Medical Physiology, 3rd Edition

Regulation of Inducible Gene Expression by Signal-Transduction Pathways

How do cells activate previously quiescent genes in response to environmental cues? How are such external signals transduced to the cell nucleus to stimulate the transcription of specific genes? Transcription factors may be thought of as effector molecules in signal-transduction pathways (see Chapter 3) that modulate gene expression. Several such signaling pathways have been defined. Lipid-soluble steroid and thyroid hormones can enter the cell and interact with specific receptors that are themselves transcription factors. However, most cytokines, hormones, and mitogens cannot diffuse into the cell interior and instead bind to specific receptors that are located on the cell surface. First, we consider three pathways for transducing signals from cell-surface receptors into the nucleus: a cAMP-dependent pathway, a Ras-dependent pathway, and the JAK-STAT pathway. Next, we examine the mechanisms by which steroid or thyroid hormones act via nuclear receptors. Finally, we discuss how transcription factors coordinate gene expression in response to physiological stimuli.

cAMP regulates transcription via the transcription factors CREB and CBP

cAMP is an important second messenger (see p. 57) in the response to agonists binding to specific cell-surface receptors. Increases in [cAMP]i stimulate the transcription of certain genes, including those that encode a variety of hormones, such as somatostatin (see pp. 993–994), the enkephalins (see p. 315), glucagon (see pp. 1050–1053), and vasoactive intestinal peptide (see Table 41-1). Many genes that are activated in response to cAMP contain within their regulatory regions a common DNA element called CRE (cAMP response element) that has the consensus sequence 5′-TGACGTCA-3′. Several different transcription factors bind to CRE, among them CREB (cAMP response element–binding protein), a 43-kDa member of the bZIP family. As shown in Figure 4-13, increases in [cAMP]i stimulate protein kinase A (PKA) by causing dissociation of the PKA regulatory subunit. The catalytic subunit of PKA then translocates into the nucleus, where it phosphorylates CREB and other proteins. Activation of CREB is rapid (30 minutes) and declines gradually during a 24-hour period. This phosphorylation greatly increases the affinity of CREB for the coactivator CBP, which is a 245-kDa protein containing two domains, one that binds to phosphorylated CREB and another that activates components of the basal transcriptional machinery. Thus, CBP serves as a “bridge” protein that communicates the transcriptional activation signal from CREB to the basal transcriptional machinery. In addition, because CBP has intrinsic HAT activity (see p. 84), its recruitment by CREB also results in chromatin remodeling that facilitates gene transcription. The result of phosphorylation of CREB is a 10- to 20-fold stimulation of CREB's ability to induce the transcription of genes containing a CRE.

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FIGURE 4-13 Regulation of gene transcription by cAMP. Phosphorylated CREB binds CBP, which has a transactivation domain that stimulates the basal transcriptional machinery. In parallel, phosphorylation activates phosphoprotein phosphatase 1 (PP1), which dephosphorylates CREB, terminating the activation of transcription. R, regulatory subunit of protein kinase A.

How is the transcriptional signal terminated? When [cAMP]i is high, PKA phosphorylates and activates phosphoprotein phosphatase 1 in the nucleus. When cAMP levels fall, the still-active phosphatase dephosphorylates CREB.

Receptor tyrosine kinases regulate transcription via a Ras-dependent cascade of protein kinases

Many growth factors bind to cell-surface receptors that, when activated by the ligand, have tyrosine kinase activity (see pp. 70–71). Examples of growth factors that act through such receptor tyrosine kinases (RTKs) are epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin, insulin-like growth factor type 1 (IGF-1), fibroblast growth factor (FGF), and nerve growth factor (NGF). The common pathway by which activation of RTKs is transduced into the nucleus is a cascade of events that increase the activity of the small GTP-binding protein Ras (see pp. 68). This Ras-dependent signaling pathway culminates in the activation of mitogen-activated protein kinase (MAPK), which translocates to the nucleus, where it phosphorylates a number of nuclear proteins that are transcription factors. Phosphorylation of a transcription factor by MAPK can enhance or inhibit binding to DNA and can stimulate either transactivation or transrepression. imageN4-12 Transcription factors that are regulated by the Ras-dependent pathway include c-Myc, c-Jun, c-Fos, and Elk-1. Many of these transcription factors regulate the expression of genes that promote cell proliferation.

N4-12

Transcription Factors Phosphorylated by MAPK

Contributed by Peter Igarashi

The following table summarizes some transcription factors that MAPK phosphorylates, together with the site of phosphorylation on the transcription factor, and the effect.

Transcription Factor

Site

Effect

c-Myc

Ser-62

Stabilizes protein

c-Jun

Ser-243 (Ser-63/Ser-73 in the activation domain are phosphorylated by a distinct Ras-dependent kinase)

Inhibits DNA binding

c-Fos

Ser-374 (direct)
Ser-362 (indirect via ribosomal S6 kinase, which is activated by MAPK)

Stimulates transrepression

p62TCF = Elk-1

Multiple

Stimulates transactivation and possibly also DNA binding

C/EBPβ = LAP or NF–IL-6

Thr-235

Stimulates transactivation

ATF-2

Thr-69 and Thr-71 (via p38 and JNK MAPK)

Stimulates transactivation

ATF-2, activating transcription factor 2; C/EBPβ, CCAAT/enhancer-binding protein β; JNK, c-Jun N-terminal kinases.

Tyrosine kinase–associated receptors can regulate transcription via JAK-STAT

A group of cell-surface receptors termed tyrosine kinase–associated receptors lack intrinsic tyrosine kinase activity (see pp. 70–71). The ligands that bind to these receptors include several cytokines, growth hormone, prolactin, and interferons (IFN-α, IFN-β, and IFN-γ). Although the receptors themselves lack catalytic activity, their cytoplasmic domains are associated with the Janus kinase (JAK) family of protein tyrosine kinases.

Binding of ligand to certain tyrosine kinase–associated receptors activates a member of the JAK family, which results in phosphorylation of cytoplasmic proteins, among which are believed to be latent cytoplasmic transcription factors called signal transducers and activators of transcription (STATs). When phosphorylated on tyrosine residues, the STAT proteins dimerize and thereby become competent to enter the nucleus and induce transcription.

A well-characterized example of the JAK-STAT pathway is the activation of interferon-responsive genes by IFN-α and IFN-γ. IFN-α activates the JAK1 and Tyk2 kinases that are associated with its receptor (Fig. 4-14A). Subsequent phosphorylation of two different STAT monomers causes the monomers to dimerize. This STAT heterodimer enters the nucleus, where it combines with a third 48-kDa protein to form a transcription factor that binds to a DNA sequence called the IFN-stimulated response element (ISRE). In the case of IFN-γ (see Fig. 4-14B), the receptor associates with the JAK1 and JAK2 (rather than Tyk2) kinases, and subsequent phosphorylation of a single kind of STAT monomer causes these monomers to dimerize. These STAT homodimers also enter the nucleus, where they bind to the DNA at IFN-γ response elements called IFN-γ activation sites (GASs), without requiring the 48-kDa protein.

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FIGURE 4-14 JAK-STAT pathway. A, Binding of a ligand such as IFN-α to a tyrosine kinase–associated receptor causes JAK1 and Tyk2 to phosphorylate themselves, the receptor, and two different STAT monomers. The phosphorylation of the STAT monomers leads to the formation of a heterodimer, which translocates to the nucleus and combines with a third protein (p48). The complex binds to ISRE and activates gene transcription. B, Binding of a ligand such as IFN-γ to a tyrosine kinase–associated receptor causes JAK1 and JAK2 to phosphorylate themselves, the receptor, and two identical STAT monomers. The phosphorylation of the STAT monomers leads to the formation of a homodimer, which translocates to the nucleus. The complex binds to the GAS response element and activates gene transcription.

Nuclear receptors are transcription factors

Steroid and thyroid hormones are examples of ligands that activate gene expression by binding to cellular receptors that are themselves transcription factors. Members of the steroid and thyroid hormone receptor superfamily, also called the nuclear receptor superfamily, are grouped together because they are structurally similar and have similar mechanisms of action. After these hormones enter the cell, they bind to receptors in the cytoplasm or nucleus. Ligand binding converts the receptors into active transcription factors. The transcription factors bind to specific regulatory elements on the DNA, called hormone response elements, and activate the transcription of cis-linked genes. The family of nuclear receptors includes receptors that bind glucocorticoids (GR), mineralocorticoids (MR), estrogens (ER), progesterone (PR), androgens (AR), thyroid hormone (TR), vitamin D (VDR), retinoic acid (RAR), lipids (peroxisome proliferator–activated receptor, PPAR), and 9-cis-retinoic acid (retinoid X receptor, RXR) as well as bile acids (bile acid receptor, FXR; see p. 964) and xenobiotics (steroid and xenobiotic receptor, SXR; constitutive androstane receptor, CAR; see p. 956).

With the exception of the thyroid hormones, the hormones that bind to these receptors are lipophilic molecules that enter cells by diffusion and do not require interaction with cell-surface receptors. The thyroid hormones differ in that they are electrically charged and may cross the cell membrane via transporters.

Modular Construction

The nuclear receptors have a modular construction consisting of an N-terminal transactivation domain, a DNA-binding domain, and a C-terminal ligand-binding domain (see pp. 71–72). These receptors bind to specific DNA sequences through two zinc fingers (see p. 82), each of which contains four cysteine residues rather than the two histidines and two cysteines that are typical of many other zinc finger proteins (see Fig. 4-9A). Particularly important for DNA recognition is the P-box motif in the hormone receptor, a sequence of six amino acids at the C-terminal end of each finger. These P boxes make base pair contacts in the major groove of DNA and determine the DNA-binding specificities of the zinc finger.

Dimerization

GR, MR, PR, ER, and AR bind to DNA as homodimers (see Table 3-6). The recognition sites for these receptors (except for ER) consist of two 6-bp DNA sequences that are separated by three other base pairs. The 6-bp DNA sequences, commonly called half-sites, represent binding sites for each zinc-finger monomer.

In contrast, other nuclear receptors preferentially bind to DNA as heterodimers formed with RXR, the receptor for 9-cis-retinoic acid. Examples of such heterodimers are VDR/RXR, TR/RXR, RAR/RXR, and PPAR/RXR (see Table 3-6). Interestingly, these heterodimers work even in the absence of the ligand of RXR (i.e., 9-cis-retinoic acid). Only the non-RXR part of the dimer needs to be occupied by its hormone ligand. These heterodimers recognize a family of DNA sites containing a DNA sequence such as 5′-AGGTCA-3′, followed by a DNA spacer and then by a direct repeat of the previous 6-bp DNA sequence. Moreover, because VDR/RXR, TR/RXR, and RAR/RXR may each recognize the same 6-bp sequences, binding specificity also depends on the length of the spacer between the direct repeats. The VDR/RXR, TR/RXR, and RAR/RXR heterodimers preferentially recognize separations of 3 bp, 4 bp, and 5 bp, respectively, between the repeats of 5′-AGGTCA-3′. This relationship forms the basis for the so-called 3-4-5 rule.

Activation of Transcription

Ligand binding activates nuclear receptors through two main mechanisms: regulation of subcellular localization and interactions with coactivators. Some nuclear receptors, such as GR, are normally located in the cytoplasm and are maintained in an inactive state by association with a cytoplasmic anchoring protein (Fig. 4-15A). The protein that retains GR in the cytoplasm is a molecular chaperone, the 90-kDa heat shock protein hsp90. GR must bind to hsp90 to have a high affinity for a glucocorticoid hormone. When glucocorticoids bind to the GR, hsp90 dissociates from the GR and exposes a nuclear localization signal that permits the transport of GR into the nucleus. The receptor must remain hormone bound for receptor dimerization, which is a prerequisite for binding to the GRE on the DNA. Other receptors, such as TR, are normally already present in the nucleus before binding the hormone (see Fig. 4-15B). For these receptors, binding of hormone is evidently not essential for dimerization or binding to DNA. However, ligand binding is necessary at a subsequent step for transactivation.

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FIGURE 4-15 Transcriptional activation by glucocorticoid and thyroid hormones. A, The binding of a glucocorticoid hormone to a cytoplasmic receptor causes the receptor to dissociate from the chaperone hsp90 (90-kDa heat shock protein). The free hormone-receptor complex can then translocate to the nucleus, where dimerization leads to transactivation. B, The binding of thyroid hormone to a receptor in the nucleus leads to transactivation. The active transcription factor is a heterodimer of the TR and the RXR.

Although nuclear receptors may stimulate gene expression by interacting directly with components of the basal transcriptional machinery, full transcriptional activation requires coactivators that interact with the receptor in a ligand-dependent manner. More than 200 coactivators may interact directly or indirectly with nuclear receptors via mechanisms that include the following:

1. Recruitment of basal transcriptional machinery. Coactivators that belong to the SRC (steroid receptor coactivator)/p160 family bind only to the ligand-bound form of the receptor. On binding to the nuclear receptor, SRC/p160 coactivators recruit a second coactivator, CBP (see p. 84), which then promotes recruitment of the basal transcriptional machinery. Nuclear receptors also bind in a ligand-dependent manner to the coactivator TRAP220, a component of Mediator (see p. 84), which is part of the basal transcriptional machinery.

2. Binding to a chromatin-remodeling complex. Nuclear receptors also interact with Brg1 (Brahma-related gene 1), the central motor component of the chromatin-remodeling complex SWI/SNF (see p. 84).

3. Histone acetylation. Several coactivators have enzymatic activities that mediate chromatin remodeling. Both SRC-1 and CBP have intrinsic HAT activity.

4. Histone methylation. The coactivator CARM1 is a methyltransferase that methylates specific arginine residues in histones, thereby enhancing transcriptional activation.

5. Ubiquitination. Nuclear receptors recruit components of the ubiquitin-proteasome pathway (see pp. 33–34 and 88) to the promoter region of nuclear receptor target genes. Ubiquitination appears to promote transcript elongation.

Repression of Transcription

Nuclear receptors sometimes function as active repressors, perhaps acting by alternative mechanisms. First, a receptor may form inactive heterodimers with other members of the nuclear receptor family. Second, a receptor may compete with other transcription factors for DNA-binding sites. For example, when the TR—without bound thyroid hormone—interacts with its own DNA response element, the TR acts as a repressor. In addition, the TR receptor isoform TRα can dimerize with one of the retinoic acid receptor isoforms (RXRβ) to interfere with binding of ER to its response element. This competition may be one of the mechanisms by which retinoids inhibit estrogen-induced alterations in gene expression and growth in mammary tissue. Finally, nuclear receptors can also inhibit gene transcription by interacting with corepressors, such as N-CoR, Sin3A, and Sin3B. These corepressors can recruit HDACs that enhance nucleosome assembly, which results in transcriptional repression.

Box 4-2

Role of a Chimeric Transcription Factor in Acute Promyelocytic Leukemia

Correct regulation of gene expression involves both transcription factors and the DNA regulatory elements to which they bind. Abnormalities of either can and do result in abnormal regulation of gene expression, which is often manifested as disease. An example of a transcription factor abnormality is acute promyelocytic leukemia (APL), a hematological malignant state in which cells of the granulocyte lineage (promyelocytes) fail to differentiate. Normally, retinoic acid (RA) binds to retinoic acid receptor-α (RARα), a member of the steroid and thyroid hormone receptor superfamily. RARα forms heterodimers with retinoid X receptor (RXR) and binds to retinoic acid response elements (RAREs) that are present in genes involved in cell differentiation. In the absence of RA, RARα/RXR heterodimers bind to RAREs and recruit the corepressor N-CoR, which in turn recruits HDACs that inhibit gene transcription. Binding of RA to RARα leads to dissociation of N-CoR, which permits binding of the coactivator CBP (see p. 89) and activation of RARα-responsive genes that promote cell differentiation. Ninety percent of patients with APL have a translocation affecting chromosomes 15 and 17, t(15;17), that produces a chimeric transcription factor containing the DNA- and hormone-binding domains of RARα, fused to the promyelocytic leukemia protein (PML). The PML/RARα chimeric protein also binds to RA and forms heterodimers with RXR but has an abnormally high affinity for N-CoR. At physiological levels of RA, N-CoR remains bound to PML/RARα, blocking promyelocytic differentiation. However, high concentrations of RA induce dissociation of N-CoR and permit differentiation. This mechanism explains why high concentrations of exogenous RA can be used to induce clinical remissions in patients with APL.

Physiological stimuli can modulate transcription factors, which can coordinate complex cellular responses

In response to physiological stimuli, some transcription factors regulate the expression of several genes (see Table 4-3). As an example, we discuss how oxygen concentration ([O2]) controls gene expression.

When chronically exposed to low [O2] (hypoxia), many cells undergo dramatic changes in gene expression. For example, cells switch from oxidative metabolism to glycolysis, which requires the induction of genes encoding glycolytic enzymes. Many tissues activate the gene encoding the vascular endothelial growth factor (VEGF), which stimulates angiogenesis (see p. 481) and improves the blood supply to chronically hypoxic tissues. The kidney activates the gene encoding erythropoietin (see pp. 431–433), a hormone that stimulates red cell production in the bone marrow. These changes in gene expression promote survival of the cell or organism in a hypoxic environment. A key mediator in the response to hypoxia is a transcription factor called hypoxia-inducible factor 1α (HIF-1α).

HIF-1α (Fig. 4-16A) belongs to the bHLH family of transcription factors (see p. 83). In addition, it contains a PAS domain imageN4-7 that mediates dimerization. HIF-1α binds to DNA as a heterodimer with HIF-1β. HIF-1β is expressed at constant levels in cells, but the abundance of HIF-1α changes markedly in response to changes in [O2]. At a normal [O2] (normoxia), HIF-1α levels are low. Under hypoxic conditions, the abundance of HIF-1α increases. HIF-1α together with HIF-1β binds to an enhancer, called a hypoxia response element, that is present in many genes activated during hypoxia, including genes encoding glycolytic enzymes, VEGF, and erythropoietin.

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FIGURE 4-16 Regulation of HIF-1α by oxygen. A, In the presence of oxygen, HIF-1α is hydroxylated on proline and asparagine by hydroxylases. B, Hydroxylation of HIF-1α promotes its degradation and inhibits its interaction with coactivators. C, In hypoxic conditions, dehydroxylation of HIF-1α promotes its stabilization and transcriptional activity.

The cell regulates the abundance of HIF-1α by hydroxylation—a post-translational modification—at specific proline and asparagine residues. Oxygen activates the prolyl and asparaginyl hydroxylases (see Fig. 4-16A). Proline hydroxylation stimulates the interaction of HIF-1α with VHL, a protein that targets HIF-1α for proteasomal degradation (see Fig. 4-16B). Asparagine hydroxylation inhibits the interaction of HIF-1αwith the transcriptional coactivator CBP. Because both of these hydroxylations reduce transcriptional activity, normoxic conditions lower the expression of HIF-1α target genes.

In contrast, under hypoxic conditions, the hydroxylases are inactive, and HIF-1α is not hydroxylated on proline and asparagine residues. HIF-1α accumulates in the nucleus and interacts with CBP, which activates the transcription of downstream target genes, including VEGF and erythropoietin (see Fig. 4-16C). The net result is a system in which the expression of multiple hypoxia-inducible genes is coordinately and tightly regulated through post-translational modification of a common transcriptional activator.