The process of the visual signal transduction is a classical example of a G protein–mediated signaling cascade and is well characterized (Figure 30-2) (Wald, 1968; Travis et al., 2007; von Lintig et al., 2010). Absorption of a photon by rhodopsin-bound 11-cis-retinal results in isomerization of the chromophore to the all-trans form, a process that induces the protein to undergo several conformational changes through a series of short-lived intermediates. One of the protein intermediates (metarhodopsin II, R^∗ in Figure 30-2) interacts with another membrane protein named transducin. Transducin is a G protein; its interaction with R^∗ leads to an exchange of a transducin-bound guanosine diphosphate (GDP) for a guanosine triphosphate (GTP). In the GTP-bound (activated) state, transducin activates an enzyme called phosphodiesterase. Phosphodiesterase catalyzes the breakdown of cyclic guanosine monophosphate (cGMP), which keeps sodium channels in the plasma membranes of rod outer segments in the open state, to an inactive product, GMP. Because the level of cGMP in rod outer segments in the dark is high (~0.07 mM), the sodium channels are open and the membranes of the cells are depolarized. Activation of phosphodiesterase following illumination results in lower levels of cGMP and leads to closing of the sodium channels and to hyperpolarization of the plasma membrane. The process of visual transduction is regulated further at several levels, including phosphorylation of rhodopsin intermediates, enzymatic hydrolysis of retinal from metarhodopsin II, and termination of the interaction between activated rhodopsin and transducin by the protein arrestin.

FIGURE 30-2 Early events in transduction of the visual signal. c, Cyclic; *GDP,* guanosine diphosphate; *GTP,* guanosine triphosphate; R^∗, metarhodopsin II.

Bleached rhodopsin can be regenerated in the dark by 11-cis-retinal freshly supplied to the photoreceptors from the adjacent retinal pigment epithelium (RPE) cells (Figure 30-3), which take up vitamin A from blood and store it in the form of all-trans-retinyl esters. These storage species are enzymatically converted in RPE cells to 11-cis-retinal, which is then transported across the interphotoreceptor matrix to

FIGURE 30-3 Schematic drawing of cells in the retina that are active in use and metabolism of retinoids. C, Cone; M, Müller cell; R, rod.

CLINICAL CORRELATION

Potential Therapeutic Uses of Ligands for Retinoid Nuclear Receptors

By virtue of their ability to modulate the rate of transcription of a variety of genes, retinoids can be potent therapeutic agents. Retinoic acid and synthetic ligands that activate retinoic acid receptor (RAR) are currently used in therapy and chemoprevention of several types of cancer, most notably promyelocytic leukemia. The ability of retinoid X receptor (RXR) to serve as a common partner for several nuclear receptors, such as RAR, vitamin D₃ receptor (VDR), thyroid hormone receptor (TR), liver X receptor (LXR), and peroxisome proliferator–activated receptor (PPAR), suggests that retinoid derivatives that are selective toward RXR might be useful in treating a variety of disorders. For example, it was reported that RXR-selective retinoids enhance sensitivity to insulin in mouse models of type 2 diabetes and obesity (Mukherjee et al., 1997). It was suggested that this antidiabetic activity is mediated by heterodimers of RXR with PPAR. It was also reported that retinoids increase the expression of apolipoproteins A-I and A-II in a human hepatoblastoma cell line, an activity that was ascribed to RXR-RAR heterodimers (Vu-Dac et al., 1996). Because apo-A–containing HDL is known to have a protective effect against coronary artery disease, these observations suggest that RXR ligands are potentially clinically useful in protecting against cardiovascular disease. It was recently reported that treatment of obese mice with all-trans-RA leads to weight loss and to enhanced insulin sensitivity (Berry and Noy, 2009). These effects were attributed to the ability of RA to activate both RAR and PPAR, which in turn enhance energy use and inhibit adipocyte differentiation.

photoreceptors. The metabolism and transport of retinoids in the eye are discussed later in this chapter.

Regulation of Cell Proliferation and Differentiation by Retinoic Acids

Retinoids have profound effects on the differentiation and growth of a variety of normal and neoplastically transformed cells. One striking example is the differentiation pattern of HL-60 cells, which originated from a human promyelocytic leukemia. These cells differentiate into macrophages when treated with 1,25-dihydroxyvitamin D₃ or with phorbol esters. In contrast, when treated with RA, HL-60 cells differentiate into granulocytes, and this is followed by an arrest in cell proliferation. Other examples include the ability of RA to enhance neuronal differentiation or, in contrast, to inhibit the differentiation of fibroblasts into adipocytes. Retinoids also control the formation of particular patterns, such as digit development, during embryogenesis (Hoffman and Eichele, 1994). In addition, studies of isolated cells, animal models, and humans have demonstrated that retinoids can inhibit cancer development and, in some cases, induce transformed cells to revert to a normal phenotype. Indeed, RA is successfully used in treatment of human acute promyelocytic leukemia and other cancers (Soprano et al., 2004).

Many of the effects of retinoids on cells are due to the ability of the vitamin A metabolites all-trans-RA and 9-cis-RA to modulate the rate of transcription of genes, including those that encode growth factors, transcription factors, enzymes, extracellular matrix proteins, protooncogenes, and binding proteins. By regulating the expression of such proteins, RA controls a complex array of metabolic pathways and cellular behaviors.

ACTIVATION OF Retinoid Nuclear Receptors

Retinoic acids regulate gene expression by activating specific transcription factors, termed retinoid receptors, which are members of a superfamily of the nuclear hormone receptor ligand-inducible transcription factors. Retinoid receptors bind to DNA recognition sequences (response elements) in regulatory regions of target genes, and upon binding of RA, facilitate the rate of transcription of these genes (Germain et al., 2006a, 2006b).

Structure of Retinoid Nuclear Receptors

Like other nuclear hormone receptors, retinoid receptors are composed of several functional domains (Figure 30-4). The N-terminal region of the receptors (A/B domain) which contains a basal, or ligand-independent, activation function termed AF-1. The DNA-binding domain (domain C) contains zinc-fingers responsible for the association of the receptor with DNA (see Chapter 37 for more information on the role of zinc finger motifs in DNA binding by proteins). Domain D is a hinge region that confers flexibility to the protein molecule. Domain E, termed the ligand binding domain, contains the ligand-binding pocket and is responsible for ligand-induced transcriptional activation by the receptors. The ligand-binding domain also contains regions that mediate the interactions of retinoid receptors with a variety of other proteins (see subsequent text). The C-terminal region, which is present in some but not all nuclear receptors, is termed the F domain, and its function is unknown at present.

Types of Retinoid Receptors: RAR, PPARβ/δ), and RXR

All-trans-RA enhances the transcription of target genes by activating three RA receptors (RARα, RARβ, and RARγ) (Germain et al., 2006a) and a nuclear receptor termed peroxisome proliferator–activated receptor β/δ (PPARβ/δ) (Schug et al., 2007). The partitioning of the all-trans-RA hormone between these receptors is regulated by specific RA-binding proteins that selectively deliver it to particular receptors (see later). RARs and PPARβ/δ control the expression of a distinct array of target genes. As the binding proteins determine whether the hormone will be targeted to RAR or PPARβ/δ in a specific cell, cellular responses to RA vary depending on the relative expression levels of these proteins. A third type of retinoid receptors, termed the retinoid X receptors (RXRα, RXRβ, RXRγ), can be activated by the 9-cis-isomer of RA (9cRA). However, 9cRA is not found in all tissues that express RXR and it is uncertain whether this compound serves as the physiological ligand for this receptor (Calleja et al., 2006; Germain et al., 2006b).

RAR and PPARβ/δ Heterodimers

Like other nuclear hormone receptors, RAR, PPARβ/δ, and RXR bind to their DNA response elements as dimers. Dimerization, which is stabilized by strong interactions between the ligand-binding domains and by weaker interactions between the DNA-binding domains of the two monomers, serves to increase the specificity of binding of receptors to particular DNA sequences as well as the strength of their interactions with response elements. Mirroring dimer formation by the proteins, DNA response elements for retinoid receptors are usually arranged as two direct repeats of the same hexanucleotide sequence. RAR and PPARβ/δ homodimers do not readily form. Instead, these receptors associate with RXR with a high affinity to form heterodimers, and these RXR-RAR and RXR-PPAR heterodimers serve as the transcriptionally active species (Durand et al., 1992; Green and Wahli, 1994).

RXR Homo- and Heterodimers

In addition to heterodimerization with RAR and PPARβ/δ, RXRs can also interact with other members of the hormone receptor family. For example, they can form heterodimers with the vitamin D₃ receptor (VDR); the thyroid hormone receptor (TR); the peroxisome proliferator–activated receptors PPARα and PPARγ, which are activated by long-chain fatty acids and some of their metabolites; and with liver X receptor (LXR), which responds to cholesterol metabolites (Figure 30-5; also see Chapters 18 and 31 for more information on transcriptional control by fatty acids and vitamin D). In addition, RXR can bind to DNA and regulate the transcription of target genes as a homodimer (RXR-RXR). RXR has also been reported to form active heterodimers with some orphan nuclear receptors (i.e., proteins that belong to the superfamily of nuclear receptors but for which the ligand is unknown). Heterodimers of RXR with other nuclear receptors can respond to the individual ligands of the two partners and consequently the transcriptional activities of RXR-heterodimers are regulated by more than one type of ligand. RXRs thus function as “master regulators” of several signaling nutrients and hormones as they converge at the genome to regulate gene expression (see Figure 30-5).

FIGURE 30-5 Heterodimerization partners of retinoid X receptor (RXR) and their respective ligands.

Role of Coregulatory Proteins

Modulation of gene transcription by nuclear receptors depends critically on binding of activating ligands, which controls the association of the receptors with coregulatory proteins (Hsia et al., 2010) (Figure 30-6). In the absence of ligands, nuclear receptors associate with proteins that function as transcriptional corepressors. These proteins display enzymatic activities that catalyze deacetylation of histones, resulting in a more compact chromatin structure that leads to repression of transcriptional rates. Upon ligand binding, nuclear receptors undergo a conformational change (Moras and Gronemeyer, 1998) that leads to dissociation of corepressors and recruitment of transcriptional coactivators. Some coactivators catalyze histone acetylation, thereby loosening the structure of the chromatin (Hsia et al., 2010), whereas others, such as components of the Mediator complex, interact with the general transcription machinery and stabilize the recruitment of RNA polymerase II to the target gene promoter (Ito and Roeder, 2001; Rachez and Freedman, 2001). Ligand-activated receptors thus facilitate the transcription of their target genes.

FIGURE 30-6 Transcriptional regulation by retinoid receptors. In the absence of ligand, the receptors associate with proteins that compact chromatin structure and repress transcription. Binding of ligands results in a conformational change that leads to an exchange of corepressors with coactivators. Coactivators modify chromatin to loosen up its structure. Subsequently, a different class of coactivators, termed the Mediator complex, bridge between the receptors and the general transcription machinery and enhance transcriptional rates.

Unlike other receptors, including RAR and PPARβ/δ, the association of RXR with corepressors is weak, suggesting that ligand-dependent activation of this receptor might operate via a different mechanism. Indeed, it was demonstrated that RXR is unique in that, in the absence of its ligand, it exists as a transcriptionally silent homotetramer and that ligand binding results in rapid dissociation of RXR tetramers to the active dimeric species (Kersten et al., 1995, 1998; Gampe et al., 2000). Ligand-induced dissociation of RXR tetramers thus seems to be the first step in activation of this unusual receptor.

Other Retinoids and Their Functions

In addition to retinal and RA, other retinoids are endogenously present in a variety of tissues. The functions of these derivatives are not completely understood but some of them have been shown to be biologically active. For example, retinol itself as well as the metabolite 14-hydroxy-retro-retinol (see Figure 30-1), have been implicated in regulating lymphocyte physiology (Ross and Hammerling, 1994; Chiu et al., 2008). The mechanisms by which these compounds support lymphocyte growth are incompletely understood. They do not associate with any of the known nuclear retinoid receptors and may function by other signaling pathways.

Another biologically active retinoid is 3,4-didehydroretinol, also known as vitamin A₂. It is abundant in freshwater fish, where its metabolite 11-cis-dehydroretinal can serve as a ligand for visual pigments. In humans, 3,4-didehydroretinol was reported to accumulate in tissues of individuals with psoriasis and several other disorders of keratinization (Vahlquist and Torma, 1988). 3,4-Didehydroretinoic acid was found in chick limb buds, where it can affect development, presumably by activating retinoid nuclear receptors (Thaller and Eichele, 1990). It was reported that oxidized retinoid metabolites such as 4-oxo-RA avidly bind to RARβ and are important in determining the positions at which particular digits develop in early embryos (Pijnappel et al., 1993). In addition, it has been demonstrated that some proteins are modified by covalent retinoylation (Takahashi and Breitman, 1991). At present, the effects of retinoylation on the functions of proteins modified in this fashion are not known.

Absorption, Transport, Storage, and Metabolism of Vitamin a and Carotenoids

Absorption and Metabolism of Vitamin a in the Intestines

Two major forms of vitamin A are present in the diet: retinyl esters, which are derived from animal sources, and carotenoids, mainly β-carotene, which originate from plants (see Figure 30-1). Retinyl esters are hydrolyzed in the intestinal lumen to yield free retinol and the corresponding fatty acid (Figure 30-7). Retinyl ester hydrolysis requires the presence of bile salts that serve to solubilize the retinyl esters in mixed micelles and to activate the hydrolyzing enzymes. Several enzymes that are present in the intestinal lumen may be involved in the hydrolysis of dietary retinyl esters. Carboxylester lipase is secreted into the intestinal lumen from the pancreas and has been shown in vitro to display retinyl ester hydrolase activity. In addition, a retinyl ester hydrolase that is intrinsic to the brush border membrane of the small intestine has been characterized in rats and humans (Rigtrup and Ong, 1992). The different hydrolyzing enzymes are activated by different types of bile salts and have distinct substrate specificities. For example, whereas the pancreatic carboxylester lipase is selective for short-chain retinyl esters, the brush border membrane enzyme preferentially hydrolyzes retinyl esters containing a long-chain fatty acid such as palmitate or stearate. Following hydrolysis, retinol diffuses into the enterocytes in a concentration-dependent manner. In contrast, uptake of carotenoids is mediated by transporters (During and Harrison, 2007).

FIGURE 30-7 Reaction catalyzed by retinyl ester hydrolase.

Absorbed β-carotene is centrally cleaved into two molecules of retinal by β-carotene 15,15′-monooxygenase (BCMO1) (Figure 30-8). This enzyme is most highly expressed in the intestinal mucosa but is also found in liver, kidney, lungs, retina, and the brain. In addition, activities that catalyze eccentric cleavage have been reported. The amounts of carotenoids that can pass intact from intestinal cells into blood vary considerably between different species. In the rat, very limited amounts of carotenoids pass into the circulation. In humans, 60% to 70% of absorbed β-carotene is cleaved in the intestine, with the remainder transferred intact into blood and deposited in several tissues such as liver and adipose tissue. The serum level of carotenoids reflects dietary intake, suggesting that a significant fraction of newly absorbed carotenoids are exported from enterocytes into blood without being metabolically converted within these cells.