Cloning of the human RoDH-related short chain dehydrogenase gene and analysis of its structure

We have previously characterized the first human NAD(+)-dependent short chain dehydrogenase capable of oxidizing all-trans-retinol and androgens, and found only in the liver and skin. In a search for related human enzymes, we identified a partial open reading frame, which exhibited >60% sequence identity to human RoDH-4. The full-length cDNA for this enzyme was determined in our laboratory by 5'-RACE PCR and was found to be identical to the recently reported novel type of oxidative human 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD). Analysis of the genomic structure revealed that the gene for RoDH-like 3alpha-HSD has four translated exons and, possibly, a fifth exon that codes for the 5'-untranslated region. The gene for RoDH-4 appears to have only four exons. The positions of exon-intron boundaries and the sizes of the protein coding regions are identical in 3alpha-HSD and RoDH-4. Moreover, both genes are mapped to chromosome 12q13, and are located in a close proximity to each other. Both genes appear to have satellite pseudogenes. Thus, RoDH-4 and 3alpha-HSD genes share similar structural organization and cluster on human chromosome 12, near the gene for 11-cis retinol dehydrogenase.     

13-cis-retinoic acid competitively inhibits 3 alpha-hydroxysteroid oxidation by retinol dehydrogenase RoDH-4: a mechanism for its anti-androgenic effects in sebaceous glands?

Retinol dehydrogenase-4 (RoDH-4) converts retinol and 13-cis-retinol to corresponding aldehydes in human liver and skin in the presence of NAD(+). RoDH-4 also converts 3 alpha-androstanediol and androsterone into dihydrotestosterone and androstanedione, which may stimulate sebum secretion. This oxidative 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) activity of RoDH-4 is competitively inhibited by retinol and 13-cis-retinol. Here, we further examine the substrate specificity of RoDH-4 and the inhibition of its 3 alpha-HSD activity by retinoids. Recombinant RoDH-4 oxidized 3,4-didehydroretinol-a major form of vitamin A in the skin-to its corresponding aldehyde. 13-cis-retinoic acid (isotretinoin), 3,4-didehydroretinoic acid, and 3,4-didehydroretinol, but not all-trans-retinoic acid or the synthetic retinoids acitretin and adapalene, were potent competitive inhibitors of the oxidative 3 alpha-HSD activity of RoDH-4, i.e., reduced the formation of dihydrotestosterone and androstandione in vitro. Extrapolated to the in vivo situation, this effect might explain the unique sebosuppressive effect of isotretinoin when treating acne.     

Xanthine dehydrogenase processes retinol to retinoic acid in human mammary epithelial cells

Retinoic acid is considered to be the active metabolite of retinol, able to control differentiation and proliferation of epithelia. Retinoic acid biosynthesis has been widely described with the implication of multiple enzymatic activities. However, our understanding of the cell biological function and regulation of this process is limited. In a recent study we evidenced that milk xanthine oxidase (E.C. 1.17.3.2.) is capable to oxidize all-trans-retinol bound to CRBP (holo-CRBP) to all-trans-retinaldehyde and then to all-trans-retinoic acid. To get further knowledge regarding this process we have evaluated the biosynthetic pathway of retinoic acid in a human mammary epithelial cell line (HMEC) in which xanthine dehydrogenase (E.C. 1.17.1.4.), the native form of xanthine oxidase, is expressed. Here we report the demonstration of a novel retinol oxidation pathway that in the HMEC cytoplasm directly conduces to retinoic acid. After isolation and immunoassay of the cytosolic protein showing retinol oxidizing activity we identified it with the well-known enzyme xanthine dehydrogenase. The NAD+ dependent retinol oxidation catalyzed by xanthine dehydrogenase is strictly dependent on cellular retinol binding proteins and is inhibited by oxypurinol. In this work, a new insight into the biological role of xanthine dehydrogenase is given.     

Xanthine dehydrogenase processes retinol to retinoic acid in human mammary epithelial cells

Retinoic acid is considered to be the active metabolite of retinol, able to control differentiation and proliferation of epithelia. Retinoic acid biosynthesis has been widely described with the implication of multiple enzymatic activities. However, our understanding of the cell biological function and regulation of this process is limited. In a recent study we evidenced that milk xanthine oxidase (E.C. 1.17.3.2.) is capable to oxidize all-trans-retinol bound to CRBP (holo-CRBP) to all-trans-retinaldehyde and then to all-trans-retinoic acid. To get further knowledge regarding this process we have evaluated the biosynthetic pathway of retinoic acid in a human mammary epithelial cell line (HMEC) in which xanthine dehydrogenase (E.C. 1.17.1.4.), the native form of xanthine oxidase, is expressed. Here we report the demonstration of a novel retinol oxidation pathway that in the HMEC cytoplasm directly conduces to retinoic acid. After isolation and immunoassay of the cytosolic protein showing retinol oxidizing activity we identified it with the well-known enzyme xanthine dehydrogenase. The NAD⁺ dependent retinol oxidation catalyzed by xanthine dehydrogenase is strictly dependent on cellular retinol binding proteins and is inhibited by oxypurinol. In this work, a new insight into the biological role of xanthine dehydrogenase is given.     

Kinetic analysis of human enzyme RDH10 defines the characteristics of a physiologically relevant retinol dehydrogenase

Human retinol dehydrogenase 10 (RDH10) was implicated in the oxidation of all-trans-retinol for biosynthesis of all-trans-retinoic acid, however, initial assays suggested that RDH10 prefers NADP(+) as a cofactor, undermining its role as an oxidative enzyme. Here, we present evidence that RDH10 is, in fact, a strictly NAD(+)-dependent enzyme with multisubstrate specificity that recognizes cis-retinols as well as all-trans-retinol as substrates. RDH10 has a relatively high apparent K(m) value for NAD(+) (~100 microm) but the lowest apparent K(m) value for all-trans-retinol (~0.035 microm) among all NAD(+)-dependent retinoid oxidoreductases. Due to its high affinity for all-trans-retinol, RDH10 exhibits a greater rate of retinol oxidation in the presence of cellular retinol-binding protein type I (CRBPI) than human microsomal RoDH4, but like RoDH4, RDH10 does not recognize retinol bound to CRBPI as a substrate. Consistent with its preference for NAD(+), RDH10 functions exclusively in the oxidative direction in the cells, increasing the levels of retinaldehyde and retinoic acid. Targeted small interfering RNA-mediated silencing of endogenous RDH10 or RoDH4 expression in human cells results in a significant decrease in retinoic acid production from retinol, identifying both human enzymes as physiologically relevant retinol dehydrogenases. The dual cis/trans substrate specificity suggests a dual physiological role for RDH10: in the biosynthesis of 11-cis-retinaldehyde for vision as well as the biosynthesis of all-trans-retinoic acid for differentiation and development.     

Retinol dehydrogenase 10 but not retinol/sterol dehydrogenase(s) regulates the expression of retinoic acid-responsive genes in human transgenic skin raft culture

Retinoic acid is essential for skin growth and differentiation, and its concentration in skin is controlled tightly. In humans, four different members of the short-chain dehydrogenase/reductase (SDR) superfamily of proteins were proposed to catalyze the rate-limiting step in the biosynthesis of retinoic acid (the oxidation of retinol to retinaldehyde). Epidermis contains at least three of these enzymes, but their relative importance for retinoic acid biosynthesis and regulation of gene expression during growth and differentiation of epidermis is not known. Here, we investigated the effect of the four human SDRs on retinoic acid biosynthesis, and their impact on growth and differentiation of keratinocytes using organotypic skin raft culture model of human epidermis. The results of this study demonstrate that ectopic expression of retinol dehydrogenase 10 (RDH10, SDR16C4) in skin rafts dramatically increases proliferation and inhibits differentiation of keratinocytes, consistent with the increased steady-state levels of retinoic acid and activation of retinoic acid-inducible genes in RDH10 rafts. In contrast, SDRs with dual retinol/sterol substrate specificity, namely retinol dehydrogenase 4 (RoDH4, SDR9C8), RoDH-like 3α-hydroxysteroid dehydrogenase (RL-HSD, SDR9C6), and RDH-like SDR (RDHL, SDR9C4) do not affect the expression of retinoic acid-inducible genes but alter the expression levels of several components of extracellular matrix. These results reveal essential differences in the metabolic contribution of RDH10 versus retinol/sterol dehydrogenases to retinoic acid biosynthesis and provide the first evidence that non-retinoid metabolic products of retinol/sterol dehydrogenases affect gene expression in human epidermis.     

Microsomes convert retinol and retinal into retinoic acid and interfere in the conversions catalyzed by cytosol

Rat liver microsomes converted retinol into retinal and retinoic acid. The production of retinal was observed over a range of substrate concentrations (10-100 microM), but retinoic acid was detected only at retinol concentrations of 50 microM or higher. At 50 microM retinol, the rate of microsomal retinal production was 2-fold greater than that of cytosol, but the rate of retinoic acid synthesis was 4-fold less than that of cytosol. Retinal was also converted into retinoic acid by rat liver microsomes, but at a rate 2-5% of that catalyzed by cytosol. Microsomes also interfered with the conversion of retinol and retinal into retinoic acid by rat liver cytosol. A 50% decrease in the cytosolic rates of retinoic acid production from retinol or retinal was caused by microsomal to cytosolic protein ratios of 0.1 and 0.5, respectively. Under the incubation conditions, which included NAD in the medium, addition of microsomes to cytosol did not affect the elimination half-life of retinol or retinoic acid, but did decrease the elimination half-life of retinal by 2-fold. These data show that retinal synthesis from retinol does not necessarily reflect retinoic acid synthesis and suggest that liver microsomes sequester free retinol and convert it into retinal primarily for elimination, rather than to serve as substrate for cytosolic retinoic acid synthesis.     

Biosynthesis of all-trans-retinoic acid from retinal. Recognition of retinal bound to cellular retinol binding protein (type I) as substrate by a purified cytosolic dehydrogenase.

An NAD-dependent rat liver cytosolic dehydrogenase accepted as substrate retinal generated in situ by microsomes from retinol bound to excess CRBP (cellular retinol binding protein, type I). This activity, which was not retained by anion-exchange chromatography at pH 9.15, was designated P1. P1 activity increased 2.5-fold, with no statistically significant change in its K or Hill coefficient, in liver cytosol from rats fed a retinoid-deficient diet. Orally dosed retinoic acid partially suppressed the increase. Activities chromatographically similar to hepatic P1 were observed in cytosols from rat kidney and testes. P1, purified from rat liver cytosol, had a pI of approximately 8.3, migrated as a tetramer (214 kDa) on a Sephadex G-200 column, and had a subunit molecular mass of 55 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With free retinal it catalyzed a maximum rate of retinoic acid synthesis of 265 nmol/min/mg of protein and exhibited allosteric kinetics with a K of 0.76 +/- 0.35 microM and a Hill coefficient of 1.5 +/- 0.13 (mean +/- S.D., n = 4). Substrate inhibition was noted with retinal concentrations greater than 6 microM. The purified enzyme not only recognized retinal generated by microsomes as substrate, but also recognized retinal bound to CRBP. The rates of retinoic acid synthesis from CRBP-retinal, with a series of increasing apoCRBP concentrations, exceeded the rates that would be supported by the free retinal present. The CRBP-retinal complex exhibited allosteric kinetics (K, 0.13 microM; Hill coefficient, 1.75; averages of duplicates) in the presence of excess apoCRBP (the ratio total CRBP/total retinal at each concentration of retinal was 2). This enzyme is likely to play a significant role in retinoic acid synthesis in vivo, because it participates in the synthesis of retinoic acid from a physiologically occurring form of retinol (holoCRBP), reflects retinoid status, and is distributed in extrahepatic tissues in addition to liver. These results also suggest a novel role for CRBP in retinoid metabolism, facilitating the conversion of retinal into retinoic acid.     

Holocellular retinol binding protein as a substrate for microsomal retinal synthesis

Holocellular retinol binding protein (holo-CRBP) was substrate for retinal synthesis at physiological pH with microsomes prepared from rat liver, kidney, lung, and testes. Four observations indicated that retinal synthesis was supported by holo-CRBP directly, rather than by the unbound retinol in equilibrium with CRBP. First, the rate of retinal synthesis with holo-CRBP exceeded the rate that was observed from the concentration of unbound retinol in equilibrium with CRBP. Second, NADP was the preferred cofactor only with holo-CRBP, supporting a rate about 3-fold greater than that of NAD. In contrast, with unbound retinol as substrate, similar rates of retinal formation were supported by either NAD or NADP. Third, the rate of retinal synthesis was not related to the decrease in the concentration of unbound retinol in equilibrium with holo-CRBP caused by increasing the concentration of apo-CRBP. Fourth, the rate of retinal synthesis increased with increases in the concentration of holo-CRBP as a fixed concentration of unbound retinol was maintained. This was achieved by increasing both apo-CRBP and holo-CRBP, but keeping constant the ratio apo-CRBP/holo-CRBP. Retinal formation from holo-CRBP displayed typical Michaelis-Menten kinetics with a Km about 1.6 microM, less than the physiological retinal concentration of 4-10 microM in the livers of rats fed diets with recommended vitamin A levels. The Vmax for retinal formation from holo-CRBP was 14-17 pmol min-1 (mg of protein)-1, a rate sufficiently high to generate adequate retinal to contribute significantly to retinoic acid synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of 9-cis-retinoic acid in vivo. The roles of different retinol dehydrogenases and a structure-activity analysis of microsomal retinol dehydrogenases

Retinoic acid is generated by a two-step mechanism. First, retinol is converted into retinal by a retinol dehydrogenase, and, subsequently, retinoic acid is formed by a retinal dehydrogenase. In vitro, several enzymes are suggested to act in this metabolic pathway. However, little is known regarding their capacity to contribute to retinoic acid biosynthesis in vivo. We have developed a versatile cell reporter system to analyze the role of several of these enzymes in 9-cis-retinoic acid biosynthesis in vivo. Using a Gal4-retinoid X receptor fusion protein-based luciferase reporter assay, the formation of 9-cis-retinoic acid from 9-cis-retinol was measured in cells transfected with expression plasmids encoding different combinations of retinol and retinal dehydrogenases. The results suggested that efficient formation of 9-cis-retinoic acid required co-expression of retinol and retinal dehydrogenases. Interestingly, the cytosolic alcohol dehydrogenase 4 failed to efficiently catalyze 9-cis-retinol oxidation. A structure-activity analysis showed that mutants of two retinol dehydrogenases, devoid of the carboxyl-terminal cytoplasmic tails, displayed greatly reduced enzymatic activities in vivo, but were active in vitro. The cytoplasmic tails mediate efficient endoplasmic reticulum localization of the enzymes, suggesting that the unique milieu in the endoplasmic reticulum compartment is necessary for in vivo activity of microsomal retinol dehydrogenases.     

Comparative functional analysis of human medium-chain dehydrogenases, short-chain dehydrogenases/reductases and aldo-keto reductases with retinoids

Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto reductase) superfamilies have been reported to catalyse the conversion between retinol and retinaldehyde. Estimation of the relative contribution of enzymes of each type was difficult since kinetics were performed with different methodologies, but SDRs would supposedly play a major role because of their low K(m) values, and because they were found to be active with retinol bound to CRBPI (cellular retinol binding protein type I). In the present study we employed detergent-free assays and HPLC-based methodology to characterize side-by-side the retinoid-converting activities of human MDR [ADH (alcohol dehydrogenase) 1B2 and ADH4), SDR (RoDH (retinol dehydrogenase)-4 and RDH11] and AKR (AKR1B1 and AKR1B10) enzymes. Our results demonstrate that none of the enzymes, including the SDR members, are active with CRBPI-bound retinoids, which questions the previously suggested role of CRBPI as a retinol supplier in the retinoic acid synthesis pathway. The members of all three superfamilies exhibit similar and low K(m) values for retinoids (0.12-1.1 microM), whilst they strongly differ in their kcat values, which range from 0.35 min(-1) for AKR1B1 to 302 min(-1) for ADH4. ADHs appear to be more effective retinol dehydrogenases than SDRs because of their higher kcat values, whereas RDH11 and AKR1B10 are efficient retinaldehyde reductases. Cell culture studies support a role for RoDH-4 as a retinol dehydrogenase and for AKR1B1 as a retinaldehyde reductase in vivo.     

Analysis of the NADH-dependent retinaldehyde reductase activity of amphioxus retinol dehydrogenase enzymes enhances our understanding of the evolution of the retinol dehydrogenase family

In vertebrates, multiple microsomal retinol dehydrogenases are involved in reversible retinol/retinal interconversion, thereby controlling retinoid metabolism and retinoic acid availability. The physiologic functions of these enzymes are not, however, fully understood, as each vertebrate form has several, usually overlapping, biochemical roles. Within this context, amphioxus, a group of chordates that are simpler, at both the functional and genomic levels, than vertebrates, provides a suitable evolutionary model for comparative studies of retinol dehydrogenase enzymes. In a previous study, we identified two amphioxus enzymes, Branchiostoma floridae retinol dehydrogenase 1 and retinol dehydrogenase 2, both candidates to be the cephalochordate orthologs of the vertebrate retinol dehydrogenase enzymes. We have now proceeded to characterize these amphioxus enzymes. Kinetic studies have revealed that retinol dehydrogenase 1 and retinol dehydrogenase 2 are microsomal proteins that catalyze the reduction of all-trans-retinaldehyde using NADH as cofactor, a remarkable combination of substrate and cofactor preferences. Moreover, evolutionary analysis, including the amphioxus sequences, indicates that Rdh genes were extensively duplicated after cephalochordate divergence, leading to the gene cluster organization found in several mammalian species. Overall, our data provide an evolutionary reference with which to better understand the origin, activity and evolution of retinol dehydrogenase enzymes.     

Characterization of a microsomal retinol dehydrogenase gene from amphioxus: retinoid metabolism before vertebrates

Amphioxus, a member of the subphylum Cephalochordata, is thought to be the closest living relative to vertebrates. Although these animals have a vertebrate-like response to retinoic acid, the pathway of retinoid metabolism remains unknown. Two different enzyme systems - the short chain dehydrogenase/reductases and the cytosolic medium-chain alcohol dehydrogenases (ADHs) - have been postulated in vertebrates. Nevertheless, recent data show that the vertebrate-ADH1 and ADH4 retinol-active forms originated after the divergence of cephalochordates and vertebrates. Moreover, no data has been gathered in support of medium-chain retinol active forms in amphioxus. Then, if the cytosolic ADH system is absent and these animals use retinol, the microsomal retinol dehydrogenases could be involved in retinol oxidation. We have identified the genomic region and cDNA of an amphioxus Rdh gene as a preliminary step for functional characterization. Besides, phylogenetic analysis supports the ancestral position of amphioxus Rdh in relation to the vertebrate forms.     

Enzymes and binding proteins affecting retinoic acid concentrations

Free retinoids suffer promiscuous metabolism in vitro. Diverse enzymes are expressed in several subcellular fractions that are capable of converting free retinol (retinol not sequestered with specific binding proteins) into retinal or retinoic acid. If this were to occur in vivo, regulating the temporal-spatial concentrations of functionally-active retinoids, such as RA (retinoic acid), would be enigmatic. In vivo, however, retinoids occur bound to high-affinity, high-specificity binding proteins, including cellular retinol-binding protein, type I (CRBP) and cellular retinoic acid-binding protein, type I (CRABP). These binding proteins, members of the superfamily of lipid binding proteins, are expressed in concentrations that exceed those of their ligands. Considerable data favor a model pathway of RA biosynthesis and metabolism consisting of enzymes that recognize CRBP (apo and holo) and holo-CRABP as substrates and/or affecters of activity. This would restrict retinoid access to enzymes that recognize the appropriate binding protein, imparting specificity to RA homeostasis; preventing, e.g. opportunistic RA synthesis by alcohol dehydrogenases with broad substrate tolerances. An NADP-dependent microsomal retinol dehydrogenase (RDH) catalyzes the first reaction in this pathway. RDH recognizes CRBP as substrate by the dual criteria of enzyme kinetics and chemical crosslinking. A cDNA of RDH has been cloned, expressed and characterized as a short-chain alchol dehydrogenase. Retinal generated in microsomes from holo-CRBP by RDH supports cytosolic RA synthesis by an NAD-dependent retinal dehydrogenase (RalDH). RalDH has been purified, characterized with respect to substrate specificity, and its cDNA has been cloned. CRABP is also important to modulating the steady-state concentrations of RA, through sequestering RA and facilitating its metabolism, because the complex CRABP/RA acts as a low K_m substrate.     

Microsomal retinal synthesis: retinol vs. holo-CRBP as substrate and evaluation of NADP, NAD and NADPH as cofactors.

Holo-CRBP (cellular retinol binding protein) is recognized specifically by an NADP-dependent microsomal retinol dehydrogenase and protects retinol from conversion into retinal by NAD and NADPH dependent dehydrogenases. The synthesis of retinal from free retinol is catalyzed by both NADP- and NAD-dependent pathways, with the former being the preferred one (Km of 4 vs. 22 microM for retinol, and Vmax/Km of 33 vs. 9, respectively). NADPH does not support quantitatively significant retinal synthesis from physiological concentrations of retinol or holo-CRBP, if an NADPH regenerating system is used to prevent NADP formation.     

NAD+-dependent retinol dehydrogenase in liver microsomes

A microsomal NAD+-dependent retinol dehydrogenase is being described with optimal activity at physiological pH. The enzyme was present in liver microsomes of rats and also in a strain of deermice which lacks the cytosolic retinol dehydrogenase. Unlike the latter enzyme, the microsomal retinol dehydrogenase was not inhibited by either ethanol or 4-methylpyrazole; its activity was insensitive to CO and not oxygen dependent, in contradistinction with that of the microsomal cytochrome P-450 and NADPH-dependent retinol oxidase. Chronic ethanol consumption resulted in an increased activity of the microsomal retinol dehydrogenase which may contribute to hepatic retinol depletion, especially in view of the insensitivity of the enzyme to ethanol inhibition.     

Characterization of a microsomal retinol dehydrogenase gene from amphioxus: retinoid metabolism before vertebrates

Amphioxus, a member of the subphylum Cephalochordata, is thought to be the closest living relative to vertebrates. Although these animals have a vertebrate-like response to retinoic acid, the pathway of retinoid metabolism remains unknown. Two different enzyme systems — the short chain dehydrogenase/reductases and the cytosolic medium-chain alcohol dehydrogenases (ADHs) — have been postulated in vertebrates. Nevertheless, recent data show that the vertebrate-ADH1 and ADH4 retinol-active forms originated after the divergence of cephalochordates and vertebrates. Moreover, no data has been gathered in support of medium-chain retinol active forms in amphioxus. Then, if the cytosolic ADH system is absent and these animals use retinol, the microsomal retinol dehydrogenases could be involved in retinol oxidation. We have identified the genomic region and cDNA of an amphioxus Rdh gene as a preliminary step for functional characterization. Besides, phylogenetic analysis supports the ancestral position of amphioxus Rdh in relation to the vertebrate forms.     

Cloning of the human RoDH-related short chain dehydrogenase gene and analysis of its structure

We have previously characterized the first human NAD⁺-dependent short chain dehydrogenase capable of oxidizing all-trans-retinol and androgens, and found only in the liver and skin. In a search for related human enzymes, we identified a partial open reading frame, which exhibited >60% sequence identity to human RoDH-4. The full-length cDNA for this enzyme was determined in our laboratory by 5′-RACE PCR and was found to be identical to the recently reported novel type of oxidative human 3α-hydroxysteroid dehydrogenase (3α-HSD). Analysis of the genomic structure revealed that the gene for RoDH-like 3α-HSD has four translated exons and, possibly, a fifth exon that codes for the 5′-untranslated region. The gene for RoDH-4 appears to have only four exons. The positions of exon–intron boundaries and the sizes of the protein coding regions are identical in 3α-HSD and RoDH-4. Moreover, both genes are mapped to chromosome 12q13, and are located in a close proximity to each other. Both genes appear to have satellite pseudogenes. Thus, RoDH-4 and 3α-HSD genes share similar structural organization and cluster on human chromosome 12, near the gene for 11-cis retinol dehydrogenase.     

Retinol forms retinoic acid via retinal.

Hepatic cytosol from normal deermice having cytosolic alcohol dehydrogenase (ADH+) also displays retinol dehydrogenase activity and converts retinol to retinoic acid, whereas cytosol from ADH- deermice lacks these enzyme activities and does not produce retinoic acid. Furthermore, microsomes from either strain do not convert retinol to retinoic acid. However, when cytosol from ADH- animals is added to the microsomes, retinoic acid is produced. The obligatory role of retinal as an intermediary step in retinoic acid formation is further shown by isotopic dilution of retinoic acid formed from labeled retinol upon addition of unlabeled retinal. Microsomal retinol dehydrogenase also catalyzes the reduction of retinal to retinol, thereby explaining the decrease in retinoic acid production from retinol in liver cytosol of ADH+ deermice when microsomes are added. Thus, the results of this study indicate that retinal is an obligatory intermediate in the hepatic production of retinoic acid from retinol and that cytosolic and microsomal retinol dehydrogenases play a key role in this process.     

Identification of the major oxidative 3alpha-hydroxysteroid dehydrogenase in human prostate that converts 5alpha-androstane-3alpha,17beta-diol to 5alpha-dihydrotestosterone: a potential therapeutic target for androgen-dependent disease

Androgen-dependent prostate diseases initially require 5alpha-dihydrotestosterone (DHT) for growth. The DHT product 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), is inactive at the androgen receptor (AR), but induces prostate growth, suggesting that an oxidative 3alpha-hydroxysteroid dehydrogenase (HSD) exists. Candidate enzymes that posses 3alpha-HSD activity are type 3 3alpha-HSD (AKR1C2), 11-cis retinol dehydrogenase (RODH 5), L-3-hydroxyacyl coenzyme A dehydrogenase , RODH like 3alpha-HSD (RL-HSD), novel type of human microsomal 3alpha-HSD, and retinol dehydrogenase 4 (RODH 4). In mammalian transfection studies all enzymes except AKR1C2 oxidized 3alpha-diol back to DHT where RODH 5, RODH 4, and RL-HSD were the most efficient. AKR1C2 catalyzed the reduction of DHT to 3alpha-diol, suggesting that its role is to eliminate DHT. Steady-state kinetic parameters indicated that RODH 4 and RL-HSD were high-affinity, low-capacity enzymes whereas RODH 5 was a low-affinity, high-capacity enzyme. AR-dependent reporter gene assays showed that RL-HSD, RODH 5, and RODH 4 shifted the dose-response curve for 3alpha-diol a 100-fold, yielding EC(50) values of 2.5 x 10(-9) M, 1.5 x 10(-9) M, and 1.0 x 10(-9) M, respectively, when compared with the empty vector (EC(50) = 1.9 x 10(-7) M). Real-time RT-PCR indicated that L-3-hydroxyacyl coenzyme A dehydrogenase and RL-HSD were expressed more than 15-fold higher compared with the other candidate oxidative enzymes in human prostate and that RL-HSD and AR were colocalized in primary prostate stromal cells. The data show that the major oxidative 3alpha-HSD in normal human prostate is RL-HSD and may be a new therapeutic target for treating prostate diseases.