ORIGINAL PAPER
Dexamethasone-induced suppression of steroidogenic acute regulatory protein gene expression in mouse Y-1 adrenocortical cells is associated with reduced histone H3 acetylation
Wei-Ping Fon . Pi-Hsueh S. Li
Received: 17 October 2007/ Accepted: 8 November 2007/Published online: 27 November 2007 @ Humana Press Inc. 2007
Abstract In this study, we investigated the effect of dexamethasone on the expression of steroidogenic acute regulatory protein (StAR) and the acetylation of histone H3 in mouse Y-1 adrenocortical tumor cells. Treatment of Y-1 cells with increasing concentrations (0.001-50 µg/ml) of dexamethasone for 24 h suppressed 8-Br-cAMP (0.5 mM)- stimulated StAR mRNA and protein levels and progester- one production in a dose-dependent manner. Treatment of Y-1 cells with 8-Br-cAMP (0.5 mM) for 1-24 h resulted in a marked increase in StAR mRNA levels. This increase was associated with an increase in progesterone produc- tion. StAR mRNA was down-regulated by dexamethasone at times greater than 3 h. To evaluate dexamethasone effect on the endogenous StAR gene, chromatin immunoprecip- itation assays were performed in combination with polymerase chain reaction. 8-Br-cAMP increased histone H3 acetylation within the proximal region of the StAR gene promoter and coincubation with dexamethasone blocked this effect. Dexamethasone had no effect on glu- cocorticoid receptor mRNA expression. These results demonstrate that dexamethasone repression of 8-Br-cAMP- stimulated StAR gene expression in Y-1 cells is accom- panied by reductions in histone H3 acetylation associated with the StAR gene promoter.
Keywords Mouse Y-1 adrenocortical cells . Dexamethasone · Steroidogenic acute regulatory protein · Corticosteroidogenesis · Histone H3 acetylation
Introduction
Evidence from studies in vivo [1] and in vitro [2] suggests that the adrenal cortex is capable of self-suppression. Experiments in vivo show that raising the plasma glucocorticoid concen- tration either by administration of glucocorticoid [3] or by stressing the animal [4] reduces the capacity of the adrenal cortex to secrete glucocorticoids. Experiments in vitro using adrenal gland tissue [5], freshly isolated adrenocortical cells incubated briefly [6] and cultured fetal [7], adult [8], and tumorous [9] adrenocortical cells show that a variety of cor- ticoids can suppress adrenocortical function.
It has been shown that acute self-suppression of corticos- teroidogenesis in isolated adrenocortical cells is at least partly mediated by the degradation of end-product glucocorticoids [10]. This degradation occurs through an increase in adrenal 5a-reductase activity [11], an enzyme that degrades corticosterone to 5x-dihydrocorticosterone and 33,5x-tetra- hydrocoticosterone, and 11ß-hydroxysteroid dehydrogenase activity [12], an enzyme that catalyzes the conversion of corticosterone to 11-dehydrocorticosterone. The presence of glucocorticoid receptor (GR) in the adrenal cortex [13] pro- vides additional evidence for a direct glucocorticoid suppressive effect on the adrenal gland. Dexamethasone was found to inhibit the corticotropin (ACTH)-induced accumu- lation of cytochrome P450 side-chain cleavage (P450scc) and cytochrome P450 17x-hydroxylase (P450c17) mRNAs at a transcription level in bovine adrenocortical cells in primary culture [14]. The inhibitory effect of dexamethasone is med- iated by the GR. Thus, the direct actions of glucocorticoids on the corticosteroid-producing cells of the adrenal gland may contribute to the adrenal suppression seen with therapeutic glucocorticoid administration. A short feedback loop may exist, thereby providing an additional regulatory pathway modulating the rate of glucocorticoids produced.
The biosynthesis of steroid hormones-glucocorticoids, mineralocorticoids, progesterone, estrogens, and andro- gens-begins from cholesterol. The rate limiting step and the main site for regulation by physiological stimuli in the activation of steroidogenesis is the delivery of cholesterol from the mitochondrial outer membrane to the inner mem- brane where the P450scc enzyme is located [15]. The transport of cholesterol in steroidogenic cells is thought to be mediated by steroidogenic acute regulatory protein (StAR) [16]. The strongest evidence for the critical role of StAR in regulating steroidogenesis has been demonstrated in patients suffering from congenital lipoid adrenal hyperplasia, in which both adrenal and gonadal steroid biosynthesis are markedly impaired due to mutations in the StAR gene [17]. An essentially identical phenotype is observed in StAR null mice [18]. The StAR has been identified as a phosphoprotein [19] that is rapidly induced by tropic hormones, such as gonadotropins and ACTH [20, 21]. StAR expression and regulation by tropic hormones has been examined in great detail [21]. Thus, the regulation of StAR expression would then be a key mechanism in the regulation of steroidogenesis.
Gene transcription is regulated by acetylation and deacetylation of histones [22]. Eukaryotic transcription is a highly regulated process, and acetylation is now known to play a major role in this regulation. DNA typically exists in vivo as a repeating array of nucleosomes, in which 146 bp of DNA are around a histone octamer that consists of two each of histone protein H2A, H2B, H3, and H4. This chromatin structure could be affected by histone acetylation. Histone acetylation eliminates the positive charge of 8-amino acid group on lysine residue, which could lead to destabilization and consequent dissociation of nucleosomes, thus allowing access of transcription factors and RNA polymerase to the DNA to promote transcription. Thus, histone acetylation could be thought of as an excellent marker of gene activity. Christenson et al. [23] first demonstrated that cAMP-stim- ulation of StAR gene transcription in MA-10 Leydig cells was associated with acetylation of histone H3 specifically within the proximal region of the StAR gene promoter.
The purpose of this study was to assess the suppressive effect of dexamethasone on StAR gene expression and pro- gesterone production in mouse Y-1 adrenocortical cells and to elucidate the mechanism underlying its suppressive effect.
Results
Effect of treatment with dexamethasone on the expression of StAR mRNA and protein and the production of progesterone
To investigate the effects of dexamethasone on the expression of StAR mRNA and protein, Y-1 cells were
incubated for 24 h with or without increasing concentra- tions (0.001-50 µg/ml) of dexamethasone in the presence of 8-Br-cAMP (0.5 mM). As shown in Fig. 1, dexameth- asone inhibited in a dose-dependent manner 8-Br-cAMP- induced mRNA levels through Northern blot analysis (mRNA size: 3.4 kb, 1.6 kb), with the minimal effective dose at 0.001 µg/ml. The inhibition of mRNA level increased with the dose, up to a maximum of 1 µg/ml. The major 3.4 kb transcript was quantified.
Results in Fig. 2a show that dexamethasone had a sig- nificant (P < 0.05) effect in suppressing 8-Br-cAMP- induced StAR protein levels. The inhibitory effect was concentration dependent, and a 24-h exposure of Y-1 cells to dexamethasone concentrations ranging from 0.001 µg/ ml to 10 µg/ml decreased StAR protein levels by 20-71%.
Dexamethasone (ug/ml)
0 0.001 0.01 0.1 1 10 50
StAR -+
«- 3.4 kb
← 2.7 kb
+ 1.6 kb
GAPDH →
+1.2 kb
100
StAR mRNA (% of control)
80
60
40
20
0
0
0.001
0.01
0.1
1
10
50
Dexamethasone (ug/ml)
A
Dexamethasone (µg/ml)
0 0.001 0.01 0.1 1
10
StAR -
+ 30 kDa
StAR protein (% of control)
100
80
60
40
20
0
0
0.001
0.01
0.1
1
10
Dexamethasone (ug/ml)
B
Progesterone (% of control)
120
100
80
60
40
20
0
0
0.001 0.01 0.1 1 10 50
Dexamethasone (µg/ml)
Results in Fig. 2b show that dexamethasone at a dose of 0.1 µg/ml or higher significantly (P < 0.05) decreased 8-Br-cAMP-induced progesterone production. The inhibited level was about 26-48% of that treated with 8-Br-cAMP alone.
In the time-course experiment, Y-1 cells were cultured with or without a constant dose of dexamethasone (10 µg/ml)
A
P1
StAR cDNA
PCR
P2
P1
StAR (540 bp)
PCR
IP
Competitor (389 bp)
B (bp)
600
M 0.2 0.4 0.8 1.6 3.2 6.412.8 25.6
500
StAR (540 bp)
400
Competitor (389 bp)
C
0.8
0.6
y = 0.7879x - 0.5863
log (StAR/competitor)
0.4
R2 = 0.9734
0.2
0
-0.2
-0.4
-0.6
-0.8
-1
-1.2
-1.4
-1
-0.5
0
0.5
1
1.5
log (amount of StAR added), attomole
in the absence or presence of 8-Br-cAMP (0.5 mM) for 1, 3, 6, 12, or 24 h. Quantitative, competitive-RT-PCR analysis (Fig. 3) revealed that StAR mRNA was down-regulated by dexamethasone at times greater than 3 h (Fig. 4a). At 6, 12, and 24 h of incubation, the amounts of 8-Br-cAMP-stimu- lated StAR mRNA were 2,880 ± 137, 4,050 ± 292, and 6,885 ± 125 amole/ug total RNA, respectively and were significantly decreased by treatment with dexamethasone (1,492 ± 458, 2,815 ± 791, and 4,390 ± 431 amole/μg total RNA, respectively). Results in Fig. 4b show that dexamethasone caused significant (P < 0.05) decreases in 8- Br-cAMP-induced progesterone production at times greater than 6 h. At 12 and 24 h of incubation, the amounts of 8-Br- cAMP-induced progesterone production were 42.3 ± 4.1 and 58.9 ± 8.0 ng/dish, respectively and were significantly decreased by treatment with dexamethasone (33.1 ± 2.0 and 42.5 ± 3.0 ng/dish, respectively).
A
Control
8-Br-CAMP
DEX +
8-Br-CAMP
M
1
3 6 12 24
1 3 6 12 24
M
1
3 6 12 24
Time (h)
- StAR
+ Competitor
8000
Control
a
StAR mRNA (amole/µg total RNA)
8-Br-CAMP
6000
DEX +8-Br-CAMP
b
a
4000
b
a
2000
b
0
1
3
6
12
24
Time (hour)
B
70
Control
a
Progesterone (ng/dish)
60
8-Br-cAMP
DEX +8-Br-CAMP
50
a
b
40
a
b
30
a
20
10
0
1
3
6
12
24
Time (hour)
Effect of treatment with dexamethasone on the acetylation of histone H3 associated with the proximal region of the mouse StAR gene promoter
To determine whether the levels of histone H3 acetylation are decreased in response to either 8-Br-cAMP or 8-Br- cAMP plus dexamethasone at the StAR promoter, we used ChIP assays with an antibody specific for the acetylated form of histone H3. Immunoprecipitated DNA was sub- jected to PCR with primers amplifying an amplicon within the proximal region of the 5’-flanking region of the mouse StAR gene. Because studies have shown that, in some cellular settings, increased acetylated histone H3, a marker
IP:
Anti-acetylated histone H3
Input
M 123
4 5 6
120
b
% of 8-Br-cAMP
100
80
c
60
a
40
20
0
Control
8-Br-CAMP
8-Br-CAMP + DEX
of transcriptional activity, can be associated with the proximal StAR promoter [24]. As shown in Fig. 5, there are no differences in the amounts of targeted sequences in inputs across non-treated and treated cells (lanes 4-6). Treatment with 8-Br-cAMP significantly (P < 0.05) stim- ulated acetylation of histone H3 within the proximal StAR promoter and this effect was inhibited 31% by coincuba- tion with dexamethasone (P < 0.05).
To determine whether inhibition of 8-Br-cAMP-stimu- lated histone H3 acetylation is accompanied by lower levels of acetylated histone H3 protein content in cells treated with the combination of 8-Br-cAMP and dexa- methasone for 24 h, we analyzed treated Y-1 cell extracts by Western blotting. As shown in Fig. 6, no significant differences were observed between treatments in total cellular acetylated histone H3.
Control 8-Br-cAMP
8-Br-cAMP + DEX
Ac-Histone H3
17 kDa
ß-actin
47 kDa
140
Ac-Histone H3 (% of 8-Br-cAMP)
120
100
80
60
40
Control
8-Br-CAMP
8-Br-CAMP + DEX
Effect of treatment with dexamethasone on the expression of GR mRNA
To determine the effects of dexamethasone on GR gene expression, Y-1 cells were treated for 24 h with or without a constant dose of dexamethasone (10 µg/ml) in the pres- ence or absence of 8-Br-cAMP (0.5 mM). RT-PCR analysis revealed that dexamethasone had no effect on GR mRNA expression (Fig. 7).
Discussion
The results of the present study clearly demonstrate the effect of glucocorticoid dexamethasone to inhibit 8-Br-cAMP- induced expression of StAR mRNA and protein in Y-I adrenocortical cells. The dexamethasone effect is dose- and time-dependent and is associated with decreases in proges- terone production. These results indicate that dexamethasone inhibits Y-1 cell steroidogenesis by down-regulating StAR mRNA and protein expression. Dexamethasone repression of StAR mRNA accumulation was demonstrated at the level
8-Br-CAMP + DEX
Control
8-Br-CAMP
·- B-Actin (800 bp)
+- GR (453 bp)
20
GR/ß-actin (arbitrary units)
1.5
1.0
0.5
0.0
Control
8-Br-CAMP
8-Br-CAMP + DEX
of chromatin, specifically a reduction in 8-Br-cAMP-medi- ated histone H3 acetylation within the proximal region of the StAR gene promoter. This observation provides a direct mechanism for the down-regulation of StAR gene expression and inhibition of progesterone production.
The studies reported here are in a good agreement with the pervious reports that glucocorticoids, either natural or synthetic, can suppress steroidogenesis at the adrenal gland by diminishing its responsiveness to ACTH in the rat [2, 3, 6, 10], guinea pig [25], domestic fowl [2], and cattle [2, 10]. The inhibitory effect of corticosterone on steroi- dogenesis of Y-1 adrenal cells has also been reported [9]. These studies confirmed evidence suggesting that the inhibitory action of glucocorticoid on corticosteroidogen- esis is at the adrenal level. This observed self-suppression of adrenocortical cells suggests the existence of a mecha- nism for the adjustment of steroidogenesis that operates in addition to the classical control exerted by the anterior pituitary.
In our studies, the effective inhibitory dose of dexa- methasone on StAR mRNA expression was at 10 ng/ml (1.94 × 10-8 M) which is less than circulating levels of
cortisol found in humans under stress, such as hypoten- sion and sepsis, and following trauma or surgery (250 ng/ ml) [26-29]. Since, greater than 90% of circulating glu- cocorticoids are bound by transcortin [30, 31], the dose (10 ng/ml) of dexamethasone, a more potent synthetic glucocorticoid, may represent an overestimate of the amount of free glucocorticoid circulating in the blood of these stressed men, and the effect seen is probably more pharmacological than physiological. Thus, the influence of glucocorticoids in normal adrenal steroidogenesis may be minimal. However, excessive production of glucocor- ticoids resulting from perturbations of the hypothalamic- pituitary-adrenal axis or the administration of large amounts of glucocorticoid for therapeutic reasons has been shown to alter the function of adrenal glands. Interpretation of corticosteroidogenesis under these con- ditions may be explained, at least in part, by a direct glucocorticoid action on adrenal cells. A negative feed- back loop may exist, thereby providing an additional regulatory pathway modulating the rate of adrenocortical steroid production.
Reports that the GR is present in the adrenal cortex [13] and that the GR antagonist RU486 reverses the inhibitory effect of glucocorticoids on ACTH-induced cortisol production of bovine adrenocortical cells [14] suggest that the effect of glucocorticoid is mediated through the GRs. Several studies have shown that in the majority of cell lines and animal tissues examined, glu- cocorticoid treatment results in a reduction in GR mRNA [32]. It seems unlikely that the above-mentioned mech- anism is effective in our present study. We found that GR mRNA was expressed in Y-1 adrenal cells and that the expression of these receptors was not affected by dexa- methasone treatment. The apparent discrepancy might be explained by the differences in experimental animal and (or) conditions used. Although no comparable data are yet available on the synthesis of the GR in Y-1 adrenal cells, it has been shown that dexamethasone at a phar- macologic dose decreased the accumulation of GR mRNA in the adrenal gland in vivo [33]. With two cul- tured cell types, human IM-9 lymphocytes and rat pancreatic acinar AR42J cells, dexamethasone decreased steady-state GR mRNA levels [34]. Dexamethasone also caused a down-regulation of the levels of GR mRNA in hepatoma tissue culture cells and rat liver in vivo [35]. In contrast to the negative regulative effects of glucocorti- coids on GR gene transcription, dexamethasone treatment of glucocorticoid-sensitive leukemia T-cell line, LEM-C7 cells, increased GR mRNA. Our results are in contrast to the down-regulation or up-regulation of GR reported in other cells and tissues. However, our results suggest that regulation of the GR by its cognate ligand may be cell-specific.
In the present study, dexamethasone also decreased 8- Br-cAMP-induced StAR mRNA and protein levels. This suggests that in Y-1 adrenal cells, one inhibitory effect of dexamethasone on steroidogenesis is at the step of StAR. Diminished levels of StAR protein would likely result in a reduction in the rate of adrenocortical cells for progester- one production, in response to ACTH, by limiting the availability of cholesterol to the P450scc complex, located in the inner mitochondrial membrane. Previous studies of the StAR protein have indicated that it has an indispensable role in steroid hormone biosynthesis [17], and it has been further postulated that this role is in cholesterol transfer to the inner mitochondrial membrane [16, 19]. It appears that the dexamethasone-induced depression in progesterone production in Y-1 adrenal cells is due to the inhibition of StAR protein synthesis. This observation is highly consis- tent with previous studies in which inhibition of steroid hormone biosynthesis has been tightly correlated with StAR synthesis. For example, agents and conditions that have been shown to result in a decrease in steroid hormone biosynthesis, such as helenalin [36], leukemia inhibitory factor [37], lipopolysaccharide [38], PGF2a [39], trans- forming growth factor-B1 [40], and heat shock [41], have all been demonstrated to decrease StAR protein content. Moreover, a StAR-dependent reduction in steroidogenesis could be manifested through a reduction on the expression and/or activity of the protein. In the present study, dexa- methasone treatment reduced StAR mRNA levels. This finding suggests that the inhibitory effect of dexametha- sone on StAR occurs as a result of a reduction in cAMP- mediated transcription of the StAR gene and/or StAR mRNA stability. The possibility that dexamethasone may exert a negative effect on StAR activity in adrenal cells through post-translational modification (e.g., phosphoryla- tion) of the protein [42] cannot be ruled out either. Furthermore, StAR gene expression is transcriptionally regulated by the orphan nuclear receptor, steroidogenic factor 1 (SF-1) [43]. The activity of SF-1 is blocked by DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenita, critical region on the X chromosome, gene 1) [44]. DAX-1 represses SF-1-mediated transactivation of StAR gene [45]. Therefore, dexamethasone-induced inhi- bition of StAR may occur via the same mechanism involving SF-1 and DAX-1. Dexamethasone has been shown to increase endogenous DAX-1 expression and concordantly decrease StAR expression in mouse adreno- cortical cells [46] and rat testicular cells [47]. This would seem to indicate that up-regulation of DAX-1 gene inhibits StAR gene expression by dexamethasone.
With regard to the direct cellular mechanisms respon- sible for the suppression of glucocorticoids on StAR gene transcription in adrenocortical cells, we focused our attention on the acetylation of histone H3 on StAR gene
promoter. Association of modified histones with chromatin can determine whether a gene is transcriptionally active or silent [48]. Quantitative ChIP assays are an important tool for analyzing interactions between histones and native chromatin [49]. Three studies have looked at the associa- tion of different proteins (including modified histones) with the StAR gene promoter or coding sequence in MA-10 Leydig cells [23], hCG-primed mouse granulosa cells [24], and FSH-stimulated porcine granulosa cells [50]. Results of the present study demonstrate that 8-Br-cAMP stimu- lation of StAR mRNA accumulation in Y-1 adrenal cells is associated with increased acetylation of histone H3 in the StAR gene promoter. Dexamethasone reduced the ability of 8-Br-cAMP to increase histone H3 acetylation in the proximal region of the endogenous StAR promoter. Acet- ylation of histone H3 at lysines 9 and 14 is linked to transcriptional activation [48]. The finding that 8-Br-cAMP stimulation of histone H3 associated with the StAR prox- imal promoter is in agreement with data for the murine gene in MA-10 cells [24]. Hiroi et al. [24] also observed that this modification occurs in MA-10 cells as early as 15 min after 8-Br-cAMP treatment. This rapid modifica- tion in MA-10 cells is accompanied by significant increases in StAR mRNA seen within 30 min. Since we detected significant histone H3 acetylation at 24 h of treatment with 8-Br-cAMP, the slower increase in StAR mRNA in Y-1 cells that was significantly increased at 6 h of 8-Br-cAMP treatment leads us to suggest that histone acetylation is minimal at 1 and 3 h of 8-Br-cAMP treatment in our model system. Our result, however, provide additional evidence for the correlation between histone H3 acetylation and StAR gene activation. Previous studies have indicated that histone acetylation and deacetylation are governed by the opposing activities of two enzyme classes: histone acetyl- transferase (HAT) and histone deacetylases (HDAC) [51, 52]. The overall acetylation status of histones depends on the dynamic equilibrium between HAT and HDAC activ- ities. Induction of HDAC activity can lead to reduced histone acetylation and decreased gene expression [49, 53]. Thus, the dexamethasone-induced repression of histone H3 acetylation at the StAR promoter may be due to an increase in HDAC activity resulting in enhanced nucleosome compaction and consequent repression of StAR gene transcription. The activation of nucleosomal histone by HAT such as p300 and CREB (cAMP response element binding protein)-binding protein (CBP) causes local unwinding of DNA and stimulates transcription [54]. Studies in A549 cells showed that dexamethasone inhibited CBP-associated HAT activity [55]. Thus, HAT may be another potential target of dexamethasone action in the regulation of StAR gene transcription. Whether dexa- methasone inhibits 8-Br-cAMP-stimulated histone acetylases or dexamethasone stimulates histone deacetylases will require
further study. In addition, dexamethasone may activate DNA methyltransferase resulting in hypermethylation and silencing of StAR gene. Due to gonadotropin-induced activation of the StAR gene did reduce dimethylation of lysine 9 of histone H3 [24].
Our Western blotting data demonstrated that 8-Br- cAMP and 8-Br-cAMP plus dexamethasone did not pro- mote a detectable change in bulk levels of acetylated histone H3 compared with control. Studies in rat and pig granulosa cells reported that FSH did not alter the levels of acetylated histone H3 when compared with control [50, 56]. These studies support the idea that total cellular levels of histone H3 acetylation are fairly constant and increases in histone H3 acetylation are restricted to target genes.
In conclusion, the results of the present study indicate that in mouse Y-1 adrenocortical cells, dexamethasone acts to repress the 8-Br-cAMP-induced expression of StAR and production of progesterone. Dexamethasone also suppresses histone H3 acetylation at the StAR promoter. These obser- vations raise the possibility that glucocorticoids in vivo may act directly on the corticosteroid-producing cells of the adrenal gland. This may contribute to the adrenal suppres- sion seen with therapeutic glucocorticoid administration.
Materials and methods
Materials
Bovine serum albumin (BSA; fraction V, fatty-acid free), 8-bromo-adenosine 3’,5’-cyclic monophosphate (8-Br- cAMP), proteinase K, salmon sperm DNA, and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO), unless otherwise stated. Dexamethasone (dexamethasone phosphate) was obtained from Narn Guang Chemical Co. (Tainan, Taiwan). Ham’s F12/ Dulbecco’s modified Eagle’s medium (F12/DMEM; 1:1), fetal calf serum (FCS), and other culture supplies were purchased from Gibco-BRL (Grand Island, NY). [a-32P] Deoxy-CTP (3,000 Ci/mmol) was obtained from NEN Life Science Products (Boston, MA). Rabbit anti-acetylated histone H3 antibody was purchased from Upstate Biotech- nology (Lake Placid, NY). Horseradish peroxidase- conjugated donkey anti-rabbit IgG antibody was purchased from Amersham Life Science (Buckinghamshire, England). Mouse StAR cDNA and StAR antiserum were provided by Dr. D.M. Stocco (Department of Cell Biology & Bio- chemistry, Texas Tech University Health Sciences Center, Lubbock, TX). Mouse glyceraldehyde-3-phosphate dehy- drogenase (GAPDH) cDNA was a generous gift from Dr. Hsiao-Sheng Liu (Department of Microbiology, College of Medicine, National Cheng Kung University, Tainan, Taiwan).
Cell culture
The mouse Y-1 adrenocortical tumor cells, a generous gift from Dr. Bernard P. Schimmer (Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontaria, Canada) were maintained in F12/DMEM supplemented with 20 mM HEPES (pH 7.4), 400 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml strepto- mycin in the presence of 10% FCS. Cells were grown in 75-cm2 tissue culture flasks at 37°℃ in a 5% CO2-air atmosphere and subcultured into 100-mm tissue culture dishes at a density of approximately 2.5 x 10° cells 24 h prior to use in experiments. The Y-1 cells have a reduced capacity for 21-hydroxylation and 11-hydroxylation reac- tions compared with primary adrenal cells. They synthesize progesterone and accumulate P450scc mRNA after stimu- lation of 8-Br-cAMP [57].
Mitochondrial protein and whole-cell extract preparation
Mitochondrial protein preparations were performed as previously described [58] with slight modifications. After addition of 1.0 ml of TSE buffer (10 mM Tris-HCI, 250 mM sucrose, 0.1 mM EDTA, pH 7.4), the cells were scraped off the dish with a rubber policeman and sonicated (50% amplitude, 10 s/cycle, 3 cycles; Dr. Hielschel Soni- cator, Model: UP-50 H; Senecoscience Prestinar, Milano, Germany). After centrifugation at 600g for 30 min at 4℃, the supernatant was collected and centrifuged at 13,800g for 30 min at 4℃. The pellet was resuspended in 300 ul of TSE buffer. Whole-cell lysates were prepared by lysing Y- 1 cells in 0.3 ml of RIPA lysis buffer (10 mM Tris-HCI, 150 mM NaCl, 5 mM EDTA, 0.1% sodium dodecyl sulfate (SDS), 1% Nonidet P-40 (NP-40), 0.25% deoxycholate, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml leupeptin, and 1 µg/ml aproti- nin). The cells were scraped off the dish with a rubber policeman and rotated for 20 min at 4℃. Cells were then passed through a 23-gauge needle to shear the DNA. After centrifugation at 13,800g for 10 min at 4℃, the superna- tant was removed. Protein concentrations in the mitochondrial protein and whole-cell lysate extracts were determined by Lowry’s method [59]. All samples were stored at -85℃ until further analysis.
Western blot analysis
Fifty micrograms of whole-cell lysates and mitochondrial protein were analyzed as previously described [58]. The protein samples were separated by 12.5% SDS-PAGE,
transferred to polyvinylidene difluoride membrane, and then the membranes were blocked in phosphate-buffered saline (PBS) containing 0.5% Tween 20 (PBST) and 4% non-fat dry milk for 1 h. The membrane was incubated with primary antibody (anti-StAR, 1:2,000 or anti-acety- lated histone H3, 1:400) in PBST with 0.1% BSA and immunoblot analysis performed using a horseradish per- oxidase-conjugated donkey anti-rabbit IgG antibody (1:3,000). Specific immunoactive bands were detected using the Western lightening Chemiluminescent Kit (NEN Life Science Products). Band intensities were determined using either the Arcus II computer-assisted image system (PDI Inc., Huntington Station, NY) or AlphaImager 2200 system (AlphaInnotech Inc., San Leandro, CA). Values obtained were expressed as integrated optical density units.
Northern blot analysis
Levels of StAR mRNA in the dose-response effect of dexamethasone were evaluated by Northern blot analysis using a mouse StAR cDNA probe as previously described [60]. Total RNA (25 µg) from each sample was fraction- ated in a 1% agarose gel and transferred onto a nylon membrane and UV cross-linked. Northern blots were pre- incubated for 1 h at 60℃ in QuikHyb solution (Stratagene, La Jolla, CA) and hybridized for 3 h at 60°℃ in the same solution containing heat-denatured [32P]-labeled StAR or GAPDH cDNA and salmon sperm DNA. Mouse StAR and GAPDH probes were labeled by random priming with [a-32P]deoxy-CTP using the Megaprime DNA Labeling System (Amersham). After hybridization, the blots were washed twice in 2x SSC/0.1% SDS at room temperature for 15 min each, and once in 0.1x SSC/0.1% SDS for 20 min at 42°C. After washing, the membrane was exposed to X-ray film at -85℃ for 24 h for autoradiographic sig- nals. The intensity of the bands on Northern blots was measured by the Arcus II computer-assisted image system. Values for StAR mRNA were normalized to values for the GAPDH.
Construction of the native and competitor templates for StAR
To quantitate the mRNA level of StAR in Y-1 cells treated with or without dexamethasone (10 µg/ml) in the presence of 8-Br-cAMP (0.5 mM) for various time intervals, we adopted standard curve quantitative, competitive (QC)- reverse transcription (RT)-polymerase chain reaction (PCR; QC-RT-PCR) method as described previously [61] using a competitive internal standard that consisted of a shortened StAR DNA fragment as illustrated in Fig. 3a.
Specific primer pairs for StAR designed according to sequences deposited in GenBank No. L36063 were as follows: sense 5’-AGATGTGGGCAAGTGTGTTTC-3’ (P1), antisense 5’-CAGGTCAATGTGGACAG-3’ (P2), and internal reverse primer 5’-CAGGTCAATGTG GTGGACAGTGAGCAGCCAAGTGAGTTTAG-3’ (IP). The IP, a 41 base-pair (bp) primer, was designed to include a sequence of 21 bp (underlined, matching the solid-lined region in Fig. 3a) that was 171 bp upstream from the P2 sequence appended to the 3’-end of P2 sequence. Thus, using P1 as the 5’-primer and P2 as the 3’-primer, a 540 bp of the StAR sequence was amplified. With the appended 21 bp of primer IP as the 3’-primer, this resulted in a 389 bp (369 bp + 20 bp from P2) amplicon. An internal region of 151 bp was deleted from the 540-bp DNA fragment.
QC-RT-PCR
Mouse StAR mRNA isolated from Y-1 cells was reverse- transcribed using oligo(dT)15 (MWG Biotech, Taipei, Taiwan) and SuperScript II M-MLV RNase H” reverse transcriptase (Invitrogen, Carisbad, CA) according to manufacturer’s instruction. Two microliter of RT product was PCR-amplified using a forward (P1) and a reverse (P2) primer. A 540-bp PCR fragment was obtained and cloned into pCRII vector (Invitrogen), which represented plasmid containing “native” sequence. Using a forward (P1) and an internal reverse (IP) primer, a DNA fragment of 389 bp was amplified by PCR and then subcloned into pCRII vector, representing plasmid containing “competitor” sequence. Both plasmids were linearized by KpnI restric- tion enzyme (Roche Molecular Biochemicals, Mannheim, Germany) and transcribed in vitro using T7 RNA Poly- merase (Promega, Madison, WI). RNase-free DNase I (Sigma) was used to remove plasmid DNA from RNA pool. In vitro-transcribed RNA was precipitated twice with 1/10 volume of 2 M sodium acetate (pH 4.0) and two volumes of 100% ethanol after phenol-chloroform extraction. The concentration of RNA was determined by 260 nm absorbance. RNA was then aliquoted and stored at -85℃ until used.
A constant amount (1.5 fentomole) of competitor and native RNA in 2 ul of diethylpyrocarbonate-treated water or 0.1 µg of unknown mRNA samples was added into 0.2-ml thin-wall PCR tubes (ABgene, Rochester, NY) containing 12 ul of 1x RT master mixture [1x RT buffer, 10 mM dithiothreitol (DTT), 0.5 µg oligo(dT)15, 2 mM deoxynucleoside triphosphate (dNTPs), and 50 U Super- Script RNase H- reverse transcriptase (Invitrogen)]. The final volume of RT mixture was 30 ul, and reverse tran- scription was performed at 42℃ for 75 min followed by
heating to 95℃ for 10 min and quick-chilled to 4℃ in a programmable thermal cycler (ABI model 2700, Perkin- Elmer Corp., Foster City, CA). RT products of unknown samples were diluted 1:100 with double-distilled water, while RT products of native and competitor RNA were diluted to 25.6 attomole and 2 attomole per 2 ul double- distilled water, respectively. The standard QC-RT-PCR assay contained 2 attomole competitor RNA and eight serial dilutions of native RNA (from 25.6 to 0.2 attomole). Two microliter of the diluted competitor RT products was added into 7 ul of 1x PCR mixture [1x PCR buffer, 0.2 mM dNTPs, 0.5 U Taq DNA Polymerase (ABgene), and 0.4 uM of primers]. This mixture was then dispensed into 0.2-ml thin-wall PCR tubes, and 2 ul of the diluted native RT products (serially diluted) or 2 ul of unknown sample RT products were added individually to each tube. The final volume of PCR mixture was 25 ul. The PCR mixture was heated for 4 min at 95℃ as the initial dena- turation. PCR amplifications were then performed under the following conditions: 30 s denaturation at 94℃, 30 s annealing at 65℃, and 40 s elongation at 72ºC for 35 cycles, followed by 5 min at 72℃ as the last primer extension. Ten microliter of PCR products were directly separated on 2% agarose gel with 1x TAE buffer (45 mM Tris-acetate, 1 mM EDTA, pH 8.0) at 100 V for 30 min using Mupid-2 Mini-Gel Electrophoresis Unit (Cosmo Bio Co. Ltd., Tokyo, Japan). The gel was then stained with ethidium bromide and photographed under UV illuminator equipped with a camera connected to a computer (Fig. 3b). The gel image was analyzed using AlphaImager software. A ratio was calculated for the intensity of native versus competitor bands on each lane of the gels. The logarithmic ratio of native to competitor was plotted against the loga- rithmic amount of initial native to produce the standard curve (Fig. 3c), and concentrations of specific mRNA transcripts were determined by comparison to the standard curve.
Chromatin immunoprecipitation (ChIP) assay
A modification of the technique described by Li et al. [62] for ChIP assay was used. Formaldehyde (Sigma) was added directly to the cell culture medium at a final concentration of 1% for 15 min (37℃) to cross-link DNA and its asso- ciated proteins. Cross-linking was terminated upon addition of glycine (125 mM final concentration) for 10 min. Cells were washed once in PBS before scraping in PBS containing protease inhibitors (1 mM PMSF, 1 µg/ml aprotinin, and 1 µg/ml leupeptin). Cells were resuspended in 400 ul SDS lysis buffer (50 mM Tris-HC1, pH 8.1, 1% SDS, 10 mM EDTA) containing protease inhibitors, fol- lowed by incubation on ice for 10 min. Samples were then
sonicated on ice for three times for 30 s each. Sonicated samples were centrifuged (13,800g, 10 min, 4℃) to spin down cell debris. The chromatin solution was diluted with 1.1 ml of dilution buffer (16.7 mM Tris-HCI, pH 8.1, 1.2 mM EDTA, pH 8.1, 0.01% SDS, 1.1% Triton X-100, 1 mM PMSF, 1 µg/ml aprotinin, and 1 µg/ml leupeptin). A supernatant fraction (50 ul) from diluted chromatin mix- tures was saved as an input control. The chromatin preparation was precleared with 80 ul of salmon sperm DNA-protein A/Sepharose (Amersham; 20% slurry) for 30 min at 4℃ with rotation. One part of the chromatin complexes was then incubated with 5 µg of anti-acetylated histone H3 antibody and rotated at 4℃ overnight. The other was incubated with normal rabbit serum as non- immune serum control. Immune complexes were collected with 60 ul of salmon sperm DNA-protein A/Sepharose (20% slurry) with rotation for 1 h at 4℃. The supernatant was collected as an unbound control. Immunoprecipitates were sequentially washed for 5 min in wash buffer A (20 mM Tris-HC1, pH 8.1, 2 mM EDTA, 0.1% SDS, 1% Triton X-100, 150 mM NaCl), wash buffer B (2 mM Tris- HCl, pH 8.1, 2 mM EDTA, 0.1% SDS, 1% Triton X-100, 500 mM NaCl), wash buffer C (10 mM Tris-HCI, pH 8.1, 1 mM EDTA, 250 mM NaCl, 1% NP-40, 1% sodium deoxycholate), and TE buffer (10 mM Tris-HC1, 0.1 mM EDTA, pH 8.0) (two times). Washed beads were extracted with 250 ul of elution buffer (1% SDS, 100 mM NaHCO3) two times. The elutes were combined in one tube, treated successively for 4 h at 65°℃ in 200 mM NaCl (20 ul) to reverse cross-links and then incubated for 40 min at 57℃ with 40 µg/ml of proteinase K in proteinase K buffer (40 mM Tris-HCl, pH 6.5, 10 mM EDTA). The DNA was extracted with phenol/chloroform, ethanol precipitated, and diluted in 10 ul double-distilled water. StAR promoter associated DNA was amplified by PCR. Each PCR mixture contained 2 ul of immunoprecipitate, unbound or input control, 0.4 uM each primer, 0.2 mM dNTPs mixture, 2.5 units of Thermus icelandicus Taq DNA polymerase (ABgene) and 1x Taq PCR buffer in a total volume of 25 ul. The primers for the mouse StAR promoter were selected to include the proximal promoter when a large number of the described transcriptional response elements reside (-68 to -153). The primers are as follows: forward, 5’-ACCTGCAGAGTCTGGTCCTC-3’ (bases -153 to -135); reverse, 5’-TCAAGTGCGCTGCCTTAAAT-3’ (bases -88 to -110). PCR amplifications were carried out under the following conditions: 30 s at 94℃, 30 s at 60℃, and 40 s at 72°℃ for 35 cycles, preceded by 4 min at 95℃ and followed by 5 min at 72℃. The PCR products were separated in 2% agarose gels. The gel was stained with ethidium bromide and then placed on a UV illuminator equipped with a camera connected to a computer. The gel image was analyzed using AlphaImager software.
RT-PCR
To determine the mRNA level of GR in Y-1 cells treated with or without dexamethasone in the presence or absence of 8-Br-cAMP for 24 h, total RNA was analyzed by RT- PCR. One hundred nanogram of RNA dissolved in steril- ized water was used for the RT. First strand synthesis was performed in 30 ul of 5x RT buffer, 10 mM DTT, 0.5 µg oligo(dT)15, 0.2 mM dNTPs, and 6.67 units of murine leukemia virus reverse transcriptase (ABgene), and 0.1 µg of total RNA. The reaction was incubated at room tem- perature for 10 min, at 42℃ for 75 min, at 95℃ for 10 min, and then at 4℃ in a GeneAmp 2700 PCR system. The primers for the mouse GR are as follows: forward, 5’-ACCTCGATGACCAAATGACC-3’; reverse, 5’-TCTGGA AGCAGTAGGTAAGGAGA-3’. Forward and reverse primers for mouse ß-actin are as follows: forward, 5’- ATCTGGCACCACACCTTCTACAATGAGCTGCG-3’; reverse, 5’-CGTCATACTCCTGCTTGCTGATCCACAT CTGC-3’. PCR reactions were performed on 2 ul of the cDNA in a final reaction volume of 25 ul in the presence of 2.5 units of Thermus icelandicus Taq DNA polymerase (ABgene). After denaturation at 95℃ for 4 min, PCR amplification was carried out by 35 cycles of incubation at 94℃ for 30 s, 58℃ for 30 s, and 72℃ for 40 s, followed by a final extension at 72℃ for 5 min. The PCR products (10 ul) were electrophoresed in a 2% agarose gel with 1 x TAE buffer (45 mM Tris-acetate, 1 mM EDTA, pH 8.0) at 100 V for 30 min using Mupid-2 Mini-Gel Electrophoresis Unit. The gel was then stained with ethidium bromide and placed on a UV illuminator equipped with a camera con- nected to a computer. The signal intensities of the PCR amplified products were quantified using AlphaImager computer software.
Radioimmunoassay (RIA) for progesterone
Quantitation of progesterone directly from aliquots of the medium was performed by RIA as previously described [63]. The antiserum to progesterone-11x-BSA (C467-B4) was supplied by Dr. J.E. Hixon (Department of Veterinary Bio- sciences, College of Veterinary Medicine, University of Illinois, Urbana, IL), and used at the dilution of 1:25,000 in 0.1 M Tris buffer (pH 7.4). The intra-assay variation, determined by duplicates of three dose levels of the control medium from cultured pig corpora luteal cells, was less than 10%. The sensitivity of the assay was 12.5 pg per assay tube.
Statistical analysis
Two means were compared using Student’s t-test. Where there were more than two means, significant differences
between means were determined by analysis of variance (ANOVA). The means were then analyzed by Fisher’s PLSD multiple comparison.
Acknowledgements We thank Dr. Hsiao-Sheng Liu for plasmid, Dr. Douglas M. Stocco for antibody and plasmid and Ms. Li-Ming Huang and Mr. Jyh-Chen Lin for technical assistance. This study was supported by a Grant (NSC91-2320-B006-065) from the National Science Council, Republic of China.
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