RETINOIC ACID
Introduction
Retinoids are derivatives of retinol that are also known as vitamin A. The structure of retinoids consists of the polyene hydrophobic tail that is attached to a cyclic 6-carbon ring. Retinoids are light-sensitive due to the presence of conjugation of the carbon-carbon double bonds in the polyene tail. Retinoids are highly hydrophobic and hence are able to pass through cell membranes via passive diffusion. Cellular growth, apoptosis, immune response, and epithelial growth are induced upon binding of retinoids to nuclear receptors that are retinoic acid receptor (RAR) and retinoid X receptor (RXR). During early embryonic development, retinoids regulate germ layer formation, body axis formation, neurogenesis, cardiogenesis and the development of pancreas, lungs and eyes (Kam et al., 2012; Zasada & Budzisz, 2019). Overall, retinoids play an important role in adult body homeostasis and embryonic development and therefore have been extensively studied.
Retinoic Acid Pathway
The substrates: Cellular Retinol-Binding Protein and Cellular Retinoic Acid-Binding Protein
Cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP) are two binding proteins which are specific for retinoids. These two proteins work as carrier proteins for members of the vitamin A family (including retinol). Each binding protein exhibits affinities for its ligands similar to the receptors' affinities for their hormones and much higher than typical enzymes for their substrates.
The capacity of biological membranes to sequester retinol exceeds the retinol concentration in vivo by at least an order of magnitude. Yet, membranes contain little retinol, and the small amount present can be attributed easily to the CRBP present. Diet-caused and/or other vitamin A fluctuations would introduce a seemingly uncontrolled influence over membrane function, membranes, and retinol repositories. Unbound retinol suffers facile nonenzymatic isomerisation and oxidation, which CRBP nearly completely arrests.
A working model of retinoic acid homeostasis
The amount of retinol absorbed may be determined by the quantity of the intracellular CRBP. Transfection of the human intestinal cell line Caco-2 with a CRBP-expressing vector increased the absorption of retinol from the medium. Retinyl ester synthesis also correlated directly with the increase in the amount of retinal absorbed. In vivo, the serum retinal binding protein (RBP) transports retinol through serum and delivers it to cells. The retinal uptake rate from serum RBP decreased in Sertoli cells when the available CRBP sites had been occupied. In this case, regeneration of apo-CRBP subsequent to retinol esterification might have affected the retinal absorption rate. Because some cells express retinol transport systems, including a retinal binding protein (RBP) receptor on their plasma membrane, simple diffusion may not always account for retinal uptake. In such cases, the amount of retinal taken up by cells would involve more than just the intracellular concentration of CRBP. Conceivably, CRBP would still contribute to uptake, perhaps by removing retinal from the inner portal of a transporter when unliganded or by blocking the internal portal when charged with retinol.
Generation of Retinoic Acid
Once formed, the CRBP-retinol complex serves as a substrate for retinyl esterification by the microsomal enzyme lecithin retinol acyltransferase (LRAT) and conversion of retinol into retinal by microsomal retinol dehydrogenase (RoDH) isozymes. CRBP directs retinol to LRAT and NADP-dependent microsomal RoDHs and prevents access to retinol of acyl-CoA:
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Retinol acyltransferase (ARAT).
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NAD-dependent microsomal dehydrogenases.
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The classical medium-chain alcohol dehydrogenases (ADHs) traditionally associated with cytosolic ethanol metabolism.
Cytosolic retinal dehydrogenase (Ra1DH) isozymes convert retinal generated from CRBP-retinol by microsomal RoDH isozymes into RA.
Influence of apo-CRBP
apo-CRBP generated during the metabolism of retinol affects retinal metabolism. The ratio apo-CRBP/holo-CRBP, by virtue of the signalling properties of apo-CRBP and the substrate properties of holo-CRBP, modulates the retinal proportion conducted into esterification vs RA biosynthesis. apo-CRBP inhibits LRAT but stimulates retinyl ester hydralase (REH) activity.
Transport and metabolism of Retanoic Acid
RA enters the nucleus where it activates the ligand-activated transcription factors, RARs (retinoic acid receptors), which function as heterodimers with unliganded retinoid X receptors (RXRs); RA undergoes metabolism initiated at two different 𝛃-ionone ring sites (at least), depending on the tissue: C4 and C18; CRABP sequesters RA in the cytosol. CRABP enhances RA metabolism, and rapid RA metabolism tempers its potency. The CRABP-RA complex serves as a low Km substrate for RA metabolism by cytochromes P450 in the post mitochondrial supernatant of rat tissues. Arresting RA metabolism through inhibition with ketoconazole or other antifungal P450 inhibitors, on the other hand, enhances its potency. In the murine teratocarcinoma cell line F9, a threefold decrease in the ED50 follows a threefold increase in the elimination t1/2 of RA. Furthermore, F9 cell mutants that overexpress CRABP metabolise RA faster than wild-type F9 cells and require higher RA concentrations to differentiate. If CRABP sequestered RA to buffer its signalling function and/or to minimise localisation in membranes, then delivering the CRABP-bound RA for metabolism could serve as a mechanism of discharging CRABP to regenerate apo-CRABP.
Figure 1
Retinoic Acid metabolism pathway
Vitamin A deficiency
RA signalling in normal development
Vitamin A , which consist of retinol , retinal and other provitamin A caratinoids, and its metabolites such as retinoic Acid are important for the normal development of the human body having major roles in the development of the neural plate, spinal cord and other tissue in the body. A deficiency in vitamin A can be brought about by several factors such as liver disorders, fat malabsorption and even malnutrition and so a healthy and balanced diet is necessary to avoid any deficiencies. Since retinoids are hydrophobic, malabsorption of fats can severly affect vitamin A absorption, also leading to a defeciency.
Retinoic acid is important for the proper orientation of the neural plate. The neural plate which is an important base for the development of the nervous system, initially develops in the anterior region in the embryo. The presence of retinoic acid allows anteroposterior patterning of the neural plate. Retinoic acid is also important in the development of the spinal cord neurons. In fact, studies have shown that embryos that are exposed to a vitamin A deficiency, are more likely to have fewer spinal cord neurons. Eye development also require retinoic acid. It is important for the activation of specific retinoic receptors that are critical in the development of the anterior eye segment. Apart from these roles, Retinoic acid is also important in proper heart, lung and pancreas development. Deficiency in vitamin A can therefore result in poor coronary vessel development, improper heart tube patterning and also abnormalities in the upper respiratory tract.
Night blindness and Xerophthalmia
Most people with a deficiency in vitamin A experience night blindness as the earliest clinical symptom. With a deficiency in vitamin A, rod photoreceptor cells in the retina fail to work properly resulting in a lack of peripheral vision when there is lack of lighting. This failure of the rod receptors result in changes on the ocular surface resulting in corneal ulcerations and necrosis, conjunctival and corneal xerosis and retinal dysfunction, resulting in night blindness.
Vitamin A deficiency may also lead to xerophthalmia. In xerophthalmia, the ocular surface changes to squamous epithelium resulting in the lack of goblet cells. The lack of these mucus secreting cells lead to dryness and inflammation of the eyes.
Retinoids in metabolic disease
Deficiency of retinoic acid can lead to various metabolic diseases such as obesity, diabetes and metabolic syndrome. This results in poor metabolism and production of energy that can be used for physiological processes. These metabolic diseases are mainly brought about by the lack of PPARβ/δ receptor activation. The PPARβ/δ receptor is a receptor that is important in lipid metabolism and glucose homeostasis. The activation of this receptor is regulated by the retinoic acid present and so if there is lack of retinoic acid, activation fails resulting in a decrease in lipid catabolism that can lead to obesity.
Retinoids in neurobiology
Retinoic acid has been found to be important in the nervous system for various process such as neurogenesis, neuronal plasticity and motor controlling functions. In fact a lot of retinoic acid receptors are found throughout the brain. A deficiency in retinoic acid can therefore result in impaired memory, learning and cognitive functions that can also lead to more serious neurodegenerative diseases such as Parkinson’s and Alzheimer’s diseases.
Retinoids in the kidney
The development of the kidneys is also dependent on the availability of Vitamin A. This vitamin is important in the regulation of the PKD1 gene. When this gene is mutated, there is a high chance that the person experiences polycystic kidney disease
Figure 2
Vitamin A deficiency: Some of the effects caused by inadequate Vitamin A intake.
Vitamin A Deficiency - Effect on Epithelial Cells
Hyperkeratosis
Hyperkeratosis is a condition caused by the thickening of the outer layer of skin. This is due to the overgrowth of a protein in the skin called keratin. Vitamin A and its derivatives (retinoids) are important for regulating epithelial cell differentiation. Studies have shown that deficiency of Vitamin A may lead to a change in the type of epithelia of the respiratory, alimentary, genitourinary tracts, the eye and exocrine glands, whereby it becomes keratinized stratified epithelium. Vitamin A deficiency in the skin was shown to cause hyperkeratosis.
Keratosis Pilaris
Keratosis pilaris (KP) is a hyperkeratotic disorder which causes keratotic papules or rough bumps on the skin, as well as perifollicular erythema which is redness around the hair follicles. The papules contain excess keratin in the follicles which lead to plugs that dilate the upper part of the follicle. The plug may extend further into the hair follicle which may cause the reduction in size of the follicular walls and oil glands. The plug contains lamellae which trap coiled brittle hairs. The skin shows mild hyperkeratosis, a decrease in the thickness of the granular layer and follicular plugging. There is also the presence of a build-up of white blood cells in the upper skin layer and surrounding the follicular areas. The outer layer of the skin may exhibit keratinisation, but these cells are not found in the follicle. Studies have shown that Vitamin A deficiency is linked to KP.
Squamous Metaplasia in Bladder
Studies found that Vitamin A deficiency caused the transformation of squamous epithelial cells in the lining of the lower urinary tract as well as of other epithelia. It also found that Vitamin A deficiency may cause tumours in the squamous cells in the lining of the lower urinary tract. Vitamin A deficiency affected the cellular differentiation of the tumours, and also the rate of abnormal growth.
Squamous Metaplasia in Uterus
The epithelial cell transformation which is caused by vitamin A deficiency occurs in the glands found in the uterus lining. A study demonstrated that the epithelial transformation and keratinisation in the uterus of a rat, was only seen in those on a vitamin A deficient diet, or in the rats on a vitamin A deficient diet whose ovaries had been removed but were given oestrogen. However, the study concluded that vitamin A deficiency was not the only cause of the change in tissue in the uterus lining of the rat, since oestrogen also leads to the change. It is not known exactly how vitamin A maintains the uterus lining, but a balance may exist between oestrogen and vitamin A which contributes to it.
Retinoic Acid Role in Differentiation and Cancer
It is currently understood that retinoic acid plays essential roles in cell development and differentiation, and cancer treatment. Lung, prostate, breast, ovarian, bladder, oral, and skin cancers have been demonstrated to be suppressed by retinoic acid.
Low doses and high doses of retinoic acid may cause cell cycle arrest and apoptosis of cancer cells. Treatment using retinoic acid was approved by the U.S. Food and Drug Administration for lymphoma and leukaemia.
In anti-cancer research, retinoic acid has been investigated and found to inhibit the markers of cell proliferation, such as cyclin D1 and human telomerase reverse transcriptase (hTERT), and growth factors, such as epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF). The biological functions inhibited by retinoic acid include tumour growth, angiogenesis, and metastasis.
The inhibitory effects of retinoic acid are achieved through activating the retinoic acid receptor (RAR) or retinoic X receptor (RXR).
RAR and RXR form heterodimers and function after ligand binding. To turn on downstream gene expression, RAR and RXR shuttle into cell nuclei and bind to the retinoic acid response elements (RARE), located in the 5’-region of retinoic acid downstream genes. The activation of the above classical pathway will lead to cell differentiation, arrest, and eventually apoptosis. In addition to the above classic pathway, retinoic acid may also regulate the downstream gene expression by modulating other transcription factors, such as nf-κb, IFN-γ, TGF-β, MAPK, etc., chromatin remodelling.
The latest finding of retinoic acid is the regulation of stem cell differentiation. Ying et al. found that retinoic acid induces the expression of lineage-specific differentiation markers Tujl and GFAP and reduces neural stem cell markers such as CD133, Msi-1, nestin, and Sox-2.
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