Hormones Can Influence Specific Biologic Effects By Modulating Transcription

The signals generated as described above have to be translated into an action that allows the cell to effectively adapt to a challenge (Figure 43-1). Much of this

NF-kB Activators Proinflammatory cytokines Bacterial and viral infection Reactive oxygen species

NF-kB Activators Proinflammatory cytokines Bacterial and viral infection Reactive oxygen species

Figure 43-10. Regulation of the NF-kB pathway. NF-kB consists of two sub-units, p50 and p65, which regulate transcription of many genes when in the nucleus. NF-kB is restricted from entering the nucleus by IkB, an inhibitor of NF-kB. IkB binds to—and masks—the nuclear localization signal of NF-kB. This cytoplasmic protein is phosphorylated by an IKK complex which is activated by cytokines, reactive oxygen species, and mitogens. Phosphorylated IkB can be ubiquitinylated and degraded, thus releasing its hold on NF-kB. Glucocorticoids affect many steps in this process, as described in the text.

Figure 43-10. Regulation of the NF-kB pathway. NF-kB consists of two sub-units, p50 and p65, which regulate transcription of many genes when in the nucleus. NF-kB is restricted from entering the nucleus by IkB, an inhibitor of NF-kB. IkB binds to—and masks—the nuclear localization signal of NF-kB. This cytoplasmic protein is phosphorylated by an IKK complex which is activated by cytokines, reactive oxygen species, and mitogens. Phosphorylated IkB can be ubiquitinylated and degraded, thus releasing its hold on NF-kB. Glucocorticoids affect many steps in this process, as described in the text.

Figure 43-11. The hormone response transcription unit. The hormone response transcription unit is an assembly of DNA elements and bound proteins that interact, through protein-protein interactions, with a number of coactivator or corepressor molecules. An essential component is the hormone response element which binds the ligand (A)-bound receptor (R). Also important are the accessory factor elements (AFEs) with bound transcription factors. More than two dozen of these accessory factors (AFs), which are often members of the nuclear receptor superfamily, have been linked to hormone effects on transcription. The AFs can interact with each other, with the liganded nuclear receptors, or with coregulators. These components communicate with the basal transcription complex through a coregulator complex that can consist of one or more members of the p160, corepressor, mediator-related, or CBP/p300 families (see Table 43-6).

AF p160

p300

adaptation is accomplished through alterations in the rates of transcription of specific genes. Many different observations have led to the current view of how hormones affect transcription. Some of these are as follows: (1) Actively transcribed genes are in regions of "open" chromatin (defined by a susceptibility to the enzyme DNase I), which allows for the access of transcription factors to DNA. (2) Genes have regulatory regions, and transcription factors bind to these to modulate the frequency of transcription initiation. (3) The hormone-receptor complex can be one of these transcription factors. The DNA sequence to which this binds is called a hormone response element (HRE; see Table 43-1 for examples). (4) Alternatively, other hormone-generated signals can modify the location, amount, or activity of transcription factors and thereby influence binding to the regulatory or response element. (5) Members of a large superfamily of nuclear receptors act with—or in a manner analogous to—hormone receptors. (6) These nuclear receptors interact with another large group of coregulatory molecules to effect changes in the transcription of specific genes.

Several Hormone Response Elements (HREs) Have Been Defined

Hormone response elements resemble enhancer elements in that they are not strictly dependent on position and location. They generally are found within a few hundred nucleotides upstream (5') of the transcrip tion initiation site, but they may be located within the coding region of the gene, in introns. HREs were defined by the strategy illustrated in Figure 39-11. The consensus sequences illustrated in Table 43-1 were arrived at through analysis of several genes regulated by a given hormone using simple, heterologous reporter systems (see Figure 39-10). Although these simple HREs bind the hormone-receptor complex more avidly than surrounding DNA—or DNA from an unrelated source—and confer hormone responsiveness to a reporter gene, it soon became apparent that the regulatory circuitry of natural genes must be much more complicated. Glucocorticoids, progestins, mineralocor-ticoids, and androgens have vastly different physiologic actions. How could the specificity required for these effects be achieved through regulation of gene expression by the same HRE (Table 43-1)? Questions like this have led to experiments which have allowed for elaboration of a very complex model of transcription regulation. For example, the HRE must associate with other DNA elements (and associated binding proteins) to function optimally. The extensive sequence similarity noted between steroid hormone receptors, particularly in their DNA-binding domains, led to discovery of the nuclear receptor superfamily of proteins. These—and a large number of coregulator proteins—allow for a wide variety of DNA-protein and protein-protein interactions and the specificity necessary for highly regulated physiologic control. A schematic of such an assembly is illustrated in Figure 43-11.

AF-1

DBD

Hinge

LBD AF-2

Receptors: Binding: Ligand: DNA element:

Steroid class

Homodimers

Steroids

Inverted repeat

RXR partnered Heterodimers 9-Cis RA + (x) Direct repeats

Orphans

Homodimers ?

Direct repeats

Figure 43-12. The nuclear receptor superfamily. Members of this family are divided into six structural domains (A-F). Domain A/B is also called AF-1, or the modulator region, because it is involved in activating transcription. The C domain consists of the DNA-binding domain (DBD). The D region contains the hinge, which provides flexibility between the DBD and the ligand-binding domain (LBD, region E). The amino (N) terminal part of region E contains AF-2, another domain important for transactivation. The F region is poorly defined. The functions of these domains are discussed in more detail in the text. Receptors with known ligands, such as the steroid hormones, bind as homodimers on inverted repeat half-sites. Other receptors form heterodimers with the partner RXR on direct repeat elements. There can be nucleotide spacers of one to five bases between these direct repeats (DR1-5). Another class of receptors for which ligands have not been determined (orphan receptors) bind as homodimers to direct repeats and occasionally as monomers to a single half-site.

There Is a Large Family of Nuclear Receptor Proteins

The nuclear receptor superfamily consists of a diverse set of transcription factors that were discovered because of a sequence similarity in their DNA-binding domains. This family, now with more than 50 members, includes the nuclear hormone receptors discussed above, a number of other receptors whose ligands were discovered after the receptors were identified, and many putative or orphan receptors for which a ligand has yet to be discovered.

These nuclear receptors have several common structural features (Figure 43-12). All have a centrally located DNA-binding domain (DBD) that allows the receptor to bind with high affinity to a response element. The DBD contains two zinc finger binding motifs (see Figure 39-14) that direct binding either as homodimers, as heterodimers (usually with a retinoid X

receptor [RXR] partner), or as monomers. The target response element consists of one or two half-site consensus sequences arranged as an inverted or direct repeat. The spacing between the latter helps determine binding specificity. Thus, a direct repeat with three, four, or five nucleotide spacer regions specifies the binding of the vitamin D, thyroid, and retinoic acid receptors, respectively, to the same consensus response element (Table 43-1). A multifunctional ligand-binding domain (LBD) is located in the carboxyl terminal half of the receptor. The LBD binds hormones or metabolites with selectivity and thus specifies a particular biologic response. The LBD also contains domains that mediate the binding of heat shock proteins, dimerization, nuclear localization, and transactivation. The latter function is facilitated by the carboxyl terminal transcription activation function (AF-2 domain), which forms a surface required for the interaction with

Figure 43-13. Several signal transduction pathways converge on CBP/p300. Ligands that associate with membrane or nuclear receptors eventually converge on CBP/p300. Several different signal transduction pathways are employed. EGF, epidermal growth factor; GH, growth hormone; Prl, prolactin; TNF, tumor necrosis factor; other abbreviations are expanded in the text.

Figure 43-13. Several signal transduction pathways converge on CBP/p300. Ligands that associate with membrane or nuclear receptors eventually converge on CBP/p300. Several different signal transduction pathways are employed. EGF, epidermal growth factor; GH, growth hormone; Prl, prolactin; TNF, tumor necrosis factor; other abbreviations are expanded in the text.

coactivators. A highly variable hinge region separates the DBD from the LBD. This region provides flexibility to the receptor, so it can assume different DNA-binding conformations. Finally, there is a highly variable amino terminal region that contains another trans-activation domain referred to as AF-1. Less well defined, the AF-1 domain may provide for distinct physiologic functions through the binding of different coregulator proteins. This region of the receptor, through the use of different promoters, alternative splice sites, and multiple translation initiation sites, provides for receptor isoforms that share DBD and LBD identity but exert different physiologic responses because of the association of various coregulators with this variable amino terminal AF-1 domain.

It is possible to sort this large number of receptors into groups in a variety of ways. Here they are discussed according to the way they bind to their respective DNA elements (Figure 43-12). Classic hormone receptors for glucocorticoids (GR), mineralocorticoids (MR), estrogens (ER), androgens (AR), and progestins (PR) bind as homodimers to inverted repeat sequences. Other hormone receptors such as thyroid (TR), retinoic acid (RAR), and vitamin D (VDR) and receptors that bind various metabolite ligands such as PPAR a P, and y, FXR, LXR, PXR/SXR, and CAR bind as heterodimers, with retinoid X receptor (RXR) as a partner, to direct repeat sequences (see Figure 43-12 and Table 43-5).

Another group of orphan receptors that as yet have no known ligand bind as homodimers or monomers to direct repeat sequences.

As illustrated in Table 43-5, the discovery of the nuclear receptor superfamily has led to an important understanding of how a variety of metabolites and xenobi-otics regulate gene expression and thus the metabolism, detoxification, and elimination of normal body products and exogenous agents such as pharmaceuticals. Not surprisingly, this area is a fertile field for investigation of new therapeutic interventions.

A Large Number of Nuclear Receptor Coregulators Also Participate in Regulating Transcription

Chromatin remodeling, transcription factor modification by various enzyme activities, and the communication between the nuclear receptors and the basal transcription apparatus are accomplished by protein-protein interactions with one or more of a class of coregulator molecules. The number of these coregulator molecules now exceeds 100, not counting species variations and splice variants. The first of these to be described was the CREB-binding protein, CBP. CBP, through an amino terminal domain, binds to phosphorylated serine 137 of CREB and mediates transactivation in response to cAMP. It thus is described as a coactivator. CBP and

Table 43-5. Nuclear receptors with special ligands.1

Receptor

Partner

Ligand

Process Affected

Peroxisome PPARa

RXR (DR1)

Fatty acids

Peroxisome proliferation

Proliferator- PPARß

Fatty acids

activated PPARy

Fatty acids Eicosanoids, thiazolidinediones

Lipid and carbohydrate metabolism

Farnesoid X FXR

RXR (DR4)

Farnesol, bile acids

Bile acid metabolism

Liver X LXR

RXR (DR4)

Oxysterols

Cholesterol metabolism

Xenobiotic X CAR

Phenobarbital

Xenobiotics

Protection against certain drugs, toxic metabolites, and xenobiotics

PXR

RXR (DR3)

Pregnanes Xenobiotics

1 Many members of the nuclear receptor superfamily were discovered by cloning, and the corresponding ligands were then identified. These ligands are not hormones in the classic sense, but they do have a similar function in that they activate specific members of the nuclear receptor superfamily. The receptors described here form heterodimers with RXR and have variable nucleotide sequences separating the direct repeat binding elements (DR1-5). These receptors regulate a variety of genes encoding cytochrome p450s (CYP), cytosolic binding proteins, and ATP-binding cassette (ABC) transporters to influence metabolism and protect cells against drugs and noxious agents.

its close relative, p300, interact directly or indirectly with a number of signaling molecules, including activator protein-1 (AP-1), signal transducers and activators of transcription (STATs), nuclear receptors, and CREB (Figure 39-11). CBP/p300 also binds to the p160 family of coactivators described below and to a number of other proteins, including viral transcription factor Ela, the p90rsk protein kinase, and RNA helicase A. It is important to note that CBP/p300 also has intrinsic histone acetyltransferase (HAT) activity. The importance of this is described below. Some of the many actions of CBP/p300, which appear to depend on intrinsic enzyme activities and its ability to serve as a scaffold for the binding of other proteins, are illustrated in Figure 43-11. Other coregulators may serve similar functions.

Several other families of coactivator molecules have been described. Members of the p160 family of coac-tivators, all of about 160 kDa, include (1) SRC-1 and NCoA-1; (2) GRIP 1, TIF2, and NCoA-2; and (3) p/CIP, ACTR, AIB1, RAC3, and TRAM-1 (Table 43-6). The different names for members within a subfamily often represent species variations or minor splice variants. There is about 35% amino acid identity between members of the different subfamilies. The p160 coactivators share several properties. They (1) bind nuclear receptors in an agonist and AF-2 transactiva-tion domain-dependent manner; (2) have a conserved amino terminal basic helix-loop-helix (bHLH) motif (see Chapter 39); (3) have a weak carboxyl terminal transactivation domain and a stronger amino terminal

Table 43-6. Some mammalian coregulator proteins.

I. 300-kDa family of coactivators

CBP CREB-binding protein p300 Protein of 300 kDa

II. 160-kDa family of coactivators

A. SRC-1 Steroid receptor coactivator 1 NCoA-1 Nuclear receptor coactivator 1

B. TIF2 Transcriptional intermediary factor 2 GRIP1 Glucocorticoid receptor-interacting protein NCoA-2 Nuclear receptor coactivator 2

C. p/CIP p300/CBP cointegrator-associated protein 1 ACTR Activator of the thyroid and retinoic acid receptors AIB Amplified in breast cancer RAC3 Receptor-associated coactivator 3 TRAM-1 TR activator molecule 1

III. Corepressors

NCoR Nuclear receptor corepressor

SMRT Silencing mediator for RXR and TR

IV. Mediator-related proteins

TRAPs Thyroid hormone receptor-associated proteins

DRIPs Vitamin D receptor-interacting proteins

ARC Activator-recruited cofactor transactivation domain in a region that is required for the CBP/p16O interaction; (4) contain at least three of the LXXLL motifs required for protein-protein interaction with other coactivators; and (5) often have HAT activity. The role of HAT is particularly interesting, as mutations of the HAT domain disable many of these transcription factors. Current thinking holds that these HAT activities acetylate histones and result in remodeling of chromatin into a transcription-efficient environment; however, other protein substrates for HAT-mediated acetylation have been reported. Histone acetylation/deacetylation is proposed to play a critical role in gene expression.

A small number of proteins, including NCoR and SMRT, comprise the corepressor family. They function, at least in part, as described in Figure 43-2. Another family includes the TRAPs, DRIPs, and ARC (Table 43-6). These so-called mediator-related proteins range in size from 80 kDa to 240 kDa and are thought to be involved in linking the nuclear receptor-coactivator complex to RNA polymerase II and the other components of the basal transcription apparatus.

The exact role of these coactivators is presently under intensive investigation. Many of these proteins have intrinsic enzymatic activities. This is particularly interesting in view of the fact that acetylation, phos-phorylation, methylation, and ubiquitination—as well as proteolysis and cellular translocation—have been proposed to alter the activity of some of these coregula-tors and their targets.

It appears that certain combinations of coregula-tors—and thus different combinations of activators and inhibitors—are responsible for specific ligand-induced actions through various receptors.

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Diabetes 2

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