INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

The SDR¹ superfamily consists of ~100 bacterial, plant, and animal enzymes ranging in size from ~25 to 38 kDa that are related in terms of tertiary structure, including conserved cofactor-binding sites and catalytic residues (1-3). But the members of the SDR superfamily have relatively few strictly conserved residues, and indeed, different members do not always share substantial amino acid identity. SDRs tend to have a multifunctional nature, i.e. they catalyze dehydrogenations/reductions of seemingly disparate substrates. In animals and bacteria, members of this superfamily catalyze the activation or inactivation of prostaglandins and many steroids. An apparent subgroup of the SDR superfamily, consisting of enzymes closely related to each other, catalyzes the metabolism of all-trans-retinol, cis-retinols, and androgens. This subfamily includes RoDH isozymes 1-3 and CRAD1 (4-7). A related SDR also catalyzes 11-cis- and 9-cis-retinol dehydrogenations (8-10).

Vertebrates require retinoid hormones, derived from the prohormone retinol (vitamin A), for vision, reproduction, embryogenesis, and maintenance of normal epithelial, bone, nerve, and immune system function (11). The retinol metabolite all-trans-retinoic acid satisfies all known retinoid functions in retinol-deficient animals, except for light transduction during vision, because it cannot undergo reduction into the opsin cofactor retinal, and spermatogenesis, because it cannot cross the mammalian blood-testis barrier in low concentrations. All-trans-retinoic acid functions through the three ligand-dependent transcription factors known as RAR, RAR, and RAR (12, 13). An all-trans-retinoic acid isomer, 9-cis-retinoic acid, controls the in vitro activity of a distinct group of receptors known as RXR, RXR, and RXR. RXRs affect the function of several receptors in the steroid/retinoid/thyroid/vitamin D superfamily of ligand-activated transcription factors, including RARs, through heterodimerization. Because receptor function depends on ligand concentrations, pathways of all-trans-retinoic acid and 9-cis-retinoic acid biosynthesis require understanding. A pathway of all-trans-retinol conversion first into all-trans-retinal and then into all-trans-retinoic acid has been established, but pathways of 9-cis-retinoic acid biosynthesis have not been determined (14). One problem with the latter has been identifying processes for enzymatically generating the putative hormone. Thus, enzymes that produce 9-cis-retinoids incur much interest.

Androgens virilize males through supporting formation, growth, and maturation of reproductive organs and secondary sex characteristics (15). Endocrine glands, including the testes, produce testosterone (4-androsten-17-ol-3-one), whereas the irreversible steroid 5-reductases of the prostate and other androgen target tissues produce the testosterone metabolite dihydrotestosterone (5-androstan-17-ol-3-one) (16). Testosterone directs internal male genital formation; dihydrotestosterone directs embryonic external sex organ development and the phenotypic changes associated with male puberty (17). Both testosterone and dihydrotestosterone function through the androgen receptor. As with the other receptors mentioned above, androgen receptor function depends on ligand concentrations. Dihydrotestosterone undergoes inactivation via reduction into 3-adiol (5-androstan-3,17-diol), catalyzed by members of the aldo-keto reductase superfamily. Dehydrogenation of 3-adiol by the SDR 17-steroid dehydrogenase generates the impotent androgen, androsterone (5-androstan-3-ol-17-one), cleared as its glucuronide (18). Pathways that regenerate dihydrotestosterone from 3-adiol occur in vivo and presumably contribute to regulating androgen receptor function (19). Until recently, however, the specific enzymes responsible for regeneration of dihydrotestosterone were not known. The reports of 3-adiol dehydrogenase activities of RoDH1 and CRAD1 provided candidates for such enzymatic activity (7, 20).

Here, we report the isolation of a cDNA that encodes a heretofore unknown SDR, CRAD2. Many tissues express CRAD2 mRNA, but liver is the quantitatively major site of expression. CRAD2 shows 3- and 17-hydroxysteroid dehydrogenase activities and catalyzes the dehydrogenation of retinols, including 9-cis-retinol. Expression of CRAD2 provides a means of altering the concentrations of active versus inactive androgens and of generating retinals from retinols.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Production of a CRAD2-specific Probe-- An 11-d.p.c. mouse embryonic gt11 cDNA library (CLONTECH) was screened through three rounds (final wash at 55 °C) with a P-labeled probe consisting of RoDH1 nucleotides 298-673 (4). DNA inserts from six positive phages were digested with EcoRI and cloned into pBluescript II SK^+/. One of the six clones, pBSK/E6, was completely sequenced by nested deletion. Despite the use of a cDNA library, the 2.2-kb insert represented a genomic clone that included exon 1 of a gene in the SDR family. A 367-base pair probe containing 55 base pairs of 5'-end untranslated region and 312 base pairs of the coding region in exon 1 of E6 was generated by polymerase chain reaction with the primers 5'-TTACTCTCTGAAAACGGGGC (sense) and 5'-TCTGTTCCCAACACGCTC (antisense).

CRAD2 cDNA Isolation-- A mouse liver gt10 library (CLONTECH) was screened with a probe consisting of nucleotides 683-1080 of CRAD1 (7). The positive plaques were then screened through two more rounds with the 367-base pair probe isolated from pBSK/E6. Phage DNA from three of five plaques isolated was digested with EcoRI. The inserts were ligated into pBluescript II SK^+/ to produce pBSK/CRAD-39, pBSK/CRAD-188, and pBSK/CRAD-218 and sequenced in both directions by dideoxy chain termination.

Expression of CRAD2-- The coding region of an SDR was digested from gt10 phage DNA containing the insert of pBSK/CRAD-218 with EcoRI and ligated into pcDNA3 to produce pcDNA3/CRAD2. CHO-K1 cells were cultured and transfected using LipofectAMINE with pcDNA3/CRAD2 or with pcDNA3 (mock) as described (7). Cell pellets were suspended in 10 mM HEPES and 10% sucrose, pH 7.5, and homogenized with a Dounce homogenizer. The homogenate was centrifuged at 800 × g for 20 min, and the supernatant protein was used for enzyme assays, unless noted otherwise. Protein concentrations were determined by the method of Bradford (21).

Enzyme Assays-- Incubations and analysis of products have been described in detail (7). Briefly, retinoid and steroid dehydrogenase assays were done at 37 °C in 0.25 ml of 50 mM HEPES, 150 mM KCl, 1 mM EDTA, and 1.6 mM NAD⁺, pH 8, with the 800 × g supernatant of mock- or pcDNA3/CRAD2-transfected CHO cells, unless noted otherwise. Retinoid dehydrogenase assays were quenched with 0.1 ml of 0.1 M O-ethylhydroxylamine and 0.35 ml of methanol, incubated at room temperature for 10 min, and extracted with 2.5 ml of hexane. The retinoids in the hexane extract were quantified by normal-phase high-performance liquid chromatography, with a detection limit ~1 pmol. 11-cis-Retinoids isomerize into more stable isomers during incubation and extraction; therefore, the sum of the retinal isomers recovered from incubating 11-cis-retinol was used to determine the rate of 11-cis-retinal synthesis (22). Steroid dehydrogenase assays were done with ³H-labeled steroids (40-101 Ci/mmol, 20,000 dpm/reaction). Reactions were extracted with methylene chloride (4 ml); the extracts were analyzed by thin-layer chromatography. ³H-Labeled steroids were detected by autoradiography. The radioactive zones were excised and counted with a liquid scintillation counter. Kinetic data were obtained under initial velocity conditions and were fit with Enzfitter using simple weighing (23).

Northern Blotting-- Northern blots were done with the mouse multiple tissue Northern blot (CLONTECH), which provides 2 µg of poly(A⁺) RNA/lane on a nylon membrane. The probe was a chemically synthesized 75-base-long oligonucleotide of nucleotides 1-75 of CRAD2. The probe was labeled with ³²P by random priming. Prehybridization was done in 10 ml of hybridization solution (50% formamide, 5× Denhardt's solution, 0.1% SDS, 100 µg/ml denatured salmon sperm DNA, and 5× saline/sodium phosphate/EDTA) at 40 °C for 4 h. Hybridization was done overnight in the same solution containing 2 × 10⁶ cpm of probe. The final wash was done at 55 °C with 1× SSC and 0.1% SDS. Signals were visualized with a Bio-Rad GS-505 Molecular Imager system.

RNase Protection Assay-- A CRAD2-specific probe was amplified by polymerase chain reaction from pBSK/CRAD-39 with the sense primer 5'-GCTTCCATACTACTCAGA (nucleotides 1135-1152) and the antisense primer 5'-CAAGATTCTATCCCACCA (nucleotides 1463-1480). The polymerase chain reaction product was subcloned into pGEM-T (Promega) and linearized with SpeI. A ³²P-labeled antisense probe was transcribed with T7 RNA polymerase (Promega) for 1 h at 37 °C in 10 mM dithiothreitol; 0.5 mM each ATP, CTP, and GTP; and 50 µCi of UTP (800 Ci/mmol). The 308-nucleotide antisense -actin mRNA probe (nucleotides 51-358) used as an internal standard was transcribed from pTRI mouse -actin (Ambion Inc.) under the same conditions. DNA templates were removed by DNase I digestion. Transcripts were purified with 5% polyacrylamide and 8 M urea gels. RNase protection assays were done with the Hybspeed^TM RPA kit (Ambion Inc.) following the manufacturer's instructions. Total RNA (50 µg) was extracted from mouse tissues with guanidinium thiocyanate/phenol/chloroform and coprecipitated with cRNA probes (1 × 10⁵ cpm for CRAD2 and 5 × 10⁴ cpm for mouse -actin) by 0.5 M ammonium acetate and 70% ethanol. Pellets were resuspended in 10 µl of hybridization buffer (Ambion Inc.) by four alternating 15-s periods of vigorous vortexing and incubation at 95 °C for 3 min. Samples were hybridized at 68 °C for 10 min. A 100-µl aliquot of RNase A/T1 mixture diluted 1:100 was allowed to digest the unhybridized probes and RNA for 30 min at 37 °C. Inactivation/precipitation mixture (150 µl) was added, and the samples were kept at 20 °C for 30 min. After centrifugation, the supernatants were removed, and the pellets were dissolved in 8 µl of gel loading buffer for denaturing gels by heating at 95 °C for 4 min. The samples were loaded onto 5% polyacrylamide and 8 M urea gels and run at ~180 V for 2 h in 1× Tris borate/EDTA. Quantitative analysis was performed with a Bio-Rad Molecular Imager PC.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

cDNA and Amino Acid Sequences-- A mouse embryonic cDNA library was screened with a probe that encoded amino acids 2-126 of RoDH1, i.e. sequence highly conserved among the three known RoDH isozymes and CRAD1 (4-7). A partial genomic clone was isolated, most likely from contamination during preparation of the commercial cDNA library. Because we have isolated genomic clones for RoDH/CRAD,² it was recognized as exon 1 of an unknown SDR with high sequence similarity to CRAD1. To obtain the complete coding region of the novel cDNA, a mouse liver cDNA library was first screened with a probe from CRAD1; the positive plaques were rescreened with a probe generated by polymerase chain reaction from the exon of the novel SDR. Two of the three positive plaques identified included complete coding regions, and all three had identical deduced amino acid sequences in areas of overlap. The amino acid sequences, however, were distinct from those of RoDH isozymes 1-3 and CRAD1. The new SDR was named CRAD2 because its substrate specificity resembled that of CRAD1 (see below).

View larger version (78K): [in this window] [in a new window]   Fig. 1.   Nucleotide and deduced amino acid sequences of CRAD2. The nucleotide sequence reflects the three clones pBSK/CRAD-188 (nucleotides 1-1344), pBSK/CRAD-218 (nucleotides 54-1180), and pBSK/CRAD-39 (nucleotides 266-1612), produced by EcoRI digestion of phage DNA. Underlining in the translated region denotes the 23 amino acids conserved in at least 70% of SDR family members. The 20 conserved in CRAD2 are boldface and underlined.

Enzymatic Activity of CRAD2-- CRAD2 expressed transiently in CHO cells had ~10-fold higher rates of activity with NAD versus NADP (Table II). CRAD2 showed cooperative kinetics with 3-adiol (3-hydroxydehydrogenase activity) and testosterone (17-hydroxydehydrogenase activity), with Hill coefficients of 1.9 ± 0.3 (mean ± S.D., n = 4) and 1.5 ± 0.3 (n = 3), respectively (Fig. 2). The V/K_0.5 value for testosterone was ~8-fold lower than that for 3-adiol, indicating much more potent androgen 3-hydroxydehydrogenase than 17-hydroxydehydrogenase activity (Table III). Michaelis-Menten kinetics were observed with androsterone, but 3-hydroxydehydrogenase activity with androsterone was 6-fold lower than with 3-adiol. CRAD2 was not saturated kinetically with 25 µM dihydrotestosterone, which had a rate of androstanedione (5-androstan-3,17-dione) formation of 3.6 nmol/min/mg. No products were detected after incubations with estradiol or corticosterone. Among the retinoid substrates, CRAD2 showed the highest rate with 11-cis-retinol and was 8-fold less efficient for all-trans-retinol. CRAD2 was not saturated kinetically with 28 µM 9-cis-retinol, which produced 9-cis-retinal at a rate of ~23 pmol/min/mg of protein.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Rate curves of CRAD2-catalyzed reactions. Representative kinetics of recombinant CRAD2 activity are shown for three of the substrates tested: 3-adiol (top panel), testosterone (middle panel), and 11-cis-retinol (bottom panel). Each point represents the average of duplicates.

View this table: [in this window] [in a new window]   Table III CRAD2 activity with steroid and retinoid substrates K0.5 values represent the averages of two separate measurements with 8-12 substrate concentrations, except those for 3-adiol and testosterone, for which four and three independent determinations were made. Duplicate determinations were made for each concentration in each experiment. Steroid reactions were done with 1-15 µg of protein for 10 min. Retinoid reactions were done with 100 µg of protein for 30 min.

Subcellular Fractionation of CRAD2-- Association of CRAD2 with membrane fractions was demonstrated by differential centrifugation. Centrifugation of the 800 × g supernatant of transfected CHO cells at 10,000 × g for 30 min partitioned 75% of the CRAD2 activity into the supernatant, measured with 3-adiol. Centrifugation of this supernatant at 100,000 × g for 2 h partitioned 89% of the recovered CRAD2 activity into the microsomal pellet.

Modulators of CRAD Activity-- Carbenoxolone, the steroidal aglycone of the licorice-derived glycyrrhizin, inhibits SDRs with IC₅₀ values in the nM to µM range (4, 5, 27-30). Carbenoxolone inhibited CRAD2 with an IC₅₀ value of 2 µM (Fig. 3). 4-Methylpyrazole inhibits the medium-chain alcohol dehydrogenase class I isozymes potently (µM K_i values), the class II isozymes modestly (K_i ~ 2 mM), and the class IV isozymes variably (mouse K_i ~ 1.5 mM and rat K_i ~ 0.2 mM). 4-Methylpyrazole inhibited CRAD2 activity with an IC₅₀ value of 5 mM. Ethanol enhanced RoDH1 and RoDH2 activities by ~30%, but 140 mM ethanol had little impact on CRAD2 activity (data not shown). Phosphatidylcholine stimulates RoDH1 activity 7-fold and RoDH2 activity 3-fold (4, 5), but 2 mM phosphatidylcholine did not enhance CRAD1 activity.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Inhibition of CRAD2 by carbenoxolone and 4-methylpyrazole. Graded concentrations of carbenoxolone (open circles; µM) or 4-methylpyrazole (open diamonds; mM) were assayed for impact on CRAD2-catalyzed metabolism. Assays were done for 30 min with 5 µM 3-adiol and 2 µg of protein from the 800 × g supernatant of transfected CHO cells. Data are the means ± S.D. of triplicates.

CRAD2 mRNA Tissue Expression-- Northern blot hybridization revealed intense expression of CRAD2 mRNA in mouse liver, with the most intense signal at 1.7 kb and two less intense signals at 3.5 and 2.9 kb (Fig. 4). Lung also showed the 1.7-kb mRNA, albeit at ~3% of the 1.7-kb liver signal. In comparison, CRAD1 was expressed very intensely in both liver and kidney, with major mRNA species at 3.5 and 2.7 kb in both tissues, a major 3.0-kb band in liver only, and a weaker 4.4-kb band in liver and kidney. RoDH isozymes 1-3 showed a single 1.7-kb band in liver by Northern blot analysis (4-7). No signals for CRAD2 were observed by Northern blotting in heart, brain, spleen, skeletal muscle, or testis, as was the case for CRAD1 and RoDH isozymes 1-3. The more sensitive RNase protection assays detected CRAD2 mRNA expression in the following (relative intensity normalized to the -actin signal): liver (100) > lung (3) > eye (2) > kidney (1.6) > brain (0.6) (Table IV). No signals were detected in testis or heart. RNase protection assays also revealed low levels of CRAD1, RoDH1, and RoDH2 in multiple tissues, but did not reveal expression of RoDH3 outside of the liver (4-7).

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Distribution of CRAD2 in mouse tissues. Top panel, RNase protection assays were done as described under "Materials and Methods" on RNA prepared from the tissues of 2-month-old male mice. Lane 1, probe; lane 2, yeast RNA; lane 3, DNA markers; lane 4, eye; lane 5, brain; lane 6, heart; lane 7, kidney; lane 8, liver; lane 9, lung; lane 10, testis. These data were normalized to the signals produced by the mouse -actin probe. Bottom panel, Northern blot hybridization was carried out as detailed under "Material and Methods" with a commercially available blot from a gel of mouse poly(A+) RNA. Lane 1, heart; lane 2, brain; lane 3, spleen; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; lane 8, testis. bp, base pairs.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

cDNA cloning of CRAD2 reveals a fifth member of a subgroup of SDRs (CRAD1, CRAD2, and RoDH1-3) whose members share amino acid sequence identities of 80-97%. Nearly 95% conservation of the first 115 N-terminal amino acid residues represents a distinguishing feature of these five members. Rat 17-HSD6, the SDR with the nearest amino acid identity to the group of five, shares only 86% identity in this area with the group of five. Human RoDH has only 83% identity in this region, and the SDR associated with 11-cis-retinol dehydrogenation has only 57% amino acid identity in this area, not markedly different from its overall identity to the group of five. Most likely, this conservation of the N terminus serves a unique, but as yet undetermined function. Similarly, amino acid residues 260-304 share ~84-98% identity among the group of five. In contrast, rat 17-HSD6 has only 76-78% identity with the group of five from amino acid residues 260 to 304.

It appears that at least two distinct subgroups of SDRs occur with activity toward retinoids. The "group of five" enzymes possibly compose a subgroup distinct from the three other enzymes most closely related in sequence, namely 17-HSD6, human RoDH, and 11-cis-RoDH. For example, both the overall difference in identity and the difference in identity in the conserved N terminus indicate that the SDR candidate for a human homologue of an RoDH isozyme represents an SDR outside of the subgroup, given the generally high species conservation of proteins involved in retinoid metabolism and/or function. The large differences between the group of five and the SDR with activity toward 11-cis-retinol also suggest that the latter lies outside of the subgroup and perhaps constitutes a subgroup of its own. The recent cloning of a cDNA that encodes a mouse SDR (RDH4) supports this supposition. RDH4, which has 9-cis-retinol dehydrogenase activity, has ~87% amino acid identity to the 11-cis-retinol dehydrogenase, but only 53% identity to mouse RoDH1.³ Moreover, the 11-cis-retinol dehydrogenase also has potent 9-cis-retinol dehydrogenase activity.²

Although the enzymes in the group of five have relatively high amino acid conservation, to the extent tested, they show substantial differences in enzymatic activity. CRAD2 discriminates between 9-cis- and 11-cis-retinols, in contrast to CRAD1, which shows similar activity (V/K_0.5 values) for these two cis-retinols (7). Recombinant RoDH isozymes have not been tested with cis-retinols. The activities with androgens also differed among the three tested. CRAD2 was most efficient as a 3-hydroxysteroid dehydrogenase, but had less efficient 3-hydroxysteroid dehydrogenase activity than either RoDH1 or CRAD1, which had K_m values ~0.1-0.2 µM for 3-adiol. CRAD1 had a V_m value (27 nmol/min/mg of protein) with 3-adiol that was 8-fold faster than that of CRAD2. Even though the experiments were done at different times and care must be exercised when comparing V_m values produced from different transfections, these rates observed for each CRAD were consistent for several transfections and therefore likely reflect inherent differences between the two. CRAD2 also differs from CRAD1 and RoDH1 in its relatively high 17-hydroxysteroid dehydrogenase activity. Neither CRAD1 nor RoDH1 had activity with dihydrotestosterone, and both had negligible activity with testosterone.

The prostate epithelial cell steroid 5-reductase reduces the C₄-ene of testosterone to produce the major biologically active androgen of prostate, dihydrotestosterone (15). The effects of dihydrotestosterone are, in turn, limited by 3-hydroxysteroid dehydrogenases (Fig. 5). 3-Hydroxysteroid dehydrogenases are members of the aldo-keto reductase superfamily (expressed in prostate, liver, kidney and several other tissues) that reduce the 3-oxo function of dihydrotestosterone to produce 3-adiol.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Possible roles of RoDH and CRAD isozymes in androgen metabolism. 5-Reductase converts testosterone into dihydrotestosterone irreversibly. The aldo-keto reductase 3-hydroxysteroid dehydrogenase (3-HSD) inactivates dihydrotestosterone by reduction into 3-adiol. 17-HSD converts 3-adiol into androsterone, which undergoes glucuronidation and elimination. RoDH/CRAD may regenerate dihydrotestosterone by oxidizing 3-adiol. Secondarily, RoDH/CRAD may oxidize androsterone into androstanedione, which may undergo reduction into dihydrotestosterone by an as yet unknown 17-HSD.

3- and 17-Hydroxysteroid dehydrogenases constitute a pathway of dihydrotestosterone inactivation. 3-Adiol binds to the androgen receptor with 5 orders of magnitude less affinity than dihydrotestosterone (31-33). 17-Hydroxysteroid dehydrogenases, members of the SDR superfamily, convert 3-adiol into androsterone, a steroid with even less androgen activity in vivo than its precursor (20). On the other hand, 3-hydroxysteroid dehydrogenases that function oxidatively would convert the relatively inactive 3-adiol and androsterone into dihydrotestosterone and androstanedione, respectively. The latter could then undergo activation into dihydrotestosterone through reduction of its 17-oxo function (Fig. 5). The three SDRs tested so far with androgens (RoDH1, CRAD1, and CRAD2) have the most efficient activity as 3-hydroxysteroid dehydrogenases of any enzymes known so far. They may contribute to androgen action by producing dihydrotestosterone from 3-adiol and androsterone, thereby "rescuing" dihydrotestosterone from inactivation and excretion. Such a role for RoDH/CRAD isozymes seems feasible in vivo because dihydrotestosterone has been detected in castrated and functionally hepatectomized rats, and 5-reductase inhibitors do not obliterate androgen activity (15). Also, despite low affinity for the androgen receptor, 3-adiol stimulates prostate growth in vivo and in organ culture, consistent with metabolism into dihydrotestosterone (19, 34, 35).

3-Adiol may not function solely as an androgen inactivation product. Pregnant mice with a null allele in the type 1 5-reductase gene failed to deliver pups on time, but instead entered prolonged labor on days ~21-22 (36). About half resorbed their fetuses or expelled dead fetuses, and the other half either died during labor or suffered massive sepsis. The defect was seemingly related solely to parturition because the birth canal suffered no apparent developmental defect, and apparently normal pups were delivered on day 19.5 by cesarean section. Dosing with 3-adiol increased the incidence of normal parturition from 27 to 93%. An equivalent dose of dihydrotestosterone was less effective, raising the incidence to 57%. These data prompted the suggestion that 3-adiol may serve as a hormone required for parturition in mice. If so, then a second function of RoDH/CRAD could involve affecting the onset of parturition through altering the 3-adiol concentration.

Observance of dual androgen/retinoid substrate SDRs provides opportunity for providing insight into the physiological interactions between retinoids and steroids. Spermatogenesis requires functional steroid and retinoid receptors (37, 38). Retinoic acids inhibit prostate epithelial cell growth (39, 40), and inhibition of all-trans-retinoic acid metabolism in the rat Dunning prostate cancer model inhibits carcinoma relapse after castration by raising all-trans-retinoic acid plasma levels (41). All-trans-retinoic acid decreases concentrations of dihydrotestosterone, 3-adiol, and androsterone in serum and seems to cause a metabolic deviation away from the 5-path in liver (39, 42). Other than causing a 3-fold decrease in androgen receptor binding (39, 43), very little is known about the mechanisms of retinoid effects on androgen activity. Indeed, very little is known in general about the extent of the retinoid/androgen interaction. Conversely, androgens affect the actions of retinoids by decreasing the mRNA of RAR ~5-fold in prostate epithelia and 15-20-fold in seminal vesicles, while increasing it 2-fold in kidney (44). The dual androgen and retinoid activities of RoDH1 and CRAD isozymes could position them as mediators of retinoid/androgen interactions. Mechanisms of such potential interactions might include direct competitive and/or allosteric effects or indirect effects through gene expression via RARs, RXRs, and/or the androgen receptor.

The 11-cis-retinol dehydrogenase activity of CRAD2 and its expression in the eye are consistent with CRAD2 contributing 11-cis-retinal for use as a rhodopsin chromophore. 11-cis-Retinoids, however, have not been demonstrated outside of the eye. Therefore, extraocular and perhaps intraocular CRAD2 may support 9-cis-retinoic acid biosynthesis by converting 9-cis-retinol, available from diet or from 9-cis--carotene metabolism (45-53), into 9-cis-retinal. Although CRAD2 was not saturated kinetically with physiological levels of 9-cis-retinol, it showed sufficient activity with low 9-cis-retinol concentrations to contribute to the pool of 9-cis-retinal, which is in the low nM range. Two other SDRs have been reported with 9-cis-retinol dehydrogenase activity. CRAD1 showed a K_0.5 value of ~5 µM and a V_m of ~10 nmol/min/mg for 9-cis-retinol (7). The second, originally reported as 11-cis-retinol dehydrogenase (8, 9), was shown subsequently also to recognize 10 µM 9-cis-retinol as substrate in a one-point assay (10). With the data currently available, it is not possible to assess the relative contributions of these three enzymes to the production of 9-cis-retinoic acid in vivo.

The exact function of CRAD2, and CRAD1 as well, may depend on loci of expression, substrate availability, hormonal influences, and other as yet unappreciated factors. Future investigations will address these issues. Finally, the variable effects of agents such as carbenoxolone and 4-methylpyrazole on different SDRs and alcohol dehydrogenases suggest that caution should be exercised in interpreting experiments in vivo using such reagents.