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Department of Biochemistry and Molecular Biology, Royal Free Hospital School of Medicine, London, United Kingdom NW3 2PF; and the Department of Cell Biology and Biochemistry, School of Medicine, Texas Tech University Health Sciences Center (D.M.S.), Lubbock, Texas 79430
Address all correspondence and requests for reprints to: Prof. B. A. Cooke, Department of Biochemistry and Molecular Biology, Royal Free Hospital School of Medicine, Rowland Hill Street, London, United Kingdom NW3 2PF. E-mail: bacooke{at}rfhsm.ac.uk
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using patch-clamping techniques, Joffre and co-workers demonstrated that chloride and potassium channels are present in mature rat Leydig cells (4). After stimulation of Leydig cells with hCG, LH, or cAMP, it was found that although there was little effect on potassium conductance, all of these ligands increased chloride conductance. These effects were independent of extracellular calcium, but were blocked by the chloride channel blocker 4-acetamido-4'-isothiocyanostilbene-disulfonic acid. They concluded from further studies (5) that a hyperpolarization-activated chloride conductance in the plasma membrane of Leydig cells can be modulated by cAMP. This nucleotide acts by modifying the kinetics of the inward current and both the kinetics and amplitude of deactivating currents. Therefore, the membrane depolarization that occurs when Leydig cells are exposed to LH/hCG can be explained by an increase in cAMP-mediated chloride conductance, allowing the chloride ions to exit the cell. Recently, Mattioli et al. demonstrated that activation of PKA and PKC mediates the depolarizing effect of LH in ovine cumulus-corona cells (6).
In initial studies of the potential role of chloride ions in steroidogenesis, chloride in the buffer medium was replaced with equimolar concentrations of a membrane-impermeant salt, gluconate. It was found that the exclusion of extracellular chloride markedly enhanced steroidogenesis in rat Leydig cells stimulated with a submaximal, but not maximal, concentration of LH (7). LH-stimulated steroidogenesis was inhibited by the anion channel blocker 4-acetamido-4'-isothiocyanostilbene-disulfonic acid. Thus, the biophysical and biochemical results suggest that chloride efflux from Leydig cells may be regulated by LH/cAMP and that this may be an important regulatory mechanism in the control of steroidogenesis.
The physical rate-limiting step in steroidogenesis is the transfer of cholesterol into the inner mitochondrial membrane, where cholesterol side-chain cleavage takes place. It has been well established (8) that cycloheximide, an inhibitor of protein synthesis, blocks cAMP-mediated cholesterol transfer to the inner mitochondrial membrane and that the acute regulation of steroidogenesis requires de novo protein synthesis. It has been suggested that the role of a newly synthesized regulatory protein could facilitate the translocation of cholesterol across the intermembrane space to the inner mitochondrial membrane. To date, there are several candidates for the acute regulatory protein (8), but one protein appears to fulfill all of the necessary criteria. This protein, which is a mitochondrially localized phosphoprotein, has recently been cloned and sequenced, and is named the steroidogenic acute regulatory protein (StAR) (9).
The aim of the present study, therefore, was to investigate the mechanisms involved in the modulation of steroidogenesis by chloride exclusion. We used the MA-10 mouse Leydig tumor cell line as a model and investigated the effects of calcium exclusion, inhibitors of protein synthesis, and time, on (Bu)2cAMP-stimulated steroidogenesis in cells incubated in chloride-free and chloride-replete medium. We also investigated the effects of chloride omission on total protein synthesis as well as the activities of 3ß-hydroxysteroid dehydrogenase (3ßHSD) and cytochrome P450 cholesterol side-chain cleavage enzyme (cytP450scc). In addition, we determined StAR, 3ßHSD, and cytP450scc protein levels by quantitative Western blotting in mitochondria isolated from cells incubated in chloride-free and chloride-replete media.
Preliminary communications of this work have been presented (10, 11, 12).
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Materials and Methods |
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Incubation media
Chloride-replete buffer had the following composition: 140
mM sodium chloride, 5 mM potassium chloride, 1
mM magnesium sulfate, 1.8 mM calcium acetate,
10 mM glucose, 10 mM HEPES, and 0.1% (wt/v)
BSA. Chloride-free buffer was prepared identically, but the chloride
salts of sodium and potassium were replaced with equimolar gluconate
salts of sodium and potassium. Calcium-free buffers were prepared in a
similar way, but with the omission of calcium acetate and the addition
of 1 mM EGTA. Calcium ions in the Waymouth’s medium were
also chelated with 1 mM EGTA. All buffers were adjusted to
pH 7.4 and had osmolarities ranging from 290–300 mosmol/liter.
Cell cultures
MA-10 cells (provided by Dr. Mario Ascoli, University of Iowa,
Iowa City, IA) were maintained in Waymouth’s medium supplemented with
15% (vol/vol) horse serum and 0.1% (vol/vol) gentamicin. Cells were
cultured at 37 C under 5% CO2 in air. When the cells were
confluent, the medium was removed, and the cells were washed twice with
15 ml PBS. They were then incubated with 4 ml 0.05% (wt/vol)
trypsin-EDTA to remove the cells from the culture dishes, followed by
40 ml Waymouth’s medium containing horse serum and gentamicin. Cells
were centrifuged at 250 x g at ambient temperature for
5 min and then resuspended in 1 ml Waymouth’s medium [containing
0.1% (wt/vol) BSA]. The numbers of viable cells were counted in the
presence of 0.4% (wt/vol) trypan blue and were plated in
75-cm2 flasks (12 x 106 cells; 20
ml/flask), 6-well plates (2 x 106 cells; 5 ml/well),
24-well plates (100,000 cells; 1 ml/well), or 96-well plates (20,000
cells; 0.25 ml/well), as indicated below. They were then cultured for
48 h.
Spent culture medium was removed from the cells and replaced with modified salts buffer [containing 0.1% (wt/vol) BSA] or Waymouth’s medium [containing 0.1% (wt/vol) BSA; as indicated in the text], and the cells were preincubated for 2 h. The media were then replaced with identical fresh buffers for a further 2–6 h in the presence and absence of (Bu)2cAMP, after which the media were acidified with 107 mM perchloric acid, and the cells and media were frozen at -20 C until assayed for progesterone. The latter was carried out by RIA (13) on the thawed medium after neutralization with 154 mM K3PO4 without extraction. Similar procedures were used for the incubations in flasks, except the media were removed for RIAs, and the cells were used for the preparation of mitochondria.
Viability of the cells
Cell viability was assayed as previously described (14). It was
found that there was no difference in the viability of the cells
incubated for 4 h in chloride-free, chloride-replete, or
Waymouth’s medium when assayed by trypan blue exclusion (98% viable)
or diaphorase histochemistry (85–90% viable).
Isolation of mitochondria
The cells were washed twice with PBS; harvested in 0.25
M sucrose, 10 mM Tris (pH 7.4), and 0.1
mM EDTA by scraping with a rubber policeman; and
homogenized. Mitochondria were isolated as described by Clark et
al. (9). The mitochondrial pellet was lyophilized before
analysis.
Western analysis
The levels of StAR present in isolated mitochondria were
quantified by Western blot analysis after separation by PAGE as
previously described (9). 3ßHSD and cytP450scc protein
levels (15) were detected on the same blots after stripping and probing
with specific antibodies to these proteins. The specific bands were
quantified using a BioImage Visage 2000 imaging system after correction
of the samples for protein loading differences and were expressed as
corrected integrated intensity, as previously described (9).
3ßHSD activity
Cells were cultured in 24-well plates in chloride-free and
chloride-replete media for 4 h. The media were then removed, and 1
µCi [3H]pregnenolone and 10 µM
pregnenolone in a total of 1 ml of either chloride-replete or
chloride-free buffer were added and incubated for 30 min. The steroids
were then extracted with chloroform (at 4 C), evaporated, reconstituted
in 50 µl ethyl acetate (containing 10 mM progesterone),
and separated by TLC using chloroform-ethanol (92:8, vol/vol). The
amounts of [3H]pregnenolone and
[3H]progesterone were quantified by cutting and scraping
the respective bands from the TLC plates, followed by scintillation
counting. 3ßHSD activities were quantified as the rate of oxidation
of pregnenolone to progesterone over unit time, corrected for the
nonenzymatic rate of pregnenolone oxidation, measured under identical
conditions in the absence of cells.
CytP450scc activity
Cells were cultured in 96-well plates in chloride-free and
chloride-replete media. They were incubated with cyanoketone (5
µM) and SU10603 (20
µM), inhibitors of 3ßHSD and
17
-hydroxylase, respectively, for 30 min.
22R-Hydroxycholesterol (50 µM) was then added
in ethanol (final concentration, 2%), and the cells were incubated for
an additional 2 h. Pregnenolone was assayed by RIA by the same
procedure as that used for progesterone.
Protein synthesis
Cells were incubated in six-well plates with chloride-replete,
chloride-free, or Waymouth’s medium containing 10 mM
methionine for 2 h. The media were removed, and the cells were
washed with the appropriate buffer (methionine-free) and then incubated
for 4 h with [3H]methionine (1 µCi/well). The
media were then removed, and the cells were washed twice in buffer
containing 10 mM unlabeled methionine and scraped in 1 ml
PBS, pH 7.4, containing 1% (wt/vol) BSA and 10 mM
unlabeled methionine. They were then homogenized. Triplicate samples
were added to paper filters (prewashed with PBS/BSA/methionine buffer),
precipitated by the addition of TCA (1 ml; 10%, wt/vol), and then
filtered and washed (twice) under vacuum. Radioactivity was estimated
by scintillation counting.
Statistics
Quadruplicate incubations were carried out for each treatment in
each experiment (except those using flasks, which were single
cultures), and all experiments were repeated at least three times.
Because the MA-10 cells were sometimes obtained from different
passages, their capacities to produce progesterone varied. When this
occurred, a typical result is given, but the statistical analysis
(ANOVA) and Student’s t test were carried out in all
experiments. P < 0.05 in a one-tailed t
test were accepted as statistically significant. The one tailed
t test was applied on the basis that our previous studies
had consistently demonstrated a positive effect of chloride exclusion
on progesterone production.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Table 1). These experiments
were carried out by substituting equimolar concentrations of
sodium/potassium gluconate in place of sodium/potassium chloride. It
was found that the potentiating effects of chloride omission were also
obtained if another substituting ion (glutamate) was used (Table 2), thus indicating that this is an effect of chloride
omission and not of the substituting ion.
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. Although
chloride omission in the calcium-free medium did cause an increase in
progesterone production, it was less marked than that in the presence
of calcium. Also, it was only statistically significant in all three
experiments with 0.05 and 0.25 mM
(Bu)2cAMP.
Kinetics of the effect of chloride omission on basal and
submaximally stimulated steroidogenesis
The removal of chloride resulted in 1.7- and 2.4-fold increases in
basal progesterone production after 2 and 6 h of incubation,
respectively (n = 3 experiments; P < 0.001).
Progesterone production in the first 1 h of incubation with 0.1
mM (Bu)2cAMP did not differ in the
cells incubated in the chloride-replete and chloride-free buffers (Fig. 1B). This was followed by a progressive difference in progesterone
production, which by 5 h had increased to 24-fold. In three
separate experiments, the mean increases in progesterone production
were 4- and 15-fold in chloride-free buffer compared with that in
chloride-replete buffer after 2 and 6 h of incubation,
respectively.
Effects of cycloheximide
The effects of the protein synthesis inhibitor, cycloheximide, on
progesterone production in the presence of Waymouth’s medium and
chloride-replete and chloride-free buffers were investigated in MA-10
cells incubated for 2 h (Fig. 2A) and 6 h
(Fig. 2B) in the presence and absence of 0.1 mM
(Bu)2cAMP. At both times, basal production of
progesterone was increased by the omission of chloride. Cycloheximide
had no effect on basal progesterone production in the presence or
absence of chloride over 2 h. However, over 6 h, the addition
of cycloheximide increased basal progesterone production from 7 ±
1 to 187 ± 27, from 13 ± 2 to 300 ± 50, and from
8 ± 2 to 63 ± 10 ng/106 cells (mean ±
SEM; n = 3 experiments) in chloride-replete,
chloride-free, and Waymouth’s media, respectively. In the absence of
cycloheximide, (Bu)2cAMP increased progesterone
production in chloride-replete and Waymouth’s media after 2 and 6
h by similar amounts, and this response was enhanced when chloride was
omitted. The addition of cycloheximide prevented the response to
(Bu)2cAMP in all media during 2-h incubation, and
after 6 h, the levels of progesterone in the presence of
(Bu)2cAMP were below the corresponding basal
levels.
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). No effects of chloride removal on protein synthesis
were found in the absence of (Bu)2cAMP. In the
chloride-replete medium, addition of 0.1 mM
(Bu)2cAMP had no significant effect on protein
synthesis. Likewise, 1 mM (Bu)2cAMP
did not stimulate [3H]methionine incorporation in the
cells incubated in Waymouth’s medium. Indeed, the only response to
(Bu)2cAMP was obtained in the absence of
chloride. The incorporation of [3H]methionine into
protein in the control cells was 10–40 x 103
dpm/106 cells·4 h.
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). Basal levels of the StAR protein were undetectable
in the cells incubated in chloride-free and chloride-replete media. In
two of the experiments, the amounts of StAR protein were measured in
mitochondria from cells incubated in Waymouth’s medium and stimulated
with 1.0 mM (Bu)2cAMP; it was found
that the level of StAR protein was 12 ± 6-fold (mean ±
range) higher than that in cells incubated in chloride-replete medium
with 0.1 mM (Bu)2cAMP.
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Progesterone production in these experiments was 120 ± 26 and 1385 ± 306 ng/106 cells·6 h, respectively (mean of three experiments ± SEM) with (Bu)2cAMP (0.1 mM) in chloride-replete medium compared with chloride-free medium, respectively. With 1 mM (Bu)2cAMP in Waymouth’s medium, progesterone production was 1375 ± 264 ng/106·6 h (mean of two experiments ± range).
Effects on 3ßHSD and cytP450sccactivities
MA-10 cells were incubated in the chloride-free and
chloride-replete media for a total of 4 h. 3ßHSD activity was
then assayed by measuring the conversion of
[3H]pregnenolone to [3H]progesterone
using a fixed incubation of 30 min. The latter was carried out in both
chloride-free and chloride-replete media. No effect of chloride
omission was found (Table 3). When 0.1 mM
(Bu)2cAMP was added during the incubation, there
was also no effect of the chloride-free buffer.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously it was demonstrated, using electrophysiological techniques, that an increase in intracellular calcium concentrations (from 10-7 to 10-6 M) activated an outward chloride current in rat Leydig cells (17). In addition, chloride channels are linked to calcium channels in some cells and are regulated by phosphorylation via PKA or PKC (2). In the present study we, therefore, investigated the potential relationship between these two anions by manipulation of extracellular chloride and calcium. It was found that when the extracellular calcium was omitted, there was a marked decrease in (Bu)2cAMP-stimulated progesterone production in both the presence and absence of chloride ions. A potentiating effect of extracellular chloride exclusion on (Bu)2cAMP-stimulated progesterone production was still found; however, the effects were less marked compared with the potentiation in calcium-replete medium. This indicates a complex relationship between calcium and chloride ions, which requires further investigation. The decreased steroidogenic capacity of the cells in the absence of calcium is in agreement with previous studies (18, 19, 20).
The above results were obtained during 2-h incubations. In longer incubations in which the cells were stimulated with 0.1 mM (Bu)2cAMP, it was found that the enhancing effect of chloride exclusion was maintained and increased continuously for up to 6 h. A 15-fold difference was obtained compared to the effect of the simple salt chloride-replete medium. Chloride removal also increased the basal production approximately 2-fold. However, to obtain a large effect with chloride ion removal, cAMP is required.
In the present study it was shown that suppression of protein translation by cycloheximide inhibited (Bu)2cAMP-stimulated steroidogenesis in both the presence and absence of chloride. This indicates that new protein synthesis is required for the effects of chloride omission on (Bu)2cAMP-stimulated steroidogenesis. During short term culture of steroidogenic cells, it is well established (and confirmed in the present study) that basal steroidogenesis is not inhibited in the presence of cycloheximide. Basal progesterone stimulated by the omission of chloride was also not inhibited by cycloheximide. This indicates that a non-cAMP-dependent pathway that is independent of protein synthesis can be stimulated by the omission of chloride. It is of interest that during 6-h incubation the basal, but not (Bu)2cAMP-stimulated, progesterone levels increased in the presence of cycloheximide. This may indicate the presence of a protein that chronically inhibits basal steroidogenesis.
An approximately 4-fold increase in overall protein synthesis was observed in chloride-depleted, (Bu)2cAMP-stimulated cells. This was measured by determining the incorporation of [3H]methionine into protein. It is possible that this increase may reflect an increase in the specific activity of the intracellular methionine pools due to an alteration in the permeability of the plasma membrane to amino acids. However, this is thought to be unlikely, because the mass of the StAR protein was also seen to increase 4-fold.
Several mechanisms for the increases in the levels of the StAR protein are possible. In addition to the cAMP-induced depolarization of the plasma membrane, mitochondrial membranes may also become depolarized by the efflux of chloride from the cells. It has been proposed that the action of StAR facilitates the transport of cholesterol from the outer to the inner mitochondrial membrane and that this involves the formation of contact points between these membranes (8). It is possible that depolarization of the outer and inner mitochondrial membranes will facilitate the formation of these contact points and, hence, the action of StAR. Another possible mechanism by which chloride depletion exerts its effects is by increased synthesis of the StAR protein. Although the exact mechanisms involved in the cAMP-induced synthesis of StAR are unknown, it has been demonstrated recently that expression of StAR can be regulated by cAMP through steroidogenic factor-1 sites in the promotor region of the StAR DNA (21). There is a requirement for phosphorylation on a threonine residue in the action of cAMP on steroidogenesis (8). This indicates that phosphoproteins in addition to StAR may be required for the steroidogenic response to (Bu)2cAMP, as the consensus phosphorylation sites in the StAR protein are serines and not threonines. It is, therefore, possible that chloride exclusion causes an increase in cAMP-mediated phosphorylation of proteins, followed by a general increase in protein synthesis. This is supported by the cAMP-dependent increase in protein synthesis in chloride-depleted cells found in the present study. In this context, it is important to note that blockage of chloride channels inhibits tyrosine kinase-mediated phosphorylation of proteins in T lymphocytes (3).
Our data indicate that chloride exclusion does not enhance 3ßHSD or cytP450scc activities or protein levels; thus, changes in these enzymes are not involved in the enhancement of steroidogenesis by chloride exclusion. The lack of effect on the protein levels of these enzymes is not surprising because the turnover of these enzymes is slow, being of the order of tens of hours, and the present studies were carried out with cells incubated for only 6 h.
In conclusion, the present studies indicate that chloride exclusion from the incubation medium of MA-10 cells enhances both basal and cAMP-mediated stimulation of steroidogenesis via the increased synthesis and/or action of the StAR protein. Further studies, particularly at the level of phosphorylation of proteins involved in the control of steroidogenesis, are required to elucidate the mechanisms involved.
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Acknowledgments |
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Footnotes |
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Received November 11, 1996.
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) and chloride-free (•) media
containing different concentrations of (Bu)2cAMP
as indicated, and incubations were continued for an additional 2
h. In B, the cells were incubated in chloride-replete and chloride-free
buffers for 2 h, and then the media were replaced by fresh
chloride-replete (
