Retinoids are essential elements in animals and indeed human. They are acquired through dietary intake of vitamin A which consists, mostly, of retinol (Gropper et al., 2008). Retinoids are mainly stored in the liver and are mobilized to tissues through the activity of various transporters such as the retinol binding protein (RBP) and transthyretin (TTR) (Quadro et al., 2004). Vitamin A is involved in many biological processes within the human body. It is required for maintaining vision, immunity, development and growth, and regulating proliferation and differentiation of some cell types (Moise et al., 2007). During normal physiological conditions, retinol is absorbed via lymphatic routs from the intestinal tract, where it undergoes first step in metabolism and is transported to target tissues for uptake and storage by specialised cells (Dew & Ong, 1997).
This system of transport involves proteins such as retinol binding protein (RBP) and transthyretin (TTR) (Yamamotoa et al., 1997). Within plasma circulation, retinoids can exist in various forms. Retinoic acid (RA) (a metabolite of retinol) for instance, binds albumin while retinol is found to bind RBP and retinyl ester forms a complex with lipoproteins (mainly chylomicrons) (Blaner, 2007). These transporting mechanisms of vitamin A and its metabolites cooperate to maintain the integrity of the system to ensure sufficient, but not excessive, amounts of retinoids reach their target tissues (Basu & Basualdo 1999). Other important players within the redinoids transport system are the lecithin-retinol acyltransferase (LRAT) and the cellular retinol-binding protein (CRBP), which are proteins that facilitate formation of retinyl ester. The main role of these enzymes is to maintain tight homeostatic balance of retinol following a high diet intake or during reduced levels of vitamin A (Moiseyev et al., 2003).
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Upon delivery to target tissue, retinol is taken up into cells through the retinol uptake system. This system of transport involves the activity of plasma transmembrane proteins, such as CBRP, which facilitate the uptake of retinol into cells (Ottonello et al., 1987). The entry of retinol into cells was thought as a simple diffusion process that occurs upon delivery by RBP (Kawaguchi et al., 2007). The suggestion of an active uptake mechanism and the existence of a transmembrane transporter for retinol was described by several research groups as early as the 1970s (Heller, 1975), but it was not until recently that this transmembrane transporter was identified as the stimulated by retinoic acid 6 (STRA6) receptor by Kawaguchi et al. (2007). These findings provided a new area of research towards targeting the uptake of retinol in potential therapies.
RETINOIDS TRANSPORT TO TARGET TISSUES
The role of chylomicrons in transporting retinyl ester
Chylomicrons are metabolized in the lymphatic system to form chylomicron remnants. Chylomicrons enter the system through thoracic duct where they are converted to remnants through the activity of lipoprotein lipase (LPL) (Redgrave 2004). Hydrolysis of chylomicron triglycerides by the LPLs to free fatty acids (FFA) facilitate remnant uptake by liver. This is due to the involvement of apolipoproteins (apolipoprotein E, apoE) in regulating receptor-mediated uptake into the liver (Cooper 1997). Chylomicrons containing retinyl esters are taken up, mostly, by the liver. In the liver, retinyl esters from chylomicrons can be stored in lipocyte cells or secreted back as retinol bound to RBP into the circulation (Goodman 1965). The remaining retinyl ester molecules that were not stored or resecreted by the liver are taken up by various tissues such as adipose tissue, muscles, bone marrow and reproductive system. This clearance of chylomicron-retinoids by extrahepatic tissue is also thought to be facilitated through LPL allowing uptake of whole particles by cells (van Bennekuma 1999). The chylomicron-source of retinoids is an important pathway in maintaining target tissues requirements. This importance is noticed, particularly, in RBP-/- mice where mild, reversible vision impairment only occurs (Quadro et al., 1999). Unless deprivation of vitamin A dietary intake occurs, RBP-/- mice will be phenotypically normal. They can maintain adequate levels of dietary vitamin A but are unable to mobilize liver-stored and tissue-stored retinyl ester. Vitamin A deficiency (VAD) appears to be affecting mice with relatively lower vitamin A intake than those with normal dietary vitamin A intake levels (Vogel et al 2002). Therefore, chylomicrons are important sources of retinoids that can compensate to some extent for the activity of RBP.
Role of Serum Albumin in transporting Retinoic acid
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When compared to retinol and retinyl ester, RA is less hydrophobic and thus cannot be absorbed in chylomicrons. RA is transported, while bound to albumin, via the portal system. This is due to its ability to prevent interaction of RBP with TTR when bound to RBP. (68)
Synthetic analogues of RA are shown to be absorbed through the portal system. The route of absorption can depend on the retinoids' lipoohilicity. Therefore, absorption can occur though nascent chylomicrons or mainly through the portal system. It can be concluded that retinoid route of absorption highly depend on its chemical properties. (69).
During fasting only 0.1-0.4% of all-trans-RA and 13-cis-RA within circulation are bound to albumin. Yet, in well-nourished animal models, this amount of circulating RA vastly contributes to the RA pools. (70)
Studies in rat showed that RA pools contribute more than 80% of liver and brain needs of RA, while other tissues acquire 5-30% of their RA from these pools. It can be noticed that all-trans-RA and 13-cis-RA are readily taken up, despite their low levels in circulation, by target cells and tissues. (71)
Therefore, albumin-bound RA transport is an important pathway for providing target tissues with retinoid.
RA dissociates from albumin and crosses through the membrane bilayers in a process similar to retinol. Synthetic RA analogues transported through the albumin pathway is considered to be of a pharmacological significance which mimics natural RA uptake. This can provide greater understanding of the process by which RA enters the cells and thus, form the basis of targeted therapies.
Circulation of retinol in plasma mediated by RBP
The unesterified form of retinol is transported as retinol bound to RBP in the blood (Kanai et al., 1968).
The synthesis and secretion of RBP takes place in the liver and other extra-hepatic tissues such as eye, kidney, lung, heart, spleen and adipose tissue (Soprano et al., 1986).
The protein TTR is also synthesised within the liver where it is thought to form a complex with RBP, prior to secretion, in the endoplasmic reticulum (ER) (Bellovinoa et al., 1996).
The RBP-TTR complex formation prevents its extensive loss through glomerular filtration (Naylor & Newcomer 1999).
Within the circulation, RBP exist as either bound or unbound to retinol (holo-RBP and apo-RBP, respectively). These forms are required for retinol export of from its liver stores.
When retinol-RBP complex reach target cells, retinol dissociates rapidly and enters the cell.
The dissociation rate of retinol does not limit the rate of uptake by cells, which can argue that the involvement of a receptor-mediated transport is unnecessary.
However, other studies showed evidence supporting the existence of a multispanning membrane receptor (STRA6) involved in mediating the uptake of retinol, from the retinol-RBP, into target cells.
The newly discovered STRA6 receptor was found to be expressed, in addition to retinal pigmented epithelium, in various blood-organ barriers. Such barriers include choroids plexi, yolk sac, and Sertoli cells.
In addition to its role in retinol uptake, the STRA6 receptor is thought to play a crucial role in development and cell differentiation. Mutations in the STRA6 gene were shown to cause several defects including anophthalmia, lung hypoplasia, diaphragmatic hernia, congenital heart defects and mental retardation.
In case of RBP dysfunction the delivery of retinoid by RBP can by compensated through the activity of chylomicrons while maintaining adequate vitamin A intake, except in the eye.
Therefore, the eye may be considered to mainly rely on STRA6 receptor-mediated uptake of retinol. Furthermore, it can be suggested that RBP and its cell surface receptor may not be required for retinol uptake.
These suggestions are supported by the fact that retinol-dependent functions are not affected by the absence of RBP except in the eye.
UPTAKE, STORAGE AND METABOLISM OF RETINOIDS
STRA6, a membrane receptor
Although a cell-surface receptor for RBP was first described by a number of groups in the mid-1970s, the molecular identity of this receptor remained elusive (summarized in Vogel et al., 1999).
The work of Kawaguchi et al. (2007) now establishes STRA6 as a cell-surface receptor for retinol- RBP that removes retinol from RBP and transports it across the plasma membrane, where it can be metabolized (see Figure 1).
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STRA6 is a member of a large group of ''stimulated by retinoic acid'' genes that encode transmembrane proteins and other proteins whose functions are largely unknown (Taneja et al., 1995).
Kawaguchi et al. (2007) provide compelling biochemical evidence that STRA6 acts as a high-affinity cell-surface receptor for RBP and propose that STRA6 is a major physiological mediator of retinol uptake by cells. Using purified RBP derivatized with an ultraviolet crosslinking agent, these investigators were able to identify STRA6 as a protein present in membrane preparations from retinal pigment epithelial (RPE) cells that binds specifically to RBP with a high affinity (Kd = 59 nM). STRA6 expression greatly enhanced retinol uptake from RBP by COS-1 cells, while RNAi knockdown of STRA6 in WiDr cells greatly diminished retinol uptake. Immunolocalization studies showed strong STRA6 expression on the basolateral membrane of RPE cells, in the blood vessels of the retina, and in the hippocampus and spleen.
Another recent report (Pasutto et al., 2007) indicates that homozygous mutations in the human STRA6 gene cause a pleiotropic, multisystem malformation syndrome. Nearly all STRA6 mutations are lethal in the perinatal period. One patient with long-term survival showed profound mental retardation, short stature, and a relatively large head.
Since these malformations are common developmental lesions associated with maternal retinoid deficiency and ablation of RARs, RXRs, or enzymes involved in retinoic acid metabolism (Sporn et al., 1994; Chambon, 2005), the report of Pasutto et al. (2007) strongly supports the notion that STRA6 indeed mediates retinoid-dependent actions within the embryo.
One of the intriguing aspects of the work of Pasutto et al. (2007) is that that the absence of RBP in humans and in mice gives rise to relatively mild phenotypes that are less severe than those observed for mutations in human STRA6.
The known human mutations of RBP cause severe biochemical retinoid deficiency but otherwise only mild clinical symptoms (night blindness and a modest retinal dystrophy; Biesalski et al., 1999).
RBP-deficient mice display several mild phenotypes including visual disturbances that resolve by 4-5 months of age if the mice are maintained on a controlled retinoid diet (Quadro et al., 1999).
For both RBP-deficient humans and mice, it has been proposed that chylomicron (postprandial) retinyl esters are able to maintain retinoid sufficiency in the absence of retinol-RBP. This raises the question of whether STRA6 may have another role (or roles) in mediating retinoid actions during development beyond its role as a receptor for RBP.
Whereas STRA6 is widely expressed in the murine embryo, in the adult, it is highly expressed in cells that compose blood-organ barriers in the brain (choroid plexus and the brain microvascular), eye (RPE), testis (Sertoli cells), kidney, spleen, and the female reproductive tract. Interestingly, computer analysis of the amino acid sequence for STRA6 had previously led investigators to postulate that STRA6 functions as a facilitative transporter of one or more nutrients (Bouillet et al., 1997).
STRA6 has also been shown to be potentially important in cancer biology. STRA6 can be synergistically upregulated by Wnt1 and retinoids in mouse mammary epithelial cells (Szeto et al., 2001), and STRA6 mRNA levels are also highly elevated in mammary gland tumors and human colorectal tumors.
Esterification and storage of retinol facilitated by LRAT
As mentioned above, LRAT plays an important role, as an enzyme, in catalyzing the formation of retinyl ester in various tissues (i.e. eye and liver). Its activity is essential for retinoids' accumulation in these tissues. For instance, retinyl esters are mainly stored in the hepatic stellate cells of the liver while visual chromophore production mainly depends on their storage in the RPE.
The absence of retinyl esters from tissues provide evidence for the importance of LRAT in Lratâˆ’/âˆ’ mice except in adipose tissue where retinyl ester levels are elevated (2-3 fold) compared to wild-type mice.
Adipocytes are considered to be important sites for the accumulation of retinyl ester in the body. They account, along with the liver, for most of the retinyl ester storage in the body.
The formation of retinyl ester in adipose tissue does not require canalization by LRAT and the enzyme which facilitates retiny ester formation is not clearly identified.
The enzyme diacylglycerol acyltransferase 1 (DGAT1) catalyze triglyceride formation from acyl-CoA and diacylglycerol. DGAT1 also catalyzes esterification of acyl-CoA-dependent retinol, exhibiting the activity of acyl-CoA-retinol acyltransferase (ARAT) in vitro. It is still believed however, that other enzymes are involved in the formation of reinyl ester within adipose tissue.
The sequestration of retinol in the form of retinyl ester mainly, allows accumulation of retinoid within tissues, limits RA generation during development, and protects against the toxic effects of vitamin A following high dietary intake levels.
Retinol and RA levels in circulation are normal in Lratâˆ’/âˆ’ mice even when these mice cannot sequester retinol in the form of retinyl ester. Therefore, RA toxicity can be seen, even when maintaining normal vitamin A dietary intake.
The upregulated Cyp26 genes, that play an important role in RA detoxification, in Lratâˆ’/âˆ’ mice, support this observation.
Among the Cyp26, other genes were found to be upregulated in Lratâˆ’/âˆ’ mice while maintaining standard vitamin A intake. These include UDP-glucuronosyltransferase-1 (UGT1) isoforms which loci are thought to be involved and regulated by RA.
Following high intake of RA or retinol, increased products levels of Î²-glucuronidation of 5,6-epoxy-RA, all-trans-RA, 4-oxo-RA and 13-cis-RA are observed in blood and bile.
In Lratâˆ’/âˆ’ mice, the increased expression of UGT1 in liver can be considered as an indication of a feedback mechanism that leads to the induction of detoxification processes mainly due to the ability of liver to sense the change in RA levels.
The role of LRAT in esterification of retinoids allows their removal from circulation and the formation of storage pools within tissues.
Therefore the delivery of retinol, and indeed retinoids with alcohol group, can be influenced through controlling the activity of LRAT and thus provide therapeutic advantages.
The formation of the retinylamides, which are pharmacologically inactive, from the retinylamine form in the liver and RPE can provide potential retinoid-based therapy.
Retinylamine, which is a potent visual cycle inhibitor, can be safely stored by LRAT via amidation, due to presence of amine group instead of the retinol-alcohol group.
The stored retinylamide can afterwards be released and hydrolyzed back into retinylamine exerting its therapeutic effects with prolonged bioavailability and low toxicity compared to other inhibitors.
The presence of LRAT is therefore important in the active uptake of retinol and retinoids with amide/hydroxyl groups, efficiently.
Uptake of retinoid by target tissues through the activity of STRA6 and CRBP
Three cellular-retinol-binding proteins, CRBP-I, II, and III, are known to facilitate the esterification of retinoids by LRAT.
The physiologic roles of LRAT and the CRBPs in the absorption and storage of retinol have been best studied in knockout animal models.
CRBP-I is expressed in the liver, kidney, and eye, and Crbp-Iâˆ’/âˆ’ mice display lower levels of hepatic retinyl esters and reduced numbers of lipid droplets in hepatic stellate cells than wild-type mice.
Crbp-Iâˆ’/âˆ’ mice succumb more easily to symptoms of VAD than wild-type mice if they are maintained on a vitamin A-restricted diet (91).
CRBP-II is expressed primarily in the intestine, and its lack in Crbp-IIâˆ’/âˆ’ mice results in decreased levels of stored retinoids and increased neonatal mortality, especially during dietary vitamin A restriction (92).
CRBP-III is expressed in the heart, muscle, adipose, and mammary tissues (93). Crbp-IIIâˆ’/âˆ’ mice have difficulty incorporating retinol into milk.
In some cases, the loss of one form of CRBP can be partly compensated for by the upregulation of other CRBPs, as is the case of the upregulation of CRBP-III in the absence of CRBP-I and vice versa (94).
Although the studies of Crbpâˆ’/âˆ’ mice demonstrate that the absence of these proteins results in a much less severe metabolic phenotype than that observed with Lratâˆ’/âˆ’ mice, these studies do show that like LRAT, the CRBPs have an important role in facilitating retinol uptake, esterification, and storage by cells (20,32,91- 93).
The critical role of LRAT in catalyzing the esterification and thus the uptake and storage of retinol suggests that this enzyme may be a useful target for assuring the specific uptake of a retinoid therapeutic agent by a target tissue.
Coupled with the hydrolysis of the esters, this potentially provides a way to localize release of the drug within a specific cell type or tissue.
The release of stored retinoid drugs from their ester pools depends on the activity of retinyl ester hydrolases in the respective target tissues.
Manipulating this reaction may provide a means for a controlled delivery of therapeutic retinoids, which, in principle, would allow for the usage of lower administered doses and hence lower toxicity.
Moreover, the expression of LRAT is subject to regulation by RA (95, 96).
STRA6 was described as an RA-responsive gene (63), and modulation of the levels of STRA6 in WiDr colon adenocarcinoma cells by RA leads to the increased uptake of retinol in these cells (62).
As a result, induction of the expression of LRAT or STRA6 using RA could increase the delivery of retinol to target tissues.
Indeed, coadministration of RA effectively increases the delivery of retinol to lungs (97,98).
In addition, the expression of CRBP-I is also upregulated via RAR action (99), whereas CRBP-II expression is controlled via RXR (100).
In fact, agonists of RXR have been shown to affect the esterification of retinol in certain cell types (101).
Possibly, ectopic expression of STRA6, LRAT, and/or CRBP via gene therapy or transcriptional regulation could help ensure the delivery of retinoids to specific target tissues or cell types.
Both LRAT and CRBP are downregulated in many tumor cell types (102-117); therefore, less retinol is available for oxidation to produce RA in cancer cells.
Upregulation of the retinoid pool in tumor cells might lead to higher endogenous levels of RA at the tumor site enabling the anticarcinogenic activities of this agent.
Future studies could examine the feasibility and therapeutic effectiveness of increasing the intracellular retinoid pool through the upregulation of the enzymes and accessory proteins involved in the generation of retinyl esters.
Retinoic acid receptors (RARs) and metabolism of retinoid
Nuclear retinoic acid (RA) receptors (RARs) consist of three subtypes, ïƒ¡ï€ (NR1B1), ïƒ¢ï€ (NR1B2) and ïƒ£ï€ (NR1B3) encoded by separate genes [Germain et al., 2006a; Germain et al., 2006c], which function as ligand-dependent transcriptional regulators heterodimerized with retinoid X receptors (RXRs).
For each subtype, there are at least 2 isoforms, which are generated by differential promoter usage and alternative splicing and differ only in their N-terminal regions. Activation of RARs by cognate ligands triggers transcriptional events leading to the activation or repression of subsets of target genes involved in cellular differentiation, proliferation and apoptosis ([Bour et al., 2006], and references therein).
The compounds that bind RARs and modulate their activity are referred to as retinoids. This generic term covers molecules that include natural vitamin A (retinol) metabolites and active synthetic analogs. Retinoids are hydrophobic, lipid-soluble, and of small size, so that they can easily cross the lipid bi-layer of cell membranes. Natural retinoids, exemplified by all-trans RA, are produced in vivo from the oxidation of vitamin A [Chambon, 2005; Sporn et al., 1994] (Figure 1).
An isomerization product of RA, 9-cis RA, also binds RARs with high affinity, but whether this compound is a natural bioactive retinoid remains controversial [Germain et al., 2006b].
Beyond the natural compounds, major research efforts in retinoid chemistry have been directed towards the identification of potent synthetic molecules and led to the generation of several classes of compounds with a panel of activities ranging from agonists to antagonists, selective or not to RAR subtypes [de Lera et al., 2007] (Figure 1).
Note that for (B) and (D) in Figure 1, a given ligand may be considered as selective for a certain RAR subtype when it exhibits an affinity difference greater than 100-fold between its primary target and other receptors (see the recommended usage of terms in the field of nuclear receptors [Germain et al., 2006c]).
RARs have a well-defined domain organization and structure, consisting mainly of a central DNA-binding domain (DBD) linked to a C-terminal ligand-binding domain (LBD). In the past 20 years, it has been established that the basic mechanism for transcriptional regulation by RARs relies on DNA binding to specific sequence elements located in the promoters of target genes and on ligand-induced conformational changes in the LBD that direct the dissociation/association of several coregulator complexes [Chambon, 1996; Germain et al., 2003; Laudet and Gronemeyer, 2001; Lefebvre et al., 2005].
The description of the crystallographic structures of these domains and the characterization of the multiprotein complexes that specify the transcriptional activity of RARs provided a wealth of information on how these receptors regulate transcription. However, recent years have witnessed the importance of the ubiquitin-proteasome system and that of the N-terminal domain (NTD), which also interacts with specific coregulators, despite its native disordered structure [Bour et al., 2007].
Moreover, according to recent studies, RARs are involved in other nongenomic biological activities such as the activation of translation and of kinase cascades. These kinases target RARs and their coregulators, adding more complexity to the understanding of RAR-mediated transcription.
In this review we will focus, in addition to the basic scenario (DNA and ligand binding, dynamics of coregulator exchanges at the LBD), on recent advances in the nongenomic effects of RA, and on how phosphorylation cascades, the NTD and the ubiquitin-proteasome system cooperate for fine-tuning RAR activity.