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-Hydroxysteroid Dehydrogenase Type 31
Laboratories of Ontogeny and Reproduction (P.R.P., C.G., N.F., Y.T.), of Molecular Endocrinology (X.-F.H., V.L.-T.), and of Health and Environment (D.N.), CHUQ, PCHUL; Departments of Ob/Gyn (C.H.B.), HealthPartners Regions Hospital St. Paul, Minnesota 55101; Ob/Gyn-CRBR (Y.T.) and Anatomy/Physiology (D.N., V.L.-T.), Faculty of Medicine, Laval University, Québec, Canada G1V 4G2
Address all correspondence and requests for reprints to: Dr. Yves Tremblay, Laboratory of Ontogeny and Reproduction-CRBR, Rm. T-1–58, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, 2705 Laurier Boulevard, Québec, Canada G1V 4G2. E-mail: yves.tremblay{at}crchul.ulaval.ca
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-dihydrotestosterone (5-DHT). The
pulmonary epithelial cell line A549, isolated from a human lung
carcinoma, like normal lung type II cell, produces disaturated
phosphatidylcholines and has been widely used for studying the
regulation of surfactant production. Androgen receptor has been
detected in A549 cells; however, the capacity of these cells for
androgen synthesis and metabolism has not been investigated at
molecular level. This study was undertaken to identify the
steroidogenic enzymes involved in the formation and metabolism of
androgens from adrenal C19 steroid precursors in A549 cells. When
cultured in the presence of normal FCS, A549 intact cells converted
DHEA to androstenediol, androstenedione principally to
testosterone, and 5-DHT to 5-androstane 3,17ß-diol. High
levels of 17ß-hydroxysteroid dehydrogenase (HSD) and 3-HSD
activities were detected in both cytosol and microsomes isolated from
homogenates. Analysis of A549 RNA indicated the presence of 17ß-HSD
type 4 and type 5, and of 3-HSD type 3 messenger RNAs. Very low
levels of 3ß-HSD type 1 and 5-reductase type 1 messenger RNAs and
activities were detected. With regard to active androgen formation,
there was little or no capacity for the conversion of DHEA
to 5-DHT. In contrast, androstenedione was rapidly transformed to
testosterone. The pattern of steroid metabolism was not affected by the
use of charcoal-stripped FCS or by the synthetic glucocorticoid
dexamethasone. Together, our findings show that A549 cells express a
pattern of steroid metabolism in which 17ß-HSD type 5 and 3-HSD
type 3 are the predominant enzymes. The level of androgens is regulated
at the level of catalysis in intact cells such that the intracellular
level of testosterone is stabilized, whereas 5-DHT is rapidly
inactivated by reduction to 3,17ß-diol. This pattern of androgen
metabolism has implications for the relative importance of testosterone
and 5-DHT in normal lung development and surfactant production. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dihydrotestosterone (5-DHT) have been
shown to delay pulmonary surfactant production in
glucocorticoid-treated and untreated human fetal lung tissue samples in
organ culture (e.g. Ref. 5). Similar deleterious effects of
androgens on fetal pulmonary surfactant production have been also
demonstrated in vivo in 5-DHT-treated pregnant rats (6)
and in pregnant rabbits treated with 4-MA, a potent inhibitor of the
enzyme 5-reductase (7). Current findings suggest that these steroid
effects are mediated by the interaction of testosterone or 5-DHT
with androgen receptors in lung fibroblasts and/or alveolar type II
cells.
Analyses of C19-steroid metabolism in whole lung tissue in the 60s and
70s (8, 9) provided the first evidence of the presence of a number of
steroidogenic enzyme activities. From their studies of lung slices,
Milewich et al. (10) concluded that 3-hydroxysteroid
dehydrogenase (3-HSD) and 17ß-hydroxysteroid dehydrogenase
(17ß-HSD) were the principal C19-steroid-metabolizing enzymatic
activities in the human lung with a cofactor milieu that favored
reduction of 17-ketosteroids to 17ß-hydroxysteroids. The detection of
low 5-reductase and 3ß-hydroxysteroid dehydrogenase/
5-4
isomerase (3ß-HSD) activities (10, 11, 12) suggested further that human
lung tissue may have limited ability to produce in situ, the
hormonally-active androgen 5-DHT from inactive adrenal C19-steroid
precursors.
It is now established that there are multiple isoforms of
hydroxysteroid dehydrogenases and 5-reductases. These are products
of separate genes and differ in their substrate and cofactor
specificity, subcellular and tissue localization, and catalytic
behavior within intact cells (13, 14, 15). These enzymes are widespread in
human tissues and it is currently believed that, within a
hormone-responsive tissue, the pattern of steroidogenic enzyme gene
expression determines steroid hormone action (13). Four enzymatic
activities regulate the formation and inactivation of testosterone and
5-DHT from adrenal-derived DHEA or androstenedione in
human peripheral tissues (Fig. 1
). These
activities are carried out by the enzymes 3ß-HSD, 5-reductase,
17ß-HSD, and 3-HSD. The latter two-enzymatic activities are of
particular interest with regard to regulation of active androgen levels
because they are reversible. As a result, the net direction of
17ß-HSD reaction will influence the ratio of biologically active
17ß-hydroxysteroids to inactive 17-ketosteroids within a tissue. In
fact, the 17ß-HSD and the 3-HSD activities depend on the balance
between specific isoform of each enzyme, which is believed to determine
the amount of hormone that can bind to members of the nuclear steroid
receptor superfamily and thus, ultimately, modulate the expression of
androgen responsive genes.
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-DHT formation and metabolism and to identify
genes responsible for these activities. The effect of the synthetic
glucocorticoid dexamethasone on steroid metabolism was also
investigated.
In this report, we describe an enzymology in A549 cells wherein
17ß-HSD type 5 and 3-HSD type 3 are the predominant steroidogenic
enzymes and their activities, combined with low 5-reductase,
integrated within intact cells to maintain high levels of testosterone
while rapidly inactivating 5-DHT.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-androstane-3,17ß-diol,
(40–60 Ci/mmol),
[1,2-3H]androst-5-ene-3ß,17ß-diol,
(40–60 Ci/mmol) and
5-dihydro[1,2-3H]testosterone (60 Ci/mmol)
from Amersham Pharmacia Biotech (Arlington Heights, IL);
[1,2,6,7-3H]dehydroepiandrosterone (100
Ci/mmol) from NEN Life Science Products (Boston, MA);
unlabeled steroids from Steraloids (Wilton, NH); HEPES, Bicine, NAD(P),
NAD(P)H and BSA from Sigma (St. Louis, MO); FCS from
Life Technologies, Inc./BRL (Burlington, Ontario, Canada)
and from HyClone Laboratories, Inc. (Logan, UT); Ecolume
from ICN Radiochemicals (Irvine, CA).
Cell cultures
A549 cells (CCL 185; American Type Culture Collection, Manassas, VA) were grown in DMEM-HEPES-high glucose
medium (Life Technologies, Inc., Gaithersburg, MD)
containing, 2.2 g/liter NaHCO3, 100 IU/ml penicillin, and 50 µg/ml
streptomycin (DMEMHG) supplemented with heat-inactivated (56 C/20 min)
FCS (10%, vol/vol). The medium was changed every 2 days.
Steroid metabolism by A549 cultures
Intact 105 cells in culture were incubated
with 1 ml of DMEMHG containing [3H]-steroid (2
x106 dpm; 9.6 nM) for the indicated
time periods and steroids measured in the medium. Steroids were
extracted twice with 5-ml diethyl ether, evaporated and applied to
silica gel-coated TLC plates and resolved in toluene-acetone-chloroform
(8:2:5, vol/vol). Androsterone and 5-DHT were separated on silver
nitrate-conditioned aluminiumoxid 60 F254 neutral TLC plates
(VWR, Montréal, Québec, Canada) developed with
toluene-acetone (4:1, vol/vol) (25). Experiments were repeated with
three different cultures and each time-point was assayed in
triplicate.
Effects of different batches of FCS and of dexamethasone on A549
cells
Experiments with Dex (2.5 x 10-7
M) were conducted with cells cultured in DMEMHG
supplemented with 10% (vol/vol) FCS or with dextran-coated and
charcoal treated FCS (FCS-A). Steroid stripping was done by adding
1 g of activated charcoal (Norit A, Fisher Inc.,
Burlington, Ontario, Canada) and 2 ml of 5% (wt/vol in water) of
dextran T-70 (Pharmacia & Upjohn, Baie
d’Urfé, Québec, Canada) to 100 ml of FCS. After stirring
(overnight at 4 C), charcoal was removed by two run of centrifugation
at 6000 x g, 30 min at 4 C. After a second step of
adsorption (3 h, RT), the FCS-A was filtered (0.22
µM) and stored at -20 C until used. Three
different lots of FCS were used. Intact cells
(105 cells/0.8 cm2) were
plated. The following day, the medium was changed and replaced either
with DMEMHG-FCS or DMEMHG-FCS-A. After 24h,
[3H]-androstenedione (2 x
106 dpm, 9.6 nM) was added
to the cells in the presence or absence of Dex, and incubations pursued
for 24 h. The identity of each steroid, in particular 5-DHT was
confirmed by HPLC analysis using a Shimadzu model 10A chromatograph and
a C18 column (Ultra sphere, 0.5 µM, 4.6 x
150 mm). An isocratic elution by water-acetonitrile-tetrahydrofuran
(65:23:12, vol/vol) was used as previously described (26).
Cortisol determination in FCS and FCS-A
The levels of cortisol were determined by a heterogeneous
competitive magnetic separation assay according to the manufacturer
instructions (Bayer Corp., Tarrytown, NY). The lower limit
of detection was 6.0 nM.
Subcellular fractionation
107 cells were resuspended in 1 ml of
ice-cold buffer containing 20% (vol/vol) glycerol, 1.0 mM
EDTA, and 4 mM potassium phosphate, pH 7.0 (KPBS). Samples
were homogenized by hand in an all-glass Dounce homogenizer fitted with
a B-pestle, centrifuged at 1000 x g to remove cell
debris, and again at 105,000 x g for 1 h.
Supernatants were saved as cytosol. The microsome-enriched pellets were
washed, subjected to a second round of ultracentrifugation, and
resuspended in 1 ml KPBS. Completeness of homogenization was evaluated
by light microscopy. Proteins were measured by the method of Bradford
(27).
Enzymatic assays on cytosolic and microsomal fractions
17ß-HSD activity was measured as we described previously (28, 29). Briefly, a 10-µl aliquot of cytosol or microsomes was combined
with 10 µl of reaction mixture containing 0.5 mM
nicotinamide nucleotide cofactor and 1.0 µM
3H-labeled steroid substrate in 0.08
M HEPES, pH 7.2, for both reductase and dehydrogenase
activities. Assays were run at 37 C. Reaction mixtures (total) were
transferred to the adsorbant layer of silica gel HL plates and analyzed
by TLC (30, 31). The activities of 3-HSD, 5-reductase and
3ß-HSD were respectively assayed with tritiated 5-DHT,
testosterone or DHEA. Following TLC, substrate and
products were localized by a light misting with water and scrapped into
10 ml of Ecolume for scintillation counting. Percent conversion was
calculated as the ratio of cpm recovered in product/cpm in substrate
plus products. This value was transformed into picomoles per mg protein
per 30 min incubation as described previously (32).
RNA preparation and complementary DNA probes
Cellular RNA was prepared from 107 cells
by lysis in 5 ml of Tri-Reagent, a mixture of phenol and guanidine
thiocyanate in a monophasic solution (Molecular Research Center,
Cincinnati, OH). RNA was separated from DNA and proteins by the
addition of 1 ml of chloroform. RNA was recovered by precipitation with
2.5 ml of isopropanol. RNA (25 µg) was glyoxalized, resolved by 1%
(wt/vol) agarose gel electrophoresis and transferred to
Nytran+ membrane (Schleicher & Schuell, Inc., VWR) (33). Three different membranes with cells recovered
from different passages were separately prehybridized, hybridized, and
washed under high stringency conditions (34). Each Northern blot was
successively hybridized with each of the following human complementary
(c) DNA probes: 3ß-HSD type 1 EcoRI/PvuII
1038-bp fragment (35); 17ß-HSD type 1
EcoRI/SacI 964-bp segment (35); 17ß-HSD types
2, 3, and 5 full-length fragments (1.3 kb) (13, 31); 17ß-HSD type 4
EcoRI/EcoRI 1.4-kb fragment (36); and
5-reductase types 1 (2.1 kb) and 2 (2.4 kb) both full-length (37).
Probes were labeled with (32P)deoxy-CTP to
2 x 106 dpm/ng with random primers (38).
Different human tissues were used as positive control. Term villous was
used for the 3ß-HSD-1, 17ß-HSD-1, -2, and -4; testis for the
17ß-HSD-3 and prostate for the 5-reductase.
Ribonuclease protection assay
Full-length human complementary DNA (cDNA) fragments of
17ß-HSD type 5 and 3-HSD type 3 were subcloned into the
EcoRI/BamHI of the Bluescript
KS+ (BSKS+) vector
(Stratagene, La Jolla, CA). The recombinant plasmid
carrying 17ß-HSD type 5 cDNA was linearized with HindIII
to generate a cRNA (antisense) probe of 188 nts specific for 17ß-HSD
type 5 messenger RNA (mRNA). The plasmid carrying 3-HSD type 3 was
linearized with SalI to generate a cRNA probe of 250
nucleotides (nts) specific for 3-HSD type 3 mRNA. The protected
regions correspond to nts 36–286 of the 3-HSD type 3 cDNA and to
nts 831-1019 of the 17ß-HSD type 5 cDNA. RNase protection assays for
the presence of human 17ß-HSD types 1 and 2 mRNAs were also performed
as described previously (30, 35).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-DHT and testosterone synthesis and metabolism, intact cells in
culture were exposed to tritiated DHEA, 5-DHT,
androstenedione, and testosterone for 24 h (Table 1). DHEA was converted to a
single product, androstenediol. There was no evidence of
androstenedione formation from DHEA. When 5-DHT was the
substrate, there was almost complete conversion into 3,17ß-diol.
With androstenedione as substrate, testosterone was the major androgen
formed accounting for about 44% of the total radioactivity recovered.
3,17ß-diol and androsterone were also formed, but at levels
consistent with the presence of a low level of 5-reductase activity.
This was confirmed when testosterone was tested. 3,17ß-diol
accumulated in the medium to the same low level (14 ± 1%) as
when androstenedione was the substrate (9 ± 2%).
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-DHT, and androstenedione were
metabolized principally into single products, time-course experiments
were performed in which enzymatic reactions were stopped at different
time periods (Fig. 2). Formation of a
single product accounted for DHEA and 5-DHT metabolism
with about 50% conversion into 3,17ß-diol in less than 5 h
for the latter. With androstenedione as substrate, testosterone
formation was detected within 1 h, with a small amount of
3,17ß-diol and androsterone formed in agreement with results from
Table 1
.
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-HSD, and 5-reductase activities in
A549 cytosol and microsomes-HSD and low levels of 3ß-HSD and 5-reductase. In addition,
reductase activity clearly predominated over dehydrogenase activity in
intact cells. To clarify the basis for these patterns, cells were
fractionated into cytosol and microsomes and NAD(P)-dependent
dehydrogenase and NAD(P)H-dependent reductase activities with
estradiol-17ß (E2) and estrone
(E1), testosterone and androstenedione, and
DHEA and 5-DHT as substrates assayed. The pattern of
oxidation and reduction at C17 of E2,
E1, testosterone, and androstenedione in cytosol
and microsomes is shown in Fig. 3. Most
(>75%) of the 17ß-HSD activity was recovered in the cytosol and
NADP or NADPH were the preferred cofactors. When assayed at pH 7.2,
dehydrogenase activity with E2 and testosterone
exceeded reductase activity with E1 and androstenedione. The pattern of
DHEA metabolism is shown in Fig. 4. Conversion of DHEA to
androstenedione was detected in both cytosol and microsomes. There was
a significantly greater NADPH-dependent reduction of DHEA
at the C17 position to androstenediol, consistent with the pattern
observed with intact cells. Here again, the majority of the activity
was recovered in the cytosol. Both cytosol and microsomes also
exhibited a large capacity for the reduction of 5-DHT to
3,17ß-diol (Fig. 5), predicted on
the basis of the metabolic pattern seen with intact cells. NADPH was
the preferred cofactor. In contrast, conversion of 5-DHT to
androstanedione was low. 5-reductase activity was assayed at pH 8.0
(type 1 isoform) and pH 5.0 (type 2 isoform) in microsomes and cell
sonicates. In agreement with the metabolic activity noted with intact
cells, activity was at the limit of detection in each case and
radioactivity was recovered as 3,17ß-diol consistent with the high
level of 3-HSD and 17ß-HSD type 5 (data not shown).
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-reductase types 1 and 2, and 17ß-HSD types 1 to
5, only mRNAs that corresponded to 17ß-HSD type 4 (Fig. 6, lane 2) and 17ß-HSD type 5 (Fig. 6, lane 3) were detected. The full-length cDNA probe of 17ß-HSD type 5
used in Northern blot analysis does not discriminate between 17ß-HSD
type 5 and the 3-HSDs, because more than 80% sequence identity
exists between these cDNAs (13). Thus, we designed cRNA probes for
17ß-HSD type 5 and 3-HSD type 1 and type 3. When RNase protection
assays were performed with these probes, RNA protected fragments
specific to 17ß-HSD type 5 (Fig. 7A)
and 3-HSD type 3 (Fig. 7B) were detected. We have also conducted
RNase protection analysis with 17ß-HSD type 1, type 2, and type 3 RNA
probes. Again, with this sensitive method, no corresponding mRNAs were
detected (data not shown). 5-reductase type 1 and 3ß-HSD type 1
mRNAs were detected but at very low levels by RNase protection assays
(data not shown).
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-reductase activity-reductase activity and mRNA at the limit
of detection was a surprising result considering the known effect of
5-DHT on surfactant production (21, 39). We reasoned that the low
level of 5-reductase mRNA and activity in A549 cells could be due to
a down-regulatory factor present in the culture medium. Therefore, we
compared three different lots of serum. In addition, to study the
possible effects of FCS-low molecular weight factors, we used
charcoal-stripped serum. As Dex has been shown to stimulate the
incorporation of tritium-labeled choline into DSPC in A549 cells
cultured in serum-free medium (40), we studied the effects of 0.25
µM of Dex, the optimal concentration in those studies.
The pattern of androstenedione metabolism in A549 intact cells was
independent of the serum lot (data not shown). The removal of small
molecules such as cortisol by charcoal had no major impact on the
steroidogenic pattern and no increase in DHT formation was observed
(Fig. 8A). Moreover, when Dex was added
to normal (data not shown) or charcoal-stripped FCS (Fig. 8B), there
was no significant effect on the metabolism of androstenedione. Similar
levels of 5-DHT and 3,17ß-diol were detected. Because the
amounts of 5-DHT formed from androstenedione were very low, the
identity of each metabolite formed was confirmed by HPLC analysis (Fig. 8C). The inset represents a spike experiment in which HPLC-purified
[3H]-5-DHT was added to the sample. Both the
5-DHT added and the metabolite produced from androstenedione had the
same retention time.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-DHT from the inactive precursor DHEA, we
found that under widely used cell culture conditions, DHEA
was metabolized principally to androstenediol. There was no evidence of
testosterone formation from DHEA, even though a low level
of 3ß-HSD activity was detected in vitro (Figs. 2 and 4).
When androstenedione was the substrate for intact cells in 24-h
incubations, 5-DHT was barely detectable and the major end-product
was 3,17ß-diol (Fig. 8), indicative of a slight capacity for net
5-reduction in agreement with the observations that 5-reductase
activity was at the limit of detection by direct assay in
vitro and that the level of 5-reductase mRNA was very low. Our
results of C19-steroids metabolism by intact A549 cells are in
agreement with those of Milewich (41). Thus, although, they are
androgen receptor positive (24), A549 cells cultured in normal 10% FCS
appears to have a minimal capacity for net 5-DHT formation from
DHEA.
It is of particular interest that A549 intact cells show a large
capacity to convert androstenedione to testosterone but have a very
limited relative capacity to metabolize testosterone to either
androstenedione or 5-DHT. This raises the possibility that, at least
under the culture conditions described here, testosterone rather than
5-DHT may be the active androgen acting on DSPC production in these
cells (17, 18, 19). Relevant to this, Nielsen (7) showed that testosterone
by itself, e.g. in the absence of conversion into 5-DHT,
modulated fetal rabbit lung development as measured by the production
of DSPC.
The pattern of E2, E1,
testosterone, and androstenedione metabolism in vitro (Fig. 3) with NADP and NADPH as the preferred cofactors and with the majority
of activity recovered in the cytosol is characteristic of 17ß-HSD
type 5 (42). If considered along with the mRNA analyses, these data
indicate 17ß-HSD type 5 is the predominant isoform of 17ß-HSD in
A549 cells. Our observation that 17ß-HSD type 5 activity is
reversible in vitro but acts predominantly as a reductase in
intact cells is also in accord with the report of Dufort et
al. (42). They found that when 17ß-HSD type 5 cDNA was
transfected into human embryonic 293 cells, the expressed enzyme acted
as both dehydrogenase and reductase in cell homogenates but essentially
only as reductase in intact transfected cells. The presence of other
isoforms of 17ß-HSD may account for the low level of activity with
both C18 and C19 steroids in the microsomal fraction. Microsomal
metabolism of E2, in particular, can be accounted
for by the presence of 17ß-HSD type 4. This isoform is highly
specific for E2 and androstenediol and acts
almost exclusively as a dehydrogenase (36). The more strongly favored
reduction of 5-DHT to 3,17ß-diol can be accounted for by the
presence of the 3-HSD type 3. 17ß-HSD type 5 also catalyzes
reduction of 5-DHT to 3,17ß-diol (42) and thus, would also
contribute to the rapid inactivation of 5-DHT as an active androgen
by these cells.
Our findings with A549 cells concur with the results that Milewich
et al. (10) obtained with human lung tissue and suggest that
17ß-HSD type 5 and 3-HSD type 3 play a major role in the
metabolism of androgens in the human lung. In addition, our results
establish that 17ß-HSD type 5 and 3-HSD type 3 mRNA expression, in
combination with regulation at the level of catalysis to favor
reductase activity in intact cells, can form the basis for a pattern of
metabolism that would stabilize the intracellular level of testosterone
and rapidly metabolizes 5-DHT. To our knowledge, this is the first
description of such a pattern in androgen receptor-rich cells.
It is well established that androgens influence fetal lung development.
Findings from a number of laboratories are consistent with a role for
5-DHT in the regulation of surfactant formation during fetal lung
development (5, 7, 21, 43). Because this hormone is produced locally
from testosterone in 5-DHT-dependent tissues, as it is case in the
prostate (44), we expected to find a significant level of
5-reductase activity in A549 cells. The apparent near-absence of
5-reductase mRNA and activity led us to examine the possibility that
serum factors may be influencing the levels of 5-reductase mRNA.
Because glucocorticoid receptor is present in A549 cells (18, 40, 45),
we examined the possibility that cortisol in FCS might be affecting
5-reductase activity in our cultures. Our initial experiments were
all done with cells cultured in medium supplemented to 10% with FCS.
Subsequent analysis revealed that various lots of FCS contained
cortisol at levels between 1.2 and 1.4 µM and that the
steroid could be removed by charcoal treatment (cortisol was
undetectable after the charcoal-stripping procedure). On that basis, we
compared the effects of untreated and charcoal-treated FCS as well as
the addition of the synthetic glucocorticoid dexamethasone on steroid
metabolism by intact cell. The absence of an effect of either
charcoal-stripping or dexamathasone on the metabolic pattern indicates
that cortisol and other steroids removed by charcoal treatment are not
inhibiting the expression of 5-reductase mRNA or the mRNA levels of
the other steroidogenic enzymes we assayed in our cultures.
Our findings raise questions about the nature of the active androgen
(testosterone or 5-DHT) and the mechanism of androgen action on
surfactant production. If pneumocyte type II epithelial cells in the
normal fetal developing lung or in the adult lung not only lack the
capacity to generate 5-DHT but can rapidly inactivate it as well, a
direct effect of 5-DHT on these cells is unlikely. In fact, our
results are suggestive that the testosterone produced by type II cells
could be the active androgen in the lung, and/or be transformed in
5-DHT by the lung fibroblasts before acting on surfactant synthesis.
Such a mesenchymal-epithelial interaction is consistent, for example,
with the inhibitory effect of 5-DHT on dexamethasone-stimulated
surfactant production by explant cultures of human lung tissue reported
by Torday (5). In addition, Floros and co-workers (46) have shown that
exposure of rat fetuses to 5-DHT in utero inhibits
fibroblast-pneumocyte factor (FPF) production by lung fibroblasts
cultured in vitro. More recent reports have confirmed that
lung fibroblasts can be directly affected by 5-DHT (47, 48, 49).
However, even though effects of 5-DHT administration in
vivo and in vitro have been demonstrated, there is also
evidence that testosterone may be the active androgen in the lung.
Relevant to this, Nielsen (7) showed that treatment of pregnant rabbits
with a 5-reductase inhibitor which eliminated 5-DHT from the
fetal lung, had no effect on male:female differences in
phosphatidylcholine: spingomyelin or saturated
phosphatidylcholine:spingomyelin ratios in fetal lung. In addition,
testosterone in the presence of the same 5-reductase inhibitor
inhibited the release of FPF by fetal lung fibroblasts in culture.
In summary, we have characterized a complex steroid enzymology in a
widely studied cellular model of human lung pneumocyte type II
function. Our results demonstrate a pattern of 17ß-HSD type 5 and
3-HSD type 3 activities in which testosterone level is maintained
and 5-DHT is rapidly metabolized. This has important implications
for the nature of the active androgen affecting surfactant production
in the lung.
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Footnotes |
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2 These authors contributed equally to this work.
Received December 22, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-androstane-3,17ß-diol in human
lung tissue and in pulmonary endothelial cells in culture. J Clin
Endocrinol Metab 60:244–250[Abstract]
-reductase:
two genes/two enzymes. Annu Rev Biochem 63:25–61[Medline]
-reductase 2 deficiency. Endocr Rev 14:577–593[Abstract]
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 P. R. Provost, C. H. Blomquist, R. Drolet, N. Flamand, and Y. Tremblay Androgen Inactivation in Human Lung Fibroblasts: Variations in Levels of 17{beta}-Hydroxysteroid Dehydrogenase Type 2 and 5{alpha}-Reductase Activity Compatible with Androgen Inactivation J. Clin. Endocrinol. Metab., August 1, 2002; 87(8): 3883 - 3892. [Abstract] [Full Text] [PDF]
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