Molecular and Cellular Endocrinology 246 (2006) 142–146

Non-genomic glucocorticoid effects to provide

the basis for new drug developments
In-Ho Song, Frank Buttgereit ∗
Department of Rheumatology and Clinical Immunology, Charité University Hospital, Schumannstrasse 20/21, 10117 Berlin, Germany

Abstract
Glucocorticoids act via genomic and non-genomic actions. The genomic glucocorticoid actions are well known and new details on processes of
transactivation and transrepression have been reported recently. Here we describe the current knowledge on non-genomic glucocorticoid actions
and discuss why these actions are considered to be of therapeutic relevance.
It is assumed that rapid non-genomic glucocorticoid effects are mediated by three different mechanisms: (1) physicochemical interactions with
cellular membranes (non-specific non-genomic effects); (2) membrane-bound glucocorticoid receptor (mGCR)-mediated non-genomic effects;
and (3) cytosolic glucocorticoid receptor (cGCR)-mediated non-genomic effects. With regard to the first mechanism, we discuss here lazaroids and
the novel development of drug targeting with liposomes as the carrier system for glucocorticoids. The clinical use of the latter two mechanisms is
still speculative, but intriguing ideas are being discussed in this regard.
© 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Glucocorticoids; Methylprednisolone; Lazaroids

1. Introduction 1.1. Physicochemical interactions with cellular membranes

The mechanisms of glucocorticoid actions can be divided into
genomic and non-genomic effects. Genomic effects are known
to be mediated by transactivation and transrepression processes.
These mechanisms have been recently reviewed more in detail
(Schacke et al., 2004; Perretti et al., 2003; Song et al., 2005).
Here we discuss the therapeutic relevance of rapid non-
genomic glucocorticoid effects which are assumed to be medi-
ated by three mechanisms: (1) physicochemical interactions
with cellular membranes (non-specific non-genomic effects)
(Buttgereit and Scheffold, 2002; Buttgereit et al., 2004);
(2) cytosolic glucocorticoid receptor (cGCR)-mediated non-
genomic effects (Croxtall et al., 2000); and (3) membrane-bound
glucocorticoid receptor (mGCR)-mediated non-genomic effects
(Bartholome et al., 2004). The therapeutic relevance of non-
genomic glucocorticoid actions is an issue of ongoing discus-
sion. For example, very recently, beneficial rapid non-genomic
glucocorticoid effects have been described for the first time in
vivo in the treatment of seasonal allergic rhinitis (Tillmann et
al., 2004).
∗ Corresponding author. Tel.: +49 30 450 513125; fax: +49 30 450 513917.
E-mail address: frank.buttgereit@charite.de (F. Buttgereit).
(non-specific non-genomic effects)
Rapid glucocorticoid actions (which occur within seconds)
are being considered as a result of physicochemical interactions
with cellular membranes. How can we explain these non-specific
non-genomic effects? Currently, the following hypothesis is
favoured: Glucocorticoid molecules intercalate at high concen-
trations in cellular membranes (plasma and mitochondrial mem-
branes) which alter cell functions by influencing cation transport
through the plasma membrane and by increasing the proton leak
of the mitochondria. The impaired cation cycling across and
the compromised ATP production via oxidative phosphoryla-
tion are considered to result in immunosuppressive effects and
to a reduced activity of inflammatory processes (Buttgereit and
Scheffold, 2002; Buttgereit et al., 2004). In the following sec-
tion, we would like to explain more in detail the experimental
results that have led to this theory.
The background to explain the rapid non-specific non-
genomic glucocorticoid effects is provided by considerations of
the cellular energy metabolism. Every organism and every cell
needs metabolic energy whereby ATP is the major source. Also
immune cells produce and consume certain amounts of ATP for
their housekeeping activities and for specific immune functions.
ATP-dependent immune functions include cytokinesis, migra-
tion, phagocytosis, antigen processing and antigen presentation,

0303-7207/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.mce.2005.11.012
 I.-H. Song, F. Buttgereit / Molecular and Cellular Endocrinology 246 (2006) 142–146 143

signalling and effector functions such as the synthesis of anti-
bodies, cytotoxicity and regulatory functions (Buttgereit et al.,
2000). The major sources for ATP production are glycolysis and
oxidative phosphorylation. The most important ATP-consuming
pathways are identified to be the transport of cations and syn-
thesis of macromolecules (Buttgereit et al., 2000). In the case
of energy deficit, the functions of immune cells are known to
be impaired (Meldrum et al., 1994; Karlsson and Nassberger,
1992; Sanchez-Alcazar et al., 1995).
Given this background, we have shown a clear hierarchy
of energy-consuming pathways in case of reduced energy sup-
ply: Pathways of macromolecule biosynthesis (protein synthesis
and RNA/DNA synthesis) are most sensitive to energy restric-
tion whereas cation transport ATPases are much less affected.
The mitochondrial proton leak is least sensitive to enery sup-
ply. These results were obtained by quantitating main ATP-
consuming pathways under progressively restricted mitochon-
drial ATP production by using myxothiazol, a specific inhibitor
of the electron transport chain. Obviously, processes not essen-
tial for the immediate needs of the cell will be given up before
those that are more critical for ionic integrity (Buttgereit and
Brand, 1995; Buttgereit et al., 1999).
Why are these experimental results important to under-
stand rapid glucocorticoid effects? If immune cells are treated
with high, but clinically relevant concentrations of methylpred-
nisolone (the glucocorticoid most commonly used for high-
dose therapy), then the following effects on bioenergetics
are observed within seconds (Buttgereit and Scheffold, 2002;
Buttgereit et al., 1997, 2004):
1. The glucocorticoid instantly inhibits respiration of Con
A-stimulated thymocytes and human immune cells (Fig. 1)
at concentrations that leave quiescent cells unaffec-
ted.
2. Methylprednisolone not only reverses but also prevents the
Con A-effect on respiration in a dose dependent manner.
3. Con A is known to produce a dramatic increase of cytoplas-
mic calcium concentration. This effect is clearly reduced in
the presence of therapeutically relevant drug concentrations
or even abolished at suprapharmacological doses.
4. Methylprednisolone inhibits calcium and sodium cycling
across the plasma membrane but has little effect on pro-
tein synthesis. The inhibition of cation cycling in Con
A-stimulated thymocytes by the glucocorticoid is caused
by direct effects and not by a reduction in ATP production
even though methylprednisolone reduces ATP availability to
some extent by inhibiting the reactions of substrate oxidation
and by increasing mitochondrial proton leak.
The comparison of these methylprednisolone effects with
those of myxothiazol we have reported above leads to the follow-
ing hypothetical explanation: Methylprednisolone dissolves in
membranes and affects physicochemical membrane properties
and the activities of membrane-associated proteins (Buttgereit
and Scheffold, 2002; Buttgereit et al., 2004). The resulting inhi-
bition of calcium and sodium entry across the plasma membrane
would explain (i) the decrease in ATP use (and therefore oxygen
consumption, see Fig. 1) for plasma membrane ion cycling and
(ii) the drop in cytosolic free calcium. A direct effect on the mito-
chondrial inner membrane would explain the observed increase
in proton permeability and the consequent partial uncoupling
of oxidative phosphorylation. This is suggested to result in
immunosuppression since immune cell function depends on
proper functioning of these processes.

Fig. 1. Oxygen consumption of lymphocytes as stimulated by Con A and inhibited by methylprednisolone (original trace measured using a Clark electrode). This
figure shows an original trace of oxygen consumption in thymocytes measured amperometrically with a Clark electrode. The first part of the curve reflects the basal
rate of oxygen consumption, calculated by dividing the change in oxygen saturation (delta O2) by the time (delta t). Addition of concanavalin A (Con A) leads to
a significant reater rate of oxygen consumption within seconds as reflected by the changed slope of the line. Additon of methylprednisolone leads to a reduction of
oxygen consumption. This experiment was carried out by Robert Tripmacher from our laboratory.
144 I.-H. Song, F. Buttgereit / Molecular and Cellular Endocrinology 246 (2006) 142–146
Substances which act similarly to high-dose glucocorticoids
are lazaroids (21-aminosteroids such as tirilazad). Lazaroids
intercalate into biological membranes without binding to glu-
cocorticoid receptors. In this way, they cause a stabilisation
of membranes and inhibition of destructive lipid peroxidation
which results in the prevention of oxidative cell damage. Tiri-
lazad is used in neurotraumatology (Braughler and Pregenzer,
1989; Dissemond et al., 2003; Bath et al., 2001). To our knowl-
edge, however, these drugs have not been used to investigate
their immunosuppressive effects in clinical trials.
Coming back to glucocorticoids, a very interesting approach
in the context of rapid non-specific non-genomic glucocorticoid
effects is drug targeting with liposomes as the carrier system
for glucocorticoids. Newly developed glucocorticoid-containing
liposomes accumulate selectively at the site of inflammation
where there is an increased permeability of the local vascular
endothelium. By reaching very high concentrations (>10−5 M)
at the site of inflammation for several hours, 100% utilisation of
the genomic actions and, in addition, significant therapeutically
beneficial non-genomic actions are achieved (Song et al., 2005).
Although the systemic glucocorticoid concentrations (plasma
levels) remain relatively high, they give rise to fewer adverse
reactions because the steroid is encapsulated in the liposome.
Schmidt et al. (2003) recently used prednisolone liposomes to
demonstrate the success of this innovative approach in the treat-
ment of experimental autoimmune encephalomyelitis in Lewis
rats (EAE).
Metselaar et al. (2003, 2004) have recently reported a com-
plete remission of experimental arthritis by joint targeting of
glucocorticoids with long-circulating liposomes (Song et al.,
2005). A single intravenous administration of 10 mg/kg lipo-
somal prednisolone phosphate in rat experimental arthritis and
in murine collagen type II-induced arthritis showed a profound
antiinflammatory effect which lasted for more than 1 week. In
contrast, 10 mg/kg of unencapsulated liposomal prednisolone
phosphate was less effective even after repeated daily injections.
Moreover, these authors also found the process of cartilage ero-
sion to be profoundly attenuated under this treatment. From the
mechanistic point of view, the observation is interesting that the
liposomes accumulate in the inflamed joint, i.e. in the proxim-
ity of blood vessels mainly in the synovial lining. This strongly
suggested that liposomes selectively localise in cells with phago-
cytotic capacity.
These initial observations suggest targeted delivery using
long-circulating liposomal glucocorticoids to be an effective and
novel therapeutic option in arthritis (Song et al., 2005). Hope-
fully clinical trials in humans will yield similar intriguing results
as in animal models. Future work will also have to optimise both
the lipid shells and the incorporated glucocorticoids.
1.2. Membrane-bound glucocorticoid receptors as
potential therapeutic targets
Apart from the physicochemical interactions with cellu-
lar membranes (non-specific non-genomic effects) mentioned
above, other mechanisms of rapid glucocorticoid actions are
being discussed. So called “mGCR-mediated non-genomic
effects” are suggested to be mediated by membrane-bound
glucocorticoid receptors (mGCR). The existence of such
membrane-bound glucocortioid receptors had already been
shown in amphibian neuronal membranes (Orchinik et al., 1991)
and in lymphoma cells (Gametchu et al., 1993; Chen et al., 1999;
Sackey et al., 1997). Recently, we were able to identify for the
first time mGCR in normal human peripheral blood mononuclear
cells (Bartholome et al., 2004; Buttgereit et al., 2004; Song et al.,
2005). This observation was made using the novel technique of
high-sensitivity immunofluorescent staining. The method uses
antibody-conjugated magnetofluorescent liposomes, which can
increase fluorescence signal intensity up to 1000-fold compared
with conventional methods and allows the detection of 50–100
target molecule per cell. Up to 9.2 and 12.3% of the monocytes
and B lymphocytes were positive for mGCR, respectively. T
lymphocytes never showed any significant mGCR expression
(Bartholome et al., 2004). Further experiments demonstrated
immunostimulation with lipopolysaccharide to increase signif-
icantly the percentage of mGCR-positive monocytes. In the
presence of brefeldin A, immunostimulation with lipopolysac-
charide did not increase the number of mGCR-positive mono-
cytes demonstrating the ability of brefeldin A to abrogate the
induced up-regulation of mGCR by inhibiting the secretory path-
way. We concluded that immunostimulation induces cellular
events whereby mGCR are actively upregulated and transported
through the cell.
On the basis of this data, we hypothesized that there is a
clinically relevant correlation between the number of mGCR-
positive cells and disease-related activity of the immune system.
We indeed found a strong positive correlation between the fre-
quency of mGCR-positive monocytes and various parameters
of disease activity in patients suffering from rheumatoid arthri-
tis (Bartholome et al., 2004). One way to interpret these data is
that mGCR may play a role in the aetiopathogenesis of disease.
However, we suggest being more likely that immunostimula-
tion or high disease activity stimulate mGCR expression in
immune cells such as monocytes which in turn leads to a signif-
icantly higher percentage of cells undergoing mGCR-mediated
glucocorticoid-induced apoptosis (Sackey et al., 1997). This
putative process would diminish the activity of the immune
system and could be therefore considered as a mechanism of
negative feed-back regulation. Further experiments will have
to be made in order to prove this hypothesis. A new approach
of optimising glucocorticoid therapy could be to develop drugs
selectively binding to the mGCR, but this is clearly specula-
tive. First the function of mGCR needs to be investigated in
detail. Though there is no evidence for specific signalling path-
ways associated with mGCR, this mechanism might explain the
rapid dexamethasone-induced adenylate cyclase/protein kinase
A-dependent inhibition of chloride ion secretion in bronchial
epithelium (Goulding, 2004; Urbach et al., 2002).
1.3. Rapid interaction with signalling processes via
cytosolic glucocorticoid receptors
Another hypothesis to explain rapid glucocorticoid effects is
that glucocorticoids bind to its cytosolic glucocorticoid recep-
 I.-H. Song, F. Buttgereit / Molecular and Cellular Endocrinology 246 (2006) 142–146
tor where they not only cause classical genomic, but also rapid
non-genomic effects resulting in interactions with signalling
processes. The underlying theory is the following: Arachi-
donic acid (AA) is an essential mediator which is important
for cell growth and several metabolic reactions (e.g. production
of inflammatory cytokines in the setting of the inflammatory
cascade). AA release from cell membrane phospholipids is con-
trolled by mediators which among others involve growth factors
(like insulin or PDGF), adaptor proteins such as SOS, Grb2,
the GTPase p21ras, erk2/ MAPK (mitogen activated protein
kinases), cPLA2 (cytosolic phospholipaseA2) and especially
lipocortin 1 (LC1) (Ahn et al., 1991; De Vries-Smits et al., 1992;
Lin et al., 1993; Croxtall et al., 1995, 1996, 1998).
With regard to cGCR-mediated glucocorticoid effects,
Croxtall et al. (2000). have recently reported that epidermal
growth factor (EGF) stimulated cPLA2 (cytosolic PLA2) acti-
vation with subsequent arachidonic acid release can be inhibited
by dexamethasone. This effect is considered to be a glucocor-
ticoid receptor-dependent (RU486-sensitive), but transcription-
independent (actinomycin-insensitive) mechanism.
What could be the explanation for this observed effect? To
answer this question, it should be noted that the unactivated
(unligated) cGCR is retained in the cytoplasm as a multi-protein
complex consisting of heat shock proteins and several kinases of
the MAPK signalling system (chaperones and co-chaperones),
including Src. Following glucocorticoid binding, the cGCR is
released from this complex, thus revealing domains of the recep-
tor that are able to bind specifically to glucocorticoid respon-
sive DNA elements. This mechanism is known to account for
genomic glucocorticoid effects. However, the role of the remain-
ing signalling components of the multi-protein complex is less
clearly defined. In their study, Croxtall et al. (2000) showed
that there is a rapid release of not only glucocorticoid receptor
protein, but also of Src from the multi-protein complex which
follows the binding of dexamethasone to the cGCR. However,
the release of glucocorticoid receptor and Src appear to be dis-
tinct events, since the nuclear translocation of glucocorticoid
receptor is not required for subsequent activation of lipocortin
1 and rapid inhibition of arachidonic acid release. Rather, it
appeared that it is the rapid effect of Scrd that fulfils this role.
In summary, it is suspected that the binding of glucocorti-
coids to the cGCR not only induces classical genomic effects,
but also rapidly controls intracellular signalling through other
components of the multi-protein complex. It may be possible
in the future to make therapeutical use of these glucocorticoid
effects. The clinical background for this assumption is that glu-
cocorticoids have been shown recently to activate endothelial
nitric oxide synthase in a non-genomic manner and mediated by
PI3K and Akt phosphorylation. This ultimately leads to vasore-
laxation, which might explain some of the cardioprotective
effects of glucocorticoids (Lösel and Wehling, 2003; Hafezi-
Moghadam et al., 2002). There are other examples in the field
of oncology where blocking of small molecules represent very
promising therapeutic approaches. In this regard, we would like
to mention the inhibition of IGFBP2 (insulin-like growth factor
binding protein) which enhances the invasion capacity of ovarian
cancer cells (Lee et al., 2005). Other examples include the EWS-
145
FlI1 fusion protein (Ewing sarcoma family of tumors), transloca-
tion fusion protein ETV6-NTRK3 (congenital fibrosarcoma or
cellular mesoblastic nephroma) and BCR-ABL kinase inhibitor
imatinib mesylate (chronic myeloid leukaemia). This is only a
selection to demonstrate that advances in molecular biology have
already led to the identification of quite a lot new protein targets
along with an increased understanding of the biologic role of
these proteins (Uren and Toretsky, 2005). This research is ulti-
mately designed to improve therapeutic option as, e.g. demon-
strated with tyrosine kinase inhibitors such as gefitinib which is
currently tested in phase II trials in breast cancer. Monoclonal
antibodies targeting epidermal growth factor receptor (EGRF)
represent another approach to interfere with EGRF-driven signal
transduction. For example, the chimerized monoclonal anti-
body cetuximab is designed to treat advanced colorectal cancer
(Caponigro et al., 2005). We have discussed these examples to
illustrate that the cGCR-mediated non-genomic glucocorticoid
effects could provide the basis for targeted therapies in the future.
1.4. Concluding remarks
Glucocorticoids are clinically very important drugs; how-
ever, their use is limited by side effects. This drives interesting
research in order to understand better the underlying mecha-
nisms of action. The non-genomic glucocorticoid effects we
describe here are currently in the focus of research. The ulti-
mate aim is to convert results of basic research in this regard
into novel therapeutic options to improve the benefit-risk-ratio
of glucocorticoid therapy.
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