From the Commissariat à l'Energie Atomique, Département de Biologie Moléculaire et Structurale, Biochìmìe des Régulations Cellulaires Endocrines, INSERM Unité 244, 17 rue des Martyrs, F-38054 Grenoble, France and the § Division of Endocrinology and Diabetology, Department of Internal Medicine, Faculty of Medicine, University Hospital, CH-1211 Geneva 14, Switzerland
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ABSTRACT |
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Transforming growth factor-s (TGF-
s)
constitute a family of dimeric proteins that affect growth and
differentiation of many cell types. TGF-
1 has also
been proposed to be an autocrine regulator of adrenocortical
steroidogenesis, acting mainly by decreasing the expression of
cytochrome P450c17. Here, we demonstrate that TGF-
1 has
a second target in bovine adrenocortical cells, namely the
steroidogenic acute regulatory protein (StAR). Indeed, supplying cells
with steroid precursors revealed that TGF-
1 inhibited
two steps in the steroid synthesis pathway, one prior to pregnenolone production and another corresponding to P450c17. More specifically, TGF-
1 inhibited pregnenolone production but neither the
conversion of 25-hydroxycholesterol to pregnenolone nor P450scc
activity. Thus, TGF-
1 must decrease the cholesterol
supply to P450scc. We therefore examined the effect of
TGF-
1 on the expression of StAR, a mitochondrial protein
implicated in intramitochondrial cholesterol transport.
TGF-
1 decreased the steady state level of StAR mRNA
in a time- and concentration-dependent manner. This inhibition occurs at the level of StAR transcription and depends on RNA
and protein synthesis. It is likely that the
TGF-
1-induced decrease of StAR expression that we report
here may be expanded to other steroidogenic cells in which a decrease
of cholesterol accessibility to P450scc by TGF-
1 has
been hypothesized.
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INTRODUCTION |
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Although initially identified by its ability to induce reversible
phenotypic transformation of non-neoplastic cells by stimulating anchorage-independent cell growth,
TGF-11 is now
recognized as a physiological mediator of growth and differentiation of
various cell types (1). Its major effects are on the cell cycle in
epithelial cells and on the synthesis of extracellular matrix proteins
in most cell types. TGF-
1 has also been identified as a
potential physiological autocrine/paracrine regulator of bovine
adrenocortical steroidogenic functions (2, 3). Our laboratory has shown
that bovine adrenal cortical (BAC) cells from the fasciculata zone
express specific high affinity TGF-
1 receptors (4) and
produce and secrete TGF-
1 under a latent form (5, 6).
Immunolocalization of TGF-
1 in the bovine or rat adrenal
gland revealed its presence in the glomerulosa and fasciculata zones
(5, 7).
The mechanism of TGF-1 action on adrenal steroidogenesis
is not fully understood. In TGF-
1-treated BAC cells, low
density lipoprotein uptake and metabolism are inhibited (8), and
angiotensin II binding is reduced (9). TGF-
1 also
decreases the expression of several steroidogenic enzymes. The major
target has been shown to be cytochrome P450c17 which is strongly
inhibited by TGF-
1 in bovine (10), ovine (11), and human
adrenal cells (12). In addition, TGF-
1 has been shown to
inhibit 3
-hydroxysteroid dehydrogenase isomerase (3
-HSD) (13) and
cytochrome P450 side chain cleavage (P450scc) (14) enzyme expressions,
although regulation of these enzymes seems to differ from one species
to another. The negative effect of TGF-
1 on
steroidogenesis has also been observed in other cell types such as
testicular Leydig cells (15, 16) and ovarian thecal cells (17).
Cholesterol is an obligatory precursor of steroid hormones in steroidogenic cells. Endogenous cholesterol is either synthesized from acetate or absorbed from blood, principally through LDLs in the bovine adrenal cortex, and is stored mainly as cholesterol esters in lipid droplets in the cytoplasm. The biosynthesis of steroid hormones upon ACTH stimulation starts with the transport of the substrate cholesterol from extramitochondrial stores to the inner mitochondrial membrane where the first enzyme in the pathway, P450scc, cleaves cholesterol into pregnenolone. The intramitochondrial transport of cholesterol is the rate-limiting step in steroidogenesis and the main site for regulation by physiological stimuli during acute stimulation of steroid production. Activation of this step is known to require de novo protein synthesis (18). Recently, the steroidogenic acute regulatory protein (StAR) has been implicated as the regulator of intramitochondrial cholesterol translocation (for a review see Ref. 19). StAR is synthesized as a 37-kDa labile cytoplasmic precursor that is imported into the mitochondrion, where the cleavage of the mitochondrial targeting sequence occurs, yielding a 30-kDa protein. Transfection of MA-10 Leydig tumor cell line with the mouse StAR cDNA (20) or that of COS-1 cells with the human StAR cDNA in combination with the cholesterol side chain cleavage enzyme system (21) results in enhanced steroid production. Mutations in the StAR gene were subsequently found to be responsible for lipoid congenital adrenal hyperplasia, an autosomal recessive disease in which the synthesis of all gonadal and adrenal steroids is severely impaired (22). Fifteen different mutations in the StAR gene were found, and all rendered the StAR protein inactive in functional assays (22, 23).
Closer examination of P450c17 inhibition by TGF-1 in
bovine adrenocortical cells suggested that TGF-
1 might
have an earlier target in the biosynthesis of steroids. Testing this
hypothesis, we found that TGF-
1 regulated two distinct
steps in steroid biosynthesis as follows: 1) the conversion of
pregnenolone to 17
-hydroxypregnenolone due to the inhibition of
P450c17 expression, and 2) the supply of cholesterol to P450scc,
suggesting inhibition of cholesterol transport to the inner
mitochondrial membrane. This prompted us to study the effect of
TGF-
1 on StAR expression. We report here that
TGF-
1 decreases the steady state level of StAR mRNA
in a concentration- and time-dependent manner and that this
inhibition requires transcription and translation of an intermediate
protein.
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EXPERIMENTAL PROCEDURES |
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Materials--
Synthetic 1-24 ACTH (Synacthène
Immédiat®), metyrapone, and SU10603 were obtained from
CIBA-Geigy (Basel, Switzerland). Recombinant TGF-
1 was
purchased from R & D Systems (Abingdon, UK). Ham's F12 medium,
actinomycin D, cycloheximide, 25-hydroxycholesterol, 17
-hydroxypregnenolone, and pregnenolone were purchased from Sigma
(Saint Quentin Fallavier, France). Trilostane was purchased from
Farillon (United Kingdom). The murine StAR cDNA was a generous gift
from Dr. Douglas Stocco (Texas Tech University, Lubbock).
Bovine Adrenal Fasciculata Cell Preparation-- Bovine adrenal glands were obtained from a local slaughterhouse. Fasciculata-reticularis cells were obtained by successive tryptic digestions, seeded in 10-cm plates or in 12-well plates, and grown in Ham's F12 medium supplemented with 10% horse serum (Eurobio, Les Ulis, France) and 2.5% fetal calf serum (Life Technologies, Inc., Cergy-Pontoise, France) as described previously (24). The cells were used at day 2 or 3 of primary culture.
Steroid Production--
BAC cells were incubated for 15 h
with or without TGF-1 (2 ng/ml). The medium was then
removed and replaced for 2 h with fresh medium for cortisol
production or fresh medium containing 10 µM trilostane
(3
-HSD inhibitor) and 40 µM SU10603 (P450c17 inhibitor) to measure pregnenolone production. To study steroid production from exogenous precursors, either 25-hydroxycholesterol (50 µM), pregnenolone (20 µM), or
17
-hydroxypregnenolone (20 µM) were added to the fresh
medium during the 2-h incubation.
P450scc Activity--
BAC cells were incubated for 15 h
with or without TGF-1 (2 ng/ml). The cells were scraped
and then homogenized with a Potter-Kontes homogenizer (1200 rpm, 35 strokes) in 5 mM Tris-HCl, pH 7.4, 275 mM
sucrose. The homogenate was centrifuged at 500 × g for
15 min to remove large debris and nuclei. Mitochondria were collected by centrifugation at 10,000 × g for 10 min and washed
once with the same buffer, and protein concentrations were determined
using the Bradford method (25). 50 µg of isolated mitochondria were equilibrated at 37 °C in 10 mM Tris-HCl, pH 7.4, 250 mM sucrose, 10 mM KCl, 10 mM
potassium phosphate, 5 mM MgCl2, 5 µM metyrapone (11
-hydroxylase inhibitor), 10 µM trilostane, and 200 µM
25-hydroxycholesterol in a final volume of 500 µl. A mix of
malate/isocitrate (1:1, v/v, final concentrations 10 mM)
was added to start the reaction. The reaction was carried out for 10 min and was stopped by the addition of 5 ml of dichloromethane (26).
Pregnenolone was extracted and assayed by RIA as described above.
RNA Preparation and Northern Blot Analysis--
Total RNA was
isolated from cells using the RNAgents® kit (Promega,
Charbonnières, France). 25 µg of total RNA were separated by
electrophoresis through a 1% agarose gel containing 1.9%
formaldehyde. RNA was then transferred to a Hybond-N membrane
(Amersham, Les Ulis, France). Blots were sequentially probed with a
full-length murine StAR cDNA and an 18 S rRNA probe that were
labeled by random priming with [-32P]dCTP (111 TBq/mmol, ICN Pharmaceuticals, Orsay, France) using the Radprime DNA
labeling kit (Life Technologies, Inc., Cergy-Pontoise, France).
Prehybridization and hybridization at 65 °C were performed in
Rapid-Hyb buffer (Amersham, Les Ulis, France). The blots were washed
twice at room temperature in 2× SSC (1× SSC = 0.15 M
NaCl, 15 mM sodium citrate) and 0.1% sodium dodecyl
sulfate, once in 1× SSC and 0.1% sodium dodecyl sulfate at 65 °C,
and once in 0.1× SSC and 0.1% sodium dodecyl sulfate at 65 °C.
Hybridizing bands were visualized on a
-imager (PhosphorImager,
Molecular Dynamics, Sunnyvale, CA) and quantified using the
ImageQuantTM program (Molecular Dynamics,
Sunnyvale, CA). Values for StAR mRNA were normalized to values for
the 18 S rRNA.
Statistics-- Statistical analysis was performed with Student's t test for comparison of two groups. Differences were considered significant when p < 0.05.
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RESULTS |
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TGF-1 Alters at Least Two Steps in the Cortisol
Biosynthesis Pathway--
Recently, using primary cultures of bovine
adrenocortical (fasciculata-reticularis) cells, we re-examined the
effects of TGF-
1 on several steps of the cortisol
biosynthesis pathway. Cortisol synthesis begins with the transport of
the substrate cholesterol to the first enzyme in the pathway, P450scc,
which cleaves cholesterol into pregnenolone which will then be
hydroxylated by P450c17 into 17
-hydroxypregnenolone. The subsequent
steps involve 3
-HSD, P450c21, and P450c11
enzymes. We used a
cell-permeant analog of cholesterol and some of the intermediate
substrates to determine whether inhibition by TGF-
1
could be overcome by skipping any of these steps. BAC cells were
pretreated for 15 h with or without TGF-
1 (2 ng/ml); the medium was then replaced with fresh medium containing the
steroid precursors, and cortisol production was measured after 2 h. The results shown in Table I confirm
that TGF-
1 is a very potent inhibitor of basal cortisol
production (81% inhibition). When the cells are supplied with either
25-hydroxycholesterol (a membrane-permeant analog of cholesterol) or
pregnenolone, TGF-
1-induced inhibition of cortisol
synthesis is reduced to only 39 and 33%, respectively.
TGF-
1 then must act prior to the production of pregnenolone, apparently at the level of cholesterol supply to P450scc.
When 17
-hydroxypregnenolone is supplied as substrate, TGF-
1 no longer significantly inhibits cortisol
synthesis (3%), confirming that TGF-
1 reduces the
conversion of pregnenolone into 17
-hydroxypregnenolone. This block
corresponds to the inhibition of P450c17 expression that we have
previously demonstrated (10, 27).
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TGF-1 Inhibits Pregnenolone Production and Decreases
Cholesterol Accessibility to P450scc--
To confirm that
TGF-
1 inhibits an early steroidogenic step, we measured
its effect on pregnenolone accumulation using inhibitors of 3
-HSD
(trilostane) and P450c17 (SU 10603) to block pregnenolone metabolism.
As shown in Fig. 1, pregnenolone
production is inhibited by TGF-
1 in a
time-dependent manner. This inhibition occurred as early as
6 h after treatment and reached a maximum at around 12 h in
both unstimulated and ACTH-stimulated cells (Fig. 1, A and
B, respectively). We then measured the effect of
TGF-
1 on the conversion of 25-hydroxycholesterol into
pregnenolone. Table II shows that when
this permeant cholesterol analog was supplied as a steroid precursor,
TGF-
1 no longer inhibited pregnenolone production (6%
inhibition in the presence of 25-hydroxycholesterol versus
58% in its absence). These results suggest that TGF-
1 decreases cholesterol accessibility to the inner mitochondrial membrane
rather than directly affects P450scc. This was confirmed by measuring
the level and activity of P450scc protein in mitochondria isolated from
cells treated or not treated with TGF-
1. We observed no
difference in either P450scc protein level (data not shown) or activity
in the absence of TGF-
1 (228 ± 21 ng of
pregnenolone/mg protein/h) or in its presence (241 ± 57 ng of
pregnenolone/mg protein/h).
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TGF-1 Decreases the Steady State Level of StAR
mRNA--
The recent cloning of the StAR cDNA has allowed
us to demonstrate that the corresponding protein plays a fundamental
role in the mitochondrial import of cholesterol (20). We therefore decided to investigate the effect of TGF-
1 on the steady
state level of StAR mRNA. Northern blot analysis of BAC cell total
RNA using either a full-length mouse cDNA (Fig.
2A) or a partial bovine cDNA probe (data not shown) revealed two major StAR transcripts (3 and 1.7 kb) that have been previously described (28) and a minor
transcript (1.3 kb). The 3-kb and the 1.7-kb transcripts account for 60 and 30% of StAR mRNA expression, respectively, whereas the 1.3-kb
transcript accounts for 10%. All three transcripts showed coordinate
induction, and therefore, the most abundant transcript of 3 kb was
quantified as a representative of the three transcripts. Continuous
treatment of BAC cells with TGF-
1 (2 ng/ml) caused a
time-dependent decrease in the steady state level of StAR
mRNA in both unstimulated and ACTH-stimulated cells. This decrease
was significant after 4 h of exposure to TGF-
1, and the effect was nearly maximal after 12 h and sustained for up to
24 h (85% inhibition) (Fig. 2B). Fig. 2C
shows the kinetics of StAR mRNA induction in ACTH-stimulated cells
versus control cells. We found that StAR mRNA levels in
BAC cells were markedly increased within 1 h and peaked at 2 h after ACTH treatment, returning to a basal level after 12 h of
stimulation.
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TGF-1 Does Not Modify StAR mRNA
Stability--
To determine whether the effects of
TGF-
1 on StAR mRNA levels were due to changes in
transcript stability, BAC cells were incubated in the presence or
absence of TGF-
1 (2 ng/ml) for 12 h and
subsequently treated with actinomycin D (2.5 µg/ml) for 6-24 h. The
half-life of StAR mRNA in transcriptionally arrested BAC cells was
determined to be 12 h in both control and
TGF-
1-treated cultures (Fig.
4). This suggests that
TGF-
1 does not modify the degradation of StAR mRNA
but rather affects its transcription rate.
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The TGF-1 Effect Depends on RNA and Protein
Synthesis--
To determine whether the effect of TGF-
1
on StAR mRNA levels was dependent on transcription and/or
translation, BAC cells were treated with TGF-
1 in the
presence or absence of actinomycin D (2.5 µg/ml) or cycloheximide (10 µg/ml). We found that, in the presence of either of these two
inhibitors, TGF-
1 no longer decreases StAR transcripts
levels (Fig. 5). Thus, inhibition by
TGF-
1 seems to depend on the synthesis of other
proteins.
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DISCUSSION |
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TGF-1 treatment of adrenocortical steroidogenic
cells has been shown to result in the inhibition of steroid hormone
biosynthesis, mainly attributable to a decrease in P450c17 expression
(10, 11, 27). In the present study, we show that TGF-
1
inhibits not only P450c17 expression but also that of StAR, a recently described protein implicated in intramitochondrial cholesterol translocation. Furthermore, we could demonstrate that this inhibition occurs at the level of StAR transcription and requires the
transcription and translation of new proteins.
A number of studies performed on bovine and ovine adrenocortical
fasciculata and glomerulosa cells (29-31), in porcine testicular Leydig cells (16), and in porcine ovarian thecal cells (17) have led to
the hypothesis that TGF-1 modifies cholesterol
accessibility to P450scc. In most of these works, TGF-
1
has been shown to decrease pregnenolone synthesis, which could be
restored by the addition of 22(R)- or
25-hydroxycholesterol. In the present study, we used precursor
steroids, 25-hydroxycholesterol, pregnenolone, and
17
-hydroxypregnenolone, to demonstrate that TGF-
1 has
at least two targets in BAC cells as follows: one that lies between
pregnenolone and 17
-hydroxypregnenolone, previously identified as
P450c17 (10, 27), and another that seems to affect cholesterol
accessibility to P450scc. Indeed, addition of 25-hydroxycholesterol
fully restored pregnenolone synthesis in TGF-
1-treated
cells. Pregnenolone production in isolated mitochondria from
TGF-
1-treated cells in which cholesterol was allowed to
accumulate was also inhibited (data not shown). Finally, we found that
TGF-
1 inhibits neither the level nor the activity of
P450scc. Altogether, these results clearly demonstrate that
TGF-
1 decreases cholesterol accessibility to the inner
mitochondrial membrane, in accordance with the studies mentioned above.
The only hypothesis in the literature to explain this effect was
formulated by Hotta and Baird (8) who showed that TGF-
1
induces a decrease in the number of LDL receptors, which are in part
responsible for cholesterol entry into the cell. However, we found that
TGF-
1 still inhibits pregnenolone production in the
absence of LDLs in the medium (30 ± 4% inhibition). In view of
our results, we cannot completely exclude that TGF-
1
might decrease the number of LDL receptors; however, it is clear that
TGF-
1 hits another target. We examined the effect of
TGF-
1 on the expression of StAR, a mitochondrial protein
required for steroid hormone biosynthesis, which regulates cholesterol
transfer to the inner mitochondrial membrane. Our results demonstrate
that TGF-
1 strongly inhibits StAR mRNA expression in
a time- and dose-dependent manner. Interestingly, TGF-
1-induced decrease of StAR mRNA expression
occurs with similar kinetics and EC50 as
TGF-
1 inhibition of pregnenolone synthesis. To
definitively establish a direct link between these two inhibitions, we
are currently testing whether overexpression of StAR relieves the
TGF-
1 inhibition of pregnenolone synthesis.
It is long known that a newly synthesized protein(s) is absolutely required for the acute regulation of steroidogenesis (32, 33). Several candidate proteins have been proposed, including the sterol carrier protein 2, the steroidogenesis activator protein, the peripheral benzodiazepine receptor with the diazepam binding inhibitor, and a family of proteins of approximately 30 kDa that are rapidly synthesized and phosphorylated in response to corticotropic hormone (for review, see Ref. 34). More recently, Clark et al. (20) cloned the cDNA encoding the 30-kDa phosphoprotein that was called StAR. A model has been proposed that dissociates the import to the mitochondrion and processing of StAR from its steroidogenic activity. It was shown both in vivo (23) and in vitro (35) that N-terminal truncated StAR proteins can increase pregnenolone synthesis without entering the mitochondria, probably by acting on the outer mitochondrial membrane. This suggests that StAR acts through its C-terminal domain to induce cholesterol import into the inner mitochondrial membrane.
It is clear that StAR plays a fundamental role in steroidogenesis. This
implies that the expression of such an important protein must be
tightly regulated. StAR is rapidly synthesized in response to cAMP
analogs in the Leydig tumor cell line MA-10 (36) and in ovary cells
(37) and in response to angiotensin II through a
Ca2+-dependent pathway in bovine glomerulosa
cells (38). StAR is regulated both at the mRNA and the protein
level. In particular, it has been found that the 37-kDa StAR protein
has a very short half-life of 4-6 min (39). We have performed Northern
blot analysis to study the expression of StAR mRNA in BAC cells. We
found two major transcripts (3 and 1.7 kb) that have been previously
described (28, 40) and a minor transcript (1.3 kb). This smaller
transcript has not been described before in bovine cells, but this
could be due to low sensitivity of Northern blots in the earlier
studies, as three transcripts have also been shown in both human (21) and mouse (36). All three transcripts are long enough to encode a
full-length protein. The functional significance of the different transcripts is not yet known; however, all three are coordinately induced by ACTH in BAC cells. StAR mRNA levels in BAC cells were markedly increased within 1 h of ACTH treatment, peaked at 2 h, and returned to a basal level after 12 h. This is a little
faster than in mouse cells stimulated with a cAMP analog (starting
after 2 h and peaking between 4-6 h) (36) and much faster than in ovary cells (maximum at 24 h) (37). We found that
TGF-1 significantly decreased StAR mRNA levels as
early as 4 h, reaching a maximum at 12 h in both unstimulated
and ACTH-stimulated cells. Our results show that ACTH induction of StAR
mRNA expression is faster than its decrease by
TGF-
1. The EC50 for this inhibition was
around 20 pg/ml, which corresponds to the EC50 described
for the inhibition of epithelial cell proliferation (41). The
inhibitory effect of TGF-
1 on the steady state level of
StAR mRNA was not due to an increase in its degradation, as we
found a similar half-life (12 h) for StAR mRNA in both control and
TGF-
1-treated cells. It can be surmised that
TGF-
1 inhibition is due to a decrease in StAR gene
transcription. This is the first report of a transcriptional inhibition
of StAR. Reduction of StAR mRNA expression by prostaglandin F2
in the rat ovary has been reported (42), but the mechanism was not
elucidated.
Importantly, we found that the inhibition of StAR in BAC cells is
dependent upon ongoing transcription and protein synthesis, as addition
of actinomycin D or cycloheximide completely abolishes the effect of
TGF-1. Thus the inhibition of StAR expression by TGF-
1 requires the production of additional intermediate
regulators. TGF-
1 signals through type I and type II
transmembrane serine/threonine kinase receptors (for a review, see Ref.
43). Activation of the receptor complex occurs when the type II
receptor kinase transphosphorylates the type I receptor in response to
ligand binding. This activates the type I kinase, which transiently
associates with and phosphorylates Smad proteins. Phosphorylated Smads
translocate to the nucleus and regulate transcription of
TGF-
-responsive genes. It will be interesting to see whether Smads
are involved in TGF-
1 signal transduction in BAC cells,
as it has not yet been demonstrated that Smads are implicated in all
biological responses to TGF-
. Possible candidates for the
intermediary protein include the transcription factors c-Jun and/or
c-Fos that bind to AP-1 sites. Indeed, the expression of these two
immediate-early genes is induced by TGF-
1 in many
different cell types (44, 45). Furthermore, phorbol myristate acetate
prevents StAR mRNA induction by a cAMP analog in the human ovary
and in the ovine corpora lutea (37, 42), by activating protein kinase
C, a kinase that is known to modulate responses through AP-1-binding
sites.
The StAR promoter, like that of the cytochrome P450 hydroxylases, lacks
typical cAMP-responsive elements; instead, alternate cAMP-responsive
sequences mediate the hormonal induction of these genes (46).
Furthermore, the steroid hydroxylases and StAR are all apparently
transcriptionally regulated by the orphan nuclear receptor, SF-1 (47).
It is tempting to speculate that TGF-1-induced inhibition of StAR and P450c17 expression may occur via the same mechanism involving a common regulator like SF-1. DAX-1, which also
belongs to the orphan nuclear receptor family and has been found to
inhibit SF-1 transactivation (48), is another potential target of
TGF-
1 action.
Altogether, the data reported here demonstrate that
TGF-1 is a major regulator of differentiated functions
of steroidogenic cells, modifying the two rate-limiting steps in
cortisol biosynthesis, StAR and P450c17 expression. We are currently
investigating the possibility of a common repressive mechanism of
TGF-
1 on StAR and CYP17 promoters.
The present observations support the potential role of
TGF-
1 as a negative regulator of adrenocortical
cell-differentiated functions, in balance with the major positive agent
ACTH, to control the overall homeostasis of this endocrine tissue.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Stocco (Texas Tech University, Lubbock) for the generous gift of StAR cDNA. We thank Isabelle Gaillard for skillful help in the preparation of primary cultures of adrenocortical cells.
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FOOTNOTES |
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* This work was supported in part by INSERM, the Commissariat à l'Energie Atomique (CEA/DSV/DBMS), the Ligue Nationale Contre le Cancer, and the Association pour la Recherche Contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a doctoral grant from the CEA.
¶ To whom correspondence should be addressed: INSERM Unité 244, DBMS/BRCE, CEA-G, 17 rue des martyrs, 38054 Grenoble Cedex 9, France. Tel.: 33 476 88 47 27; Fax: 33 476 88 50 58; E-mail: S.Bailly{at}geant.ceng.cea.fr.
1
The abbreviations used are:
TGF-1, transforming growth factor
1;
StAR, steroidogenic acute regulatory protein; LDL, low density
lipoprotein; 3
-HSD, 3
-hydroxysteroid dehydrogenase/isomerase; P450c17, cytochrome P450 17
-hydroxylase; P450scc, cytochrome P450
side chain cleavage; BAC cells, bovine adrenocortical cells from the
fasciculata zone; RIA, radioimmunoassay; ACTH, adrenocorticotropic hormone; kb, kilobase pairs.
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REFERENCES |
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