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Institut de Génétique et de Biologie Moléculaire et Cellulaire (E.L., P.S.-C.), Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université Louis Pasteur, 67404 Illkirch, Strasbourg, France; Department of Obstetrics and Gynecology (M.H.M.), Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2515; Department of Cell Biology & Biochemistry (D.M.S.), Texas Tech University Health Sciences Center, Lubbock, Texas 79430
Address all correspondence and requests for reprints to: Dr. Paolo Sassone-Corsi, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université Louis Pasteur, Boîte Postale 163, 67404 Illkirch, Strasbourg, France. E-mail: paolosc{at}igbmc u-strasbg.fr.
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Introduction |
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DAX-1 is an unusual member of the nuclear receptor superfamily (14) whose expression is mostly restricted to steroidogenic tissues (adrenal cortex, ovary, Leydig cells) (15, 16). In addition, DAX-1 is also expressed in testicular Sertoli cells, pituitary gonadotropes and in the ventromedial hypothalamic nucleus (15, 16). Mutations in the DAX-1 gene cause adrenal hypoplasia congenita (AHC), which is usually associated with hypogonadotropic hypogonadism (HHG) (14, 17). AHC is an X-linked disorder characterized by impaired development of the permanent zone of the adrenal cortex. After birth, only large vacuolated cells resembling fetal adrenocortical cells are present in the adrenal cortex of patients with AHC. This results in adrenal insufficiency early in infancy, with low serum concentration of glucocorticoids, mineralocorticoids and androgens, and failure to respond to ACTH stimulation (14, 17). HHG is diagnosed at the expected time of pubertal maturation in AHC patients treated with steroids and is caused by a selective defect in gonadotropins production, with all other pituitary hormones being normal (14, 17). It has been shown that both a deficit of GnRH release from the hypothalamus and pituitary impairment of LH and FSH secretion contribute to the pathogenesis of HHG in patients with AHC (18). In addition, the DAX-1 gene locus is situated in Xp21, within the critical interval delimited for the DSS (dosage-sensitive sex reversal) region (19). Male patients with a duplication of the DSS region develop as phenotypic females (19). We have recently shown that DAX-1 acts as a negative regulator of steroid production in adrenal cells by repressing the expression of StAR. DAX-1 inhibition of StAR expression is dependent upon binding to a hairpin DNA site in the StAR gene promoter (20).
Here we report that DAX-1 can affect the steroidogenic cascade at multiple levels in Y-1 adrenocortical cells, inhibiting the expression of enzymes involved in different steps of this process.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transfections
To obtain stably transfected cell lines, Y-1 cells were
transfected by the calcium phosphate technique with pD383, which
carries the neomycin resistance gene (20), alone (Y-1/neo) or in a 1:10
ratio together with pSG.DAX-1 (14) (Y-1/hDAX-1) or
MT-REV(AB)neo (21; Y-1/RIAB). Clones were
selected and maintained in medium containing 500 µg/ml G418 (Gibco
BRL, Gaithersburg, MD).
For transient transfection experiments, Y-1, Y-1/neo and Y-1/hDAX-1 cells were seeded in six-well dishes and transfected (calcium phoshate technique) with 1 µg of human StAR [pGL1.3kb StAR (22)], P450scc [pSCCT2.3k (23)] and type II 3ß-HSD promoter [-1251 h3ßHSD-II CAT (24)] reporter constructs, 1 µg of empty pSG5 expression vector or pSG.DAX-1 (14) and 400 ng of pCH110 (Pharmacia, France) per well.
For PKA pathway studies, Y-1 clones were transfected with 1 µg of a CAT reporter plasmid (p4xCRE CAT) containing a tetramerized cAMP-responsive element (TGACGTCA) and a basal HSV thymidine kinase promoter.
CAT and luciferase assays were performed using standard techniques (14, 20), and results were normalized for ß-galactosidase activity. Each experiment was performed at least three times in duplicate.
Assay for secreted steroids
Three hundred thousand cells seeded onto 60-mm plates were
cultured in regular medium plus G418 for 24 h, and then in
DMEM/Ham’s F-10 1:1 containing 1.5% FCS for an additional 24 h.
Fresh medium containing either forskolin (Sigma; 10 µg/ml) or ethanol
vehicle was then added, and incubation was continued for an additional
16 h. Supernatants were then harvested for fluorogenic steroid
measurement, as described (25, 26). This sensitive assay detects 20-
and 21-hydroxylated steroids (25). These steroids have the property to
be highly fluorescent, with an excitation peak at 470 nm and an
emission peak at 536 nm, when they are in a solution of sulfuric acid
in ethanol (25). For the assay, 0.3 ml of culture medium were
centrifuged and extracted with 0.7 ml of methylene chloride. The
organic phase was extracted with 0.7 ml of 65% sulfuric acid in
ethanol for 1 min and then 0.5 ml of the acid were transferred into a
glass tube containing 3 ml of 65% sulfuric acid in ethanol. After 90
min, sample fluorescence was measured using a Hitachi F-2000
fluorescence spectrophotometer, with an excitation wavelength of 470 nm
and an emission wavelength of 536 nm. Corticosterone (Sigma) was used
as a standard. The total amount of steroids measured was then
normalized to the cellular protein content of secreting cells. Each
measurement was the average of triplicate cultures, and six independent
experiments were performed. The mean intraassay coefficient of
variation for three distinct samples assayed in 12 sets of triplicates
was 5.1; mean interassay coefficient of variation for the same samples
run in 10 separate assays in triplicate was 11.1.
For assay of pregnenolone and progesterone, 5 x 104 Y-1/neo, Y-1/hDAX-1 and Y-1/RIAB cells/well were seeded in triplicate in a 96-well culture plate and cultured overnight in serum-containing medium. Cells were washed twice with serum-free medium and then incubated for 2 h in serum-free medium in basal conditions or stimulated with forskolin (10 µg/ml), with and without 25 µM 22(R)-hydroxycholesterol (Sigma). Pregnenolone and progesterone in the supernatants were measured by a specific RIA, as described (11).
PKA assay
PKA activity was determined as described previously (26).
Briefly, 10 µl of cell extract (at a concentration of 2 mg/ml) were
added to a 40-µl reaction mix containing a final concentration of 20
mM Tris-HCl (pH 7.4), 10 mM magnesium acetate,
0.5 mM IBMX (Sigma), 10 mM dithiothreitol, 5
mM NaF, 1 mM cAMP (Sigma), 200 µM
[
-32P] ATP (100–200 cpm/pmol, DuPont-NEN, Boston,
MA), and 30 µM kemptide (Sigma) as substrate.
After 5 min incubation at 30 C, 25 µl of the reaction were
transferred onto Whatman P81 phosphocellulose strips. These were washed
five times in 75 mM phosphoric acid and once in 95%
ethanol. Filters were then air-dried and counted by liquid
scintillation. Each sample was assayed in triplicate. PKA activity is
expressed in units (picomoles of phosphate transferred per minute) per
milligram of cell extract.
Northern and Western blot
Northern and Western analyses were performed as previously
described (20). Anti DAX-1 2F4 mouse monoclonal antibody (15) and
anti-StAR rabbit polyclonal antiserum (11) were used for detection of
the DAX-1 and StAR proteins, respectively. Antisera directed against
P450scc and 3ß-HSD were kindly provided by G. Defaye (INSERM U244,
CENG, Grenoble, France) (27).
Statistical analysis
The Wilcoxon rank sum test was used to assess the statistical
significance of differences in steroid production (both in basal
conditions and after forskolin stimulation) between Y-1/neo cells and
Y-1/hDAX-1 clones. A value of P < 0.05 was considered
as statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), and therefore they constitute a
convenient system to study the effect of DAX-1 expression on
differentiated adrenocortical cell function. To this purpose, we have
produced stably transfected Y-1 clones that express the human DAX-1
complementary DNA under the control of the SV40 early promoter
(Y-1/hDAX-1). We also established cell lines expressing either the
neomycin resistance gene (Y-1/neo) or a cAMP-dependent protein kinase
type I
regulatory subunit (RI) harboring mutations in both sites
A and B of the cAMP-binding domain (Y-1/RIAB). Expression
of this mutated RI has been previously shown to markedly impair
steroidogenesis in Y-1 cells (21).
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). All Y-1/hDAX-1 clones produce very low
levels of fluorogenic steroids, both in basal conditions and after
stimulation of the cAMP pathway by forskolin (overall mean
(±SEM) 3.49 ± 0.4 µg/mg cell protein in basal
conditions; 5.09 ± 0.42 µg/mg cell protein,
forskolin-stimulated) (Fig. 1B). Conversely, Y-1/neo cells produce
significantly higher amounts of steroids than Y-1/hDAX-1 cells (overall
mean (±SEM) 6.19 ± 1.6 µg/mg cell protein in basal
conditions; 36.4 ± 19 µg/mg cell protein,
forskolin-stimulated). (Fig. 1B). Y-1/hDAX-1 cells steroidogenic
capacity is more severely impaired after forskolin stimulation (about
6.5 fold; P < 0.025) than in basal conditions (about
2-fold; P < 0.05), compared with Y-1/neo cells. Given
their homogeneous phenotype, one of the Y-1/hDAX-1 clones was chosen at
random for further characterization.
The PKA pathway is functional in Y-1/hDAX-1 cells
Because steroid production in adrenal cells is known to be
regulated by the cAMP pathway (2, 13), the DAX-1 effect could be due to
an impairment in the transduction of the cAMP signal. We measured PKA
activity in Y-1/hDAX-1 cells and found that it is comparable to Y-1/neo
cells (Fig. 2A). Conversely, and
consistent with previous results (21, 26), PKA activity is reduced in
the Y-1/RIAB cells, which express a dominant-negative RI
(Fig. 2A). In addition, Y-1/DAX-1 cells assume the characteristic round
shape after forskolin stimulation (not shown). This phenomenon has been
shown to require a functional PKA (2). Finally, forskolin-stimulated
expression of a CRE (cAMP responsive element) reporter plasmid, which
is activated by cAMP-responsive factors of the CREB/CREM/ATF family, is
not impaired in Y-1 DAX-1 cells (Fig. 2B). Taken all together, these
results show that the cAMP-dependent signaling pathway remains
functional in Y-1/hDAX-1 cells. In addition, expression of SF-1, which
has been shown to be a critical regulator of the expression of StAR and
of several enzymes involved in steroid biosynthesis (8), is not
impaired in Y-1/hDAX-1 cells (Fig. 2C). SF-1 levels were not affected
by forskolin stimulation in any of the cell lines (not shown).
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, histograms 1–4). Conversely,
production of both pregnenolone and progesterone is extremely low in
Y-1/hDAX-1 and in Y-1/RIAB cells (Fig. 3, histograms
9–12/17–20).
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, histograms 13–16). This
pattern of steroid secretion closely resembles that of
Y-1/RIAB cells, in which pregnenolone production was only
marginally increased by 22(R)-hydroxycholesterol (Fig. 3, histograms
21–24). These data indicate that a nearly complete block exists in
Y-1/hDAX-1 cells in the first steps of the steroidogenic cascade.
Expression of StAR, P450scc and 3ß-HSD is impaired in Y-1 cells
expressing DAX-1
To unveil the molecular basis of the steroidogenic block produced
by DAX-1, we studied the expression of StAR, P450scc, and 3ß-HSD in
Y-1/hDAX-1 cells. As we have reported earlier (20), StAR RNA and
protein are undetectable in Y-1/hDAX-1 cells and cannot be induced by
forskolin treatment. Moreover, P450scc expression is also reduced by
several fold when compared with Y-1/neo cells (Fig. 4, B and C). In addition, no 3ß-HSD
expression can be detected (Fig. 4, B and C). The impairment of the
expression of these components of the steroidogenic machinery in
Y-1/hDAX-1 cells accounts for their pattern of steroid production after
administration of 22(R)-hydroxycholesterol and indicates that DAX-1
affects steroid synthesis by repressing multiple biochemical steps in
the cascade.
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, A
and B, and Ref. 20). The activity of these promoters is also
significantly reduced in Y-1/hDAX-1 cells, compared with the Y-1/neo
cells (Fig. 5, D and E). DAX-1 has a marginal effect on the activity of
the 3ß-HSD promoter in Y-1 cells (Fig. 5C). Conversely, stimulation
of the cAMP pathway is unable to activate this promoter in Y-1/hDAX-1
cells (Fig. 5F). These results show that DAX-1 expression in Y-1 cells
can inhibit expression of StAR, P450scc and 3ß-HSD at the level of
transcription.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role of DAX-1 as a regulator of multiple steps in the steroidogenic cascade has interesting implications concerning the pathogenesis of the DSS syndrome. This syndrome is caused by a duplication of a genomic region situated in Xp21. Male subjects with a duplication of the DSS region are phenotypic females, even if a large spectrum of secondary sex characteristics is found in these patients (19). Because the DAX-1 gene locus resides inside the critical region mapped for the DSS locus (14), it is tempting to speculate that double dosage expression of DAX-1 could be responsible for sex reversal, because of an inappropriate suppression of sex steroid production by the male fetus.
The cAMP signal transduction pathway plays an essential role in
eliciting steroid hormone biosynthesis in steroidogenic tissues (1, 2, 13). It has been demonstrated that a functional cAMP-dependent PKA
protein kinase is required for steroid production in adrenal cortical
cells. Some Y-1 cell clones harbor point mutations in the regulatory
subunit of type I PKA. These mutations render the enzyme resistant to
activation by cAMP and impair steroidogenesis in these cells (2, 30).
Steroid production is also impaired in wild-type Y-1 cells transfected
with a dominant-negative PKA regulatory subunit mutated in both the A
and B sites of the cAMP-binding domain (21, and our data).
Interestingly, both basal and cAMP-stimulated steroid production are
down-regulated in these cells. This indicates that a basal level of
activation of the cAMP pathway is also essential to sustain basal
expression of the genes involved in the steroidogenic process (21).
Y-1/hDAX-1 cells are strikingly similar to Y-1 cells expressing a
mutated PKA RI regulatory subunit because both their basal and
cAMP-stimulated steroid production are impaired. Conversely, the
functionality of the cAMP pathway is not affected by DAX-1 expression
in Y-1 cells: both PKA activity and the capacity of nuclear factors to
respond to the stimulation of the cAMP pathway are not affected in
Y-1/hDAX-1 cells (see Fig. 2). A variety of agents and conditions are
known to antagonize the cAMP pathway in steroidogenic cells and
decrease steroid biosynthesis, such as heat shock (31), apoE expression
(32), lipopolysaccharide (33), phorbol esters (34), tumor necrosis
factor- (35), and PGF2 (36). It is possible that at
least some of these agents might function either by inducing DAX-1
expression or by activating its function to repress the StAR and the
P450scc promoters in steroidogenic cells.
The transcription factor SF-1 and its bovine homologue Ad4BP belong to the nuclear hormone receptor superfamily and are both related to Drosophila FTZ/F1, an orphan nuclear receptor that regulates the expression of the fushi tarazu homeobox gene (6, 7). SF-1 has been shown to regulate the expression of multiple genes involved in the steroidogenic pathway (see Ref. 8 for review). It has been reported that SF-1 can mediate the cAMP-dependent transcriptional activation of P450scc, P450c17 and P450aro (37, 38, 39). In addition, inactivation of the FTZ/F1 gene in the mouse causes the complete absence of adrenal glands and gonads (40). Interestingly, there is full overlap between the sites of expression of the SF-1 and the DAX-1 genes (15, 16). We (20) and others (41) have shown that DAX-1 can repress SF-1 mediated transactivation. This has raised the possibility that the two proteins could heterodimerize in solution (41). We have not been able to detect in vivo association of SF-1 and DAX-1 (unpublished observations). Instead, we have found that DAX-1 binds to hairpin DNA structures and that their deletion abrogates DAX-1 capacity to repress transactivation by SF-1 (20). To explain the SF-1/DAX-1 functional interactions, we favor a model that envisages that DAX-1 binding to DNA results in down-regulation of SF-1- mediated transactivation by recruitment of the transcriptional silencing domain residing in the DAX-1 C-terminus (42).
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Acknowledgments |
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Footnotes |
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2 Supported by an Italian Telethon Fellowship.
Received January 26, 1998.
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5-4 isomerase promoter by
the transcription factor steroidogenic factor-1/adrenal 4-binding
protein and phorbol ester. J Biol Chem 272:7960–7967 in
isolated rat luteal cells. Endocrinology 120:1019–1026[Abstract]
alters bovine luteal cell synthetic capacity and viability.
Endocrinology 130:854–860[Abstract]
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