Glucocorticoids (GC) are hormones synthesised in response to stress which maintain the homeostatic mechanisms. This effect on the body is mediated by binding of the glucocorticoid hormone to a ligand regulated transcription factor known as the glucocorticoid receptor (GR). GRs are expressed in every cell of the body and are very important, as they regulate genes which control vital mechanisms of the body like metabolism, immune response and development.
GCs are active therapeutics for the treatment of various inflammatory disorders and cancers. However, due to their side effects, their use is limited. Thus, identification of mechanisms of GR sensitivity and resistance is crucial for development of new therapeutic approaches and improvement of existing treatments.
The possible reason for such resistance arises from deregulation of the GR pathway. This can reduce the number of GR, alter DNA binding or increase the expression of transcription factors such as AP-1, STAT and NF-Ä¸B which have negative effects on GR function. One reason for the GR resistance could possibly be the lack of specific and reliable biomarkers of steroid resistance. Observations show that the presence of interleukins 2 and 4 (IL 2/4), reduces sensitivity of T-cells to GCs.
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Our aim here is to discuss and describe the role of the GR in inflammation and how this can be used to identify biomarkers, isolated from T lymphocytes treated with IL 2/4 and glucocorticoids that can be targeted for therapy.
SECTION 1: INTRODUCTION
Steroids are biochemical elements that are naturally present in the body and regulate physiological, metabolic and immune processes. Glucocorticoid is a type of steroid hormone secreted by the adrenal gland and is present in almost every vertebrate animal cell (So et al, 2009). GCs regulate a variety of physiological functions and maintain stress- related homeostasis. They raise blood glucose levels by the breakdown of fats and proteins, inhibit bone formation and raise blood pressure. They are widely used due to their anti-inflammatory and immune-modulation properties and thus have clinical applications. GC's are widely prescribed for the treatment of inflammatory signs and symptoms (Campagnolo et al, 2008). Most of the glucocorticoid activity in mammals is from cortisol (Fig.1; Miner et al, 2007).
Fig.1: The chemical structure of cortisol (a GC)
Glucocorticoids are the most effective intermediaries for suppressing inflammation. They act via the glucocorticoid receptor.
Dexamethasone (Fig.3) is a class of synthetic glucocorticoids which has 20-30 times more potency to treat inflammation as compared to other classes of GCs.
In our research we will thus be using dexamethasone as stimulant, along with IL 2 and 4, after the isolation of T-lymphocytes from human blood.
GR is a cytoplasmic, ligand dependent transcription factor. The human GR gene is present on chromosome 5 and consists of 9 exons. Exon 1 contains 5'UTR, exons 2-9 contain the coding sequences and exon 9 the 3'UTR. It is also known as NR3C1 which explains that GR belongs to the Nuclear Receptor subfamily 3, group C, member 1 (Charmandari et al, 2004). The receptor exhibits different effects in different parts of the body. Its primary action is to regulate gene transcription on binding to GCs. Like other steroid hormone receptors, GRs are also intracellular receptors (IRs) as they are found in the cytosol of cells. These receptors are transcription factors. Heat shock proteins (hsp) are found bound to the receptor until the hormone is present.
Fig.3: Domains of the glucocorticoid receptor.
The structure of GR consists of a DNA binding domain (DBD) and a Ligand binding domain (LBD) (Fig.3) bound to dexamethasone and TIF2 co-activator protein. The glucocorticoid hormone alone cannot show any effect (Buckingham, 2009). Its effects are expressed through the GR (Fig.4).
Fig.4: Binding of the hormone to the glucocorticoid receptor.
The figure 4 shows how GC binds to the receptor with the removal of certain heat-shock proteins that act like chaperones. The resulting GC-GR complex acts as a homodimer coactivator and diffuses into the nucleus.
Levels of control of the Glucocorticoid Receptor function:
To determine the anti-inflammatory properties of glucocorticoids, it is important to know the factors that could affect the glucocorticoid receptor function and thus reduce its proposed anti-inflammatory properties.
3.1. Ligand and DNA binding of the Glucocorticoid Receptor:
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GR is structurally similar to the androgen and progesterone receptors that contain a DNA binding domain and a ligand binding domain. The proper control of transcription relies on the assembly of GR regulatory complexes at promoter-proximal site of target gene response elements (Luecke and Yamamoto, 2005).
GC binds to the GR in the cytosol and translocates to the nucleus where it binds to DNA as a homodimer at consensus sites, termed Glucocorticoid Response Elements (GREs), usually located in the cis-regulatory region of target genes. The GR modulates the expression of numerous target genes depending on the cell type and promoter context. The magnitude and direction of GR-mediated transcriptional activity is determined by the GRE sequence, balance between co-activators and co-repressors and post-translational modifications (Liberman et al, 2009). The GR bound to the hormone binds to GRE upstream of the target gene and initiates transcription (Fig.5).
Fig.5: Transactivation by the GR.
(Lovinger D.M., 2008)
Alternatively, GR can repress transcription by binding to transcription factors like NF-Ä¸B and AP-1, and various other co-activators. This attributes to the anti-inflammatory effect of GC as it suppresses the action of inflammatory and immune modulating target genes. GR affects the activity of other transcription factors like activator protein-1 (regulates homeostasis in response to viral and bacterial infections and stress), STAT (Signal Transducer and Activator of Transcription which regulates cell growth, differentiation and survival) and NF-Ä¸B (a transcription factor controlling cellular response to stress). These effects may be attributed due to a number of different mechanisms like the competition between GR and the cAMP response element binding protein (CBP) for the transcription factors stated above. The GR to binding of transcription factors such as NF-Ä¸B, leads to the loss of DNA binding capability of the GR (Muzikar et al, 2009).
Transrepression of the GR is considered to produce positive therapeutic effect as it shows an anti-inflammatory action. Transactivation of genes is considered to show negative therapeutic effects due to its side effects.
Cofactors : Co-activators and co-repressors
Activation of GR is a complex process and involves a number of steps which includes the ability of the receptor to bind the hormone and then translocate into nucleus to identify target genes. Transcription factors are proteins that bind to DNA regulatory sequences of target genes to modify the rate of gene transcription. The pathway of these factors might involve multiple intracellular signal transductions or may directly be activated by ligands (for example GCs) or be activated within the cytoplasm. Thus these factors are able to convert environmental signals at the cell surface into long-term changes in gene transcription (Adcock, 2000; Frego and Davidson, 2006).
Co-activator molecules are large proteins that bind transcription factors to basal transcription apparatus and thus act as integrators of gene transcription. GR interacts with heat shock proteins (hsp90) as shown in Fig.4 above, and co-activators like Cyclic Adenosine Mono Phosphate (cAMP) response element binding protein (CBP) and p300. Hsp90 maintains the GR in a position that it is able to bind to the hormone and also plays an important role in the translocation of the GR complex to the nucleus (Ricketson et al, 2007). The p300-CBP complex interacts with a number of transcription factors and mediates the expression of target genes (Fig.6).
Interactions between coactivators and GR are still under investigations and a few coactivators have been identified which are BRGÂ 1 (SWIÂ /SNF) complex, theÂ P/CAFÂ (ADA/SAGA) complex,Â CBP/p300, the p160 coactivators and components of theÂ DRIPÂ (TRAP/ARC) complex (Fig.6; Jenkins et al, 2001; Chaudhuri, 2008).
Co-repressors on the other hand are proteins that bind to a transcription factor which contains a DNA binding domain and decrease the expression of the GR target genes. They interact via receptor interaction domains (RIDs) in the carboxyl terminal half of the ligand binding domain of the GR (Lee at al, 2000).
Fig.6: Effect of coactivators and corepressors on DNA transcription.
The identified corepressors are NCoR1 (Nuclear Receptor Corepressor 1), which assists nuclear receptors in down regulation of DNA expression, e.g.: Thyroid hormone and Retinoic Acid receptor Co-repressor 1 (TRAC-1), and NCoR2 (Nuclear Receptor Corepressor 2), which fixes histone deacetylases to DNA promoter regions (Wang and Simons, 2005), e.g.: SMRT (Silencing Mediator of Retinoid and Thyroid hormone receptor) (Fig.6).
Post-translational modifications of the Glucocorticoid Receptor
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Glucocorticoid signalling pathway needs to be tightly regulated to ensure the anti-inflammatory property of glucocorticoids. The function of GR can also be affected by different post translational modifications like phosphorylation, ubiquitination, sumoylation, acetylation and methylation (Kumar and Calhoun, 2008).
The human GR has five phosphorylation sites at serines 113, 141, 203, 211 and 226. These serines are usually followed by a proline and are thus called proline-directed consensus sequences (Galliher-Beckley and Cidlowski, 2009). The phosphorylation on serines is usually dependent on the ligand binding to GR. These sites can be phosphorylated by Cyclin Dependent Kinase (CDK), Mitogen Protein Kinases (MAPK), and Glycogen Synthase Kinase-3 (GSK-3) (Krstic et al, 1997).
Phosphorylation has two major effects on the GR function. First, it effects the subcellular localization of the receptor and secondly, it alters the ability of the receptor to modulate target gene transcription. It has also been suggested that hyper-phosphorylation of the GR may be the reason for the development of GR resistance in the body due to the decreased binding of GR to its target sites (Duma D. et al, 2006).
The covalent binding of ubiquitin, a multi-protein complex, to the GR results in its' degradation by proteasome. The ubiquitin-proteasome mediated pathway standardises the GC signalling system by controlling the degradation rates of GR. This is important as it degrades both short lived and regulatory proteins involved in cell cycle progression, cell surface receptor action, modulation of ion channels and antigen presentation. Thus, phosphorylation could result in the loss of proteins in the glucocorticoid receptor. It has been observed that mutation of all phosphorylated sites in GR increased the half life of the receptor and eliminated downregulation (Duma D. et al, 2006).
Addition of a Small Ubiquitin related Modifier-1 (SUMO-1) is another important post translational mechanism which can regulate GR function. Proteins like transcription factors, chromatin remodelling proteins and co-regulators which are involved in gene regulation are targets for SUMO. In sumoylation the SUMO-1 gets attached to the lysine residues of the target proteins and regulates protein stability, localization and activity of transcriptional regulators (Davies et al, 2008).
The protein recognition and SUMO linkage is a dynamic process and is catalysed by an enzyme named Ubc9. The glucocorticoid receptor has three SUMO attachment sites K277, K293 and K703. The first two attachment sites reside in the N-terminal transactivation region while the third attachment site resides in the ligand-ligand binding domain of the receptor because of which sumoylation might have a greater effect on the functioning of the receptor (Duma D. et al, 2006).
3.3.4. Acetylation and methylation
Acetylation and methylation can also regulate protein function. Even though less evidence is available in support of this, some reports provide indications for this case. Studies show that histones can be methylated and these modifications regulate gene transcription through chromatin rearrangements. Very little is known about the transcriptional activity mediated by histone methylation. Methylation of GR coactivators like p300 can modulate GR signalling by inhibiting the interaction between p300 and GR Interacting Protein (GRIP1) (Duma D. et al, 2006).
Acetylation is a very important feature as previous studies show that acetylation occurs in the DNA binding domain (DBD) of GR on amino acids 492-495. Studies show that the glucocorticoid receptor is acetylated after dexamethasone binding. The acetylation negatively regulates dexamethasone induced repression of NF-Ä¸B dependent gene expression (Ito K. Et al, 2005).
SECTION 2: INFLAMMATION PROPERTIES
Inflammation is described as "the succession of changes which occurs in a living tissue when it is injured provided that the injury is not of such a degree as to at once destroy its structure and vitality" or "the reaction to injury of the living microcirculation and related tissues" (Punchard et al, 2004). According to World Health Organisation (WHO) 2007 report, inflammation and inflammation related illnesses are the biggest challenge in current medicine as about 500 million people suffer from them (Beck et al, 2009).
Until the 19th century inflammation was regarded as an undesirable response that was harmful to the host. However, studies later suggested that inflammation is a body's own defensive and healing process. Typical signs of inflammation are as follows:
Inflammation is accompanied by a reddish appearance which is due to the accumulation of many erythrocytes in the area,
Heat sensation is caused due to the increased movement of blood through dilated vessels,
Swelling can be seen due to infiltration of cells into the damaged area, and
Pain is always felt in the damaged area which could be either due to the damage caused, or due to the inflammatory response and even due to the stretching of nerves due to swelling.
The inflammatory response of the body could be of great value, as well as threatening. The inflammatory response to tissue damage is of great value as it isolates the damaged area, immobilizes effector cells to the site and encourages healing. Sometimes, immune response causes more damage to the body than the agent itself and the conditions are described as allergies and autoimmune diseases like asthma, rheumatoid arthritis, sclerosis, etc. These diseases are debilating and incurable.
The underlying genetics and molecular biology forms the basis, to study and identify tendency of inflammatory diseases, and pharmalogical studies aim to develop novel treatments to treat these diseases. Thus, research into different ways of preventing inflammatory diseases in an effective way, with no side effects, is an important part of the industry.
The role of glucocorticoid receptor in inflammation
One of the most important pharmacological roles of glucocorticoids is their anti-inflammatory and immunosuppressive activity. The anti-inflammatory effects of glucocorticoids are mediated through negative interaction with NF-Ä¸B and AP1, which are well characterized inducers of pro-inflammatory cytokine expression (Tait et al, 2008; McMaster and Ray, 2008).
5.1. Mechanisms of Glucocorticoid action
The glucocorticoid receptor has multiple isoforms that arise due to alternative splicing and translational events of the gene NR3C1.
GRÉ‘-A: binds ligand and mediates GC action. The cell types that show negligible expression of GRÉ‘-A show minimum effect of glucocorticoids.
GRÎ² and GR-P: do not bind ligand and mediate GRÉ‘-A activity.
Fig.7: Structural organisation of the human glucocorticoid receptor É‘ protein (Source: Smoak and Cidlowski, 2004).
The figure shows three major domains namely the N-terminal transactivation domain which consists of AF-1 activation domain and helps transcriptional enhancement and its association with the basal transcription machinery (BTM). The DNA binding domain is necessary for receptor dimerization and target binding. The C-terminal domain is a binding site for hsp, coactivators and the ligand dependent activation function domain (AF-2) (Dey et al, 2001).
NF-Ä¸B antagonism mechanism
The involvement of NF-Ä¸B in inflammation has been established well in both in vivo and in vitro systems. The ubiquitous transcription factor is made up of p65, p52 in combination with p100, p50 in combination with p105, c-Rel and RelB. The p65 and p50 combination is involved in the transcriptional activation process. NF-Ä¸B exists in the cytoplasm interacting with the inhibitor IÄ¸B (Beck et al, 2009). This interaction prevents DNA binding as nuclear translocation of NF-Ä¸B is inhibited (Fig. 8)
Fig.8: Mechanisms of NF-Ä¸B antagonism (Smoak and Cidlowski, 2004).
GRÉ‘ represses the transcriptional activity of NF-Ä¸B by interacting with p65 subunit as show in the figure above.
Another proposed mechanism is the competition (mutual antagonism) of GRÉ‘ and NF-Ä¸B for mutual co-factors such as cAMP response element binding protein (CREB)-binding protein (CBP) and steroid receptor co-activator (SRC-1). CBP and SRC-1 are required for maximum transcription activity of NF-Ä¸B and GRÉ‘ (Bosscher et al, 2009). It is also required for GRÉ‘ and CBP to p65 to bind to the same region of CBP.
Studies also show that the GR interferes with the phosphorylation of RNA pol II C-terminal domain (CTD) as a result of which NF-Ä¸B is not able to bind transcription elongation factor (P-TEFb), thus inhibiting transcription (Luecke and Yamamoto, 2005).
Thus a variety of mechanisms are likely to be involved in the reciprocal antagonism between GRÉ‘ and NF-Ä¸B (Fig. 8).
AP-1 antagonism mechanism
AP-1 is an important regulator in the gene expression of many cytokine genes and is also in controlling apoptosis or programmed cell death (Johnson and Lapadat, 2002).
Fig.9: AP-1 repression mechanisms by GRÉ‘ (Smoak and Cidlowski, 2004).
GRÉ‘ represses AP-1 activity by interacting with c-Jun and prevents binding of DNA in the nucleus in a fashion more or less similar to the NF-Ä¸B repression mechanism. AP-1 interacts with a dimer composed of a Jun family member like c-Jun and a Fos protein like c-Fos. This dimer regulates gene transcription by binding to the GR. When bound to GRÉ‘ at the site of inflammatory gene promoters they repress transcription because they do not allow AP-1 to bind DNA (Smoak and Cidlowski, 2004; Kassel and Herrlich, 2007).
Another mechanism shows that GRÉ‘ facilitates the expression of MPK-1 gene which encodes for MAP kinase and prevents the activation of c-Jun N-terminal Kinases (JNK), extracellular signal-regulated kinases (ERK) and p38 in the cytoplasm which are kinases important for inflammatory responses which results in the destabilisation of the pro-inflammatory cytokines such as cyclooxygenase (COX-2) (Smoak and Cidlowski, 2004).
Because of the strong anti-inflammatory effects of GC's, they are used to treat a variety of conditions like arthritis, allergies, skin disorders, cancer, pulmonary disorders, transplant rejection and even spinal cord injury (Hardy et al, 2008). Glucocorticoid as a drug has been used for over 50 years now to treat chronic inflammatory diseases despite their side effects like suppression of the activity of the hypothalamus, pituitary and adrenal (HPA) glands (Silverman and Sternberg,2008), increased serum glucose, altered electrolyte balance, insomnia, initiation of osteoporosis and glaucoma and behavioural changes. The currently used drugs with generic name dexamethasone are Decadron which is marketed by Merck and Co., Hexadrol, Dexasone and Maxidex.
GCs are also known to be a part of the treatment of some cancers. For example, GCs in combination with ketoconazole or chemotherapy are used in prostate cancer therapy (Keith, 2008).
GCs have intense immune modulating properties, and synthetic glucocorticoids are the most potent anti-inflammatory agents currently available. However, variations in response to these drugs is important, as there have been incidences of side effects like osteoporosis which are usually irreversible and can lead to considerable morbidity. No clear evidence supports how this variation exerts these effects. Microarray technology allows gene profiling to be done to identify accurate gene expression levels within an individual and so leads to the identification of genome response to different concentrations of glucocorticoids (Donn et al, 2007). In our experiments we will be using gene microarray experiments to determine dissimilarities in GC dependent gene expression networks taking place in T cells. We also plan to isolate RNA from blood donors that will be processed by the Affymetrix GeneChip technology available in the core facility at the University of Manchester. Labelled targets derived from the mRNA of an experimental sample will be hybridised to nucleic acid probes attached to the solid support. System analysis will then be used to identify relevant changes in the gene expression profiles.
The increased understanding of how glucocorticoids act has given new insights into the pathophysiology of inflammatory diseases such as arthritis and has also opened new doors for the development of new anti-inflammatory treatments. Development of glucocorticoids with an improved therapeutic effect is now a major focus. GCs are crucial for anti-inflammatory therapy. Previous efforts of designing of glucocorticoid structures with an increased therapeutic activity have been focussing on transcriptional ways of dissociating anti-inflammatory transcriptional repression from their side effects or by the combination of drugs like prednisolone and dipyridamole (an antithrombotic drug) in order to demonstrate a dissociated activity profile without the side effects. None of the works so far have shown full success. The bottleneck in translational research into glucocorticoid resistance is the lack of specific and reliable biomarkers that can be obtained from blood samples. Alternatively, it has been observed that in the presence of interleukins 2 and 4 (IL2/4), T cells show reduced sensitivity to glucocorticoids. In our experiments we will thus be analysing GC induced transcriptome response in IL 2/4 treated T cells in order to isolate biomarkers of resistance that can be targeted for therapy.
AIMS AND OBJECTIVES
Our aim is to seek and isolate a biomarker of steroid resistance, using ex-vivo gene expression profiling of lymphocytes from healthy human volunteers and to analyse GC induced transcriptome response in IL 2/4 treated T-lymphocytes to target them for therapy and facilitate development of better drugs with lower side effects.
PLAN OF INVESTIGATION
T lymphocytes would first be isolated from blood samples taken from a minimum of five healthy volunteers. These T lymphocytes would then be stimulated with IL 2 and IL 4 for 48 hours and then with synthetic glucocorticoid dexamethasone for 6 hours. Gene expression profiling would then be carried out on these samples using gene microarray experiments.
Future investigations would involve the use of results obtained in the present experiment, to build new hypothesis with identified overlapping set of gene networks. The role of MAPK pathway in IL 2/4 induced resistance to glucocorticoids would also be determined. The knowledge obtained from translational research and the development of cost effective simple assays, will allow the development of better understanding and treatments of resistance to glucocorticoids and perhaps facilitate isolation of compounds that will target the identified pathways and result in the switch to GC sensitivity.