Our bones get more brittle with increasing age, and to add
insult to injury, the most effective therapy for another problem that is
associated with getting older, rheumatoid arthritis, often adds to the problem
by causing bone resorption. The Glucocorticoid steroids, are the best available
anti-inflammatories, and are used widely in the treatment of arthritis, as well
as other inflammatory conditions such as dermatitis and autoimmune diseases.
The Glucocorticoids, secreted by the Adrenal Cortex are powerful
anti-inflammatory compounds due to their ability to inhibit all stages of the
inflammatory response, from redness to wound healing to cell proliferation
(Ref.1). They are powerful anti-inflammatory compounds that have the ability to
inhibit all stages of the inflammatory response. They also have an essential
role in cell metabolism and got the nomenclature, from their effect of raising
the level of blood sugar (glucose) by stimulating gluconeogenesis in the liver:
the conversion of fat and protein into intermediate metabolites that are
ultimately converted into glucose. Cortisol (or Hydrocortisone) is the most
important human Glucocorticoid. It is essential for life and regulates or
supports a variety of important cardiovascular, metabolic, immunologic, and
homeostatic functions. Corticosterone, another Glucocorticoid, helps in the
regulation of the conversion of amino acids into carbohydrates and glycogen by
the liver, and stimulates glycogen formation in the tissues. All the cellular
responses to Glucocorticoids is attributed to their binding to the
intracellular GR (Glucocorticoid Receptor) (Ref.2), that, in turn, translocates
to the nucleus, that positively and negatively modulates gene expression
through diverse mechanisms. The GR is the Glucocorticoid-activated member of
the nuclear receptor superfamily of transcription factors. It mediates the
immunosuppressive and anti-inflammatory activity of these ligands in multiple
physiological systems, including the respiratory and central nervous systems.
Belonging to the family of steroid hormones, Glucocorticoids are essential for
development and survival of vertebrates (Ref.3).
Unbound GR is associated within the cytoplasm in an inactive
oligomeric complex with some regulatory proteins such as the HSP90 (Heat Shock
Protein-90 KD) which binds as a dimmer to the C-terminal domain, the HSP70
(Heat Shock Protein-70 KD), the p59 immunophilin, FKBP52 and the small p23
phosphoprotein. GRs are composed of several conserved structural elements,
including a COOH-terminal ligand-binding domain (which also contains residues
required for dimmerization and hormone-dependent gene transactivation), a
nearby hinge region containing nuclear localization signals, a central
zinc-finger-containing DNA-binding domain, and an NH2-terminal variable region
important for ligand-independent gene transactivation. The interaction between
HSP90 and GR is required to maintain the C-terminal domain in a favourable
conformation for ligand binding (Ref.4). The Gucocorticoid hormone passes
through the plasma membrane into the cytoplasm where it binds to the specific,
high-affinity GR. The resulting complex is the non-DNA-binding oligomer of the
GR in which the receptor is complexed with other proteins. Binding of hormone
agonists releases GR from its interactions with the inhibitory complex, thus
inducing a conformational change which results in unmasking of the receptor
nuclear localization signal. Upon activation, GR thereby translocates to the
nucleus and binds as a dimmer to DNA through its central domain, which is
structurally characterized by a DNA binding motif (Ref.3). The stabilization
and nuclear localization of GR is facilitated by its sumoylation by SUMO1
(Small Ubiquitin Related Modifier-1). The sumoylation process is catalyzed by
the SUMO1-conjugating E2 enzyme Ubc9 (Ref.5). GR interacts either with DNA by
targeting specific nucleotide palindromic sequences termed GRE (Glucocorticoid
Response Elements) or nGRE (Negative GRE) (Ref.6). In particular, the dimmeric
GR places its two DNA-binding fragments into adjacent major grooves of the DNA
double helix in correspondence of appropriately spaced GRE half palindromes.
Depending on the nature of the GRE, the overall process of GR binding can
result in activation or repression of genes containing GR-binding sites
(Ref.3).
Although the activity of the GR is often thought of simply
in terms of direct gene transactivation, considerable cross-talk also occurs
between the GR and a cohort of molecules to mediate their function as
transcriptional regulators. GRs can interact with coactivator complexes
including CBP (CREB-Binding Protein), p300, ACTR (Activator of Thyroid Hormone
and Retinoid Receptors), SRC1 (Steroid Receptor Coactivator-1), and PCAF
(p300/CBP Associated Factor) that possess HAT (Histone Acetyltransferase)
activities, and the SWI/SNF complex which possesses ATPdependent chromatin
remodeling activities (Ref.3 & 7). Acetylation of core histones alters
nucleosomal packing to allow increased access of transacting factors and components
of the basal transcriptional machinery to the local DNA. All these complexes
may act in concert to relieve chromatin-mediated gene repression, with the TRAP
(Thyroid Hormone Receptor Associated Protein)-GRIP (Glucocorticoid Receptor
Interacting Proteins)-ARC (Activated Recruited Cofactor) complex functioning to
recruit the core transcription machinery. The latter includes the TBP (TATA
Box-Binding Protein), the TAFs (TBP Associated Factors), the general
transcription factors TFIIA, TFIIB, TFIIE, TFIIF, TFIIH, and the enzyme, RNA
Pol II (RNA Polymerase-II). The nuclear receptors can also interact with the
corepressors NCoR (Nuclear Receptor Corepressor) and SMRT (Silencing Mediator
of Retinoid and Thyroid Hormone Receptor) thus leading to the recruitment of
the Sin3-HDAC (Histone Deacetylase) corepressor complex, possessing histone
deacetylase functions. This corepressor complex can thereby inhibit gene
transcription by counteracting the actions of the coactivator complexes
containing histone acetyltransferase activities (Ref.2 & 8).
Alternatively, GR can also modulate the expression of genes
through a GRE-independent mechanism, which is mediated in part through protein–protein
interactions of GR with other sequence-specific DNA-binding factors or
coactivators (Ref.9). The negative modulation of gene transcription operated by
Glucocorticoids occurs through non genomic mechanisms (transrepression),
mediated by inhibitory influences exerted by activated GR on the functions of
several transcription factors. This contributes to the anti-inflammatory
properties of the Glucocorticoids. Transrepression is due at least in part to
direct, physical interactions between monomeric GR and transcription factors
such as c-Jun-c-Fos and NF-KappaB (Nuclear Factor-KappaB), that synergistically
coordinate the transcriptional activation of many genes involved in
inflammatory diseases such as Asthma (Ref.10). In particular, the three main
domains of GR may contribute to interact with the p65 subunit of NF-KappaB and
with both Jun and Fos components of Activator Protein-1. The resulting
reciprocal antagonism of the transcription factors engaged in these
protein-protein associations causes an impairment of their transcriptional
properties. However, Activator Protein-1, consisting of c-Jun homodimmers can
also enhance GRE-mediated transactivation. On the other hand,
Glucocorticoid-activated GR increases DNA-binding activity of CEBP-Beta via post-translational
mechanisms involving phosphorylation at Thr(235) (Ref.11). GR can interact as a
monomer, via direct protein-protein interactions, with transcription factors
such as NF-KappaB and Activator Protein-1, activated by cytokines and other pro-inflammatory
stimuli (Ref.4). The resulting mutual repression prevents both GR and the other
transcription factors from binding to their respective DNA response elements.
In addition, Glucocorticoids repress NF-KappaB-mediated activation of
pro-inflammatory genes by reducing the levels of serine-2 phosphorylation of
the carboxy-terminal domain of RNA Pol II, which is essential for the
recruitment of this enzyme to the promoter region. Glucocorticoids also
increase the transcription and synthesis of I-KappaB and thus may inhibit
NF-KappaB by promoting its retention in the cytosol. Other products of
Glucocorticoid inducible genes responsible for NF-KappaB inhibition include the
two recently discovered proteins GILZ (Glucocorticoid-Induced Leucine Zipper)
and GITR (Glucocorticoid-Induced Tumor Necrosis Factor Receptor Family-Related
Gene), which play a crucial role in modulation of T-cell activation and
apoptosis. GR can also cooperate with transcription factors, including octamer
transcription factors Oct1 and Oct2; CREB (cAMP Response Element Binding
Protein), and STAT5 (Signal Transducers and Activators of Transcription-5), to
activate transcription. Competition for limiting co-activators of transcription
is an important determinant of the fate of the cross-talk between the GR and
other transcription factors. Both Activating Protein-1 and the GR are
co-activated by CBP-p300, and in fact overexpression of CBP or p300 reverses
the antagonism between Activator Protein-1 and the GR. Similarly,
overexpression of CBP or SRC1 reverses the transcriptional antagonism between
the GR and NF-KappaB (Ref.8 & 12).
Glucocorticoids downregulate cell proliferation by
decreasing the expression of Cyclin-D1 and the phosphorylation of Rb
(Retinoblastoma) protein and by activating p21(CIP1) (Cyclin Dependent Kinase
Inhibitor-p21). The antiproliferative effect of Glucocorticoids is mediated by
the GR and CEBP-Alpha, and both active transcription factors are required to
induce the synthesis of p21(CIP1). In human cells, including lung fibroblasts,
pulmonary and bronchial smooth-muscle cells, and peripheral-blood lymphocytes,
the GR forms a complex with CEBP-Alpha, which then binds to the CCAAT DNA
consensus sequence in the p21(CIP1) promoter (Ref.13). The Glucocorticoid
signaling interacts with other signaling pathways activated by various
cytokines, thus regulating diverse biological processes through modulating the
expression of target genes. GR represses TGF-â transcriptional activation of
the PAI-1 (Plasminogen Activator Inhibitor-1) and other genes in a
ligand-dependent manner. Glucocorticoids inhibit the TGF-â-induced expression
of ECM (Extracellular Matrix) proteins including Fibronectin and Collagen, and
proteinase inhibitors such as tissue inhibitors of Metalloproteinase. GR inhibits
transcriptional activation by both Smad3 and Smad4 C-terminal activation
domains (Ref.14). The MAPKs (Mitogen-Activated Protein Kinases) play a key role
in inflammatory cell types through transducing the response from
proinflammatory cytokine receptors to the transcriptional apparatus. MAPK
subgroups such as JNK regulate activation of the AP-1 complex required for
proinflammatory gene expression. The MAPK p38 subgroup regulates the stability
of mRNAs that encode the proinflammatory molecules TNF-Alpha, IL-6, IL-8, and
VEGF (Vascular Endothelial Growth Factor). Negative regulation of the MAPK
family by Glucocorticoids may be an additional mechanism by which the GR exerts
its antiinflammatory effects (Ref.15). The MAPK subgroups JNK, ERK1, ERK2, and
p38 are all targets of negative regulation by activated GRs. For example,
Glucocorticoids destabilize the mRNA of the proinflammatory enzyme COX2
(Cyclooxygenase-2) by inhibiting the activity of p38 (Ref.16). The GR represses
the MAPK family by inhibiting the phosphorylation step required for their
activation. The defined molecular mechanism behind this inhibition has not been
fully characterized and may be cell type and stimulus specific (Ref.9).
The therapeutic and prophylactic use of Glucocorticoids is
widespread due to their powerful anti-inflammatory, antiproliferative and
immunomodulatory activity (Ref.17). These are widely prescribed
anti-inflammatory drugs, used to treat a wide variety of inflammatory diseases,
including allergies, asthma, rheumatoid arthritis, and auto-immune diseases.
Glucocorticoids enhance the production of other anti-inflammatory molecules
such as IL-1RA (Interleukin-1 Receptor Antagonist), IL-10 (Interleukin-10),
secretory leukocyte inhibitory protein and neutral endopetidase (Ref.2).
Glucocorticoids are important mediators of the immune system and modulate the
biological activities of inflammatory cytokines. The very effective control of
airway inflammation exerted by Glucocorticoids in asthma is largely mediated by
inhibition of the transcriptional activity of several different genes encoding
pro-inflammatory proteins such as cytokines (IL-1, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-8, IL-10, IL-11, IL-13, TNF-Alpha, GMCSF, IFN-Gamma), chemokines
(IL-8, RANTES, MIP-1a, MCP-1, MCP-3, MCP-4, Eotaxin), adhesion molecules
(ICAM1, VCAM1, E-Selectin), and mediator-synthesizing enzymes (i-NOS, COX2,
cytoplasmic PLA2) (Ref.9, 10 & 16). Glucocorticoids, acting through the GR,
potently modulate immune function and are a mainstay of therapy for treatment
of inflammatory conditions including allergies, asthma, rheumatoid arthritis;
autoimmune diseases, leukemias and lymphomas (Ref.3). Common Glucocorticoids
include prednisone, dexamethasone, and hydrocortisone. Hydrocortisone is used
as an anti-inflammatory in the treatment of arthritis, as well as other
inflammatory conditions such as dermatitis and autoimmune disease. While
Glucocorticoids are widely used as drugs to treat various inflammatory
conditions, prolonged Glucocorticoid use may have adverse side effects such as
immunosuppression, fluid shifts, brain changes, and psychological changes.
Physicians are therefore very cautious about prescribing these medications,
especially for long periods of time. The search for novel Glucocorticoids with
reduced side effects has been intensified by the discovery of new molecular
details regarding the function of the Glucocorticoid receptor. These new
insights may pave the way for novel, safer therapies that retain the efficacy
of currently prescribed steroids (Ref.18).
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