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INTRODUCTION |
The acute trophic hormone-responsive steroidogenesis is dependent
on mobilization of cholesterol from cellular stores to the mitochondrial inner membrane. This is a rate-limiting and regulated step in steroidogenesis and is dependent on de novo protein
synthesis in steroidogenic cells (1, 2). Conversion of cholesterol to
pregnenolone is the first enzymatic step in steroidogenesis which is
catalyzed by the cholesterol side chain cleavage cytochrome P450
enzyme, located in the inner mitochondrial membrane (3-5). Several
factors have been proposed as essential mediators for cholesterol
delivery into this site critical for the initiation of steroidogenesis
(6). It has been demonstrated that luteinizing hormone, its
"superagonist" hCG,1 and
the analog of their second messenger (cAMP), dibutyryl-cAMP (Bt2cAMP), cause in MA-10 mouse Leydig tumor cells
increased synthesis of a series of 37-, 32-, and 30-kDa proteins, which
are closely associated with mitochondria (7-9). Inhibition of protein
synthesis in the hormone-stimulated steroidogenic cells has been shown
to decrease their response in steroid biosynthesis. It is known that the inhibitor-sensitive step is present in the mitochondria and that
cycloheximide (CHX) has no effect on the activity of cholesterol side
chain cleavage complexes or on cholesterol accumulation to the outer
mitochondrial membrane (10, 11).
A role for thyroid hormones has long been implicated in mammalian
testicular and ovarian function (12-14). Hypothyroidism is associated
with abnormalities in sexual function, such as azoospermia, oligozoospermia, and loss of libido and impotence in men, whereas in
women it causes irregular menstrual bleedings and impaired fertility
due to corpus luteum insufficiency (15). Oppenheimer et al.
(16) first demonstrated the presence of nuclear-binding sites for
T3 in the rat testis. It has also been demonstrated that
the morphological and functional development of testes is highly
dependent on thyroid hormones, especially under regulation of the
nuclear T3 receptor (17, 18). In goat (Capra
hircus) Leydig cells, T3 induces a proteinaceous
factor, sensitive to the protein synthesis inhibitors actinomycin D or
CHX, which is present in the soluble supernatant fractions of
100,000 × g, and is responsible for androgen
production (19). It was also found that T3 augments the
stimulatory effect of leutinizing hormone on androgen secretion. In
cultured human luteinized granulosa cells, T3 modulated
hCG-induced progesterone secretion, cell proliferation, and cAMP
production, whereas it had no effect on medium size follicles (20, 21).
The mechanisms underlying the T3-hCG interaction in mouse
Leydig tumor cells have so far remained elusive. Thyroid hormone
receptors have also been demonstrated in human granulosa, corpus
luteum, and rat granulosa cell nuclei (13, 22, 23). Immunocytochemical
studies with antiserum against the cellular erythroblastosis
A/T3 receptor also provide evidence for the presence of
T3 receptor protein in the rat tissues (24). These data
imply that thyroid hormones are involved in reproduction, and the
effects are known to be absolutely dependent on de novo
protein synthesis. However, nothing is still known about the nature of
the T3-induced protein(s) in the regulation of steroidogenesis.
Recently, an leutinizing hormone-induced 30-kDa mitochondrial factor,
named the StAR protein, has been purified and cloned from the MA-10
cells, and it has the necessary properties of inducing steroidogenesis
(25). The cDNA clones for StAR have also been isolated from the
human, rat, and cow, and they exhibit a high degree of homology
(26-28). The StAR gene is known to encode a 37-kDa nonphosphorylated
protein that is processed to a 30-kDa mature form via four
intermediates, with half-lives of 3-5 min in the mitochondria (29,
30). Northern hybridization analysis revealed the presence of three
StAR transcripts in the mouse (3.4, 2.7, and 1.6 kilobases) and human
(7.4, 4.4, and 1.6 kilobases), and two in the cow (3.0 and 1.8 kilobases) (25, 26, 28). In MA-10 mouse Leydig tumor cells, the
cAMP-mediated stimulation of steroidogenesis is well
correlated with StAR mRNA expression and StAR protein
synthesis (29).
Despite the potential involvement of thyroid hormones in
steroidogenesis, their precise mode of action remain to be determined. The important role of StAR in steroidogenesis prompted us to examine its involvement in the thyroid hormone stimulation, and consequently in
the mechanism of action by employing the orphan nuclear receptors SF-1
and DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita
critical region on the X-chromosome). Recent studies implicate that
SF-1 and DAX-1 play a key role in adrenal and gonadal differentiation,
development, and function (31-33). Impaired adrenal development and
function, associated with hypogonadotropic hypogonadism, appear due to
mutations of these two nuclear receptors. The complex endocrine
phenotypes with the SF-1 and DAX-1 mutations point strongly to their
interactions in a hierarchial pathway, and they may activate the target
genes in a cooperative fashion (34). Conspicuously, SF-1 knock-out mice
completely lack adrenal glands and gonads despite normal embryonic
levels of serum corticosterone (35). It is apparent that understanding
the involvement of thyroid hormones into the regulation of
steroidogenesis requires exploration of the above two transcription factors.
In light of these observations, we found it important to examine the
physiological relationship of thyroid hormone action and StAR gene
expression during steroidogenesis, using mouse Leydig tumor cells
(mLTC-1, Ref. 36) as the experimental model. The current findings
demonstrate for the first time that thyroid hormones, through induction
of the SF-1-mediated StAR gene expression, play a major role in the
regulation of steroidogenesis.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Transfection--
The mouse tumor Leydig cells
(mLTC-1, see Ref. 36) were maintained in HEPES-buffered Waymouth's
medium supplemented with 9% heat-inactivated horse serum (Life
Technologies, Inc., Paisley, Scotland, United Kingdom) and 4.5% fetal
calf serum (Biochlear, Wilts, UK) containing 0.1 g/liter gentamycin
(Biological Industries, K. B. Haemek, Israel). The cells were
subcultured at a density of 6 × 104 cells/well in
24-well plates for determining progesterone (P) production, and at
5 × 105 cells/well in 6-well plates for total RNA extraction.
Transfections were carried out at 65-75% confluency of the cells by
using FuGENE 6 transfection reagent (Boehringer-Mannheim GmbH,
Mannheim, Germany) under optimized conditions according to the
instructions of the manufacturer. Briefly, FuGENE 6 transfection reagent was diluted in serum-free Waymouth's medium and incubated at
room temperature to a final volume of 300 µl. Two µg of
pCMV119+-SF-1 construct, obtained from Dr. K. L. Parker (Duke University Medical Center, Durham, NC), and 2 µg of
pBKCMV-hDAX-1 construct, obtained from Dr. R. Yu (Northwestern
University Medical School, Chicago, IL), or their combination (1:1),
were used for transfections. Two µg of a
-galactosidase expression
vector, pSV-
-galactosidase (Promega, Madison, WI) were used as an
internal control of tranfection efficiency. FuGENE 6-DNA complex
following 15 min incubation at room temperature were distributed
dropwise to the plate containing 3 ml of regular medium.
Isolation, Purification, and Incubation of Cultured Mouse Leydig
Cells--
Adult mouse (C57 strain, 2-3 months) testicular Leydig
cells were prepared as described previously (37, 38), modified further
for better recovery. Briefly, decapsulated interstitial cells were
isolated by collagenase (0.2%, 20 min, 34° C, 95% O2, 5% CO2) dispersion in Dulbecco's modified Eagle's
medium/F-12 (1:1) (Life Technologies, Inc.) containing 25 mmol/liter
Hepes, pH 7.4, 18 mmol/liter sodium bicarbonate, and 0.2% bovine serum albumin (Sigma). After filtration through sterile nylon gauze (mesh
0.5-0.8 mm), collagenase was removed by washing, and cells were
purified by a continuous Percoll (Pharmacia, Uppsala, Sweden) gradient
(density range 1.01-1.126 kg/liter) centrifugation. The cell types
partitioned due to the various buoyant densities, and those gathered at
the zone approximately to 1.07 kg/liter of Percoll were collected,
washed, and the purity of Leydig cells was assessed by histochemical
3
-hydroxysteroid dehydrogenase reaction; about 80% were found to be
3
-hydroxysteroid dehydrogenase positive cells (39). The cells were
subcultured in growth medium supplemented with 10,000 units/liter
penicillin and 50 mg/liter streptomycin, at a density of 1 × 105 cells/well in 24-well plates, and stimulated with the
indicated substances as described above.
RNA Extraction, and Quantitative Reverse Transcription and
Polymerase Chain Reaction--
Total RNA was isolated from control and
stimulated cells by the acid guanidinium thiocyanate/phenol/chloroform
extraction method of Chomczynski and Sacchi (40). The purity of the
extracted RNA was determined by scanning with spectrophotometer at
wavelength 220-320 nm.
The isolation and amplification of the mouse (mLTC-1) StAR cDNA
were carried out by engineering primers from the mouse StAR cDNA
sequence (25). The sense primer, 5'-GACCTTGAAAGGCTCAGGAAGAAC-3', and
the antisense primer, 5'-TAGCTGAAGATGGACAGACTTGC-3', spanned bases
51
to
27 and 931 to 908, respectively, in relation to the first base of
the translation initiation codon. To evaluate the potential variation
in RT-PCR efficiency, an internal control, a 395-bp fragment of the L19
ribosomal protein gene was coamplified in each sample, using as sense
primer 5'-GAAATCGCCAATGCCAACTC-3' and as antisense primer
5'-TCTTAGACCTGCGAGCCTCA-3'.
RT and PCR of the target genes were run sequentially in the same assay
tube, as described previously (41). Briefly, equal amounts of total RNA
from the different experimental groups (2 µg/sample) were reverse
transcribed using avian myeloblastosis virus reverse transcriptase
(Finnzymes, Espoo, Finland) and the antisense primers. The cDNAs
generated were further amplified by PCR using the primer pairs
mentioned above. The total reaction volume was 50 µl which contained
1 nmol/liter of each oligo primer, 200 µmol/liter of a deoxy-NTP
mixtures including [
-32P]CTP, 20 units of RNasin, 12.5 units of avian myeloblastosis virus-reverse transcriptase-RT, and 2.5 units of Dynazyme-DNA polymerase in 1 × PCR buffer (10 mmol/liter
Tris-HCl, 50 mmol/liter KCl, 1.5 mmol/liter MgCl2, and
0.1% Triton X-100, pH 8.8) (Finnzymes). The reaction was initiated at
50° C for 15 min (RT) followed by denaturation at 97° C for 5 min. Then the PCR was run with a variable number of cycles of
amplification defined by denaturation at 96° C for 1.5 min,
annealing at 55° C for 1.5 min, and extension at 72° C for 3 min
(PTC-200, Peltier Thermal Cycler, MJ Research, Watertown, MA). The
number of PCR cycles examined was 16 to 40, and 16 cycles were chosen
for further analysis (data not shown). A final cycle of extension at
72° C for 15 min was included. To examine the PCR products, a
25-µl aliquot of each reaction was analyzed by gel electrophoresis on
a 1.2% agarose gel. The molecular sizes of the amplified products
(StAR and L19) were determined by comparison with the molecular weight
markers run in parallel with RT-PCR products. The gels were then vacuum
dried and exposed to Kodak x-ray films (XAR-5, Eastman Kodak,
Rochester, NY) at 4° C for 1 to 3 h, and autoradiograms were
analyzed for StAR mRNA expression. The relative levels of different
signals were quantitated by densitometry (Tina 2.0 Package,
Straubenhardt, Germany).
Generation of a StAR Competitor (StAR-2) and Competitive
RT-PCR--
For the generation of a competitor, full-length StAR
cDNA produced by RT-PCR, as described previously, was subcloned
into the pGEM T-vector following instructions of the manufacturer
(Promega). The identity of the inserted fragment was confirmed by
sequencing with fluorescent dye termination reaction (Prism Ready
Reaction Dye Termination Cycle Sequencing Kit) using an automated
sequencer (Perkin-Elmer).
Taking advantage of the presence of two AvaI sites in the
StAR cDNA, the subcloned product was subjected to restriction
endonuclease digestion, followed by separation in 1.2% agarose gel
electrophoresis. The existing AvaI fragment of 407-bp from
the full-length StAR cDNA was excised from the gel. The remaining
fragments (StAR cDNA subcloned into pGEM T-vector) were subjected
to blunt-end ligation by the Klenow fragment of DNA polymerase I and
the strand was recircularized by T4 DNA ligase, therefore generating a
StAR cDNA containing a 407-bp deletion, inserted into the cloning
site of the pGEM T-vector. Transcription of the ApaI
linearized template by means of the Sp6 RNA polymerase (Promega)
generates a sense cRNA of approximately 570 bp length, which was used
as the StAR competitor (StAR-2, Fig.
1).

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Fig. 1.
Schematic representation of the mouse StAR
and StAR-2 cDNAs. StAR-2 cDNA was generated from the
full-length mouse StAR cDNA by deleting a 407-bp fragment
(hatched rectangle) using two AvaI sites. This
shortened cDNA fragment was used as a template to generate the cRNA
competitor (StAR-2) by means of Sp6 RNA polymerase. The corresponding
positions of the sense and antisense PCR primers in the cRNA are
illustrated by arrows.
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To confirm the validity and reliability of the quantitative results
obtained by RT-PCR, StAR mRNA levels were further assessed by
competitive PCR. For this procedure, decreasing concentrations (800-1.56 ng) of the StAR competitor (StAR-2) cRNA were amplified together with a fixed amount of target RNAs (2 µg of total RNA from
control and T3 stimulated samples), using the same primer pairs as above. Aliquots of 25 µl of the RT-PCR products were analyzed and the levels of StAR mRNA were evaluated as described above.
Northern Hybridization Analysis--
Twenty micrograms of the
total RNA isolated from control and treated groups were resolved on
1.2% denaturing formaldehyde agarose gel and transferred onto
Hybond-N+ nylon membrane (Amersham Int., Aylesbury, UK) by
employing the capillary transfer method. The StAR and SF-1 cDNA
probes were labeled with [
-32P]dCTP (1800 Ci/mmol)
using the Prime-a-Gene labeling method (Promega). The labeled probes
were purified by using Sephadex G-50 nick columns (Pharmacia).
Prehybridization and hybridization were carried out under stringent
conditions as described previously (42). After hybridization, the
membranes were washed twice at room temperature for 20 min with 2 × SSC containing 0.1% SDS, followed by 2 h at 42° C with
0.1 × SSC and 0.1% SDS until removal of the background counts.
To examine the variation in StAR and SF-1 mRNA levels, the
membranes were subjected to rehybridization with a cDNA probe of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The membranes were
then exposed to x-ray films (Kodak XAR-5) for 60-72 h at
80° C.
The relative mRNA levels of StAR in different transcripts, SF-1,
and GAPDH expression were quantitated as above.
Isolation of Nuclei and T3 Binding
Incubations--
Isolation of nuclei from the cells was carried out
according to the procedure described elsewhere (43, 44), with minor modifications. In brief, the cells were lysed by ultrasonication (Bransonic 12, Soest, The Netherlands) in SMNaT buffer (0.25 mol/liter sucrose, 10 mmol/liter MgCl2, 50 mmol/liter
NaHSO3, 10 mmol/liter Tris buffer, pH 7.0) containing 1%
Triton X-100 under ice. The homogenate was centrifuged at 3000 rpm for
15 min at 4° C. The crude pellets containing nuclei were again
sonicated, washed twice, and examined microscopically with or without
stain (acetorcin, 2% orcin in 50% acetic acid). The pellet was
finally washed in SMNaT buffer without Triton X-100 and resuspended in
SMCT incubation buffer (0.32 mol/liter sucrose, 3 mmol/liter
MgCl2, 2 mmol/liter dithiothreitol, 10 mmol/liter Tris
buffer, pH 7.0).
[125I]Iodo-T3 (1080-1320 µCi/µg) binding
to the mLTC-1 cell nuclear membrane preparations was performed
according to the procedure described earlier (19, 44), slightly
modified for optimum binding. Briefly, a nuclear membrane preparation
(25 µg of DNA/incubation) was incubated with a fixed concentration of
[125I]iodo-T3 (100,000 cpm) either in the
absence (total) or presence of 600-fold excess of unlabeled
T3 (nonspecific) in a final volume of 400 µl. For the
competitive inhibition studies, 25 µg of DNA were incubated with
100,000 cpm of [125I]iodo-T3 together with
varying concentrations of the unlabeled hormone (1.5-1,500
pmol/liter). The incubation was carried out at 37° C for 2 h,
and terminated by adding 2 ml of ice-cold 40% polyethylene glycol
(Mr 6,000). After centrifugation at 3,000 rpm
for 20 min at 4° C, the supernatant was discarded and the pellet
washed twice with the incubation buffer, and finally radioactivity in
the pellet was determined in a
-counter (1260 Multigamma II, LKB
Wallac, Turku, Finland).
Determination of Progesterone (P) and Testosterone (T)--
The
concentrations of P and T in the media were assessed, after extraction
with diethyl ether, with specific RIAs as described previously (45,
46).
Statistical Analysis--
The data are presented as the
mean ± S.E. from representative cultures carried out in
triplicate or quadruplicate. Statistical analysis was performed using
the Statview program following ANOVA (Fisher's protected least
significant differences test) as noted. A p value less than
0.05 was considered statistically significant.
 |
RESULTS |
T3-induced P Production Requires Protein
Synthesis--
The induction of P production by T3 was
evaluated for the dependence of on-going protein synthesis. The
inhibitors of protein synthesis, actinomycin D and CHX (10 mg/liter
each), the former preventing incorporation of methionine into protein
and the latter inhibiting RNA synthesis, were examined separately. The
cells stimulated with T3 (37.5 pmol/liter) for 8 h
showed a significant (4.0 ± 0.2-fold) increase in P production
(Fig. 2). Treatment with actinomycin D or
CHX remarkably diminished (p < 0.0001)
T3-induced P production, suggesting that the T3
action is dependent on intact transcription and translation of the
target cells. Actinomycin D or CHX alone exhibited no effect on the
basal P production.

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Fig. 2.
The stimulatory effect of T3 on P
production by the mLTC-1 cells requires on-going protein
synthesis. The mLTC-1 cells were stimulated for 8 h in the
presence or absence of T3 (37.5 pmol/liter), and
actinomycin D (ACT. D) or CHX (10 mg/liter each). P
concentrations were determined from the media by RIA. The data
represent the mean ± S.E. of six independent experiments in
triplicates. The asterisks represent significance of
differences in the following comparisons: control versus
T3, T3 versus T3 + actinomycin D, and T3 versus T3 + CHX, (****, p < 0.0001).
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Effect of T3 on StAR mRNA Expression and P Production
by mLTC-1 Cells--
The results summarized in Fig. 2 represent the
time-dependent increase in steady-state levels of StAR
mRNA and P production in response to T3 (37.5 pmol/liter) stimulation. The T3-induced StAR mRNA
expression was significant (p < 0.05) as early as 30 min, and the magnitude of the response gradually increased up to a
3.6 ± 0.4-fold maximum by 8-10 h (Fig.
3A). The StAR mRNA levels
declined sharply thereafter, reaching another plateau between 16 and
24 h. The cAMP analog 8-Br-cAMP, a well known inducer of the StAR
gene expression through activation of the PKA signal transduction
pathway (29, 47, 48), was used as a positive control stimulus. It could
be clearly seen that a 4-h stimulation with 8-Br-cAMP (1 mmol/liter)
elevated the StAR mRNA and P levels about 3.4-fold, which was
comparable to that achieved by T3 stimulation in 4 h.
The P levels at each time point were determined and expressed as fold
increases, and they paralleled those occurring in StAR mRNA
expression (Fig. 3B). The accumulation of P in the
medium was significant within 1 h (p < 0.0001),
and it gradually increased reaching a plateau between 7 and 9 h,
and then followed the pattern of StAR expression. The concordant time
courses of the two T3 responses demonstrate a direct
correlation between StAR mRNA expression and P production.

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Fig. 3.
The time response pattern of
T3-stimulated StAR mRNA expression and P production of
the mLTC-1 cells. Total RNA was extracted from control and
T3 (37.5 pmol/liter)-treated cells and subjected to RT-PCR
analysis as described under "Experimental Procedures." A
395-bp fragment of the ribosomal protein L19 gene was coamplified in
each sample to correct for the variation in RT-PCR efficiency. The
RT-PCR products were resolved in 1.2% agarose gels, which were dried,
and exposed to x-ray films. A representative autoradiogram showing the
time response (0-24 h) of the T3-induced StAR mRNA
expression, and a 4-h response to 8-Br-cAMP treatment (1 mmol/liter)
was used as the positive control (Panel A). Panel
B shows the arbitrary densitometric units of the StAR mRNA
responses, and the P production of the same samples, both expressed as
fold-increases over the control (CON) level. The arbitrary
densitometric unit value of each band was corrected with the
corresponding L19 bands. The results are the mean ± S.E. of three
independent experiments in duplicate. The asterisks
represent significance of differences in comparison to control; *,
p < 0.05; ****, p < 0.0001; , the
first time point with significant elevation from control; ,
significant reduction in comparison to maximal T3
stimulation.
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Since the StAR mRNA expression and P production are clearly
stimulated by T3 in a time-dependent manner, we
next examined the dose-response pattern of the T3 effects.
The results presented in Fig. 4 show that
when the cells were stimulated with increasing concentrations of
T3 (0.015-1500 pmol/liter) for 8 h, a
dose-dependent effect was observed on StAR mRNA
expression and P production. T3 induced an increasing
response in StAR mRNA levels, as determined by quantitative RT-PCR
(Fig. 4A). Increased StAR mRNA expression was detected
(p < 0.05) at concentrations
1.5 pmol/liter,
the half-maximal stimulation occurring at 16.5 pmol/liter, and maximum increase at doses
37.5 pmol/liter of T3 (Fig.
4B). The dose-response pattern of P production was similar
with significant response at T3 level
1.5 pmol/liter (Fig. 4C), further indicating a direct link
between the T3 effect on steroidogenesis and StAR
expression.

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Fig. 4.
Dose-response patterns of mLTC-1 cell StAR
mRNA levels and P production in response to stimulation with
T3. Details of the RT-PCR analysis are described under
"Experimental Procedures" and in the legend of Fig. 3. Panel
A, a representative autoradiogram presenting the effects of
increasing doses of T3 (0.015-1500 pmol/L) on StAR
mRNA expression following a 8-h stimulation. Panel B,
the response of the StAR mRNA in arbitrary densitometric units, as
corrected with intensity of the corresponding L19 amplicon. Panel
C, the concomitant P production as determined from the culture
media. The data represent the mean ± S.E. of five independent
experiments in duplicate. The asterisk represents
significance of difference in comparison to control: *,
p < 0.05. , the first time point with significant
increase over control.
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Competitive RT-PCR Approach for Quantitation of the
T3-induced StAR mRNA Expression--
To verify the
accuracy and validity of the quantitative RT-PCR results, we also
examined the competitive PCR approach. A homologous cRNA (StAR-2) was
included into the RT-PCR reaction to determine the sensitivity of our
assay system in competitive conditions. The results of the competitive
PCR are presented in Fig. 5, where fixed
amounts of total RNA (2 µg) either from control or T3
(37.5 pmol/liter)-stimulated samples were allowed to compete with
varying quantities (800-1.56 ng) of the cRNA competitor (StAR-2). As
expected, amplification of decreasing amounts of the competitor with
fixed amounts of total RNA provided signals of decreasing intensity. Conversely, the StAR signals gradually decreased as the concentrations of competitor increased, indicating a specific nature of the
competitive PCR reactions (Fig. 5A). The ratios of
intensities of the StAR/StAR-2 amplification products in both cases
were corrected for the constant intensity of L19 band as
illustrated on the graph of Fig. 5B. It could be clearly
seen that T3 induced about 3.6-fold increase in StAR
mRNA expression as compared with control. The experiment further
confirmed the previous results obtained by quantitative RT-PCR,
indicating that T3 exerts a specific action on StAR
mRNA expression.

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Fig. 5.
Schematic representation of the competitive
RT-PCR procedure to verify the quantitative accuracy of the
T3-induced stimulation of the StAR mRNA levels.
Decreasing concentrations of StAR-2 cRNA (800-1.56 ng) were added to a
fixed concentration of total RNA (2 µg) from control and treated
samples. To correct for the experimental variations, a fragment of the
L19 gene was coamplified in each sample. The RT-PCR products were
analyzed as described under "Experimental Procedures" and in the
legend of Fig. 3. Panel A, representative autoradiograms
showing the intensity of the StAR, StAR-2, and L19 amplicons in control
and T3-stimulated samples. Panel B represents
the arbitrary densitometric unit ratio of StAR/StAR-2 amplicons from
both groups, corrected for the corresponding intensity of the L19
bands. The situation where the amplicon ratio is 1 (horizontal
line) indicates the StAR mRNA concentration in the two
experimental groups (control and T3 stimulation). The
results presented are the mean ± S.E. of five independent
experiments.
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T3 and hCG Have Additive Effects on StAR mRNA
Levels--
The potential interactions of T3 and hCG were
assessed on StAR mRNA expression, since the mechanisms which
underlie their actions involve two separate, i.e. nuclear
and plasma membrane receptor systems, respectively. The results
presented in Fig. 6A
demonstrate the effects of hCG (0.7 pmol/liter), T3 (37.5 pmol/liter), and their combination in StAR mRNA levels. The cells
stimulated for 8 h either with hCG or T3 alone showed
about 4.0- and 3.6-fold StAR mRNA levels, respectively. The
combination of hCG + T3 evoked an additive effect
(p < 0.0001) of StAR message accumulation, which was
about 6-fold over nonstimulated cells. In additional experiments,
T3 also exhibited a similar additive effect when the cells
were incubated with 8-Br-cAMP in the presence of T3 (data
not shown).

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Fig. 6.
Effects of hCG, T3, and their
combination on StAR mRNA expression and P production in mLTC-1
cells. The cells were stimulated in the absence (CON)
or presence of hCG (0.7 pmol/liter), T3 (37.5 pmol/liter),
or their combination for 8 h. Two micrograms of total RNA from
each group were used in RT-PCR. The RT-PCR products were analyzed as
described under "Experimental Procedures" and in the legend of Fig.
3. Panel A, a representative autoradiogram showing the
effects of hCG, T3, and hCG + T3 on StAR
mRNA expression. Panel B, the arbitrary densitometric
unit values of each band as corrected to the corresponding L19 band.
The results are expressed as fold increase over control
(CON) value and represent the mean ± S.E. of three
independent experiments. Panel C, the cells were stimulated
for 8 h without (CON) or with half-maximal doses of hCG
(0.3 pmol/liter), T3 (16.5 pmol/liter), or hCG + T3. P concentrations were determined from the media by RIA.
The values are the mean ± S.E. of three experiments in
triplicate. Different letters above the bars
indicate these groups are significantly different at a level
p < 0.0001.
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To corroborate the observed effect of hCG and T3 in StAR
expression we also determined their response in P production.
Stimulation of cells with these hormones at ED50 doses, hCG
(0.3 pmol/liter), and T3 (16.5 pmol/liter), either
separately or in combination, clearly increased P accumulation and
followed a similar pattern as evidenced with StAR mRNA expression
(Fig. 6C). These data further suggest a close coordination
between the stimulated levels of StAR expression and P production.
Specificity of Thyroid Hormone Binding to Nuclear Membrane
Preparations and on StAR mRNA Expression--
To examine the
presence of thyroid hormone receptors, competitive inhibition
experiments were carried out under optimized conditions with nuclear
membrane preparations of the mLTC-1 cells. Fig.
7A shows that at increasing
concentrations (1.5-1500 pmol/liter) of unlabeled T3,
triiodothyroacetic acid (TRIAC), and thyroxine (T4),
T3 demonstrated the highest affinity for these binding
sites.

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Fig. 7.
Specificity of thyroid hormone binding to
nuclear membrane preparations and on StAR mRNA expression.
Panel A, competitive inhibition of
[125I]iodo-T3 binding to nuclei of mLTC-1
cells by T3, TRIAC, and T4. The nuclei were
prepared from the cells and subjected to the nuclear receptor binding
assay as described under "Experimental Procedures." The results are
the mean ± S.E. of four experiments in triplicate. Panels
B and C, induction of StAR mRNA expression by
thyroid hormones. Total RNA was extracted from different groups of
mLTC-1 cells after 8 h incubation without (CON) or with
T3, TRIAC, and T4 (each 37.5 pmol/liter). The
RT-PCR analyses were carried out using 2 µg of total RNA from each
group, and the RT-PCR products were analyzed as described under
"Experimental Procedures" and in the legend of Fig. 3. A
representative autoradiogram demonstrating the action of thyroid
hormones on StAR mRNA levels is presented in Panel B.
The intensities of the StAR mRNA amplicons (arbitrary densitometric
units) after correction for the intensity of the corresponding L19
bands, are presented in Panel C. The values are the
mean ± S.E. (n = 4). Different letters
above the bars indicate that these groups are
significantly different at a level p <0.001.
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The results presented so far clearly demonstrate that T3
modulates StAR gene expression and P production. We next examined whether other thyroid hormones, i.e. TRIAC and
T4, would induce StAR mRNA expression. As shown in Fig.
7, B and C, all three thyroid hormones exerted
significant increases on StAR mRNA levels in 8 h; however,
TRIAC and T4 were less potent than T3.
Effect of T3 on Dispersed Leydig Cell T Production--
To
assess functional relevance of the thyroid hormone effects observed
with the murine Leydig tumor cells, adult mouse primary Leydig cells
were stimulated in the absence or presence of two concentrations of
T3 (7.5 and 75 pmol/liter) and hCG (0.7 pmol/liter) for
8 h. As shown in Fig. 8,
T3 at both doses significantly augmented the T production,
which was 1.19 ± 0.07- and 1.8 ± 0.21-fold higher, respectively, over nonstimulated cells. The cells were simultaneously incubated with hCG (0.7 pmol/liter) for comparison, which also showed a
clear stimulation of T production.

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Fig. 8.
Effect of T3 on T production in
isolated mouse Leydig cells. Dispersed adult Leydig cells were
cultured for 24 h before stimulation, as described under
"Experimental Procedures." After washing, the cells were stimulated
in the absence (CON) or presence of T3 (7.5 and
75 pmol/liter) and hCG (0.7 pmol/liter) for 8 h. T concentrations
were determined from the media by RIA. Different letters
above the bars indicate that they are significantly
different at a level p <0.01.
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Northern Hybridization Analysis of StAR mRNA and T3
Stimulation--
Besides RT-PCR, it was also possible to show the
effect of T3 on StAR expression by Northern blot analysis.
Total RNA was extracted from control and T3 (37.5 pmol/liter)-stimulated mLTC-1 cells, and probed with the StAR cDNA.
The full-length mouse StAR cDNA probe hybridized with two major RNA
transcripts of 3.4 and 1.6 kb in size, of which the latter corresponds
to the functional StAR protein (Fig. 9).
In addition, two minor transcripts of 2.7 and 1.4 kb sizes were
detected when the membranes were exposed for longer time (72 h). The
intensities of all the transcripts increased in parallel, 3-6-fold,
with T3 stimulation.

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Fig. 9.
Northern hybridization analysis of StAR
mRNA expression in control and T3-treated mLTC-1
cells. Twenty micrograms of total RNA from control and
T3-stimulated samples (both in triplicate) were probed with
the mouse full-length StAR cDNA, as described under "Experimental
Procedures." The apparent molecular weights of the different
transcripts are shown on the right. The autoradiogram was
exposed for 3 days. The expression of GAPDH mRNA level of each
fraction demonstrates equal loading of RNA (lower panel).
Similar results were obtained from four independent experiments.
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Expression of SF-1 and DAX-1 in mLTC-1 Cells, and Their Correlation
to the Levels of T3-induced StAR Expression and P
Production--
The involvement of thyroid hormone in the regulation
of StAR gene expression and steroidogenesis was assessed in the cells expressing SF-1, DAX-1, or their combination with transient
transfections. The results presented in Fig.
10 show that SF-1 expression
concomitantly elevated T3 (7.5 and 75 pmol/liter, 8 h)-mediated StAR mRNA levels, which were about 2.2-fold higher, as
compared with non-transfected cells. In contrast, cells expressing
DAX-1 displayed remarkably diminished (p < 0.0001)
T3-induced StAR expression and P production. The magnitude
of the SF-1-mediated response was attenuated following T3
stimulation when the cells were cotransfected with SF-1 and DAX-1 (Fig.
10), indicating that SF-1 is an essential regulator of thyroid
hormone-mediated StAR gene expression. The accumulation of P levels in
the media from non-transfected, SF-1, DAX-1, and SF-1 + DAX-1
expressing cells with increasing concentrations of T3 (0.15-150 pmol/liter) exhibited a
dose-dependent response, while DAX-1 drastically inhibited
the augmenting effect of T3 on P production (Fig.
11).

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Fig. 10.
Effects of SF-1, DAX-1, and SF-1 + DAX-1
co-expression on T3-stimulated StAR mRNA
expression. The mLTC-1 cells were transfected with expression
plasmids of SF-1, DAX-1 (2 µg each), or their combination, as
described under "Experimental Procedures." After 48 h, the
cells were stimulated in the absence and presence of two concentrations
of T3 (7.5 and 75 pmol/liter). Total RNA was extracted from
the control and treated groups and subjected to RT-PCR analysis as
described under "Experimental Procedures" and in the legend of Fig.
3. Panel A, a representative autoradiogram showing the
effects of T3 (0, 7.5, and 75 pmol/liter; 8 h) on StAR
mRNA expression from nontransfected (NT), SF-1, DAX-1,
and SF-1 + DAX-1 expressing cells. Panel B, the arbitrary
densitometric unit values of each band were quantitated and normalized
with the corresponding L19 bands. The data presented are the mean ± S.E.; n = 4. The asterisks represent
significant differences in response to T3 stimulation, as
compared with the SF-1 transfected cells; ****, p < 0.0001.
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Fig. 11.
Expression of SF-1, DAX-1, and SF-1 + DAX-1
on T3-stimulated P production in mLTC-1 cells. The
cells were transfected with SF-1, DAX-1, or their combination as
described under "Experimental Procedures" and in the legend of Fig.
10. After 48 h, the cells were stimulated for 8 h with
increasing concentrations of T3 (0.15-150 pmol/liter). The
media were collected from each group of treatments and P levels were
determined from the media by RIA. The results are expressed as fold
increases over respective controls. These data are representative of
four independent experiments in triplicates. NT,
non-transfected control cells.
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Effect of SF-1 and DAX-1 Expression on the Levels of Endogenous and
T3-stimulated SF-1 mRNA--
Northern hybridization
analysis revealed that the level of SF-1 mRNA in mLTC-1 cells
increased significantly (p < 0.0001) upon transfection
of the SF-1 expression plasmid. In contrast, the SF-1 levels were
significantly suppressed by DAX-1 and SF-1 + DAX-1 co-expression (Fig.
12). Given the direct link between StAR
expression and steroidogenesis, we examined the
T3-stimulated SF-1 mRNA expression, which clearly
showed a dose-dependent elevation, with a 2.9 ± 0.3-fold maximum in comparison to non-stimulated cells, favoring the
indirect steroidogenic effect of T3 rather than a direct
one (Fig. 13).

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Fig. 12.
Expression of SF-1, DAX-1, and SF-1 + DAX-1
on the endogenous SF-1 mRNA levels. The cells used for
comparison were either non-transfected (NT) or transfected
with SF-1, DAX-1, or their combination (each in triplicate), as
described under "Experimental Procedures" and in the legend of Fig.
10. Forty-eight hours later, RNA was extracted and subjected to
Northern blot analysis with SF-1 cDNA probe using 20 µg of the
total RNA. Panel A, a representative autoradiogram showing
expression of the SF-1 mRNA. The GAPDH mRNA expression of each
fraction is indicated for equal RNA loading. Panel B
represents the arbitrary densitometric unit values which are the
mean ± S.E. of four independent experiments in triplicate. The
asterisks represent significant difference from the control
SF-1 expression; **, p < 0.01; ****, p < 0.0001.
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Fig. 13.
Effect of increasing doses of T3
on SF-1 mRNA expression in mLTC-1 cells. The cells were
stimulated in the absence or presence of increasing doses of
T3 (0.15-150 pmol/liter) for 8 h, and followed by
total RNA extraction. Twenty micrograms of RNA from control and
T3-stimulated samples were hybridized with SF-1 cDNA
probe as described under "Experimental Procedures." Panel
A, a representative autoradiogram demonstrating the response of
SF-1 mRNA expression to T3 stimulation (each in
duplicate). The GAPDH mRNA level of each fraction demonstrates
equal loading of RNA. Panel B, the arbitrary densitometric
unit values represent the mean ± S.E. of three separate
experiments in duplicates; **, p < 0.01; ****,
p < 0.0001.
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DISCUSSION |
The acute production of steroid hormones is initiated by
translocation of cholesterol from the outer to the inner mitochondrial membrane, an event that has recently been found to be mediated by the
StAR protein. StAR is a novel 30-kDa protein associated with
mitochondria, and its level is rapidly induced in response to trophic
hormone or cAMP stimulation in steroidogenic cells (8). The present
experiments were designed to explore the regulation of the StAR gene
expression by thyroid hormones, and in particular by T3,
biologically the most potent thyroid hormone. Our data demonstrate the
presence of thyroid hormone receptors in nuclei of the mLTC-1 mouse
Leydig cells where T3 increases the steady-state levels of
StAR mRNA and steroid production. The most intriguing results
presented here are the parallel temporal and dosage patterns of the
T3-induced StAR expression and steroid production which provide evidence that T3 stimulation of the StAR gene
expression and steroidogenesis are linked to a common regulatory
cascade. In addition, the T3 and hCG effects on StAR
mRNA expression and P production appear to be additive in these
cells. The physiological relevance of these findings was assessed by
T3-stimulated T production of primary mouse Leydig cells.
Collectively, to our knowledge, this is the first demonstration of
involvement of thyroid hormones in StAR gene expression, which may
provide the mechanism for the known importance of thyroid function for
that of the gonads.
The classical mechanism of stimulation of steroidogenesis occurs
through trophic hormone-stimulated cAMP production, and concerns both
acute and chronic effects on steroid production (25, 29). This
regulatory cascade involves the transport of cholesterol to
mitochondria, which is the first step in the biosynthesis of all
steroid hormones. It has been demonstrated that during cholesterol transport both outer and inner mitochondrial membranes become closely
associated and form "contact sites" which are dependent on the
transfer of phospholipids (49-51). The increase in contact sites is
closely associated with the stimulation of steroidogenesis. The trophic
hormone or cAMP-induced StAR gene transcription requires on-going
protein synthesis which is responsible for the acute regulation of
steroidogenesis (1, 32). Clark et al. (29) have demonstrated
that in MA-10 cells, Bt2cAMP coordinately stimulates StAR
protein and StAR mRNA expression which were consistent with stimulated steroidogenesis. Our results document that T3
promotes StAR gene expression and P production in a time- and
dose-dependent manner.
The hormone-induced StAR protein is processed from a short-lived 37-kDa
precursor protein that is sensitive to cycloheximide (25, 52). Epstein
and Orme-Johnson (30) isolated a mitochondrial phosphoprotein in
adrenal glomerulosa cells that fulfills the criteria of a regulator of
steroidogenesis and resembles the StAR protein. Recent studies also
implicate that phosphorylation of the serine residue at codon 194/195
of StAR modulates steroidogenic activity of the StAR protein (53). It
has been shown that blockade of the StAR protein synthesis at the
translational level prevents the cholesterol transport into adrenal
glomerulosa cells (54). In mechanically dispersed testicular Leydig
cells, T3 induced significant androgen production by
generating a 52-kDa soluble protein, also sensitive to the protein
synthesis inhibitors (19). Our findings clearly demonstrate that
T3 stimulation of P production in mLTC-1 cells is inhibited
by actinomycin D or CHX, indicating the involvement of acute protein
synthesis in the regulation of steroidogenesis, which seems to be
mediated by StAR protein. Moreover, T3 markedly and in
additive fashion augmented the hCG-induced expression of StAR mRNA
and P production. This is in keeping with the different mechanisms of
action of T3 and leutinizing hormone/hCG, and suggest that
these effectors employ different cis- and/or trans-activators of the
StAR gene.
A pertinent question arising from the T3-induced StAR
expression concerns its biological relevance. To explore this
particular aspect, other thyroid hormones, TRIAC and T4,
were examined in the same manner for StAR mRNA expression and
competitive nuclear binding studies. The results also demonstrate the
specificity for T3 of the thyroid hormone-binding sites in
mLTC-1 cell nuclei, whereas TRIAC and T4 were shown to
induce weaker responses in both StAR mRNA expression and in
inhibition of [125I]iodo-T3 binding. However,
a single class of high-affinity receptors was observed in these cells.
The affinity (Kd
0.486 nmol/liter) of
T3-binding sites (not illustrated) to the nuclear preparations of mLTC-1 cells was comparable to that reported in conventional target tissues for thyroid hormones (15, 19, 55). The
physiological response of thyroid hormone was assessed in purified
adult mouse testicular Leydig cells, where T3 and hCG both
were equipotent in inducing T production.
The crucial role of StAR protein in the regulation of steroidogenesis
has been demonstrated in StAR knock-out mice (56), and in patients
suffering from lipoid congenital adrenal hyperplasia, which was found
to be due to mutations in the StAR gene (57). Furthermore, Northern and
Western analyses have demonstrated that expression of the StAR gene is
involved in regulating steroidogenesis in MA-10 cells (27, 29). Our
present model of the mechanisms of T3-induced StAR mRNA
expression and steroidogenesis reflects similar phenomena, indicating
that T3-induced StAR protein is directly involved in the
acute regulation of P production. In addition, four StAR transcripts
were detected in the mLTC-1 cells, two major (3.4 and 1.6 kb) and two
minor (2.7 and 1.4 kb) ones, and all of them were increased upon
T3 stimulation. The molecular sizes of the different
transcripts corresponded to those previously reported in different
species (25-28). It is also possible that the minor and inconsistent
band at 1.4 kb is a splice variant of the StAR gene with differential
polyadenylation signal or length of the poly(A) tail.
With respect to the interaction of T3 with StAR expression,
its mechanism of action is of considerable interest. Stocco and collaborators (29, 52) and others (27, 54) have provided ample evidence
that StAR expression is predominantly regulated by the cAMP second
messenger system, although other factor(s) might also be involved.
There is a conspicuous lack of cAMP response elements in the mouse and
human StAR promoter sequences (47, 58). This is not surprising since it
seems to be a common feature of many cAMP-regulated genes, including
the cytochrome P450 steroid hydroxylase genes (59). It has been
implicated that the orphan nuclear receptors SF-1 and DAX-1 have
pivotal roles in the regulation of reproductive endocrine functions at
multiple levels during development and differentiation, including the
expression of steroidogenic enzymes (33, 60, 61). Studies also indicate
that the mutations associated with DAX-1 and SF-1 cause adrenocortical
insufficiency and gonadal abnormalities associated with
hypogonadotropic hypogonadism (31). In addition, DAX-1 binds to DNA
hairpin structures and suppresses the transcriptional activity of SF-1
and StAR in Y-1 adrenocortical tumor cells (62, 63). On the other hand,
the SF-1 knock-out mice provided intriguing information pertaining to
the role of SF-1 as a key regulator of endocrine function and expression of the steroidogenic enzymes (35). The nucleotide sequences
of the 5'-flanking region of the mouse StAR gene (3.6 kb) revealed the
presence of two SF-1-binding sites at positions
135 and
42, and one
DAX-1-binding site at
24 position. The importance of the SF-1-binding
sites for StAR expression has been further confirmed by the deletion of
those sequences from the mouse StAR promoter, which significantly
diminished its activity (47). Two potential binding sites are also
located at positions
890 and
42 for the nuclear hormone receptors
which resembles the SF-1 consensus motif, whereas neither the human nor
the mouse StAR promoter sequences contain specific thyroid hormone
receptor recognition sites (47, 58). For this reasons, and since
T3 augmented the StAR expression of the cells
constitutively over-expressing SF-1, T3-stimulated SF-1
expression does not totally explain its effect on StAR, and additional
facts are apparently involved.
The cells expressing SF-1 did not only respond with increased StAR
mRNA expression and P production, but these levels of expression were also under the regulation of T3. In contrast, DAX-1
expression drastically repressed the potential effects of
T3 on StAR gene expression and P production, whereas it
markedly diminished the SF-1-mediated expression. Strikingly, in a
dose-dependent manner, T3 stimulation
significantly elevated the levels of SF-1 mRNA expression, whereas
DAX-1 profoundly inhibited the endogenous SF-1 level. These data
strongly suggest that T3 has no direct action on StAR gene
in the regulation of steroidogenesis, rather an indirect effect through
the modulation of SF-1 expression. The precise mechanisms of thyroid
hormone action in steroidogenesis may be multifactorial, and remains to
be established. On the basis of the present observations, it is
tempting to propose that the mechanism of thyroid hormone action on
StAR gene expression, which consequently regulates steroidogenesis, is
at least in part mediated by SF-1. The regulation of SF-1 function
together with the post-translational modifications, i.e.
phosphorylation and the interactions of SF-1 with coactivator(s)
involved in T3-induced StAR gene expression, will be an
area of obvious interest in our future investigations.