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Department of Physiology, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland
Address all correspondence and requests for reprints to: Prof. Ilpo T. Huhtaniemi, M.D., Ph.D., Department of Physiology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. E-mail: ilpo.huhtaniemi{at}utu.fi
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Abstract |
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Introduction |
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The rate-limiting, committed, and regulatable step in steroid hormone biosynthesis is the transport of cholesterol from the outer to the inner mitochondrial membrane, and it is dependent on de novo protein synthesis (13). Recently, a novel LH-induced 30-kDa mitochondrial factor was purified and cloned from MA-10 mouse Leydig tumor cells, named the steroidogenic acute regulatory (StAR) protein (14). The mature StAR protein is synthesized from a larger 37-kDa precursor protein whose expression is restricted to steroidogenic cells and is acutely involved in the regulation of steroidogenesis (15, 16). In MA-10 mouse Leydig tumor cells, (Bu)2cAMP coordinately stimulates StAR protein and StAR messenger RNA (mRNA) expression, which were consistent with stimulated progesterone (P) production (17). Inhibition of protein synthesis by actinomycin D in hormone-stimulated MA-10 cells has been shown to inhibit StAR gene transcription, whereas CHX has no effect on the steady state level of StAR mRNA (18).
Several lines of evidence propose that multiple second messenger systems, including cAMP, protein kinase A (PKA), protein kinase C (PKC), and Ca2+, are involved in the induction of StAR gene expression in adrenal and gonadal cells (19, 20, 21, 22). However, clear-cut data on involvement of the Ca2+ messenger system in hormone-stimulated StAR expression in Leydig cells are still lacking. Ramnath et al. (23) demonstrated in MA-10 cells that the modulation of steroidogenesis is affected by the omission of Ca2+ from the incubation medium in the presence or absence of chloride ions. To gain molecular insight into the mechanisms, the crucial role of Ca2+ messenger system in StAR protein expression and steroidogenesis has yet to be investigated in mouse Leydig tumor cells (mLTC-1) (24).
The physiological action of K+, especially in adrenal cells, is thought to involve the opening of plasma membrane Ca2+ channels, facilitating the influx of Ca2+ for activation of steroid hormone biosynthesis (25). In adrenal glomerulosa cells, ample evidence exists for the requirement of Ca2+ for the steroidogenic action of K+ (26, 27, 28). It has also been reported that K+ opens voltage-operated T- and L-type Ca2+ channels, where the low threshold T-type channels greatly accelerate this process (29, 30). In human H295R adrenocortical choriocarcinoma cells, StAR protein expression and aldosterone synthesis have been shown to be induced by angiotensin II (Ang-II)-, K+-, and PKC-dependent pathways (19, 31, 32). Much progress has been made in understanding these mechanisms in adrenal cells, but definitive evidence for the role of K+ in hCG-stimulated StAR expression in Leydig cells is lacking.
The mechanism of the Ca2+ effect that modulates hCG action in steroidogenesis needs further clarification. Studies indicate that members of the orphan nuclear receptor family are involved in the development and differentiation of endocrine functions at multiple levels, including steroid hormone biosynthesis (33, 34). In accordance, SF-1 and DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X-chromosome) play pivotal roles in regulation of the cytochrome P450 steroid hydroxylase genes during development and the differentiated function of the adrenal glands and gonads (35, 36). In addition, mutations of these two receptors cause multiple abnormalities of adrenal development and function associated with hypogonadotropic hypogonadism, strongly pointing out their interactions in a common developmental pathway, with activation of target genes in cooperative fashion (37, 38). It has also been demonstrated that the 5'-flanking region of the mouse StAR gene exhibits two SF-1-binding sites at positions -135 and -42 (18), and one DAX-1-binding site at position -24, whereas the human StAR promoter possesses two putative DAX-1-binding sites at positions -27 and -61 (39), but their importance has yet to be confirmed. It is noteworthy that DAX-1-binding sites in the human promoter are conserved and form hairpin loops, whereas these regions in the mouse are ineffective to form a potential hairpin loops. The interaction of these factors with StAR in regulating steroidogenesis presents the opportunity to understand in more depth the development of the steroidogenic machinery.
In light of the above observations, the present investigation was aimed to evaluate the mechanisms of Ca2+ messenger system in the hCG-mediated StAR mRNA, StAR protein expression, and P synthesis. The involvement of extracellular Ca2+ and K+ potentially augmented the hCG responses without altering the basal level of steroidogenesis. The use of Ca2+ chelators and transmembrane channel blocker allowed assessment of the role of Ca2+ mobilization and the rapid increase in [Ca2+]i after hCG stimulation. Our data clearly show that alterations of [Ca2+]i in response to hCG stimulation are highly correlated with the expression level of SF-1, a key component in the coordinate regulation of StAR expression and steroidogenesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-isopropyl-
[(N-methyl-N-homoveratryl)-
-aminopropyl]3,4-dimethoxyphenylacetonitrile
hydrochloride), Ca2+ ionophore (A 23187), fura-2/AM,
3-isobutyl-1-methylxanthine, sodium lauorylsarcosine, guanidine
thiocyanate, 2-mercaptoethanol, antifoam A, EGTA, and EDTA were
purchased from Sigma Chemical Co. (St. Louis, MO).
45CaCl2 (37 megabecquerels/1 mCi) was purchased
from DuPont-New England Nuclear (Boston, MA),
[-32P]UTP (800 Ci/mmol) and
[-32P]deoxy (d)-CTP (1800 Ci/mmol) were obtained from
Amersham (Aylesbury, UK). Gentamycin was purchased from Biological
Industries (Bet-Haemek, Israel), HEPES-buffered Waymouth’s medium was
obtained from Life Technologies (Paisley, Scotland), and
cell culture plasticware was obtained from Greiner Labortechnik
(Frickenhausen, Germany). All other chemicals used in this study were
of analytical grade and obtained from commercial sources.
Cell culture, steroid incubation, and transfection experiments
The mouse tumor Leydig cell line (mLTC-1) was maintained in
HEPES-buffered Waymouth’s medium supplemented with 9%
heat-inactivated horse serum (Life Technologies) and 4.5%
FCS (Bioclear, Devizes, Wilts, UK) containing 0.1 µg/liter
gentamycin. The cells were cultured on 9-cm diameter plates in regular
medium at 37 C under a humidified atmosphere of 95% air and 5%
CO2. During splitting, the viability of the cells was
examined with 0.4% trypan blue dye exclusion, and cells were plated at
a density of 6 x 104 cells/well in 24-well plates for
determining P production. A total of 5 x 105
cells/well in 6-well plates were used for
45Ca2+ incorporation studies and total RNA
extractions. The cells were plated about 24 h before carrying out
the stimulations.
On the day preceding an experiment, the cells were washed twice with 0.01 mol/liter PBS, and the medium was replaced with serum-free Waymouth’s medium supplemented with 0.1% BSA (fraction V, Sigma Chemical Co.) containing 0.25 mmol/liter 3-isobutyl-1-methylxanthine. The influence of Ca2+ on hCG-induced StAR mRNA, StAR protein, and P levels was determined in Ca2+-free medium. The additives (stimulators, inhibitors, etc.) were constituted fresh, diluted, applied to the cells in serum-free medium, and incubated for 4 h under a controlled atmosphere. At the end of stimulations, P content was measured from the diethyl ether-extracted incubation medium by a specific RIA, as described previously (40).
The mLTC-1 cells were subcultured at a density of 1.5 x 106 cells/6-cm diameter plate in regular medium. Transfection studies were conducted by FuGENE 6 transfection reagent (Boehringer Mannheim GmbH, Mannheim, Germany) at a 60–70% confluence of cells under optimized conditions, according to the instructions of the manufacturer. Briefly, the FuGENE 6 transfection reagent was diluted in serum-free medium and incubated at room temperature in a final volume of 300 µl. Two micrograms of the pCMV119+-SF-1 construct (obtained from Dr. K. L. Parker, Durham, NC) and 2 µg pBKCMV-hDAX-1 construct (obtained from Dr. R. Yu, Chicago, IL) were used for transfections. To control the transfection efficiency, 2 µg of a ß-galactosidase expression vector, pRSV-ß-galactosidase (Promega Corp., Madison, WI), were used. After 15-min incubation of the FuGENE 6-DNA complex at room temperature, it was distributed dropwise to the plate containing 3 ml regular medium. Stimulation studies were conducted between 40–48 h of transfection.
RNA extraction, and quantitative RT-PCR
Total RNA was extracted from control and stimulated cells by the
single step acid guanidinium thiocyanate-phenol-chloroform extraction
method of Chomczynski and Sacchi (41). The purity of the extracted RNA
was determined by spectrophotometric scanning at a wave length 220–320
nm.
The isolation and amplification of the mouse (mLTC-1) StAR complementary DNA (cDNA) were carried out by designing primers from the mouse StAR cDNA sequence (14). The sense primer, 5'-GACCTTGAAAGGCTCAGGAAGAAC-3', and the antisense primer, 5'-TAGCTGAAGATGGACAGACTTGC-3', spanned bases -51 to -27 and 931–908, respectively. 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 the sense primer 5'-GAAATCGCCAATGCCAACTC-3' and as the antisense primer 5'-TCTTAGACCTGCGAGCCTCA-3'.
The target genes were amplified by RT and PCR, and run sequentially in the same assay tube, as described previously (42). 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. Each reaction contained 1 nmol/liter of each oligo primer, 200 mmol/liter of dNTP mixtures containing [32P]dCTP, 20 U RNasin (Promega Corp.), 12.5 U avian myeloblastosis virus reverse transcriptase, and 2.5 U Dynazyme-DNA polymerase in 1 x 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, Espoo, Finland). 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 the steps 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, Inc., Cambridge, MA). Sixteen to 40 PCR cycles were examined, 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 mol wt markers run in parallel with the RT-PCR products. The gels were then vacuum dried for 45–60 min and exposed to Kodak x-ray films (XAR-5, Eastman Kodak Co., Rochester, NY) at 4 C for 1–3 h, and the autoradiograms were analyzed for StAR mRNA expression after correcting for the variation in RT-PCR efficiency with the corresponding L19 bands. The relative levels of different signals were evaluated by densitometry (Tina 2.0 Package, Straubenhardt, Germany).
Isolation of mitochondria, SDS-PAGE, and immunodetection of StAR
protein
After stimulation of the mLTC-1 cells, they were washed with
0.01 mol/liter PBS and scraped off using a rubber policeman into a
buffer containing 10 mmol/liter Tris-Cl (pH 7.2), 250 mmol/liter
sucrose, 0.1 mmol/liter EDTA, and 1.0 mmol/liter
phenylmethylsulfonylfluoride. The cells were homogenized at 4 C (1200
rpm, 30 strokes) with a Potter-Elvehjem homogenizer fitted with a
serrated pestle. Mitochondrial preparations were obtained by
differential centrifugation. The homogenate was centrifuged at 500
x g for 25 min to remove broken cell debris and nuclei, and
the resulting supernatant was further centrifuged at 1000 x
g for 25 min. The pellet containing mitochondria was washed
twice at 9000 x g for 15 min each time in the same
buffer.
The mitochondrial protein (12 µg/lane) was solubilized in sample buffer [25 mmol/liter Tris-Cl (pH 6.8), 1% SDS, 5% ß-mercaptoethanol, 10% glycerol, and 0.01% bromophenol blue] and loaded onto a 12% SDS-polyacrylamide gel (Mini Protean II System, Bio-Rad), as described by Laemmli (43), with minor modifications. Electrophoresis was performed at 200 V for 1 h, and the proteins were electrophoretically transferred onto a nitrocellulose membrane (Hybond, Amersham). The membranes were incubated in a blocking buffer (Tris-buffered saline containing 0.1% Tween-20 and 5% nonfat dry milk) for 2 h at room temperature, followed by incubation with antipeptide antibodies of the 30-kDa StAR protein generated in rabbit against amino acids 88–98 (obtained from Dr. D. M. Stocco, Lubbock, TX) (14). The incubation with the antibodies was carried out overnight at 4 C. The membranes were washed three times (10 min each time) in Tris-buffered saline buffer and incubated for 1 h at room temperature with horseradish peroxidase-labeled donkey antirabbit IgG (Amersham). The membranes were washed as stated above, the immunodetection of StAR protein was revealed by the ECL Western blotting detection kit (Amersham), and the membranes were exposed for 1–3 min to Fuji Photo Film Co. Ltd. x-ray films (Fuji Photo Film Co., Ltd., Tokyo, Japan). The immunospecific bands were quantified by phosphorimager and densitometry.
Northern hybridization analysis of StAR mRNA expression
Twenty micrograms of the total RNA obtained from control and
stimulated samples were resolved on 1.2% formaldehyde denaturing
agarose gel and transferred onto Hybond-N+ nylon membranes
(Amersham) by employing the capillary transfer method. An antisense
complementary RNA probe corresponding to bases -27 to 931, a
NotI fragment of the StAR cDNA, was produced by in
vitro transcription (Promega Corp.) with T7 RNA
polymerase, dNTPs, and [-32P]UTP (Amersham). For
preparing an SF-1 cDNA probe, an EcoRI-PstI
fragment of the SF-1 cDNA was labeled with [-32P]dCTP
using the Prime-a-Gene labeling method (Promega Corp.).
The labeled probes were purified using Sephadex G-50 nick columns
(Pharmacia, Uppsala, Sweden). Prehybridization and hybridization were
carried out under stringent conditions, as previously described (44).
In brief, prehybridization was performed for at least 6 h at 65 C
in a solution containing 50% formamide, 3 x SSC (150 mmol/liter
sodium chloride and 50 mmol/liter sodium citrate, pH 7.0), 5 x
Denhardt’s solution, 1% SDS, 0.1 µg/liter heat-denatured calf
thymus DNA, and 100 mg/liter yeast transfer RNA. Hybridization was
performed at 66 C in the same solution after addition of the
32P-labeled probe and was continued for an additional
16 h. In the case of the cDNA probes SF-1 and human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), prehybridization and
hybridization were carried out at 42 C. The membranes were washed twice
at room temperature for 20 min each time with 2 x SSC containing
0.1% SDS, followed by 2 h at 66 C with 0.1 x SSC and 0.1%
SDS until removal of the background counts. Membranes were then exposed
to x-ray films (Kodak XAR-5) for 36–48 h at -80 C. To normalize the
variation in StAR and SF-1 mRNA levels, the membranes were subjected to
rehybridization with a cDNA probe of GAPDH. The relative mRNA levels of
StAR in the different transcripts and SF-1 and GAPDH expression were
quantitated as described above.
Determination of
45Ca2+ uptake
Radiolabeled calcium was used to determine the dependency of the
hCG effects on extracellular Ca2+, as described previously
(45) with minor modifications. Briefly, before stimulation, the cells
were washed twice with 0.01 mol/liter PBS. At time zero,
45Ca2+ (4 µCi/well) was added in
Ca2+-free medium (final concentration of Ca2+,
1.5 mmol/liter) to each well for 2 h at 37 C in the absence or
presence of hCG, K+, and hCG or K+ in the
presence of verapamil (10 µmol/liter). Two hours later, the cells
were washed, collected in 0.5 ml 0.1 N NaOH, vigorously
mixed with the scintillation liquid, and finally measured for
radioactivity by liquid scintillation spectrometer (1215 RACKBETA II,
Wallac Oy, Turku, Finland).
Determination of intracellular
[Ca2+]i
The [Ca2+]i was monitored in mLTC-1
cells with the fluorescent probe for Ca2+, fura-2/AM. After
recovery from trypsinization shock, the cells were washed and incubated
at a concentration of 1 x 106/ml in
Ca2+-free medium containing 4 µmol/liter fura-2/AM for 45
min at 37 C. The unincorporated fura-2/AM was washed away after 45 min,
and the cells were sedimented and resuspended in Ca2+-free
medium. The fluorescence intensity of the fura-2/AM was determined in a
Hitachi F 2000 spectrophotometer (Tokyo, Japan) by employing excitation
at 340 nm (5-nm bandpass) and emission wavelength at 505 nm. The
effects of digitonin (50 µmol/liter) and EGTA (4 mmol/liter) were
carried out for maximum and minimum fluorescence intensities.
Statistical analysis
The data were analyzed by one-way ANOVA, followed by Fisher’s
least significant difference tests, using the StatView 4.5 program
(Abacus Concepts, Inc., Berkeley, CA) fitted for the Macintosh
computer. The results were expressed as the mean ±
SEM, and P < 0.05 was considered
statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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show
that increasing concentrations of hCG (0–1000 µg/liter)
significantly elevated the StAR mRNA levels in a
concentration-dependent manner, with a maximum 2.8 ± 0.4-fold
increase at 4 h, in Ca2+-free medium. Significant
changes in StAR message were detected at concentrations of 1 µg/liter
hCG or more, with half-maximal stimulation at 22 µg/liter and a
maximum increase at 50 µg/liter or more. Interestingly, an increase
in the extracellular Ca2+ concentration to 1.5 mmol/liter
in the presence of hCG markedly augmented (1.7 ± 0.3-fold) the
hCG-stimulated StAR mRNA levels, indicating that there is
a component dependent on Ca2+ in hCG action. A 4-h
stimulation with hCG exhibited a quantitatively similar dose-dependent
increase in P production at doses of 1 µg/liter or more, with maximum
stimulation of 5.6 ± 0.4 over the value in nonstimulated cells.
Likewise, the presence of 1.5 mmol/liter Ca2+ additively
increased the hCG effect (P < 0.0001) on P production,
resulting in an approximately 2-fold higher response than with
saturating dose of hCG (50 µg/liter) alone (Fig. 1C). Cells
stimulated with varying concentrations of Ca2+ (0.1–4
mmol/liter) and a fixed concentration of hCG (50 µg/liter) displayed
a dose response to Ca2+ in P production, reaching a maximum
at 1–2 mmol/liter of Ca2+, whereas the basal level of P
was not altered (data not shown). The stimulatory effect of
Ca2+ on hCG-induced P production was completely abolished
(P < 0.0001) by the Ca2+-chelating agents
EGTA and EDTA (4 mmol/liter each; Fig. 2). The diminished effect on P production
was correlated with the concentration of Ca2+ in the
extracellular medium, further suggesting the requirement for
extracellular Ca2+ in potentiation of hCG action (Fig. 2).
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Effect of Ca2+ channel blocker verapamil on
hCG-induced StAR mRNA expression
To corroborate the observed effect of extracellular
Ca2+, we next examined the effect of verapamil, a
voltage-sensitive transmembrane Ca2+ channel blocker. The
results summarized in Fig. 3 demonstrate
that this substance, when added at concentrations of 0.1–100
µmol/liter to the hCG and Ca2+ incubations inhibited StAR
mRNA expression in a dose-dependent fashion, indicating the importance
of extracellular Ca2+ mobilization through the
transmembrane channels for the modulation of hCG action. The
ED50 of the verapamil effect was 4 µmol/liter (Fig. 3).
The blocking of extracellular Ca2+ entry on hCG-mediated P
production followed a qualitatively similar pattern as that observed
for StAR mRNA expression (data not shown). These data imply that
Ca2+ entry from the extracellular space is responsible for
modulation of the hCG action on StAR mRNA expression and
steroidogenesis.
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illustrates that interaction of 4
mmol/liter K+ with 50 µg/liter hCG clearly enhances StAR
mRNA expression and P production. The augmenting effect of
K+ on hCG-induced responses was significantly diminished
(P < 0.0001) by 10 µmol/liter verapamil or 4
mmol/liter EGTA (Fig. 4, A–C). The interaction of K+ was
progressively Ca2+ dependent, as removal of the latter from
the extracellular medium significantly prevented the K+
effect (Fig. 4). These data demonstrate the direct correlation of
hCG-stimulated StAR expression and steroidogenesis with extracellular
Ca2+.
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, the increase in [Ca2+]i by A 23187 (1
µmol/liter) was followed by a significant increase (P
< 0.01) in StAR mRNA expression. Addition of A 23187 to hCG-stimulated
cells augmented StAR mRNA expression about 1.5-fold in an additive
manner, whereas EGTA or verapamil prevented the A 23187 action.
Involvement of A 23187 produced a modest, but consistent, elevation in
P production (data not shown).
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).
Introduction of K+ (4 mmol/liter) into this system also
significantly (P < 0.001) increased
45Ca2+ uptake, which was further responsive in
the presence of hCG (data not shown). The addition of verapamil (10
µmol/liter) strongly diminished the actions of hCG and K+
on 45Ca2+ uptake. The results of this study
were further confirmed by determining [Ca2+]i
in fura-2/AM-loaded cells in response to hCG. It can be seen from Fig. 6B that initially there was no change in
[Ca2+]i in the fura-2/AM-loaded cells, which
provide the basal level of [Ca2+]i, an
average of 215 ± 18 nmol/liter. An increase of 10–14% in
[Ca2+]i occurred after hCG addition; this
reached a plateau within 1–3 min and was sustained for at least 10
min. Addition of digitonin (50 µmol/liter) or EGTA (4 mmol/liter)
caused marked responses of maximum and minimum
[Ca2+]i, respectively. Under the present
experimental conditions, cells were maintained outside the
spectrophotometer and were found to be viable under microscopic
observation. These data imply that [Ca2+]i
increases during hCG stimulation due to mobilization of extracellular
Ca2+.
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, anti-StAR protein antibody revealed a
2.5 ± 0.3-fold increase in a 30-kDa immunoreactive protein in
response to hCG (50 µg/liter) stimulation by 4 h. Interestingly,
extracellular Ca2+ significantly (P <
0.0001) modulated the hCG-induced StAR protein content (1.6 ±
0.2-fold), whereas CHX (10 mg/liter) markedly prevented the stimulatory
action of hCG on StAR protein expression (Fig. 7, A and B). Addition of
EGTA to this incubation reduced the Ca2+-specific increase
in hCG. Interaction of K+ (4 mmol/liter) in hCG-induced
StAR protein expression was qualitatively similar (Fig. 7, C and
D).
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, the full-length mouse StAR hybridized
with four transcripts at 3.4, 2.7, 1.6, and 1.4 kb, among which the
1.6-kb band encodes the functional StAR protein (17). Inclusion of
Ca2+ in the incubation medium resulted in 2- to 2.5-fold
induction of StAR gene expression over hCG (50 µg/liter) stimulation,
whereas Ca2+ alone failed to show an effect on basal StAR
mRNA expression. The minor and inconsistent band at 1.4 kb was detected
with longer exposure of the membranes, and it is possibly a splice
variant of the StAR gene. Addition of EGTA (4 mmol/liter) and verapamil
(10 µmol/liter) to this system markedly (P < 0.0001)
suppressed the StAR transcript levels. Involvement of Ca2+
in the hCG-mediated action coordinately increased (3- to 5-fold) all
StAR transcripts.
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). Addition of Ca2+ to hCG
stimulation further augmented (P < 0.0001) SF-1 mRNA
expression. The efficiency of transfection achieved in the experiments
was verified with a constant amount of ß-galactosidase message.
Removal of Ca2+ from the extracellular medium by EGTA (4
mmol/liter) or blocking of the transmembrane Ca2+ entry by
verapamil (10 µmol/liter) diminished the Ca2+-dependent
increase in hCG on SF-1 expression, suggesting the involvement of SF-1
in the Ca2+-mediated hCG action.
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). Expression of SF-1 in these cells
moderately increased StAR mRNA levels by 4 h, but markedly
augmented the hCG-induced increase (2.0 ± 0.2-fold) in StAR
expression compared with that in mock-transfected cells. The hCG
response was further responsive to Ca2+ addition (Fig. 11). In contrast, the magnitude of the
SF-1-mediated hCG response of StAR mRNA expression was remarkably
diminished (P < 0.0001) by concomitant DAX-1
overexpression, indicating a role for SF-1 as an essential regulator of
hCG-induced StAR gene expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role of Ca2+ as a second messenger of hormone action has been implicated in diverse cellular functions along with gonadal steroidogenesis (3, 6, 23). In Leydig cells, the steroid hormone biosynthesis is regulated by LH/hCG through activation of multiple second messenger systems (4, 8). The present experiments were designed to elucidate the precise involvement of extracellular Ca2+ in the hCG-stimulated StAR gene expression and steroidogenesis in mLTC-1 mouse Leydig tumor cells. Our data allowed us to conclude that the functional response of hCG involved in steroidogenesis is not dependent of extracellular Ca2+, whereas the addition of Ca2+ markedly enhanced hCG activity. The increase in [Ca2+]i from extracellular space after hCG stimulation is responsible for inducing SF-1-dependent StAR expression that is intimately associated with increased steroidogenesis. Collectively, these results provide evidence for the molecular involvement of the Ca2+ messenger system in hCG-induced StAR expression and steroidogenesis in mouse Leydig cells.
Multiple levels of regulation impinge on StAR action, i.e. the PKA- and PKC-dependent pathways, the Ca2+ messenger systems, as well as other factors and processes that are responsible for the mobilization of cholesterol from the outer to the inner mitochondrial membrane. Recently, it has been demonstrated that phosphorylation of the serine residue at codon 194/195 of the StAR protein modulates the functional activity of the StAR intimately associated with steroidogenesis (52).
Regulation of StAR expression and aldosterone synthesis have also been demonstrated by the Ca2+-mobilizing agonists Ang-II and K+ in adrenal glomerulosa cells (19, 22, 23). It is noteworthy that in H295R adrenocortical cells, the inhibition of Ang-II and K+ functions markedly prevented aldosterone synthesis without altering the induction of StAR expression (19, 32). Our results demonstrate that extracellular Ca2+ significantly enhanced the stimulatory action of hCG on StAR mRNA and protein levels together with P production, without altering their basal levels. The stimulatory effect of hCG on StAR expression and steroidogenesis is independent of extracellular Ca2+, whereas the addition of Ca2+ potentially augments this action. In accordance, experiments with Ca2+ inhibitors exhibited modest, but consistent, attenuation of the hCG-induced responses, suggesting that [Ca2+]i could be effectively affected by them. The increases in [Ca2+]i and 45Ca2+ uptake were primarily dependent on hCG action, as blockade of the transmembrane Ca2+ passage by verapamil reduced the modulatory action of hCG. The concentration of verapamil (10 µmol/liter) used in subsequent experiments showed a specific antagonistic effect without nonspecific inhibition, as determined by its involvement in basal P production during a 4-h incubation period (data not shown). These observations support the contention of a critical role of Ca2+ in potentiating LH/hCG action involved in steroidogenesis (4, 9).
With respect to the potential interaction with Ca2+ in StAR expression, the studies on K+ deserve a note. K+ has been regarded as a Ca2+-mobilizing agent in adrenal cells through opening of T- and L-type transmembrane Ca2+ channels that are progressively involved in steroid hormone biosynthesis (25, 27). Our findings in mLTC-1 cells demonstrate that K+ markedly increased the levels of hCG-stimulated StAR mRNA and protein, in close coordination with acute P production. Furthermore, the inducible effect of K+ on StAR expression was drastically suppressed when Ca2+-chelating agents or a specific Ca2+ channel blocker were included in the extracellular medium. Both agents significantly diminished the stimulatory actions of Ca2+ and K+, strongly suggesting the requirement of the extracellular Ca2+ for modulation of the hCG response. It is worth mentioning that neither Ca2+ nor K+ alone had a noticeable effect on StAR expression and P production, whereas they only potentiated the hCG action. This is expected, because the activation of PKC by phorbol ester (12-O-tetradecanoylphorbol 13-acetate) has been demonstrated to induce StAR expression without affecting steroidogenesis in MA-10 mouse Leydig tumor cells (32). The results of our study clearly show that the interaction of K+ is highly Ca2+ dependent and apparently facilitates the increase in [Ca2+]i upon hCG stimulation that is responsible for StAR expression and acute P production.
The receptor binding of LH/hCG results in activation of the adenylate cyclase and phospholipase C signal transduction cascades (53, 54, 55). The steroidogenesis in Leydig cells is controlled by blood-borne endocrine influences, mainly from the pituitary gland, as well as by intratesticular autocrine and paracrine impulses (56). As LH/hCG plays a key role in the stimulation of Leydig cell steroidogenesis, a pertinent question is whether hCG is capable of increasing [Ca2+]i, which then could amplify the gonadotropin effect on StAR expression. In adrenal glomerulosa cells, ionomycin-mediated calcium clamping has recently been shown to modulate StAR protein expression (22). Our findings document that the addition of hCG to fura-2/AM-loaded mLTC-1 cells exhibited a moderate elevation in [Ca2+]i within 30–40 sec, with a plateau between 1–3 min. The marginal increase in [Ca2+]i after hCG stimulation is possibly due to inefficient coupling of the LH receptor to phospholipase C, which is consistent with previous findings (7). In addition, the involvement of A 23187, primarily altering [Ca2+]i, clearly augmented StAR gene expression in an additive fashion with hCG. It is important to note that hCG was unable to stimulate the inositol phosphate pathway in MA-10 mouse Leydig tumor cells (57). In contrast, in rat Leydig cells, hCG markedly increased [Ca2+]i after a lag period of 2–3 min, and this was sustained for at least 15 min (9). It has also been shown in these cells that K+ clearly enhanced [Ca2+]i through the voltage-sensitive Ca2+ channels in the plasma membrane. The inclusion of digitonin and EGTA in this experimental paradigm represented maximum and minimum levels of [Ca2+]i, respectively, suggesting a physiological response of the cellular machinery. Tomic et al. (7) recently demonstrated that in rat Leydig cells, LH transiently mobilizes functional Ca2+ in an all or none manner. Our observations clearly show that the change in [Ca2+]i upon hCG stimulation, originating from extracellular space, is responsible in inducing the hCG function involved in StAR expression.
The predicted role of StAR in steroidogenesis prompted us to examine
the potential involvement of the orphan nuclear receptors, SF-1 and
DAX-1. Recent studies demonstrated that these factors play a key role
in the regulation of endocrine functions during development, at
multiple levels of reproductive functions, and in the expression of
steroidogenic enzymes (58, 59). In addition, DAX-1 binds to DNA hairpin
structures and diminishes the transcriptional activity of SF-1 and StAR
in adrenocortical cells (39, 60). Of great significance is the finding
that SF-1 knockout mice present with a complete lack of adrenal glands
and gonads, which is pertinent to the role of SF-1 as a key regulator
in adrenal and gonadal developmental function (61). Our findings
reinforce the idea that inhibition of endogenous SF-1 expression by
DAX-1 remarkably attenuated StAR gene expression and P production,
consistent with the previous findings. Recently, it has also been
demonstrated that the expression of SF-1 was not impaired in Y1 cells
that overexpress DAX-1 (62). The omission of Ca2+ from the
extracellular medium or blocking of transmembrane Ca2+
uptake in relation to hCG resulted in a marked inhibition of
Ca2+-mediated increase involved in SF-1 mRNA expression.
The involvement of Ca2+ progressively modulated the
hCG-induced SF-1 expression that consequently influences the following
regulatory cascade of steroidogenesis, i.e. extracellular
Ca2+
hCG[Ca2+]iSF-1StARacute
steroidogenesis.
Collectively, the present data suggest that extracellular Ca2+ provides the source of [Ca2+]i, subsequently augmenting the hCG activity, and is involved in the regulation of StAR gene expression and P production. The interaction of Ca2+ with hCG presumably increases the abundance of StAR expression, primarily through elevations in transcription of the StAR gene. These findings further confirm and extend the idea of a crucial involvement of SF-1 and Ca2+ in the hCG-mediated StAR expression and the acute regulation of steroid hormone biosynthesis.
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Acknowledgments |
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Received October 1, 1998.
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