From the Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, New York 14214
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We report here a mouse cDNA that encodes a
316-amino acid short-chain dehydrogenase that prefers
NAD+ as its cofactor and recognizes as substrates
androgens and retinols, i.e. has steroid 3- and
17
-dehydrogenase and cis/trans-retinol catalytic
activities. This
cis-retinol/androgen
dehydrogenase type 2 (CRAD2) shares close amino
acid similarity with mouse retinol dehydrogenase isozyme types 1 and 2 and CRAD1 (86, 84, and 87%, respectively). CRAD2 exhibits cooperative
kinetics with 3
-adiol (3
-hydroxysteroid dehydrogenase activity)
and testosterone (17
-hydroxysteroid dehydrogenase activity), but
Michaelis-Menten kinetics with androsterone (3
-hydroxysteroid
dehydrogenase activity), 11-cis-retinol,
all-trans-retinol, and 9-cis-retinol, with
V/K0.5 values of 1.6, 0.2, 0.1, 0.04, 0.005, and not saturated, respectively. Carbenoxolone
(IC50 = 2 µM) and 4-methylpyrazole
(IC50 = 5 mM) inhibited CRAD2, but neither
ethanol nor phosphatidylcholine had marked effects on its activity.
Liver expressed CRAD2 mRNA intensely, with expression in lung, eye,
kidney, and brain (2.9, 2, 1.6, and 0.6% of liver mRNA,
respectively). CRAD2 represents the fifth isozyme in a group of
short-chain dehydrogenase/reductase isozymes (retinol dehydrogenases 1-3 and CRAD1), closely related in primary amino acid sequence (~85%), that are expressed in different quantities in various tissues, have different substrate specificities, and may serve different physiological functions. CRAD2 may alter the amounts of
active and inactive androgens and/or convert retinols into retinals.
These data expand insight into the multifunctional nature of
short-chain dehydrogenases/reductases and into the enzymology of
steroid and retinoid metabolism.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 HybspeedTM 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
The deduced amino acid sequence of CRAD2 contained 20 of the 23 amino acids conserved in ~70% of SDRs, including the cofactor-binding residues G36(X)2SGXG, the L109XNNAG sequence (unknown function), and the catalytic residues Y175(X)3K (Fig. 1). All three RoDH isozymes, CRAD1, and CRAD2 show high sequence conservation in their first 115 N-terminal amino acids. The largest difference occurs between CRAD2 and RoDH2, which differ in six amino acid residues, four of which represent nonconservative changes (T13N, Q22K, N71S, and R104T). CRAD2 differs from CRAD1 by only five amino acids of the first 115, four of which are nonconservative (E20V, N43T, Q68E, and A92T). RoDH1, identical to RoDH3 in this area, and CRAD1 also differ by only five amino acids of the first 115 N-terminal residues: three changes are nonconservative (T13N, Q22K, and N71S). This region contains the cofactor-binding site, the conserved SDR sequence LVNNAG, and a putative membrane-anchoring sequence.
|
|
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/K0.5 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.
|
|
|
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 Ki values), the class II isozymes modestly (Ki ~ 2 mM), and the class IV isozymes variably (mouse Ki ~ 1.5 mM and rat Ki ~ 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.
|
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).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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/K0.5
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 Km values ~0.1-0.2 µM for 3
-adiol. CRAD1 had a Vm
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
Vm 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.
|
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
K0.5 value of ~5 µM and a
Vm 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.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant DK47839.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF056194.
To whom correspondence should be addressed: 140 Farber Hall,
School of Medicine and Biomedical Sciences, 3435 Main St., SUNY, Buffalo, NY 14214. Tel.: 716-829-2032; Fax: 716-829-2661.
1
The abbreviations used are: SDR, short-chain
dehydrogenase/reductase; RoDH, retinol dehydrogenase; CRAD,
cis-retinol/androgen dehydrogenase; RAR, retinoic acid
receptor; RXR, retinoid X receptor; kb, kilobase(s); CHO, Chinese
hamster ovary; 17-HSD6, 17
-hydroxysteroid dehydrogenase type
6.
2 X. Chai and J. L. Napoli, unpublished results.
3 A. Romert and E. Eriksson, personal communication.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|