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Molecular Control of Immune/Inflammatory Responses: Interactions Between Nuclear Factor-B and Steroid Receptor-Signaling Pathways

Laboratory of Signal Transduction, Molecular Endocrinology Group, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709

	Abstract

Top Abstract I. Introduction II. Nuclear Factor-{kappa}B (NF... III. Steroid Hormones/Receptors:... IV. NF-{kappa}B and GR... V. Interactions Between NF... VI. Summary/Conclusions Addendum/Erratum References

 

I. Introduction

II. Nuclear Factor-B (NF-B)

A. Introduction

B. NF-B is a dimeric transcription factor

C. The regulatory subunit IB is an inhibitor of NF-B

D. Activation and function of NF-B

E. The transcription factor NF-B interacts with multiple transcription factors and transcriptional co-factors

F. Transgenic animals suggest a complex role for NF-B family members in immunity and development

III. Steroid Hormones/Receptors: Glucocorticoids and the Glucocorticoid Receptor (GR)

A. General background

B. Glucocorticoid mechanism of action: the GR

C. Glucocorticoid physiology

D. GR/NF-B interactions

E. GR interacts with other transcription factors and transcriptional cofactors

IV. NF-B and GR Antagonism: Physiological Significance?

V. Interactions Between NF-B and Other Steroid Hormone Receptors

A. Androgen receptor (AR)

B. Estrogen receptor (ER)

C. Progesterone receptor (PR)

VI. Summary/Conclusions

	I. Introduction

Top Abstract I. Introduction II. Nuclear Factor-{kappa}B (NF... III. Steroid Hormones/Receptors:... IV. NF-{kappa}B and GR... V. Interactions Between NF... VI. Summary/Conclusions Addendum/Erratum References

 
THE mammalian immune and inflammatory responses are complex physiological processes that are critically important to the homeostasis and ultimate survival of an organism. Their coordinate regulation must be assured to allow appropriate and timely immune reaction without an overreaction that might cause damage to the host. Two cellular signaling pathways that have been identified as important regulators of immunity and inflammation are the nuclear factor (NF)-B and glucocorticoid-mediated signal transduction cascades.

Glucocorticoid receptor- (GR) and NF-B are inducible transcription factors with diametrically opposed functions in the regulation of immune and inflammatory responses. NF-B is known to mediate the transcriptional activation of a variety of cytokines and cytokine-induced genes involved in immunity. GR, long known to function as a suppressor of immunity and inflammation, inhibits the expression of many of the same cytokines and cytokine-induced genes that are activated by NF-B. However, because these genes have no identifiable glucocorticoid responsive elements (GRE) either within their promoters or their intragenic regions, the mechanism by which glucocorticoids repress these genes was unclear.

Recent research indicates that GR and NF-B physically interact (1, 2, 3) and function as mutual transcriptional antagonists (1, 4). These findings have rekindled interest in, and encouraged re-examination of, these interactions as the basis for mutual functional antagonism of GR and NF-B. While physiological processes in a variety of tissues and organ systems are regulated by glucocorticoids and are likely to be modulated by GR interactions with the ubiquitous NF-B, the immune/inflammatory response has been the primary focus of current research on NF-B/GR antagonism. Since both these transcription factors are known to be potent regulators of the immune system, elucidation of the mechanisms by which GR and NF-B negatively interact not only provides the basis for understanding their role in the precise control of normal immune response, but also opens up the possibility of novel therapeutic intervention in immune pathology.

The purpose of this review is to describe our current knowledge of NF-B and GR-signaling pathways, including overviews of the NF-B and steroid hormone receptor families of transcription factors and their regulatory proteins, their general mechanisms of action, the cell types in which they function, and their regulated genes. Special consideration will then be given to the newest findings concerning the interactions between these two transcription factors, and the physiological significance of these findings in terms of immunity and inflammation. Finally, emerging data concerning interactions between NF-B and other steroid hormone receptors will be considered.

	II. Nuclear Factor-B (NF-B)

Top Abstract I. Introduction II. Nuclear Factor-{kappa}B (NF... III. Steroid Hormones/Receptors:... IV. NF-{kappa}B and GR... V. Interactions Between NF... VI. Summary/Conclusions Addendum/Erratum References

 
A. Introduction
NF-B was identified more than a decade ago by Sen and Baltimore (5) as an enhancer-binding protein controlling Ig-light chain gene expression in B cells. This seminal paper identified the NF-B protein as an activity in electrophoretic mobility shift assays that specifically retarded the migration of -light chain enhancer DNA containing the 10-mer sequence 5'-GGACTTCC-3'. Research that followed this initial finding indicated that this -enhancer binding activity was only found in nuclear extracts from B cells that were at an appropriate stage to express Ig -light chain, and showed that NF-B was an essential factor for the function of the -light chain enhancer (6, 7). Because of this apparent B cell specificity, NF-B was considered to be a tissue-restricted transcription factor. However NF-B was later found to be expressed not only in B cells, but in other immune cells as well. In non-B cell lymphocytes, NF-B was found to be an inactive protein sequestered in the cytoplasm, which could be activated with phorbol ester and lipopolysaccharide, rather than as the constitutively active nuclear protein seen in B cells (8, 9, 10). Of particular interest was the finding that, in T cells with latent human immunodeficiency virus (HIV) infection, NF-B could be activated by a variety of compounds that cause lymphokine secretion. Once activated, NF-B enhanced the expression of HIV in these cells (10). This exciting finding led to a proliferation of research in the fledgling field of NF-B research.

Today, NF-B is recognized as a ubiquitously expressed transcription factor that can be activated in a wide variety of cell types. Despite the discovery that NF-B enjoys a wide pattern of cellular expression, the particular importance of NF-B to cells of the immune system remains. In immune cells, NF-B has now been shown to positively regulate the expression of a wide variety of genes involved in mammalian immune and inflammatory responses, including cytokines, cell adhesion molecules, complement factors, antiapoptotic factors, and immunoreceptors (for a detailed list, refer to Table 1).

View this table: [in this window] [in a new window]   Table 1. Genes transcriptionally regulated by NF-B

B. NF-B is a dimeric transcription factor

View larger version (35K): [in this window] [in a new window]   Figure 1. The general mechanism of NF-B activation. Unactivated NF-B heterodimers are retained in the cytoplasm by the inhibitory NF-B subunit IB. Cytoplasmic NF-B is associated with the catalytic subunit of protein kinase A (PKAc). In response to an activating signal, IB kinase (IKK) phosphorylates IB, targeting it for ubiquitination and subsequent degradation via the proteasome, while PKAc phosphorylates the p65 subunit of of NF-B. This activation process exposes a nuclear localization signal on p65, allowing the activated NF-B heterodimer to move into the cell nucleus where it interacts with B-responsive elements in the chromatin and modulates gene transcription.

View larger version (37K): [in this window] [in a new window]   Figure 2. Schematic overview of the Rel family of transcriptional activators. All family members contain a highly conserved Rel homology domain of approximately 300 amino acids, which contains sequences required for dimerization, nuclear localization, and DNA recognition. Dorsal and Dif are Drosphila gene products, while all others identified are mammalian. The p50 and p52 transcriptional activators are proteolytically cleaved from the p105 and p100 precursor molecules, respectively. They lack the transactivation domain that is present in all other Rel proteins.

Since p50 and p52 lack the transactivation domain found in the other Rel family members, it is not clear whether homodimers of these proteins have any transcriptional activity in native environments. One study (38) indicates that p52 homodimers can directly transactivate when associated with the IB-like protein bcl-3. In general, however, it is believed that an NF-B dimer must possess at least one Rel transactivation domain to activate transcription in vivo. The most widely accepted function for p50 and p52 homodimers is as transcriptional repressors that block NF-B sites in the DNA from transcriptionally active NF-B dimers (39). Evidence for a repressive function of p50 homodimers was found in human T cells, where p50 homodimers constitutively expressed in the nucleus bind DNA and repress NF-B-dependent transcription. This repression was overcome by overexpression of bcl-3, which has been shown to prevent binding of p50 homodimers to NF-B sites in vitro and to activate NF-B-mediated transcription in cultured cells (35, 40). Whether this observed bcl-3-mediated increase in transcription is entirely due to its repressive effect on p50 homodimers is unclear, however, because bcl-3 can function as a transactivator at NF-B sites when it trimerizes with otherwise transcriptionally inactive p52 homodimers (38).

The composition of NF-B dimers determines sequence specificity by combining different DNA recognition loops in the Rel homology domains of different subunits. For example, p65 homodimers preferentially bind DNA enhancer motifs that are not well recognized by p65/p50 heterodimers or p50/p50 homodimers (31). Such variety in dimerization may contribute to the cell type specificity of NF-B response. For example, cell type differences in NF-B activity might depend on which subunits of NF-B are expressed (and in what ratios) and on the NF-B activating signals to which these different cells respond. Dimer composition may also affect interactions with inhibitory/regulatory proteins such as the various members of the IB family of proteins (22, 28, 32, 35, 41, 42, 43). The ability to modulate gene expression in a cell type-specific or temporally regulated manner by altering the expression patterns of various Rel subunits affords NF-B-mediated signal transduction the potential for both great flexibility and tight control.

Several gene knockout mice that are deficient in one or more specific NF-B subunits have been generated. The phenotypes of these transgenic animals vary widely and will be discussed in detail in a later section of this review. When taken as a whole, the information gleaned from NF-B knockouts solidifies the importance of NF-B to immune function, inflammatory response, and development.

p65/p50: the "Classical" NF-B. The prototypical and most thoroughly studied NF-B dimer is the p65/p50 heterodimer. When these two subunits are coexpressed at comparable levels in the cell, their affinity for each other is higher than the affinity of either homodimer, and therefore the heterodimer is preferentially formed (22). This heterodimer has a high affinity for the consensus NF-B DNA sequence 5'-GGGRNNYYCC-3' (22, 28) and is generally considered to be the predominant, inducible (i.e., activatable by extracellular signals, nonconstitutive) form of NF-B in most cells. For this reason, throughout the remainder of this text, the term NF-B will refer to the "classical" NF-B p65/p50 heterodimer unless otherwise specified.

As the transcriptionally active subunit of the prototypical NF-B heterodimer, the structure of the p65 (Rel A) polypeptide has been extensively studied. The transactivation function of p65 has been mapped to a 120-amino acid region of the C terminus, which contains two distinct and independent transactivation domains (27). The first domain comprises 30 amino acids at the extreme C terminus and is known as TA₁, whereas TA₂ is located within the adjacent 90 amino acids of the C-terminal region of p65. These transactivation domains share a common sequence motif that is induced to form an -helical conformation when bound to target molecules (27, 44). Phorbol ester-induced phosphorylation of the TA₂ domain between amino acids 442 and 470 has been shown to enhance the transcriptional activity of p65 (44). Recent crystallographic data (45) have also revealed the structure of DNA-bound p65/p50. These data demonstrate that the subunits dimerize via ß-sheet sandwich structures in the C-terminal dimerization domains, and contacts with the DNA consensus sequence are made using loops from the ends of the N- and C-termini. Beg et al. (46) have shown that the nuclear localization sequence located in the middle of the p65 polypeptide is the target for interaction with the inhibitory subunit IB.

C. The regulatory subunit IB is an inhibitor of NF-B
Not long after the initial identification of NF-B, Baeuerle and Baltimore (47) identified an inhibitory protein of 60–70 kDa that specifically associates with NF-B dimers, forming a trimer that cannot bind DNA and is retained in the cell cytoplasm. They termed this protein IB, for inhibitor of NF-B. More recently, there have been multiple IB proteins identified with different specificities for NF-B dimers and different cell type distributions. This family of IBs now includes IB, IBß, IB, IB-R, Bcl-3, p50/p105(C-terminus), p100/p52(C-terminus), and the Drosophila melanogaster proteins Cactus and (probably) Relish (14, 15, 16, 17, 21, 24, 28, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57). The IB family of proteins is outlined schematically in Fig. 3. There is also evidence that the yeast Saccharomyces cerevisiae expresses an IB-like protein (23). IB and IBß both preferentially interact with dimers containing p65, but they are responsive to different signaling pathways in the cell and may be differentially expressed and employed in different cell types (28, 58). The p100/p52 and p50/p105 molecules contain both an NF-B-like and an IB-like region. The IB region can be cleaved from the NF-B region to yield two functional and distinct molecules or can act intramolecularly to inhibit the function of the NF-B portion of the intact molecule.

View larger version (29K): [in this window] [in a new window]   Figure 3. The IB family of regulatory proteins. All family members contain three to seven ankyrin repeat domains, which mediate associations with NF-B. These proteins also contain a C-terminal PEST sequence and a variable acidic region in the C terminus. p105 And p100 are precursor proteins that have IB-like structure and activity in the C-terminal portion of the molecule.

D. Activation and function of NF-B
Constitutive NF-B activity in B cells is largely attributable to the p50-cREL heterodimer (28, 59, 60). However, in most other cell types, NF-B activity is largely inducible, and the prototype p65/p50 NF-B is retained in the cytoplasm of an unstimulated cell by its association with IB. When an extracellular stimulus, such as a cytokine (e.g., tumor necrosis factor-), viral protein, lipopolysaccharide, or an oxidative stressor activates the NF-B-signaling pathway, IB is phosphorylated at serines 32 and 36 (61). Phosphorylated IB is a target for ubiquitination at lysines 21 and 22 (21, 62, 63), which leads to rapid removal of IB via the proteosomal degradation pathway and, consequently, unmasking of the p65 nuclear localization sequence and movement of NF-B to the nucleus of the cell (64, 65). [There are two different but related ser/thr kinases, identified by several groups and now termed IKK-1 and IKK-2, that are generally accepted to be responsible for the inducible phosphorylation of IB (66, 67, 68, 69). A very recent study indicates that these IKKs have a greater phosphorylation activity on IB when it is bound to NF-B, thus explaining the ability of active IKK and free IB to coexist within a cell and regulate NF-B (70). Regulation of the activity of these kinases provides an indirect mechanism for the regulation of NF-B activation.]

Although not yet completely understood, the activation of NF-B involves not only the dissociation of IB and translocation to the nucleus, but also phosphorylation of the p65 subunit by PKAc. This PKAc is associated with the NF-B/IB heterotrimer in the cytoplasm and is believed to be held inactive by the association with IB. Once IB dissociates, the PKAc becomes active, phosphorylates serine 276 of p65 (at a consensus PKA site in the Rel domain of the protein), and dramatically increases the transcriptional activity of NF-B (71). Recent studies show that this PKA-mediated phosphorylation of p65 is required for recruitment of CREB-binding protein and the closely related p300 (CBP/p300), a transcriptional coactivator discussed below, by NF-B p65 (72). There is evidence that other protein kinases may also be important regulatory proteins for p65. For example, casein kinase II has been shown to mediate the interleukin-1 (IL-1)-stimulated phosphorylation of p65 in fibroblasts and hepatoma cells (73). It is possible that the phosphorylation required for p65 activation is mediated by various kinases in a cell type-specific manner.

The classical p65/p50 NF-B heterodimer is a major inducer of inflammatory genes, and there are a large number of proinflammatory, extracellular signals that can activate NF-B. These stimuli include viruses [such as herpes simplex virus and adenovirus] and viral proteins; bacterial products like lipopolysaccharide; inflammatory cytokines like TNF, IL-1, and IL-2; and a variety of DNA-damaging agents and oxidative stressors (reviewed in Ref. 22). Once activated, the nuclear NF-B binds to cognate NF-B sites in the chromatin and modulates gene expression. A variety of NF-B-responsive genes involved in immune response and inflammation due to these extracellular proinflammatory signals are induced by activated NF-B. New synthesis of IB causes retention of NF-B in the cytoplasm and attenuation of NF-B-mediated transcriptional activation and provides a feedback mechanism for modulating the extent and duration of inflammatory responses by the cell.

Genes that are known to be regulated by NF-B are listed in Table 1. In addition to the well studied role of NF-B in the immune system (including lymphocyte development, inflammatory response, and host defense mechanisms), more recent work has also implicated NF-B as an important regulator of apoptosis and embryonic development (74).

E. The transcription factor NF-B interacts with multiple transcription factors and transcriptional cofactors
With the advent of yeast two-hybrid screening techniques, a variety of enhancer binding transcription factors have been found to interact with a variety of nuclear proteins. Many of these interacting nuclear proteins have been identified as transcriptional cofactors, which have the ability to either enhance or repress the transactivation function of transcription factors (for example, see Refs. 75, 76, 77). As knowledge in the field of transcriptional regulation expands, a consensus is forming that transcription factors do not usually function alone as independent activators of transcription. Rather than the classical view that these proteins bind DNA and then directly contact the basal transcriptional machinery, the current understanding is that DNA-binding transcription factors often interact simultaneously with multiple nuclear proteins that are either transcription factors themselves or accessory proteins. The resulting large transcription-regulatory complexes interact with the basal transcription apparatus to modulate gene expression. The combination of transcription-regulatory proteins "piled up" on the promoter of a gene allows for cell type, developmental, promoter, and stimulus-driven specificities in gene expression regulated by a transcription factor. Examples of such promoter complexes are depicted in reports by Merika et al. (75), which deals with the "enhanceosome" complex, and Shibata et al. (78), which examines complexes containing members of the nuclear receptor superfamily.

As the list of proteins that interact with NF-B and alter its transactivation function grows, it is becoming clear that NF-B is no exception to this general rule of transcription-regulatory complex formation. The complexity and potential flexibility of NF-B effects in the cell are suggested both by the broad range of NF-B-interacting proteins that have been identified and by the fact that some of these proteins appear to have context-dependent effects, acting antagonistically with NF-B in some cases and synergistically in others. The p65 subunit of NF-B has been shown to directly interact in vitro with the general transcription factors TFIIB and TATA-binding protein (TBP) (79). A functional interaction between TBP and p65 (as well as c-Rel), which activates NF-B-dependent transcription, has been shown in cultured insect and mammalian cells (79, 80, 81, 82). These data reveal a potentially important mechanism for NF-B-mediated regulation of the basal transcriptional apparatus.

As previously mentioned, Bcl-3 was originally identified to be an inhibitory subunit of NF-B and is now known to function as a specific transcriptional coactivator of the p52 and p50 members of the Rel family (38). HMG-1 (high mobility group-1) coactivates NF-B-mediated transcription, and SRC-1 specifically coactivates NF-B p50 (83, 84). Members of the CCAAT-enhancer binding protein (C/EBP) family of bZIP proteins (C/EBP, C/EBPß, and C/EBP) bind multiple members of the Rel family (p65, p50, and Rel). While C/EBP and C/EBP function as corepressors (85), C/EBPß can either repress or synergize with NF-B. On the acute-phase response element of the rat angiotensinogen gene, NF-B and C/EBPß antagonize by competing for overlapping binding sites in the DNA (86), while on the promoters for IL-6 and IL-8, NF-B and C/EBPß activate transcription synergistically apparently by cooperative DNA binding (87, 88). Members of the signal transducers and activators of transcription (STAT) family of transcriptional regulators have also been shown to interact with NF-B. STAT6, normally a coactivator protein, has also been shown to function as a corepressor for NF-B, presumably by competing with NF-B for overlapping binding sites in the promoter region of the E-selectin gene (89). Conversely, STAT1 and NF-B have been shown to synergistically activate transcription of proinflammatory genes in mouse fibroblasts, although the mechanism of this interaction is not well defined (90). The transcriptional coactivator protein CBP/p300 has been shown to interact with many transcription factors, and its interactions with p65 are a focus of much current research. Not only has CBP/p300 been shown to be a coactivator of p65-mediated transactivation (72, 91), but the specific interaction between p65 and CBP/p300 has been shown to be required for assembly of the "enhanceosome," an assembly of transcriptional cofactors that synergize and mediate transcription of interferon-ß (75).

In addition to interaction with a variety of transcriptional cofactors, NF-B is also capable of interaction with other nuclear proteins that are themselves activatable transcription factors. An example of such an interaction is found between NF-B and AP-1 (Fos-Jun). When these two dimeric transcription factors interact via the Rel homology domain of p65 and the bZIP region of Fos and Jun, the transactivation function of each factor is potentiated (92). NF-B and the GR provide another example of NF-B/transcription factor interaction. For years, the mechanism by which glucocorticoids exerted their potent antiinflammatory effects was not understood. It is now clear that NF-B and the ligand-dependent GR can directly interact. In contrast to the potentiation of function with NF-B and AP1, the result of the GR/NF-B interaction is usually mutual transcriptional antagonism. (For angiotensinogen and probably several other hepatic acute-phase reactant genes, however, it appears that NF-B and GR positively interact at the acute phase response element to activate transcription (93, 94, 95).) NF-B/GR antagonism provides a fundamental mechanism for the regulation of many immune and inflammatory responses and therefore will be discussed in detail in a separate section of the text.

Other members of the steroid receptor family have also been found to interact with NF-B. Androgen receptor (AR) and NF-B have been shown to be mutual antagonists in cultured cell systems, and evidence for such antagonism in rat liver in vivo has also been demonstrated (4, 96). For progesterone receptor (PR) and estrogen receptor (ER), there is also evidence of negative interaction with NF-B, although the reciprocal antagonism of NF-B p65 by PR and ER appears to be a cell type-specific phenomenon (4, 97, 98).

F. Transgenic animals suggest a complex role for NF-B family members in immunity and development
As mentioned earlier, the generation of transgenic mice with deficiencies in specific NF-B subunits has underscored the importance of this family of transcription factors in development and the immune system. The phenotypic severity of the different knockout animals ranges from deficiencies in specific immune responses to embryonic lethality. The most dramatic Rel family knockout is p65/RelA, whose phenotype is embryonic lethal due to massive apoptosis in the liver. In addition, cultured embryonic fibroblasts from these animals demonstrate profound deficiencies in inducible NF-B activity, but unaffected basal NF-B activity. These findings point to p65 as being critical for activatable, but not constitutive, NF-B activity and suggests a significant role for p65 in both embryonic development and regulation of apoptosis (99).

The remainder of Rel knockout animals display normal embryonic development, but all show serious impairment of immune function. Immune deficiencies vary with each Rel protein knocked out, and in different cell types with a single type of Rel knocked out, supporting the idea of cell type specificities in the function and expression of the Rel family members. Knockouts of c-Rel (100) have profoundly impaired T cell function and proliferation as well as lymphoid hyperplasia. Knocking out Rel B results in multiorgan inflammation, T cell deficiencies, an inability to differentiate dendritic cells that cannot be compensated by expression of other Rel subunits, myeloid hyperplasia, and extramedullary hemopoiesis (28, 100). p50/p105 Knockouts exhibit B cells deficient in antibody production and proliferation (74). Interestingly, p105 knockouts (which express functional p50 but no inhibitory portion of the precursor molecule and consequently have enhanced p50 activity) suffer chronic organ inflammation, susceptibility to bacterial infection, and lymphoid hyperplasia. These different and opposing phenotypes in B cells, T cells, and macrophages suggest that the p50 homodimer functions as either a transactivator or transrepressor in a cell type-specific manner (101). Knockouts of p100 (which express functional p52 but no inhibitory portion of the precursor) die in the early postnatal period from gastric hyperplasia and exhibit overactive immune responses such as lymphocyte overproliferation and increased cytokine production (102). Animals that lack bcl-3 expression can neither mount T helper responses nor antigen prime B or T cells (103).

The creation of double knockouts in Rel family members has revealed some unexpected phenotypes and suggests that Rel members may have redundant functions, again speaking to the importance of maintaining Rel function for proper immune response and development. For example, when p50 and p52 are both disrupted, mice are incapable of maturing osteoclasts and B cells. This leads to a phenotype of severe osteopetrosis and abnormal splenic/thymic architecture (104). Double p50/RelB knockouts die in the early postnatal period from massive organ inflammation that is far more severe than seen in a single-knockout animal (105).

	III. Steroid Hormones/Receptors: Glucocorticoids and the Glucocorticoid Receptor (GR)

Top Abstract I. Introduction II. Nuclear Factor-{kappa}B (NF... III. Steroid Hormones/Receptors:... IV. NF-{kappa}B and GR... V. Interactions Between NF... VI. Summary/Conclusions Addendum/Erratum References

 
A. General background
Steroid hormones have long been recognized to be essential modulators of a wide array of cellular processes and intercellular communication, and the prominent role of steroids in the normal physiology and development of an animal have made them a focus of endocrine research since the middle of this century (reviewed in Ref. 106). While there is some evidence that "non classical" mechanisms of steroid hormone action may be important in special circumstances (106, 107, 108), the widely accepted, classical mechanism by which these lipophilic cellular signaling molecules are now understood to function is via a cognate cellular receptor molecule. These receptors bind steroid hormones and function as ligand-dependent modulators of gene expression. Until the mid-1980s, the majority of the knowledge concerning steroid hormone receptors was derived via standard biochemical techniques where the receptors were isolated and characterized using radiolabeled ligands (109). However, the field of steroid hormone receptor biology has rapidly accelerated since the successful cloning of the receptors (e.g., Refs. 109, 110, 111, 112, 113) and the subsequent application of molecular biological techniques to gain an understanding of steroid hormone receptor function. The explosion of data in the steroid hormone receptor field over the past 15 yr has afforded us an understanding of the steroid receptors, their similarities and differences, and their role in transducing steroid hormone signals into cellular responses.

The steroid hormone receptors are a family of structurally similar, modular proteins that include GR forms and ß, PR forms A and B, mineralocorticoid receptors (MR), and ER forms and ß. Steroid hormone receptors belong to the nuclear receptor superfamily. All members of the nuclear receptor superfamily are believed to share a common ancestry, and all function via a similar mechanism of action, as ligand-dependent, DNA-binding transcription factors that interact with the basal transcriptional apparatus. Other classes of nuclear receptors include the thyroid, retinoid, and orphan receptors (114). Orphans are receptors with structural/sequence similarity to other nuclear receptors but for whom a ligand and/or function has not been identified.

Members of the steroid/thyroid/retinoid receptor superfamily share a variable amino-terminal transactivation domain, a central and well conserved DBD, and a moderately conserved carboxy-terminal domain responsible for binding ligand (110, 114). Within the steroid hormone receptor family, ER is the least conserved member. It has a highly homologous DBD but differs significantly from the other steroid receptors in primary structure outside this region. Because ER differs from the other steroid receptors and shares homology with thyroid hormone receptors, it is considered by some to be a separate subfamily within the steroid hormone receptor family. A schematic diagram comparing the primary structure of the steroid hormone receptors is found in Fig. 4.

View larger version (40K): [in this window] [in a new window]   Figure 4. Schematic diagram of the steroid hormone receptors: primary structure. A, The general structure of a steroid hormone receptor includes a variable transactivation domain in the amino terminus, a well conserved central DBD, and a moderately conserved steroid-binding domain in the carboxy terminus. B, Primary structure of each human steroid hormone receptor. GR and GRß are the result of alternative splicing of the same mRNA transcript. PR A and B forms result from alternate sites of transcription initiation. ER and ERß are believed to arise from separate genes. [Data are derived from Refs. 111–113 and 264–269.]

Glucocorticoids are lipophilic steroid hormones and, as such, are found in circulation associated with "carrier" proteins such as corticosteroid-binding globulin (in the case of endogenous steroids) and albumin (for both endogenous and synthetic steroids) (116). The most widely accepted mechanism for glucocorticoid entry into the cell is by free diffusion of these lipophilic molecules across the lipid bilayer of the cell into the cytoplasm. However, there is some evidence (107, 108, 117) suggesting that glucocorticoid entry into cells is a regulated process involving specific membrane-associated receptors distinct from the classical intracellular GRs that are discussed in detail here. These membrane-associated receptors are believed to signal via G proteins and may also mediate the rapid, nongenomic effects of glucocorticoids that have been observed, particularly in neuronal tissues (108, 118).

In the absence of glucocorticoid hormone, classical GR is retained in the cytoplasm in an inactive (i.e., DNA binding-incompetent) state by its association with the regulatory heat shock proteins hsp 90 and hsp 56 (119, 120). The inactive conformation of GR exhibits high affinity for ligand. Once inside the cell, glucocorticoid binds the high-affinity cytoplasmic GR and induces a poorly understood process known as receptor activation. Activation of GR involves a change in receptor conformation, dissociation from regulatory heat shock proteins, and hyperphosphorylation. Activated receptor rapidly translocates to the cell nucleus and binds to specific DNA sequences as a homodimer (121, 122, 123, 124). The DBD/dimerization domain of GR has been well characterized (125) and consists of two zinc ions coordinated with eight cysteine residues to form two peptide loops called zinc fingers. Each zinc finger is followed by an amphipathic -helix. GR DBDs bind cooperatively to specifically spaced target half-sites in the DNA, and this specific DNA association induces receptor dimerization. The subunits of GR then interact with adjacent major grooves of the DNA via their amphipathic -helices. The specific palindromic sequences in the promoter regions of glucocorticoid-responsive genes to which activated GR dimer binds are known as GREs, or glucocorticoid responsive elements. The consensus GRE sequence is: 5'- GGTACA nnnTGTTCT-3' (126). Once bound to a GRE, homodimeric GR induces or increases transcription of the target gene, presumably by interacting with the basal transcription apparatus (127). In addition to its function as a ligand-dependent activator of transcription, GR also functions as a negative regulator of transcription in a specific subset of glucocorticoid-responsive genes. Many of these glucocorticoid-repressed genes contain a negative GRE (nGRE). nGREs are less well defined than positive GREs, and their gene-repressive function appears to be context dependent. The mechanism of action for GR on an nGRE likely involves displacement of a positive regulatory protein from the promoter (126, 128, 129).

Recently, research in our own and other laboratories has focused on a variant form of human GR, termed GRß, which arises from alternative splicing of the GR mRNA transcript. Originally cloned along with the GR isoform in 1985 (130), GRß was largely ignored because it was not found to bind ligand or activate transcription of glucocorticoid-responsive reporter genes (130, 131). GRß is identical to GR through the first 727 amino acids but diverges in the carboxy terminus, where it lacks the last 50 amino acids found in GR but has an additional 15 nonhomologous amino acids past the point of divergence. As with GR, GRß is widely expressed in adult and fetal human tissues but differs from GR in that it is localized to the cell nucleus independent of the presence of ligand. The most recent results from transient transfection assays demonstrate that human (h) GRß functions as a dominant negative regulator of hGR transactivation (Refs. 132, 133 ; R. H. Oakley and J. A. Cidlowski, unpublished observations). Although the extent to which hGRß functions as a dominant negative in vivo is not yet clear (132, 133, 134), these studies suggest that tissue- or cell type-specific expression patterns of hGRß and hGR may function to modulate the glucocorticoid responsiveness of tissues.

C. Glucocorticoid physiology
Glucocorticoids are synthesized in the zonae fasciculata/reticularis of the adrenal cortex and released into circulation in response to a wide range of stressful stimuli (e.g., starvation, pain, surgery, trauma, emotional stress, extreme heat or cold, and cellular damage). Their release is orchestrated by the hypothalamic-pituitary-adrenal (HPA) axis, where hypothalamic CRH acts on the pituitary to cause release of ACTH, and ACTH then stimulates the adrenal gland to release glucocorticoid.

Glucocorticoids have a vast array of functions within the body, and an organism cannot survive without them (135). One of the first identified functions of glucocorticoids, and the one from which their name was derived, is as an important regulator of intermediary metabolism. Their main role in intermediary metabolism is to up-regulate the process of gluconeogenesis in the liver, kidney, and skeletal muscle, which results in elevated levels of blood glucose. Glucocorticoids also modulate fat, protein, and glutamine metabolism, as well as bone turnover. The importance of glucocorticoids in regulation of metabolism is underscored by their role in the "metabolic syndrome," a collection of metabolic disorders including hyperlipidemia, hyperinsulinemia, insulin resistance, and hypertension, which is believed to be caused in part by hypersensitivity to cortisol (136, 137). Immune cell apoptosis is also known to be a glucocorticoid-regulated process (138), and there are glucocorticoid effects on the central nervous system that are as yet poorly understood. In addition to broad effects in the adult organism, glucocorticoids are known to have profound effects on fetal development and parturition and are especially important to fetal lung maturation.

Some of the most dramatic and clinically relevant effects of glucocorticoids are those on the immune system. Glucocorticoids are potent suppressors of immune response and inflammation. These characteristics have made synthetic glucocorticoids the drug of choice for therapeutic intervention in a broad range of autoimmune and inflammatory disorders. The benefits of glucocorticoids in treatment of rheumatoid arthritis were first recognized nearly 60 yr ago (139). Currently, a variety of synthetic glucocorticoids are employed in the treatment of systemic lupus erythematosus, inflammatory bowel disease, psoriasis, eczema, and asthma (3, 140, 141, 142). They are also used to suppress the host immune system and prevent rejection during organ transplantation. Interestingly, glucocorticoids enjoyed widespread use in the clinic, based primarily on empirical evidence of their efficacy, even before much information was available concerning their mechanisms of antiinflammatory and immunosuppressive action. Today, multiple mechanisms of antiinflammatory and immunosuppressive glucocorticoid action have been put forth in the literature, but the relative importance of these mechanisms is still unclear, and there is a good possibility that additional mechanisms are yet to be discovered.

The general mechanisms by which glucocorticoids exert their cellular effects have been elucidated over the past 15 yr, yet specifically understanding the immunosuppressive mechanism of glucocorticoid action has proven to be a conundrum. Although glucocorticoids are capable of reducing the number of lymphocytes both by redistribution of peripheral lymphocytes to the lymph nodes and by inducing lymphocyte apoptosis and growth suppression in the thymus (143, 144, 145), these functions are insufficient to explain the potent immunosuppressive action of glucocorticoids. Glucocorticoids are also known to suppress the expression of proinflammatory cytokines, which are key regulators of the immune response. As outlined above, glucocorticoids are now known to act via a cytosolic receptor that functions as a ligand-dependent transcription factor, modulating the expression of genes with GREs in their promoters. However, the majority of proinflammatory genes (such as cytokines) that are suppressed by glucocorticoids have no such responsive elements in their promoters, which might explain the role of glucocorticoids in their regulation. It was not until the role of NF-B in the immune system and interactions between NF-B and GR were identified that feasible mechanisms for the powerful glucocorticoid-mediated immune suppression could be proposed.

D. GR/NF-B interactions
GR activation results in altered expression of many genes that affect a variety of cellular processes. A listing of genes that are regulated by glucocorticoids can be found in Table 2. Brief inspection of Table 2 indicates that many genes involved in immune and inflammatory responses in the cell, particularly proinflammatory cytokines and cell adhesion molecules, are modulated by glucocorticoids.

1. Evidence for direct NF-B/GR interactions. It has been determined that GR and the p65 subunit of NF-B physically interact, and the consensus is that this (presumably direct) physical interaction involves the Rel homology domain of p65 and the DBD of GR (Refs. 1, 2, 3, 4, 146 and our unpublished observations). It has also been demonstrated that the physiological antagonism of GR and NF-B is based on a mutual transcriptional antagonism, rather than an alteration in the expression levels of these two transcription factors in the cell (4). Initial hypotheses concerning NF-B/GR antagonism focused on the possibility that the physical interaction between these two factors impaired the DNA-binding functions of each and therefore blocked the ability of each to activate transcription. However, subsequent studies on this question have provided contradictory results, and little conclusive evidence exists suggesting that the DNA-binding function of either transcription factor is impaired under conditions in which a mutual transcriptional antagonism occurs (2, 3, 147, 148). In fact, we have observed that NF-B p65 is capable of blocking GR transactivation of a reporter gene (4), but cannot interfere with homologous down-regulation of GR (a process that requires GR to bind intragenic GREs; J. C. Webster and J. A. Cidlowski, unpublished observations). This argues against a mechanism of repression that involves a simple blockage of DNA binding.

To further address the question of mutual transcriptional repression, other possible mechanisms that might drive the NF-B/GR antagonism were considered by several groups. One approach taken by our group was to functionally dissect both the GR and NF-B to determine which subunits/domains of these transcription factors are required for mutual transcriptional repression. A summary of these findings is presented in Fig. 5. First, it was determined that the p65 subunit, but not the p50 subunit, of the NF-B heterodimer could repress GR-mediated transactivation of a reporter gene. The ability of a series of GR deletion mutants (missing portions of the ligand-, DNA binding-, or transactivation domain) to negatively interact with p65 was then assessed. These experiments showed that multiple domains of GR are required to repress p65 transactivation. While the importance of the DBD of GR to the p65-GR interaction was already known (1, 3), the data demonstrated that deletion of either one of the two zinc fingers that are found in this domain, as well as deletion of the steroid-binding domain or large portions of the transactivation domain of GR, also abolishes the repressive effect of GR on NF-B. Interestingly, the reciprocal repression of GR by p65 was less selective, since all transcriptionally active mutants of GR were repressed by p65. These findings suggested that the reciprocal physiological antagonism between NF-B and GR might not be based on entirely reciprocal mechanisms of transcriptional antagonism. One mutant GR that was examined is both constitutively nuclear and transcriptionally active (since it has no ligand-binding domain and is ligand independent), yet it is still repressed by p65, suggesting that the mechanism of p65-GR antagonism does not involve cytoplasmic sequestration of the factors, but rather occurs within the nuclear compartment (4).

View larger version (16K): [in this window] [in a new window]   Figure 5. Role of GR domains in p65/GR mutual transcriptional antagonism. Any transcriptionally active mutant of GR is repressed by p65. However, deletions in the GR DBD or the steroid-binding domain, as well as large deletions of the transactivation domain, all prevent GR-mediated repression of p65 transactivation.

One possible mechanism by which cofactors might mediate GR-p65 antagonism is as a target for competition. A limiting cellular pool of a common transcriptional cofactor, required by both GR and NF-B for full transactivation, might result in repression of one transcription factor’s basal or induced activity when the other transcription factor was activated and had bound up all available cofactor. While no one cofactor has been identified that would occupy this role, one candidate currently being examined in both our own and other laboratories (146) is CBP/p300. The recent proliferation of yeast two-hybrid assays has demonstrated that many transcription factors (such as steroid hormone receptors and nuclear receptors), cofactors (such as SRC-1), and proteins of the basal transcriptional machinery (RNA polymerase II, TATA-binding protein, and TFII-B, for example) can interact with CBP, and a view of CBP as a central "adapter/integrator" protein, which brings a complex set of transcriptional proteins together on the DNA, is forming (78, 79, 150, 151). Both GR (see below) and NF-B interact with CBP/p300, making CBP an attractive candidate for a role as mediator of NF-B/GR antagonism, either by functioning as an adapter/cointegrator or as the limiting pool of a mutually required transcriptional cofactor. This idea of CBP functioning as a mediator of transcription factor antagonism was first proposed by Kamei and co-workers (150), who provided evidence that nuclear receptor-mediated repression of AP-1 activation is a result of competition for limiting amounts of CBP.

While it seems logical to consider the role of cofactor (in particular, CBP) competition in NF-B/GR antagonism, in vitro studies by Caldenhoven et al. (1) demonstrate that GR-mediated repression of an NF-B reporter is overcome by increased expression of p65 and therefore argue against such a mechanism. Another potential problem with the hypothesis that CBP might be limiting is that activation of NF-B or GR should globally inhibit all receptors that require CBP for their activity. This simple model of transcription factor competition for CBP would apparently not allow for the specific nature of the NF-B/GR antagonism. However, the more complex proposal that CBP might function as a cointegrator and mediate the antagonism of NF-B and GR by bringing the two factors together in a specific inactive conformation is promising in that it allows for a specific repressive effect even when faced with both the relatively promiscuous interactions of CBP/p300 and the in vitro data that show that GR repression of NF-B can be titrated out by overexpression of p65.

3. Other mechanisms of NF-B/GR interaction. Another potential mechanism of NF-B/GR antagonism which does not involve direct physical interaction of GR and NF-B requires further examination. Recent work from our laboratory (J. C. Webster and J. A. Cidlowski, unpublished observations) indicates that NF-B increases the expression of hGRß, the endogenous dominant negative inhibitor of hGR transactivation. Therefore, a cell in which NF-B is activated may have a higher ratio of GRß:GR and, consequently, impaired GR transactivation. This would provide a dual mechanism of GR repression by NF-B: directly by mutual transcriptional antagonism and indirectly by increasing the relative amounts of a dominant negative regulator of GR. Such a mechanism would be reminiscent of a dual mechanism of NF-B repression by GR (directly by transcriptional antagonism and indirectly by inducing IB), which has been identified and is discussed in Section IV of this manuscript.

4. Evidence for a functional antagonism of GR by NF-B. That two independent mechanisms of GR repression by NF-B likely exist within the same cell suggests that maintaining negative control on GR-signaling pathways is of physiological importance. Although most interest in NF-B/GR antagonism is focused on the role of GR in dampening NF-B actions, the antagonism of GR function by NF-B is probably equally important to the physiology of an organism. While not yet supported by direct evidence, it may be that glucocorticoid-responsive tissues with an activated inflammatory response (mediated by an activated NF-B) become somewhat resistant to glucocorticoid signaling because GR function is blocked.

The repression of GR by NF-B may also be relevant in patients with steroid-resistant asthma. The pathology of asthma is caused by chronic inflammation of the airway epithelium, and the treatment of choice for controlling this inflammation is administration of glucocorticoid (152, 153). However, a subpopulation of asthmatics do not respond to antiinflammatory treatment with glucocorticoids. Some of these steroid-resistant asthmatics have an abnormally low number of GRs, and others demonstrate reduced ligand binding affinity (presumably a reversible effect due to the actions of IL-1 and IL-4), which can explain the lack of glucocorticoid responsiveness (154, 155). Other steroid-resistant asthmatics show no defects in their GRs or in steroid absorption or clearance (152). While there are data that suggest that abnormal AP-1 (activator protein 1)-GR interactions reduce GR DNA binding in these patients (152, 156), it will also be important to determine whether these individuals have any abnormality in NF-B regulation that leads to excessive NF-B activation. It is conceivable that a chronically high level of NF-B activity would lead not only to chronic inflammation, but also to glucocorticoid resistance by blocking of the GR-signaling pathway. Should abnormal NF-B regulation be found in these patients, targeting both NF-B- and GR-signaling pathways simultaneously could provide a promising approach to effective antiinflammatory therapy in this otherwise refractory population.

E. GR interacts with other transcription factors and transcriptional cofactors
As discussed, GR binds to specific regions of the chromatin and alters the basal level of transcription for responsive genes. However, as with NF-B, GR is now known to interact with a variety of nuclear regulatory proteins and other transcription factors that can modulate its function and alter its interaction with DNA and/or the basal transcription machinery. Interactions with different cofactors/transcription factors may contribute to cell type specificities in glucocorticoid responsiveness and GR-mediated gene expression.

1. Transcriptional cofactors and GR. Some proteins function as transcriptional coactivators for the GR, enhancing, or in some instances, enabling the transcriptional activity of ligand-activated GR. Examples of proteins recently identified as GR coactivators are CBP/p300 (cAMP-response element binding protein), GRIP1/TIF2 (GR-interacting protein-1 in mouse, transcriptional intermediary factor 2 in humans), HMG-1 proteins, 14–3-3 eta, STAT 5 (signal transducer and activator of transcription-5), GRIP 170, hRPF1, RAP 46, and SRC-1 (steroid receptor coactivator-1) (76, 78, 127, 157, 158, 159, 160, 161, 162, 163, 164, 165). Proteins that interact with steroid and other nuclear receptors and serve as transcriptional corepressors, including N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoid and thyroid hormone receptors), have also been identified (78, 164, 166, 167). While it has not yet been conclusively determined whether any of these recently identified corepressors specifically interact with GR, it is likely that as more corepressors are identified and their mechanisms of action are clarified, corepressors that specifically affect GR-mediated transcription will be identified.

2. Transcription factors and GR. In addition to interacting with transcriptional cofactors, GR has been shown to interact with other proteins that are themselves independent transcription factors. This "cross-talk" has been shown to alter the transcriptional properties of both GR and the other transcription factors. For example, GR cross-talk is observed with members of the ubiquitous OTF (octamer transcription factor) family of constitutive transcriptional activators (168). Oct-1 and Oct-2 interactions with GR in some cell types and promoter contexts enhance the transcriptional activity of both factors via a cooperative binding mechanism, while in other contexts Oct-1/GR physical interactions result in functional interference (169). Interaction with GR represses Oct-2A transactivation, possibly due to competition for transcriptional cofactors (170), and transcriptional repression of the bovine PRL (PRL3) gene by GR has been shown to be enhanced by interactions with Oct-1 and another homeodomain protein, Pbx (171). GR has also been shown to repress the activity of the transcription factor GATA-1 via direct physical interaction in mouse erythroleukemia cells (172) and to synergize with the developmentally regulated transcription factor hepatocyte nuclear factor-1 in liver and liver-derived cell lines (173).

3. AP-1 and GR. A particularly important physical interaction occurs between GR and the ubiquitous dimeric transcription factor activator protein-1 (AP-1). GR can interact with both Fos and Jun subunits of the AP-1 heterodimer (although it appears that Fos may be the preferential target for GR) and alter the interaction of both transcription factors with DNA, resulting in reciprocal repression of AP-1 and GR transactivation functions. GR also similarly interacts with Fos and Jun homodimers to alter transcriptional properties of both factors (174, 175, 176, 177, 178, 179). AP-1 (Fos-Jun) is a proinflammatory transcription factor that is induced by a variety of cytokines and by phorbol ester (180). Like NF-B, AP-1 has been shown to activate transcription of genes involved in inflammatory diseases such as rheumatoid arthritis and asthma (180, 181, 182), and AP-1 and NF-B have been shown to act synergistically in the induction of some proinflammatory genes in lung epithelium (182). In addition, many inflammatory genes that are repressed by glucocorticoids but do not have nGREs in their promoters do carry sites for AP-1 as well as for NF-B (183). These data suggest that AP-1 repression by GR is another important mechanism of antiinflammatory and immunosuppressive action by glucocorticoids. It is important to bear in mind that, since NF-B and AP-1 can synergize in the proinflammatory pathway, GR repression of AP-1 may be an important indirect mechanism for suppressing NF-B-mediated immune responses.

4. GR and other steroid hormone receptors. It has also been demonstrated that GR can interact with other steroid receptors, resulting in altered transcriptional activity of genes responsive to each reporter. In some cases, the mechanism of interaction is known to involve heterodimerization. For example, GR and MR heterodimerize, and coexpression of GR and MR in a given cell may influence the cell type-specific pattern of gene activation (184). When AR and GR heterodimerize, mutual transcriptional inhibition results (185). GR and progesterone receptor A (PR-A) have also been found to interact. Although the mechanism of interaction is not yet clear, it has been demonstrated that PR-A can function as a dominant negative regulator of GR transactivation (186).

	IV. NF-B and GR Antagonism: Physiological Significance?

Top Abstract I. Introduction II. Nuclear Factor-{kappa}B (NF... III. Steroid Hormones/Receptors:... IV. NF-{kappa}B and GR... V. Interactions Between NF... VI. Summary/Conclusions Addendum/Erratum References

 
NF-B is a key proinflammatory and proimmune transcription factor, and ligand-activated GR is a potent suppressor of immunity and inflammation. The negative cross-talk that has been demonstrated between these two transcription factors with opposing functions has clear implications for the importance of these two transcriptional modulators in immune regulation. Given their profound effects on immunity and inflammation, it is conceivable that NF-B/GR mutual antagonism provides the primary control mechanism by which an organism maintains homeostasis in situations where host defense is activated. The signals that activate NF-B, such as bacterial or viral infection and oxidative stress, are perceived as stressful stimuli by the organism. These stimuli also activate the HPA axis, with the end result being the release of high levels of glucocorticoid into circulation. Therefore, both the pro- and antiinflammatory pathways are activated by these environmental stressors. The presumption is that GR-mediated immunosuppression serves to limit the cellular damage that would be caused by an excessive immune response.

While a strong case can be made for the importance of NF-B in glucocorticoid regulation of immunity and inflammation, it is important to maintain perspective and consider NF-B/GR effects within the context of all known GR-mediated effects on immune function. For example, AP-1 may also be a key mediator of these glucocorticoid effects. As discussed above, AP-1 regulates expression of proinflammatory genes both independently and synergistically with NF-B, and the well described antagonism of AP-1 by GR suggests that glucocorticoid effects on immune/inflammatory cascades are mediated through AP-1 dependent pathways in much the same manner that they are mediated by NF-B. AP-1/GR antagonism also suggests that proinflammatory genes whose transcription is synergistically activated by AP-1 and NF-B might be even more sensitive to glucocorticoid-mediated suppression than those that are activated by only one of these two transcription factors. There is also evidence that GR can directly regulate the expression of some proinflammatory cytokines and related genes via transcriptional and posttranscriptional mechanisms. For example, the IL-8 gene has been shown to be repressed in a fibrosarcoma cell line via a GRE in its 5'-flanking sequ