP binds. Classically, Ras proteins are
thought to act to link signals from receptor tyro-
sine kinases to a variety of intracellular processes
controlling such cellular functions as cell prolifer-
ation and di€erentiation. Whether DEX can act
via Dexras1 within the human trabecular mesh-
work is currently not known but o€ers an ad-
ditional potential mechanism by which GCs may
alter TM cell function.
6.6. Glucocorticoid Responsiveness Unrelated to the
Glucocorticoid Response Element (GRE)
Huss et al. (1996) have reported recently that
CYP3A23, a major glucocorticoid-inducible
member of the CYP3A family, is induced by
DEX in a unique manner. In characterizing the
promoter region of the gene, they identi®ed two
binding sites. However, neither site contained a
consensus GRE and gel-shift analysis demon-
strated a lack of GR association. In addition, a
novel steroid signaling pathway has recently been
discovered (Kliewer et al., 1998) in which certain
steroids including DEX, can regulate gene ex-
pression independent of the glucocorticoid recep-
tor. The new orphan nuclear receptor, PXR, has
been shown to selectively bind certain pregnane
steroids, such as pregnenolone and synthetic glu-
cocorticoids (e.g., DEX) with high anity that
leads to the formation of a heterodimer with the
retinoic acid receptor RXR. This complex binds
to two-6 nucleotide direct repeats arranged as a
hormone response element in responsive genes
 E€ects of glucocorticoids on the trabecular meshwork
(e.g., the CYP3A gene family) and activates gene
transcription. It is currently unknown whether
the trabecular meshwork expresses PXR. It may
be possible that one or more of the GC-mediated
e€ects on the TM are due to glucocorticoids
interacting with PXRs in the TM.
7. NON-GENOMIC STEROID EFFECTS
Although the most common ``classical'' steroid
e€ects occur via soluble ligand-activated nuclear
receptors which alter gene expression, there is
also evidence for nongenomic steroid e€ects. In
contrast to the classical steroid action, the nonge-
nomic steroid e€ects are very rapid (occurring
with seconds or minutes) and do not require pro-
tein or RNA synthesis (i.e., they are insensitive to
actinomycin D and cycloheximide). Nongenomic
steroid action occurs at physiological levels of
steroids (nanomolar to subnanomolar concen-
trations) and often involves several second mes-
senger systems including the Na/H antiporter, IP3
metabolism, and/or calcium mobilization.
Examples of nongenomic steroid action include
neurosteroid e€ects on the GABA receptor com-
plex, aldosterone e€ects on the Na/H antiporter
in lymphocytes and vascular smooth muscle cells,
and progesterone e€ects on oocyte maturation
and the spermatozoon acrosome reaction
(Wehling, 1995; Nemere et al., 1993). Saturable
and speci®c membrane binding sites for certain
steroids have been identi®ed, and there is a good
association between binding anity, binding
speci®city, and biological responses. Membrane
binding sites for glucocorticoids (i.e., cortisol and
DEX) have been identi®ed in the rat liver and in
lymphoma cells (Wright and Paine, 1995;
Gametchu et al., 1993). It has been suggested that
the non-genomic, rapid response to steroids may
modulate the classical steroid genome response.
To date, no one has determined whether there
are non-genomic e€ects of glucocorticoids or glu-
cocorticoid metabolises on the trabecular mesh-
work or whether the TM contains membrane
binding sites for corticosteroids. There has been
at least one report of a rapid (within hours)
increase in intraocular pressure in POAG patients
treated systemically with DEX (Weinreb et al.,
649
1985). Whether this IOP elevation was due to the
genomic or nongenomic e€ects of dexamethasone
is currently unknown.
8. THE POTENTIAL ROLE OF THE
GLUCOCORTICOID RECEPTOR COMPLEX
IN GLAUCOMA
8.1. Glucocorticoid Receptor Expression
Since the expression level of glucocorticoid
receptor varies in a tissue speci®c manner, it is
possible that this expression pattern may be sub-
ject to major regulatory variations. One mechan-
ism to regulate the expression of the
glucocorticoid receptor appears to be glucocorti-
coids themselves. Glucocorticoids down-regulate
the expression of the glucocorticoid receptor in
many cell lines and in tissues or cells in intact ani-
mals and healthy humans (Burnstein et al., 1991;
Burnstein and Cidlowski, 1992; Burnstein et al.,
1994; Silva et al., 1994). The negative e€ect of
glucocorticoids on glucocorticoid receptor ex-
pression may represent a short-loop feedback
mechanism which protects tissues from excessive
glucocorticoid levels (Bamberger et al., 1996). It
is possible that trabecular meshwork cells in glau-
coma patients are unable to activate the short-
loop feedback mechanism or alternately the
mechanism may be active but less ecient. In
either case, the GR complex would continue to be
present and trabecular meshwork cells would con-
tinue to respond to glucocorticoids and not be
able to self-regulate GR levels. This would lead to
a hypersensitivity within the trabecular meshwork
and could result in the morphological and physio-
logical changes seen in glaucoma.
8.2. Intracellular Hormone Availability And 11b
b-Hy-
droxysteroid Dehydrogenase
Cortisol is the natural glucocorticoid in man,
and approximately 90±95% of circulating cortisol
is bound to the serum binding proteins transcor-
tin (cortisol binding globulin) and albumin. For
many of the synthetic glucocorticoids, such as
dexamethasone, there is a lower amount of serum
650 R. J. Wordinger and A. F. Clark
protein binding which, along with an increased
receptor anity and resistance to metabolic inac-
tivation, accounts for their greater biological ac-
tivity. Glucocorticoids not complexed with serum
binding proteins permeate the cellular membranes
and bind to soluble intracellular glucocorticoid
receptors. Glucocorticoids which have di€used
through the cell membrane must gain access to
the glucocorticoid receptor complex to exert their
e€ects.
In addition to plasma glucocorticoid steroid
levels, corticosteroid-binding globulin (CBG), and
receptor density, glucocorticoid availability within
a given cell is also dependent on the enzyme 11b-
hydroxysteroid dehydrogenase. This enzyme cata-
lyses the interconversion of physiological gluco-
corticoids with 11-keto metabolites. Two distinct
isozymes (11b-HSD1 and 11b-HSD2) have been
characterised (Whorwood et al., 1995). The 11b-
HSD2 isozyme is NAD-dependent and is local-
ised in mineralocortcoid responsive tissues. This
isozyme rapidly inactivates glucocorticoids thus
rendering these tissues responsive to mineralocor-
ticoids such as aldosterone and unresponsive to
cortisol (Seckl and Chapman, 1997). Of interest
to our discussion is the 11b-HSD1 isozyme which
is a NADPH-dependent oxidoreductase and is
widely distributed in non-mineralcorticoid target
tissues and generates physiological glucocorti-
coids (e.g., corticosterone) from inert 11-ketocor-
ticosteroids (Agarwal et al., 1989). Recently,
Stokes et al. (1998) reported that 11b-HSD1 is
expressed in human trabecular meshwork cells. It
is thus possible that the expression of this isozyme
may alter the sensitivity of the trabecular mesh-
work to glucocorticoids and hence play a role in
steroid hypertension and POAG.
Interestingly, Kralli et al. (1995) recently ident-
i®ed a yeast transporter protein (LEM1) that
actively and speci®cally exports glucocorticoids
from the cell. While not currently identi®ed, a
mammalian homologue(s) may play an important
role in regulating the cellular availability of gluco-
corticoids and thus be involved in glucocorticoid
sensitivity. If the human homologue to LEM1 is
expressed in trabecular meshwork cells and was
defective or somewhat less ecient in glaucoma,
intracellular levels of glucocorticoids would not
be regulated and the trabecular meshwork tissue
would have a greater glucocorticoid sensitivity
and activity.
8.3. Hormone Binding Anity
The potency of the GR as a transcriptional reg-
ulator correlates with its hormone-binding a-
nity. One important function of the GR complex
is to maintain the receptor in a ligand-friendly,
high anity conformation. It is apparent that the
expression level of hsp 90 can in¯uence GR func-
tion. High levels of hsp 90 are found in target tis-
sues that are particularly glucocorticoid sensitive
(Vamvakopoulos, 1993). However, a direct link in
vivo between elevated hsp 90 levels and glucocor-
ticoid function has not been ®rmly established
(Bamberger et al., 1996). In addition, the exact
roles of hsp 70 and hsp 56 are not understood. In
glaucomatous trabecular meshwork tissue, it is
possible that hsp 90 expression is elevated thus
leading to changes in glucocorticoid function.
Elevated levels of hsp 90 would render the human
trabecular meshwork more glucocorticoid sensi-
tive and could lead to multiple changes in tissue
structure.
8.4. Hormone-Induced Conformational Change and
Dissociation from the Hsp Complex
The induction of a conformational change in
the GR is believed to be the most important con-
sequence of ligand binding (Tsai and O'Malley,
1994). Following the conformational change, the
glucocorticoid receptor dissociates from the hsp
complex. Schmidt et al. (1985) reported the exist-
ence of a heat-stable protein which enhanced the
eciency of glucocorticoid-induced glucocorticoid
receptor/hsp dissociation. The cDNA and/or
amino acid sequence of this protein has not yet
been determined. However, elevated expression or
activity of this protein in human trabecular mesh-
work tissue would lead to enhanced glucocorti-
coid-induced glucocorticoid/hsp dissociation and
enhanced availability of the glucocorticoid recep-
tor for translocation to the nucleus and sub-
sequent interaction with nuclear factors.
 E€ects of glucocorticoids on the trabecular meshwork 651

Fig. 9. Expression of the human glucocorticoid receptor (GR) gene is the result of alternative transcriptional splicing to
form two GR speci®c isoforms. The GRa isoform consists of exons 1-9a and translates into a protein of 777 amino acids.
The GRb isoform consists of exons 1-8 plus exon 9b and translates into a protein of 742 amino acids (adapted from
Bamberger et al., 1996).

8.5. The GRb

b Isoform
In 1985, human GR cDNA was cloned and
two homologous receptor isoforms were predicted
(Hollenberg et al., 1985). It was later demon-
strated that alternative splicing is responsible for
generating the isoforms which were termed GRa
and GRb (Bamberger et al., 1995; Oakley et al.,
1996) (Fig. 9). The human GR gene contains 10
exons and both the GRa and GRb isoforms con-
tain the ®rst 8 exons of the gene but the last 2
exons (9a and 9b) are spliced into the respective
mRNA (Fig. 9). The a and b isoforms have the
same ®rst 727 amino acids. However, GRb di€ers
from GRa in the COOH terminus. The last 50
amino acids of the GRa isoform are replaced by
a unique 15 amino acid sequence. Since early stu-
dies indicated that GRb did not bind ligand and
was not capable of activating glucocorticoid sen-
sitive promoters (Hollenberg et al., 1985; Giguere
et al., 1986), most of the e€orts towards under-
standing the molecule mechanisms involved in
GC action were directed to GRa.
Recently however, Bamberger et al. (1995) and
Oakley et al. (1996) raised the possibility that the
GRb isoform has biological signi®cance. They
demonstrated that GRb can antagonize/inhibit
the e€ects of ligand-activated GRa if both iso-
forms are expressed within the same cell. Using
dexamethasone, Oakley et al. (1996) showed that
GRb does not bind dexamethasone, was found
primarily in the nucleus, and in the absence of
GRa was transcriptionally inactive at the level of
the GRE. Bamberger et al. (1997a) also showed
that GRb does not alter the anity of the GRa
for GC ligands. These studies raise the possibility
that the GRb isoform binds to the GRE in the
absence of ligand and acts as an endogenous in-
hibitor of glucocorticoid action by inhibiting the
e€ects of hormone-activated GRa (Fig. 10). In
addition, Bamberger et al. (1997b) recently
demonstrated inhibition of mineralocorticoid ac-
tivity by GRb. Other evidence in favor of a phys-
iological role for GRb in human cells includes the
®ndings that both GRb mRNA transcripts
(Bamberger et al., 1995; Oakley et al., 1996;
Hecht et al., 1997) and GRb protein (de Castro et
al., 1996; Hecht et al., 1997; Oakley et al., 1997)
have been reported to be present in a wide variety
of human cell lines and tissues.
There have been di€erences reported as to the
cellular localization of GRb. Using isoform-
speci®c anti-GRb polyclonal antibodies, Oakley
et al. (1997) reported that GRb was localized to
the nucleus independent of glucocorticoid treat-
ment. However, de Castro et al. (1996) had pre-
viously demonstrated that both GRa and GRb
were distributed in the cytoplasm and the nucleus
in the absence of the hormonal ligand, and also
showed translocation into the nucleus after the
652 R. J. Wordinger and A. F. Clark

addition of dexamethasone. Hecht et al. (1997)
produced a monoclonal antibody raised against
the 15 unique COOH-terminal amino acids of
GRg. Using CLL and HeLa cells, they reported
that the antibody recognized a speci®c immuno-
reactive band in the cell cytosol.
An interesting study recently was published by
Leung et al. (1997) which supports a physiological
role for GRb. The authors reported that GRb
may account for glucocorticoid insensitive
asthma. Using PMMC cells from peripheral
blood of glucocorticoid insensitive asthmatics,
they reported that the GR ligand and DNA bind-
ing defect was due to cytokine-induced expression
of GRb. They concluded that GRb acts as an in-
hibitor of normal GRa as originally suggested by
Bamberger et al. (1995). They also demonstrated
that a combination of IL-2 and IL-4 sustained
the abnormality. They further showed that a com-
bination of the cytokines, but not the individual
cytokine, induced signi®cantly greater GRb ex-
pression in normal cells. The mechanism by
which a combination of IL-2 and IL-4 enhances
the expression of GRb is currently unknown but
raises the possibility that other cytokines and/or
growth factors may act to control expression of
the GRa and GRb isoforms in both normal and
pathological tissues including the trabecular
meshwork.
Preliminary experiments in our laboratory indi-
cate that mRNA for the GRb isoform is
expressed in cultured human TM cells and in TM
tissues (Wordinger and Clark, unpublished obser-
vation). Thus the existence of two human gluco-
corticoid receptor isoforms that may potentially
exert opposite e€ects on cells complicates our
understanding of the glucocorticoid receptor-
mediated transcription complex. In addition,
knowledge of these isoforms demands a re-evalu-
ation of the concept of glucocorticoid sensitivity
in target tissues. For example, it will be important
to determine relative amounts of the glucocorti-
coid receptor isoforms in normal and glaucoma-
tous trabecular meshwork samples and whether
the isoform expression is subject to regulatory
processes (e.g., growth factors, cytokines). If the
human trabecular meshwork is responding as if in
a glucocorticoid hypersensitivity state, it may be
traced to reduced GRb expression or altered ac-
tivity and hence a reduced ability of a tissue to
down-regulate the ligand-bound GRa activity.
Consequently, an abnormally low expression of
GRb or the expression of an altered or defective
GRb isoform would cause tissues to become

Fig. 10. The mechanism of action of the GRb isoform is illustrated. This mechanism suggests that the GRb isoform pre-
vents the activated form of GRa from binding to glucocorticoid response element (GRE) in the promoter region of a tar-
get gene and thus blocks glucocorticoid action.
 E€ects of glucocorticoids on the trabecular meshwork
hypersensitive to the e€ects of glucocorticoids
(Bamberger et al., 1996; Bamberger et al., 1995).
In the case of glaucoma, even a mild form of gen-
eralized glucocorticoid hypersensitivity may lead
to abnormalities in tissues that are more sensitive
to glucocorticoids under physiological conditions.
It is therefore signi®cant that there have been sev-
eral reports of altered glucocorticoid sensitivity in
POAG patients (Becker et al., 1973; Rosenberg
and Levene, 1974; Bigger et al., 1975; Foon et al.,
1977).
Not all evidence supports a physiological role
for GRb in human cells. Hecht et al. (1997) using
isoform-speci®c antibodies against the GRb iso-
form and Western immunoblotting demonstrated
a relatively low endogenous expression. They also
reported via immunoprecipitation that GRb was
bound to hsp 90. The authors could not however,
demonstrate that GRb inhibited the e€ects of
dexamethasone-activated GRa on a glucocorti-
coid-responsive reporter gene. They concluded
that the low expression of GRb and GRb-hsp 90
interaction in the presence of ligand as well as a
lack of inhibition of hormone-activated GRa
challenges the concept that the GRb isoform is a
dominant negative inhibitor of GRa activity
(Bamberger et al., 1995; Oakley et al., 1996).
They speculate that GRb would not have a sig-
ni®cant physiological function at the observed ex-
pression levels. Also, Bamberger et al. (1997c)
have demonstrated that the transrepressive e€ect
of glucocorticoids on the IL-2 gene is exclusively
mediated by GRa in Jurkat cells. The e€ect was
not antagonized by GRb. They suggested that a
cell type-speci®c pattern of GRb-mediated anti-
glucocorticoid activity may be present. Clearly,
the physiological importance of and the mechan-
ism(s) by which GRb can have anti-transactivat-
ing e€ects in certain cells but not others, remains
to be determined. Also, while the GRb isoform is
expressed in human cells and tissues, it may not
be universally expressed across species. Otto et al.
(1997) reported the absence of GRb expression in
mice.
653
8.6. Alternate Isoform of the Estrogen Receptor (ERR2)
Recently Trapp and Holsboer (1996) have
identi®ed another endogenous receptor which acts
as a cell-speci®c inhibitor of glucocorticoid recep-
tor-mediated gene expression. The ERR2 receptor
was originally characterized as a nuclear orphan
receptor which contained a high degree of hom-
ology to the human estrogen receptor (ERR1).
Trapp and Holsboer (1996) demonstrated that
ERR2 functions as a potent repressor of gluco-
corticoid receptor-mediated transcriptional ac-
tivity. Similar to GRb, ERR2 failed to inhibit
without co-expression of GRa. But unlike GRb,
ERR2 does not compete for GRE binding in the
nucleus. The authors also showed that the inhibi-
tory e€ect of ERR2 was not due to the formation
of a ERR2-GRa complex. The exact mechanism
of ERR2-mediated suppression remains un-
known, but it has been suggested that ERR2
competes with GRa for essential transcription
factors.
9. THE POTENTIAL ROLE OF NUCLEAR
TRANSCRIPTION FACTORS IN
GLAUCOMA
9.1. Activating Protein-1 (AP-1)
Not all actions of glucocorticoids are necess-
arily mediated by the activated glucocorticoid
receptor binding to glucocorticoid response el-
ements (GREs) in the nucleus. The activated,
ligand bound form of the glucocorticoid receptor
is also capable of binding other transcription fac-
tors and thereby inhibiting their function. A good
example of this type of interaction is the binding
of GR to the AP-1 transcription factor (Jonat et
al., 1990; Yang-Yen et al., 1990; Schule et al.,
1990). The AP-1 complex generally consists of c-
fos/c-jun heterodimers which bind to the TPA re-
sponse element (TRE) of a number of important
proliferation and pro-in¯ammatory genes. A
number of di€erent growth factors and phorbol
esters stimulate TRE genes via the AP-1 tran-
scription complex. The activated GR can bind to
the AP-1 complex and inhibit its binding to TRE
promoters, thereby preventing the expression of
654 R. J. Wordinger and A. F. Clark
Fig. 11. Interaction of the activated glucocorticoid recep-
tor-ligand complex (GRa*) with NF-kB and AP-1 tran-
scription factors. Glucocorticoids are known to increase
the synthesis of IkB which binds to NF-kB thus prevent-
ing cellular action and blocking transcription of speci®c
genes such as growth factors (GFs), cell adhesion mol-
ecules (CAMs), and cytokines. In addition, GRa* can
bind directly to AP-1 (cfos/cjun) thus preventing AP-1
from binding to the transcriptional element (TRE) on tar-
get genes such as collagenase.
TRE genes (Fig. 11). This is one proposed mech-
anism for the anti-in¯ammatory activity of gluco-
corticoids which involves the inhibition of
collagenase expression during scar formation and
in arthritis.
We hypothesize that a similar mechanism may
be occurring in the trabecular meshwork. The
activated glucocorticoid receptor may directly
bind to the AP-1 protein complex and thereby
inhibit the expression of collagenase and other
matrix metalloproteinases. It has been demon-
strated that certain factors known to stimulate
TRE genes (IL-1, serum, and phorbol esters) are
able to stimulate MMP and tPA expression in
cultured TM cells (Alexander et al., 1991). In ad-
dition, the potent glucocorticoid dexamethasone
inhibits TM cell MMP and tPA expression
(Samples et al., 1993, Snyder et al., 1993). This
may be one mechanism which is responsible for
the increased extracellular matrix material depos-
ited in the TM during corticosteroid-induced ocu-
lar hypertension and glaucoma (Clark, 1995a;
Johnson et al., 1990; Johnson et al., 1997; Rohen
et al., 1973; Rohen, 1983).
9.2. NF-k
kB/Ik
kB
A second important transcription factor
a€ected by glucocorticoids is NF-kB. NF-kB is
actually a family of transcriptional regulatory
dimeric proteins related to the rel oncogene which
control the expression of a number of genes for
cytokines, growth factors, adhesion molecules,
acute phase proteins, transcriptional factors, and
other biological messengers. NF-kB regulated
genes are activated by a wide variety of stimuli
including cytokines, mitogens, viruses, as well as
physical stress and oxidative stress (Baldwin,
1996; Siebenlist et al., 1994). Cells respond very
rapidly to these stimuli because NF-kB is pre-
made and complexed in the cytosol with an in-
hibitory protein IkB. Upon stimulation of the
cell, IkB becomes phosphorylated which marks
IkB for degradation and frees NF-kB to translo-
cate to the nucleus and activate NF-kB regulated
genes. Like NF-kB, there are multiple forms of
IkB.
Glucocorticoids appear to inhibit NF-kB tran-
scription in several di€erent ways. The activated
form of the glucocorticoid receptor can directly
bind to the NF-kB protein complex, prevent the
complex from being translocated into the nucleus,
and thereby inhibit activation of NF-kB regulated
genes (Ray and Prefontaine, 1994; Scheinman et
al., 1995a). More importantly, glucocorticoids
also have been demonstrated to activate IkB tran-
scription, and increased levels of IkB bind NF-kB
and hold this complex in the cytosol (Scheinmann
et al., 1995b; Auphan et al., 1995). Many of the
anti-in¯ammatory and immunosuppressive e€ects
of glucocorticoids can be explained by this latter
discovery.
 E€ects of glucocorticoids on the trabecular meshwork
It is quite possible that at least some of the
e€ects of glucocorticoids on the TM are mediated
through inhibition of the expression of NF-kB
regulated genes. Exposure of cultured human TM
cells to dexamethasone appears to down-regulate
the expression of speci®c cell adhesion molecules
(Dickerson, Steely and Clark, unpublished obser-
vations) which are normally regulated by NF-kB.
This may account for the apparent loss of cells
from the trabecular meshwork in corticosteroid
glaucoma (Clark, 1995a; Rohen et al., 1973). In
addition, the expression of many growth factors
is regulated by NF-kB, and glucocorticoid treat-
ment may inhibit the expression of one or more
factors which are essential for proper TM cell
function. This hypothesis is supported by prelimi-
nary evidence that suggests that the expression of
mRNAs for speci®c growth factors in cultured
TM cells is suppressed by DEX treatment
(Wordinger et al., unpublished observation).
10. THE GLC1A/MYOC GENE AND
GLAUCOMA
In an attempt to identify proteins involved in
the generation of ocular hypertension, several lab-
oratories have examined the altered protein pro-
®les of trabecular meshwork cells cultured in the
presence or absence of glucocorticoids (Polansky
et al., 1991, 1994; Tripathi et al., 1990; Partridge
et al., 1989; Kawase et al., 1994). There appears
to be a major induction of a 55 kDa cellular pro-
tein in both cultured human (Polansky et al.,
1991, 1994, 1997) and porcine (Tripathi et al.,
1990) trabecular meshwork cells treated with glu-
cocorticoids for 2±3 weeks as demonstrated by
SDS- and 2-dimensional polyacrylamide gel elec-
trophoresis. It has been reported that this same
protein is secreted into the culture media as a 64±
68 kDa glycoprotein (Polansky et al., 1994). This
protein has been variably labeled GIP 55
(Polansky et al., 1991, 1994, 1997), Cort-GP
(Tripathi et al., 1990) or TIGR (trabecular mesh-
work inducible glucocorticoid response, Chen et
al., 1993; Polansky et al., 1997; Nguyen et al.,
1998). The gene for the protein may be regulated
secondarily by glucocorticoids because its induc-
tion is not immediate and generally takes 7±10
655
days of glucocorticoid exposure. The dose-re-
sponse for expression of the protein is also some-
what unusual. Whereas the anity (Kd) of the
human trabecular meshwork glucocorticoid
receptor for the potent glucocorticoid dexametha-
sone has been reported to be 3 nM (Weinreb et
al., 1981; Clark et al., 1994), the EC50 for TIGR
induction appears to be approximately 10-fold
higher at 30 nM (Polansky et al., 1991, 1994).
Although the biological function of this TM
protein is currently not known, it has been
suggested to be associated with steroid-induced
ocular hypertension for the following reasons: (1)
it is induced and expressed in TM cells exposed
to glucocorticoids, (2) the delayed time of induc-
tion closely matches the propensity of topical
ocular glucocorticoid administration to raise
intraocular pressure, and (3) there appears to be a
correlation between expression of this protein
with doses of glucocorticoids required to elevate
intraocular pressure (Polansky et al., 1991, 1994).
Further support for the involvement of this TM
protein in steroid-induced ocular hypertension
comes from studies showing its DEX-induced ex-
pression in the TM of ocular hypertensive per-
fusion cultured human eyes and in the TM of
monkeys treated daily for one year with cortisol
acetate (Clark et al., submitted for publication).
A number of laboratories have been evaluating
glucocorticoid e€ects on the trabecular meshwork
as a model to better understand primary open
angle glaucoma. This approach has been sup-
ported further by the recent discovery of the ®rst
glaucoma gene (GLC1A) which is responsible for
autosomal dominant juvenile glaucoma, a rare
form of POAG (Stone et al., 1997). Several candi-
date genes for this locus (GLC1A) were exam-
ined, and the glucocorticoid-induced TM
response (TIGR) gene mapped to the center of
the GLC1A locus on chromosome 1q24 (Stone et
al., 1997). These two genes have been found to be
identical (Ortego et al., 1997; Fingert et al., 1998;
Nguyen et al., 1998). A number of mutations in
this gene have been identi®ed as being involved
with heritable forms of juvenile as well as adult
onset POAG (Stone et al., 1997; Adam et al.,
1997; Alward et al., 1998). Approximately 5% of
patients with the most common form of glau-
coma, adult-onset POAG, have mutations in the
656 R. J. Wordinger and A. F. Clark
GLC1A gene (Stone et al., 1997; Alward et al.,
1998). The GLC1A gene is not uniquely expressed
in the trabecular meshwork. This gene also is
expressed in a ciliary epithelial cell cDNA library
(Escribano et al., 1995) and is identical to the
myocilin gene (MYOC) recently isolated and
characterised from a human retinal cDNA library
(Kubota et al., 1997). The GLC1A (MYOC) gene
has signi®cant sequence homology to non-con-
ventional myosin and olfactomedin and has been
suggested to be a cytoskeletal associated protein
in retinal photoreceptor cells (Kubota et al.,
1997). Interestingly, the vast majority of the
GLC1A mutations cluster to the third exon which
encodes the portion of myocilin which is homolo-
gous to olfactomedin.
The original reports of myocilin expression in
the trabecular meshwork indicated that this pro-
tein was a secreted glycoprotein (Polansky et al.,
1991, 1994, 1997; Nguyen et al., 1998). However,
recent work on the localisation of myocilin in cul-
tured trabecular meshwork cells indicates that it
also is expressed intracellularly in punctate depos-
its surrounding the nucleus (Fig. 12) (Clark et al.,
1998) and is associated with intracellular vesicles
(Stamer et al., 1998). Myocilin also appears to co-
localize with the microtubule motor protein, kine-
sin (Clark et al., unpublished observations). It is
interesting to speculate that myocilin may be
involved with or responsible for the increased for-
mation of intracellular vesicles seen in DEX-trea-
ted cultured human trabecular meshwork cells
(Wilson et al., 1993), and ultrastructural localis-
ation studies are currently being done to deter-
mine if this is correct. Immunolocalization studies
of myocilin in trabecular meshwork tissue from
normal and glaucomatous individuals show a
punctate, intracellular distribution, and its rela-
tive expression appears to increase in glaucoma-
tous trabecular tissue (Lutjen-Drecoll et al.,
1998).
Recent studies have revealed the genomic struc-
ture of myocilin (Kuboto et al., 1998; Nguyen et
al., 1998; Fingert et al., 1998). The myocilin gene
contains three exons and is localized on chromo-
some 1q24. There are a number of interesting
regulatory sites upstream of the myocilin gene,
several of which appear to be glucocorticoid regu-
latory elements. De®nitive identi®cation of these
sites as authentic GREs will require additional
studies such as DNA footprinting or generation
of speci®c reporter gene constructs. The ®rst exon
of myocilin shares sequence homology with non-
muscle myosin and has a consensus signal
sequence as well as a leucine zipper. The sequence
of the third exon is homologous to olfactomedin,
a secreted glycoprotein found in the extracellular
compartment of olfactory tissues. There appears
to be only a single myocilin gene and studies to
date suggest that myocilin is expressed as a single
transcriptional mRNA product of 2.5 kb.
Several isoforms of myocilin are expressed in
cultured trabecular meshwork cells. Two to four
immunoreactive protein spots of myocilin (Mr
56±59 kDa and pl 5.15±5.30) are seen in high res-
olution 2D-polyacrylamide gels of proteins
extracted from DEX-treated TM cells (Clark et
al., 1998). Recombinant myocilin is expressed in
insect cells as an intact 504 amino acid protein as
well as a protein in which its signal sequence is
cleaved (Nguyen et al., 1998). In addition, a lar-
ger 66 kDa isoform is seen secreted into the
media of DEX-treated cultured human TM cells
which is immunoreactive with anti-myocilin anti-
bodies (Nguyen et al., 1998). It is suggested that
this larger isoform of myocilin is due to post-
translational glycosylation because the myocilin
gene contains a number of consensus glycosyla-
tion sites. Additional research is needed to chemi-
cally characterize the di€erent isoforms of
myocilin proteins.
The role myocilin may play in regulating IOP
may be more complicated than initially appears.
For example, recent evidence suggests that the
increased expression of myocilin in DEX-treated
TM cells is not responsible for increased out¯ow
resistance (Underwood et al., 1998). As men-
tioned in Section 4.3.7, there is a decrease in
hydraulic conductivity in DEX-treated TM cells
grown on ®lters. DEX treatment also increases
myocilin expression in the system. However, con-
conmitant treatment with myocilin antisense oli-
gonucleotides inhibits myocilin expression but
does not alter the DEX-induced decrease in
hydraulic ¯ow (Underwood et al., 1998).
 E€ects of glucocorticoids on the trabecular meshwork 657

Fig. 12. Immuno¯uorescent localisation of myocilin (GLC1A) within cultured human trabecular meshwork cells.
(A) Myocilin is expressed in discrete vesicle-like structures surrounding the nucleus. (B) Myocilin (Texas Red) is expressed
within the CLAN geodesic dome-like micro®laments (Oregon Green) of DEX treated trabecular meshwork cells.

11. FUTURE RESEARCH TRENDS 11.1. Glucocorticoid Responsiveness

Although a great deal of research has been
done on the e€ects of glucocorticoids on the tra-
becular meshwork and the role glucocorticoids
play in ocular hypertension, there are still a num-
ber of important issues to resolve and questions
to be answered. We suggest that the following
areas would be of critical importance for future
studies.
Why are some individuals classi®ed as steroid
responders and why are almost all glaucoma
patients steroid responders? In order for a tissue
to respond to glucocorticoids (a) glucocorticoids
must be present within the target cell at sucient
levels to elicit a response, (b) the glucocorticoid
receptor must be expressed at physiological levels
with speci®c heat shock protein chaperones, and
(c) a target gene must be active and not perma-
658 R. J. Wordinger and A. F. Clark
nently silenced. If one of these features is missing,
defective, or inecient, the system as a whole
cannot be activated or will be activated at a
reduced level. Conversely, hypersensitivity to glu-
cocorticoids (e.g., steroid responders) may result
from system components that are up-regulated or
from naturally occurring control mechanisms that
are faulty.
What aspects of the system can be examined
and/or controlled within trabecular meshwork
cells of POAG patients? One area of study should
be heat shock protein expression. Is the ex-
pression altered in POAG? For example do ster-
oid responders express higher levels of hsp90 or
other components of the glucocorticoid receptor
complex? High levels of hsp90 have been reported
in target tissues that are particularly glucocorti-
coid sensitive. Is this the case in the trabecular
meshwork? Is there a di€erence in the binding of
the glucocorticoid ligand to the receptor complex
and/or translocation of the ligand-receptor
complex to the nucleus? What co-activators for
glucocorticoid action are normally present and
functioning within the trabecular meshwork and
are these proteins potential control mechanisms
which can be manipulated?
11.2. GR Isoforms
Is the GRb isoform or ERR2 expressed in tra-
becular meshwork cells and do they play a role in
responsiveness to glucocorticoids? A number of
important issues need to be studied with regard
to the physiological role of the GR isoforms in
general and their signi®cance in glaucoma. A
question remains as to whether the GRa and
GRb isoforms may be expressed in di€erent
ratios in di€erent cells and in normal or patho-
logical conditions. There is con¯icting data as to
the role of the GRb isoform. It may be possible
that the inhibitory e€ect of GRb-mediated repres-
sion of GRa is not a universal phenomena in all
human cells but is regulated depending on the cell
context at any given time.
It is also quite likely that the molecular inter-
actions between GRa and GRb as well as the in-
teractions with hsp 90 or other hsp is much more
complex than previously recognized. We are in
need, for example, of studies to determine if
GRa/GRb heterodimers are formed in normal
human cells or under pathological conditions.
Also, if these heterodimers are shown to exist, are
they of physiological importance and what might
be their role at the level of the GRE sequence?
The potential also exists, as demonstrated with
the mineralocorticoid receptor (Bamberger et al.,
1996), that the GRb isoforms may interact with
other steroid receptors and/or other proteins and
transcription factors (e.g., AP-1, NF-kB). These
types of interaction would add an additional level
of complexity to our understanding of the mol-
ecular mechanisms involved in GC response.
Finally we must recognize and acknowledge
that the interaction(s) between GRa and GRb
may di€er depending on cellular context. For
example, there may be an alteration in the ratio
of GRa/GRb in di€erent developmental, di€eren-
tiation or pathological circumstances. The cell
context may dictate an alteration in glucocorti-
coid response. Also the report by Leung et al.,
1997 raises the possibility that glucocorticoid
insensitivity is related to overexpression of GRb.
It is likewise possible to speculate that glucocorti-
coid hypersensitivity might also be related to the
ratio or molecular interactions of the isoforms.
Lastly, Leung et al., 1997 raises the possibility
that cytokine and growth factors may also in¯u-
ence the speci®c expression of the GR isoform
and hence the level of cellular response to GC
ligands.
11.3. Gene Regulation
Upon translocation to the nucleus the gluco-
corticoid-receptor dimerizes and binds to gluco-
corticoid response elements within the promoter
regions of target genes. In addition, we now
know that single glucocorticoid response elements
(sGRE) exist and can be functional. Are sGREs
present in glucocorticoid target genes within the
trabecular meshwork and what role might they
perform? We also know that negative glucocorti-
coid response elements (nGRE) exist and can
function to down-regulate or inhibit target genes.
Which target genes in the trabecular meshwork
express nGREs and are inhibitory from a func-
 E€ects of glucocorticoids on the trabecular meshwork
tional standpoint? Examination of sGREs and
nGREs will expand our understanding of the nor-
mal control mechanisms that are active within the
trabecular meshwork.
In addition to the type 1 and type 2 mechan-
isms, are there additional novel mechanisms avail-
able within the trabecular meshwork cell to
control and/or modify glucocorticoid action? If
novel mechanisms exist are they physiologically
signi®cant? For example do glucocorticoids as-
sociate with RNA-binding matrix protein within
the nucleus of the trabecular meshwork cell? Is
this association signi®cant? Could this be an ad-
ditional mechanism by which trabecular mesh-
work cells in¯uence RNA processing or RNA
stability and hence their ability to respond to glu-
cocorticoids? Would novel mechanisms be pre-
sent, absent, or modi®ed within the POAG
trabecular meshwork?
11.4. Functional Cooperation Between Signaling
Pathways
Another exciting avenue for future research lies
in the functional cooperation of the glucocorti-
coid receptor with other signaling pathways.
There is now information indicating ``cross-talk''
can occur between steroid signaling pathways and
growth factor/cytokine signaling pathways which
utilize tyrosine kinases or serine-threonine
kinases. Does functional cooperation between sig-
naling pathways exist within the human trabecu-
lar meshwork? It is known that the aqueous
humor contains numerous growth factors
(Tripathi et al., 1991; Tripathi et al., 1994). We
have shown that human trabecular meshwork
cells express numerous growth factor receptors
(Wordinger et al., 1998) and also produce growth
factors (Wordinger et al., 1996) which may be uti-
lized via autocrine/paracrine signaling pathways
(Wordinger et al., 1998). Which potential growth
factors/cytokines should be examined to deter-
mine if functional cooperation exists with the glu-
cocorticoid receptor? Is it possible that steroid
responders or glucocorticoid hypersensitivity is a
re¯ection of functional cooperation between sig-
naling pathways or that secondary signaling path-
ways have been activated?
659
Can glucocorticoids act in the human trabecu-
lar meshwork via the newly identi®ed Dexras1
gene? Since Ras proteins are known to act as
molecular switches, especially linking signals eli-
cited from growth factors, can glucocorticoids
activate Ras pathways within the trabecular
meshwork? What physiological role would the
Dexras1 gene play within the trabecular mesh-
work with regards to normal control mechan-
isms?
11.5. Trabecular Meshwork Cytoskeleton and
Extracellular Matrix
Are CLANs (cross-linked actin networks) pre-
sent in TM tissue and do they play a direct role
in ocular hypertension? Is the glucocorticoid-
mediated extracellular matrix deposition in the
trabecular meshwork due to increased ECM syn-
thesis, decreased degradation or both? What is
the composition of the ECM deposits? Are the
ECM deposits abnormally assembled or merely
an over abundance of normal ECM material?
11.6. Steroid Inducible GLC1A Gene
What is the function of the steroid inducible
GLC1A glaucoma gene, where is it located in the
TM, and what is its relevance to other forms of
glaucoma? These discoveries have opened a very
exciting area of research and demonstrate that at
least one subset of glaucoma involves genetic
defects in the trabecular meshwork. We hope to
soon discover: What is the function of the
GLC1A (MYOC) protein? How does increased
expression (e.g., glucocorticoid induction) or mu-
tations in GLC1A cause elevated intraocular
pressure? How is GLC1A di€erentially regulated
by glucocorticoids since only a subset of people
treated with glucocorticoids develops ocular
hypertension?
11.7. Growth Factors and Growth Factor Receptors
Growth factors present in the aqueous humor
maintain the normal function of the structures
660 R. J. Wordinger and A. F. Clark
that border the anterior chamber of the eye
including the trabecular meshwork (Tripathi et
al., 1991; Tripathi et al., 1994). However, the
exact role growth factors play in the cell biology
of the trabecular meshwork has not been studied
extensively. Human trabecular meshwork cells are
capable of responding to numerous growth fac-
tors since they express functional growth factor
receptors (Wordinger et al., 1998) and also
express alternate spliced isoforms of growth fac-
tor receptors (Wordinger et al., 1999). It is thus
possible that growth factors presented to the tra-
becular meshwork cells via the aqueous humor
(paracrine) or produced locally via autocrine
mechanisms may maintain the normal structure
and function of the trabecular meshwork. Any
condition or agent(s) which alters the normal ex-
pression of growth factors or growth factor recep-
tors by trabecular meshwork cells, may lead to an
abnormal microenvironment within the tissue and
pathological alterations.
Can glucocorticoids e€ect trabecular meshwork
growth factor and growth factor receptor ex-
pression and hence play a role in ocular hyperten-
sion? Are there potential links between growth
factors and growth factor receptors and POAG?
This is an important area for future research
since previous reports in other tissues have indi-
cated that glucocorticoids can indeed e€ect ex-
pression of growth factors.
12. CONCLUSIONS
This review has concentrated on the e€ects of
glucocorticoids on the human trabecular mesh-
work. Both in vivo and in vitro studies suggest an
association of glucocorticoids with primary open
angle glaucoma. Glucocorticoids alter trabecular
meshwork cell morphology and gene expression
of speci®c cellular components. Of importance to
the generation of ocular hypertension and glau-
coma, glucocorticoids e€ect cell cytoskeletal el-
ements, cell adhesion molecules, and constituents
of the extracellular matrix.
The interaction of glucocorticoids with the glu-
cocorticoid receptor is complex. Speci®c mechan-
isms involve the translocation of the activated
glucocorticoid receptor to the nucleus and GREs,
sGREs, and nGREs as well as co-activators to
elicit gene expression. Novel mechanisms of glu-
cocorticoid action in addtion to classic mechan-
isms may be important within the trabecular
meshwork. Nuclear transcription factors associ-
ated with the GRE complex also may be involved
in glaucoma. The GLC1A gene locus and the ex-
pression of myocilin in the trabecular meshwork
are exciting new areas of discovery that will help
to dissect the pathway(s) regulating aqueous
humor out¯ow.
Finally we have suggested new areas of future
research which we believe will advance our under-
standing of the role glucocorticoids play in glau-
coma. We should be encouraged by the progress
that has been made in this area but much work
remains to be accomplished. Only then will we
fully understand the cell and molecular aspects of
glaucoma and be able to initiate new and innova-
tive therapeutic interventions.
AcknowledgementsÐThe authors would like to acknowledge
and thank the following organizations for their ®nancial
support of our research e€orts: The National Glaucoma
Program, a program of the American Health Assistance
Foundation, Rockville, MD. (Grants NGR-96413 and NGR-
98412), The Glaucoma Research Foundation, San Francisco,
CA, and Alcon Laboratories, Fort Worth, TX. We also wish
to acknowledge and thank Mr. Alfredo Portales Jr. for his
medial illustrations which have greatly enhanced this
manuscript. In addition we thank Dr. Mitchell D. McCartney
for kindly providing Fig. 1.
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