Genomic Actions of Retinoic Acid

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Apart from the effects on vision, most of the effects of vitamin A deficiency (Section 2.4) involve derangements of cell proliferation and differentiation (squamous metaplasia and keratinization of epithelia), dedifferentiation, and loss of ciliated epithelia. Retinoic acid has both a general role in growth and a specific morphogenic role in development and tissue differentiation. These functions are the result of genomic actions, modulating gene expression by activation of nuclear receptors. Both deficiency and excess of retinoic acid cause severe developmental abnormalities.

Retinoic acid has a specific morphogenic role in limb development. There is a small concentration gradient of retinoic acid across the developing limb bud and a gradient of retinoic acid binding protein in the opposite direction, suggesting that the resultant relatively steep gradient of free retinoic acid may be the important factor determining the pattern of development (Thaller and Eichele, 1987,1990). It may also be important in the development of the central nervous system. CRABP has a strictly delimited anatomical localization in the developing mouse brain and is only expressed transiently, between days 11 to 14 of gestation (Momoi et al., 1990).

At pharmacological levels, retinoic acid enhances the expression of uncoupling protein 1 (thermogenin) in brown adipose tissue and decreases the expression of leptin in white adipose tissue, suggesting that it may have an effect on energy homeostasis, but it is not known whether or not the effects are relevant at physiological levels (Kumaretal., 1999; Villarroyaetal., 1999). Retinoic acid also induces synthesis of glucokinase in pancreatic f -islet cells. Increased metabolism of glucose as a result of glucokinase activity is responsible for initiating insulin secretion in response to a rise in blood glucose concentration, and retinoic acid increases the secretion of insulin by pancreatic islets in culture (Cabrera-Valladares et al., 1999).

Retinoic acid may either enter the target cell from the circulation or may be formed intracellularly by oxidation of retinol. A number of tissues - but not muscle, kidneys, small intestines, liver, lungs, or spleen - have a cellular retinoic acid binding protein (CRABP) that is distinct from CRBP. Testis and uterus contain CRBP and CRABP; both retinol and retinoic acid are essential in the functions of these organs. Although retinoic acid will support testosterone synthesis, it will not support spermatogenesis, nor will it support placental development in vitamin A-deficient animals (Appling and Chytil, 1981). Similarly, retinol and retinoic acid have different actions on bone cells in culture, suggesting that both have functions in normal bone development, in addition to antagonizing the actions of vitamin D when retinoic acid is present in excess. Retinol inhibits collagen synthesis, whereas retinoic acid stimulates the synthesis of noncollagen bone proteins (Dickson et al., 1989).

The role of these intracellular binding proteins seems to be to transport retinoic acid into the nucleus. Unlike receptor proteins, which bind their ligand in the nucleus and then interact with regulatory elements on DNA, the CRBPs do not enter the nucleus or interact with DNA and nucleoproteins.

2.3.2.1 Retinoid Receptors and Response Elements There are two families of nuclear retinoid receptors: the retinoic acid receptors (RARs), which bind all-frans-retinoic acid and the retinoid X receptors (RXRs), so-called because their physiological ligand was unknown when they were first discovered. It is now known to be 9-ds-retinoic acid, which also binds to and activates RARs. As well as being the activating ligand for RXR, 9-ds-retinoic acid also increases the rate of catabolism of its receptor, which may be important in the regulation of the various hormonal responses that require formation of RXR heterodimers (Figure 2.6).

14-Hydroxyretroretinol, 4-oxoretinol, and anhydroretinol, as well as possibly other retinoids, also bind to and activate the RAR family of receptors at physiological concentrations, but do not bind to the RXR family. 4-Oxoretinol is formed from all-frans-retinol in differentiating cells; 10% to 15% of intracellular retinol may be oxidized to 4-oxoretinol during an 18-hour period, whereas there is no formation of all-frans-retinoic acid or 9-ds-retinoic acid. 4-Oxoretinol induces differentiation of cells in culture (Achkar et al., 1996). In the developing Xenopus embryo, 4-oxoretinaldehyde is the major retinoid, acting as a precursor of both 4-oxoretinol and 4-oxoretinoic acid, both of which activate the RAR (Blumberg et al., 1996). This developmental role of 4-oxoretinoids may explain the observation that pregnant vitamin A-deficient rats resorb the fetuses around day 15 of gestation if they are provided with retinoic acid, but not if they are provided with retinol (Wellik and DeLuca, 1995). 14-Hydroxyretroretinol and 13,14-dihydroxyretroretinol act as growth promoters for retinol-deficient cells in culture, but do not induce differentiation; by contrast, anhydroretinol has growth-inhibiting activity.

COOH

all frans-retinoic acid

COOH

all frans-retinoic acid

~CH3 9-c/s-retinoic acid

14-hydroxy-retroretinol an hydro-ret ¡no I

COOH

RAR-RXR heterodimers

RXR homodimers

(X)

CO

RXR-calcitriol-R heterodimers

RXR-COUP heterodimers

CO

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RXR-thyroid hormorie-R heterodimers

RXR-PPAR heterodimers

Figure 2.6. Interactions of all-ira«s- and 9-CT's-retinoic acids (and other active retinoids) with retinoid receptors. COUP, chicken ovalbumin upstream promoter.

Figure 2.6. Interactions of all-ira«s- and 9-CT's-retinoic acids (and other active retinoids) with retinoid receptors. COUP, chicken ovalbumin upstream promoter.

The two families of receptors differ from each other considerably, and RARs show greater sequence homology with thyroid hormone receptors than with RXRs. RARs act only as heterodimers with RXRs; homodimers of RARs have poor affinity for retinoid response elements on DNA. The liganded CRABP(II) enhances the binding of the RAR-RXR heterodimer to response elements on DNA and amplifies the effect of the receptor dimer (Delva et al., 1999).

RXR forms active homodimers and also form heterodimers with the cal-citriol (vitamin D) receptor (Section 3.3.1), the thyroid hormone receptor, the peroxisome proliferation-activated receptor (PPAR, whose physiological ligand is a long-chain polyunsaturated fatty acid or an eicosanoid derivative), and the chicken ovalbumin upstream promoter (COUP) receptor - an orphan receptor whose physiological ligand is unknown (Mangelsdorf and Evans, 1995; Glass, 1996; Glass et al., 1997). Formation of RXR heterodimers seems to be required for DNA binding of calcitriol, thyroid hormone, and PPAR receptors. Interestingly, there is no requirement for a ligand bound to the RXR for this to occur. An unliganded RXR can form active heterodimers (Wendling et al., 1999).

In the presence of all- trans-or9-cis-retinoic acid, the receptor heterodimers are transcriptional activators. However, the heterodimers will also bind to DNA in the absence of retinoic acid, in which case they act as repressors of gene expression (Fujita and Mitsuhashi, 1999).

There are three isoforms of each receptor type: RARa, RARp, RARy, RXRa, RXRp, and RXRy. They are encoded by different genes, with different tissue-specific expression, and different expression at different times during development. There is a greater conservation of amino acid sequence between species for any one type of retinoid receptor than between the different receptor types in the same species, suggesting that the receptor types evolved separately a considerable time ago. In addition, there are two different subforms of each (RARa, RARy ,RXAa, RXRp, and RXRy) and four subforms of RARp. These arise by differential splicing of the RNA transcript (Rowe and Brickell, 1993).

RARa and RXRp have widespread distribution in tissues; RARp and RARy are expressed to different extents in different tissues during development. RARa and RXRy have tissue-specific distribution.

Studies with knockout mutant mice lacking one or another of the retinoid receptors suggest that there is some degree of redundancy or overlap between the receptor subtypes, and that RARy is especially important in the teratogenic actions of retinoids (Section 2.5.1.1; Mark et al., 1999; Maden, 2000):

• RARa0 mice show no congenital abnormalities, but have a high postnatal mortality.

• RARp0 mice show no detectable effects.

• RARy0 mice have widespread congenital abnormalities, as seen in severe vitamin A deficiency.

• RARa0 mice show the same teratogenic effects of retinoic acid excess as wild-type mice.

• RARp0 mice show the same teratogenic effects of retinoic acid excess as wild-type mice.

• RARy0 mice show some, but not all, of the teratogenic effects of retinoic acid excess.

• RXRa0 mice show abnormalities of the eye and heart.

• RXR^0 mice are morphologically normal, but the males are sterile.

The multiplicity of possible combinations of homodimers and heterodi-mers of RAR and RXR subtypes, and the various possible RXR heterodimers with other receptors, permits a wide variety of active retinoid receptor complexes that bind to different response elements on DNA. Unlike most hormone response elements on DNA, which are palindromic and bind a symmetrical receptor homodimer, the most common type of retinoid response element is a direct repeat: purine-G-(G or T)-T-C-A-(Xn)-purine-G-(G or T)-T-C-A, in which the spacer (Xn) is commonly 5 base pairs, but may be 1 or 2. There are also more complex retinoid response elements, including palindromic and inverted palindromic repeats, as well as hexameric motifs with variable spacing. This means that a wide variety of different genes may be regulated differently in response to retinoids.

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