Steroid Sulfotransferase 2A1 Gene Transcription Is Regulated by Steroidogenic Factor 1 and GATA-6 in the Human Adrenal
Karla J. Saner,
Takashi Suzuki,
Hironobu Sasano,
John Pizzey,
Clement Ho,
Jerome F. Strauss, III,
Bruce R. Carr and
William E. Rainey
University of Texas Southwestern Medical Center (K.J.S., B.R.C., W.E.R.), Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, Dallas, Texas 75390-9032; Tohuku University School of Medicine (T.S., H.S.), Department of Pathology, Sendai, Japan 980-8574; Kings College London (J.P.), London, United Kingdom SE1 1UL; and University of Pennsylvania Medical Center (C.H., J.F.S.), Center for Research on Reproduction and Womens Health, Philadelphia, Pennsylvania 19104
Address all correspondence and requests for reprints to: William E. Rainey, Ph.D., Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9032. E-mail: william.rainey{at}utsouthwestern.edu.
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ABSTRACT
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Sulfonation is a phase II conjugation reaction responsible for the biotransformation of many compounds including steroids, bile acids, and drugs. Humans are presently known to express at least five cytosolic sulfotransferase (SULT) enzymes, of which only two are hydroxysteroid SULT, SULT2A1, commonly known as steroid sulfotransferase, and the cholesterol sulfotransferase SULT2B1. SULT2A1 is highly expressed in the adrenal where it is responsible for the sulfation of hydroxysteroids including conversion of dehydroepiandrosterone to dehydroepiandrosterone sulfate and in the liver where it is responsible for sulfation of bile acids and circulating hydroxysteroids. Little is known concerning the transcriptional regulation of human SULT2A1 in adrenal. Herein we demonstrate the role of two transcription factors, steroidogenic factor 1 (SF1) and GATA-6, in the regulation of SULT2A1 transcription. These transcription factors were quantified by real-time RT-PCR in normal human adrenal tissue. Transient transfection assays with deleted and mutated SULT2A1 promoter constructs allowed for the determination of specific SF1 and GATA binding cis-regulatory elements necessary for transactivation of SULT2A1 promoter, and binding was confirmed by EMSA analysis. Both SF1 and GATA-6 were positive regulators of SULT2A1 promoter constructs. These data support the hypothesis that adrenal SULT2A1 expression is regulated by SF1 and GATA-6.
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INTRODUCTION
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SULFONATION IS A phase II conjugation reaction responsible for the biotransformation of many compounds including bile acids, steroids, and drugs. Conjugation of a compound with a charged sulfate moiety generally increases aqueous solubility and subsequent renal excretion from the body (1). Humans are presently known to express at least five cytosolic sulfotransferase (SULT) enzymes, of which only two are hydroxysteroid SULT, SULT2A1, commonly known as steroid sulfotransferase, and the cholesterol sulfotransferase SULT2B1 (2). This differs significantly from what occurs in common laboratory animals. Rats, for example, have five characterized hydroxysteroid SULT isoforms, which are expressed at higher levels in female rats (3). The expression of human SULT2A1 occurs predominantly in the liver and adrenals and does not appear to be sex regulated. Due to its high levels in the liver, SULT2A1 is the proposed mechanism for bile acid sulfonation of secondary bile acids such as lithocholic acid and chenodeoxycholic acid (1, 4). The production of sulfonated steroids is very high in the adrenal such that the production of dehydroepiandrosterone sulfate (DHEA-S) is quantitatively the most abundant hormone secreted by the human adrenal (5). Circulating levels of DHEA-S are likewise very high in the adult adrenal due to both the high production rate and the relatively low clearance rate from the blood (6). SULT2A1 has been localized by immunohistochemistry to the dehydroepiandrosterone (DHEA)-producing zona reticularis of the adrenal cortex where it catalyzes the conversion of DHEA to its sulfated form (7, 8). Although the enzymatic activity of SULT2A1 has been studied in some detail, little is known about the regulation of human SULT2A1 expression in any tissue.
Determining the mechanisms that regulate SULT2A1 gene expression may also aid in understanding the dynamic production of DHEA and DHEA-S throughout the human lifespan. The fetal adrenal produces large amounts of DHEA and DHEA-S, which in turn are further metabolized to estrone and estradiol via aromatase and 17ß-hydroxysteroid dehydrogenase type I, respectively (9). After birth, the infant adrenal undergoes restructuring from the fetal to adult phenotype and ceases to produce significant amounts of DHEA or DHEA-S (10). The process known as adrenarche occurs between ages 68, and the adrenal again begins to produce DHEA and DHEA-S in large amounts (11). DHEA and DHEA-S production increases in both sexes until the mid-20s when it peaks, with men having higher production levels than females. After this point, production rates decline and again reach basal levels in both males and females around the seventh decade (10). The regulation of adrenal DHEA and DHEA-S production throughout the lifespan remains to be elucidated.
Regulation of adrenal development and steroidogenesis is mediated in part by the orphan nuclear receptor steroidogenic factor 1 (SF1, NR5A1) (12, 13). This transcription factor is crucial to adrenal development as SF1 knockout mice fail to develop a functional adrenal (14). SF1 is also a critical factor in the regulation of adrenal cytochrome P450 enzymes. SF1 has been shown to bind the DNA consensus sequence CAAGGTCA and LXXLL-related motifs (15).
The GATA family of transcription factors have been demonstrated to be potent regulators of transcription in a wide variety of tissues including the heart and gonads (16, 17, 18, 19, 20). Recent studies indicate that GATA-6 is highly expressed in the adult and fetal adrenal cortex (21) and is capable of regulating transcription of steroidogenic enzymes, including SULT2A1 (22). Several studies have demonstrated that a related family member, GATA-4, is capable of regulating transcription of several genes encoding steroid-metabolizing enzymes in the gonads through interactions with SF1 (23, 24) and is also present in the human fetal but not adult adrenal (21). In addition, a second, longer GATA-6 isoform has recently been identified which utilizes an alternate upstream initiation codon from that which was previously reported (25).
Herein we demonstrate that SF1 is able to activate transcription of the human SULT2A1 gene in a dose-dependent manner using specific cis-elements on the SULT2A1 promoter. We further demonstrate that both isoforms of GATA-6 are present in the human adrenal and can also regulate adrenal SULT2A1 gene transcription both alone and in combination with SF1 by binding to their respective nuclear response elements on the regulatory region of the SULT2A1 gene.
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RESULTS
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The Human Adrenal Expresses High Levels of SULT2A1 and SF1
The human steroid sulfotransferase enzyme is highly expressed in both the adrenal and liver (7, 26, 27). To determine whether SULT2A1 gene expression correlated to SF1 (NR5A1) expression, we quantified both SF1, and as a comparison, its liver homolog LRH (liver receptor homolog, NR5A2) in adrenal and liver tissues. As shown in Fig. 1
in a log scale, mRNA levels of SULT2A1 were 9.2 attomol/µg 18S RNA in the adrenal and 11.4 attomol/µg 18S RNA in the liver. Use of real-time RT-PCR demonstrated that mRNA, like enzyme activity, is highly expressed in both tissues. SF1, which is known to play a crucial role in adrenal and gonadal development (14), was highly expressed in the adrenal (3.83 attomol/µg 18S RNA) but expression was almost undetectable in the liver (0.02 attomol/µg 18S RNA). The converse is true of the SF1 homolog LRH, which regulates the expression of rat 7
-hydroxylase (28), a crucial rate-determining enzyme in bile acid biosynthesis (29). LRH expression in the liver was 1.62 attomol/µg 18S RNA, which was almost 50 times higher than that of SF1 (0.03 attomol/ µg 18S RNA). These results indicate that both SF1 and LRH may play a tissue-specific role in regulation of SULT2A1 gene expression, and we decided to further characterize the role of SF1 in adrenal regulation of SULT2A1.

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Fig. 1. Quantification of SULT2A1, SF1, and LRH mRNA Levels in Human Adrenal and Liver
Data represent the mean ± SEM of at least five independent deoxyribonuclease-treated RNA samples and are expressed in attomoles of mRNA per µg of 18S RNA. Note that the data are presented with a log scale.
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SF1 Can Activate SULT2A1 Transcription
Although SF1 has been previously shown to act as a potent activator of several genes involved in steroidogenesis (30), little is known about the regulation of SULT2A1 transcription. We tested the effect of SF1 on SULT2A1 transactivation in vitro using the nonsteroidogenic human embryonic kidney (HEK)293 cell model to perform transient transfections. SULT2A1 was regulated in a dose-dependent manner by SF1 with maximal stimulation of 5-fold over basal with a maximal activity at a dose of 0.1 µg expression vector per well (Fig. 2A
). DAX (dosage-sensitive sex reversal-adrenal hypoplasia congenital critical region on the X chromosome, gene 1) is a known repressor of SF1-mediated activation of a number of genes (31, 32). Therefore, to determine whether the effect of SF1 on SULT2A1 gene transcription was specific, we tested the ability of DAX to repress SF1-mediated SULT2A1 transcriptional activation (Fig. 2B
). DAX was able to repress SULT2A1 activation of SF1 to levels that were below basal activity.

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Fig. 2. Effect SF1 and/or DAX on Transcriptional Activity of SULT2A1
A, Concentration-dependent effects of SF1 on SULT2A1 reporter gene activity. HEK293 cells were transfected with luciferase reporter constructs containing the SULT2A1 promoter construct at a concentration of 1 µg/well. Cells were cotransfected with the indicated amount of SF1 expression plasmid or the empty expression plasmid pcDNA 3.1 zeo. Cells were lysed and assayed for luciferase activity after 24 h. Data were normalized to cotransfected Renilla luciferase, and data shown are expressed as fold induction over basal reporter. Results represent the mean ± SEM of data from three independent experiments each performed in triplicate. Asterisks indicate that these values are statistically significant from basal levels (P < 0.0001). B, Effects of DAX on SF1 stimulation of SULT2A1 transfection. DAX completely mitigated the effect of SF1 on SULT2A1 gene transcription in HEK293 cells. Cells were transfected with SULT2A1 luciferase reporter constructs (1 µg/well) and the indicated expression vector (0.1 µg/well). Cells were lysed and assayed for luciferase activity after 24 h. Data were normalized to cotransfected Renilla luciferase, and data shown are expressed as fold induction over basal reporter. Results represent the mean ± SEM of data from at least three independent experiments each performed in triplicate.
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The Proximal SULT2A1 Promoter Contains Several Functional SF1 Binding Sites
Although production of adrenal DHEA-S varies drastically throughout the human lifespan (10, 33, 34), little is known about the regulation of SULT2A1. Examination of the human SULT2A1 promoter (1259 bp) revealed six potential SF1 binding sites (Fig. 3A
) that have been previously characterized to be a nuclear receptor-binding half-site, CAAGGTCA (12). Five of the six potential sites listed are not perfect consensus sites but were chosen by their previously demonstrated ability to bind SF1 with relatively high affinity. To narrow our focus of potential binding sites, sequential deletion constructs were created and cotransfected with SF1 expression vector to determine what region(s) of the promoter were necessary for optimal transactivation of the gene (Fig. 3B
). A small drop in transcriptional activation occurred between 1259 and 1063, implicating the 1205 SF1 binding site. However, mutational analysis of the 1205 site had no effect on SF1-mediated transactivation. A complete loss of SULT2A1 promoter activity over basal was seen with the loss of sites at 85 and 65. SF1-mediated SULT2A1 transactivation was reduced by approximately 30% by disruption of the 65 site, but activation was completely lost when the 85 site was altered (Fig. 3C
). Mutation of both the 85 and 65 sites resulted in a complete loss of activation over basal levels for SF1-mediated transactivation, indicating a compensatory role for these binding sites.

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Fig. 3. SULT2A1 Promoter Analysis
A, Schematic representation of SULT2A1 promoter with potential SF1 binding sites. Gray circles represent potential nuclear receptor half-sites, and the numbers below represent the base pair at which the site begins based on the translational start site. B, A series of pGL3 Basic reporter constructs containing progressively smaller amounts of SULT2A1 5'-flanking DNA (1 µg/well) were cotransfected in HEK293 cells with SF1 expression plasmid (0.1 µg/well). Data were normalized to cotransfected Renilla luciferase, and fold induction was calculated relative to the basal promoter control. Results represent the mean ± SEM of data from three independent experiments each performed in triplicate. C, Mutation analysis of the three putative SF1 binding sites on the SULT2A1 promoter indicating the importance of both the 85 and 65 binding sites. Asterisks indicate that the value is statistically significant from the full-length, nonmutated plasmid cotransfected with the same transcription factor (P < 0.0001).
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GATA Transcription Factors Are Present in Human Adrenal
We and others have recently demonstrated the presence of GATA-6 and GATA-4 in the human adrenal cortex and ovarian follicle, respectively (22, 35, 36, 37). Using real-time RT-PCR the expression of GATA-6 and GATA-4 transcripts was further characterized in adrenal as well as liver for comparison. Real-time RT-PCR analyses indicated that GATA-6 is present in the adrenal (
0.28 attomol/µg 18S RNA) at levels that were 14 times greater than seen for GATA-4 (0.02 attomol/µg 18S RNA). GATA-6 mRNA and protein were virtually undetectable in the liver, whereas GATA-4 mRNA was detected in the liver (Fig. 4A
).

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Fig. 4. Expression of GATA in Human Adrenal and Liver
A, Real-time RT-PCR analysis of GATA-4 and GATA-6 expression in human adrenal (n = 4) and liver (n = 4). GATA-6 levels were high in the adrenal and low in the liver, and the converse is true of GATA-4. Values are expressed in attomoles of mRNA per µg of 18S RNA. B, Immunohistochemical analysis of SULT2A1 (left panel), GATA-6 (center panel), and SF1 (right panel) in human adult adrenals. Each panel is of a serial section of the same field. M, Medulla; R, reticularis; F, fasciculata. Bar = 50 µm.
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Immunohistochemical data in the human adrenal gland support the real-time data. As previously demonstrated (7, 8), SULT2A1 shows intense staining in only the reticularis portion of the adrenal cortex (Fig. 4B
, left panel), whereas SF1 also shows intense staining throughout the cortex (Fig. 4B
, right panel). Similarly, GATA-6 exhibits a diffuse pattern of expression in the adrenal reticularis and fasciculata (Fig. 4B
, center panel), contrasting to previously published data using in situ hybridization and immunohistochemistry utilizing a different GATA-6 antibody (21).
GATA-6 Can Activate SULT2A1 Transactivation via a Binding Site on the SULT2A1 Promoter
GATA-6 has been shown previously by our group to activate transcription of the genes encoding the steroidogenic enzymes necessary for synthesizing DHEA-S in the adrenal cortex (22). However, additional transcription factors such as SF1 were required for GATA-6 stimulation of steroidogenic acute regulatory protein, cholesterol side-chain cleavage (CYP11A), and 17
-hydroxylase/17,20 lyase (CYP17), but not SULT2A1. In a nonsteroidogenic cell system devoid of cofactors such as SF1, GATA-6 is able to induce a concentration-dependent increase in SULT2A1 transactivation, with a maximal 4-fold increase at a dose of 0.3 µg per well (Fig. 5
).

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Fig. 5. Concentration-Dependent Effects of GATA-6 on SULT2A1 Reporter Gene Activity
HEK293 cells were transfected with luciferase reporter constructs containing the SULT2A1 promoter construct at a concentration of 1 µg/well. Cells were cotransfected with the indicated amount of GATA-6 expression plasmid or the empty expression plasmid pcDNA 3.1 zeo. Cells were lysed and assayed for luciferase activity after 24 h. Data were normalized to cotransfected Renilla luciferase, and data shown are expressed as fold induction over basal reporter. Results represent the mean ± SEM of data from at least two independent experiments each performed in quadruplicate. All values except 0.001 µg are significantly higher than basal (P 0.05).
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Analysis of the proximal 5'-flanking region of SULT2A1 (1259 bp) revealed three potential GATA binding sites, and each consisted of the consensus response element sequence for GATA family members, (A/T)GATA(A/G) (Fig. 6A
) (16, 38). Sequential deletion analysis revealed that GATA-6-induced SULT2A1 transactivation was lost only when the 190 GATA binding site was deleted (Fig. 6B
). Although all GATA family members are able to bind the same consensus site, GATA-6 is the only member currently identified in the human postnatal adrenal cortex (Fig. 4
) (39). It has been well established that the related factor GATA-4 can synergize with SF1 to amplify transcriptional activation of genes in the gonads (23, 24, 40, 41). Additionally, our initial experiments demonstrated that co-transfection of GATA-6 and SF1 yields an additive effect to activate transcription compared with either gene alone (22). In this study we used mutational analysis to determine whether functional SF1 binding was necessary for optimal GATA-6 activation of SULT2A1. Mutation of the 190 GATA binding site lowered SULT2A1 transactivation to almost basal levels (Fig. 7A
), which is in agreement with the promoter deletion analysis implicating only the 190 site as being necessary for GATA-6 mediated activation of SULT2A1 gene transcription. Transfection analysis using the SULT2A1 double mutant (85/65) cotransfected with GATA-6 showed a slight drop in activation compared with wild-type activation (Fig. 7A
), whereas SF1-mediated activation of the SULT2A1 promoter with a mutated GATA response element showed no change compared with wild-type activation. These data indicate that GATA-6 and SF1 can stimulate SULT2A1 reporter activity independently of one another although maximal stimulation of the wild-type promoter is achieved when both factors are present.

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Fig. 6. The Roles of GATA Binding cis-Elements in the Regulation of SULT2A1 Transcription
A, Schematic representation of SULT2A1 promoter with potential GATA binding sites. Gray triangles represent potential binding sites, and the numbers below represent the base pair at which the site begins based on the translational start site. B, Deletion analysis. A series of pGL3 Basic reporter constructs containing progressively smaller amounts of SULT2A1 5'-flanking DNA (1 µg/well) were cotransfected in HEK293 cells with GATA-6 expression plasmid (0.1 µg/well). Data were normalized to cotransfected Renilla luciferase and expressed as relative luciferase units. Results represent the mean ± SEM of data from three independent experiments each performed in triplicate. Analysis of different lengths of the SULT2A1 promoter indicate one putative GATA-6 binding site in the promoter region, at 190. The GATA-stimulated 117 deletion is significantly lower than the full-length SULT2A1 clone stimulated by GATA (P < 0.001).
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Fig. 7. Effect of SF1 and GATA-6 in SULT2A1 Gene Transcriptional in Nonsteroidogenic and Steroidogenic Cell Lines
A, Mutation analysis of the SULT2A1 promoter in nonsteroidogenic HEK293 cells. Effects of SF1 and GATA-6. SF1 and GATA-6 are able to stimulate induction of the wild-type SULT2A1 promoter (1259 bp) both separately and together. When both the 85 and 65 putative SF1 binding sites are mutated, GATA-6 stimulation is not greatly affected, but both SF1 and SF1/GATA-6 binding are abrogated. Mutation of the GATA-6 binding site effects all SF1 and GATA-6 transcriptional activation. The activity of the GATA-stimulated SULT2A1 190 mutation is significantly lower than that of its full-length counterpart (P < 0.001). B, Effect of GATA-6 on SULT2A1 gene transcription in H295R adrenocortical cells. H295R cells were transfected with luciferase reporter constructs containing the SULT2A1 promoter construct at a concentration of 1 µg/well. Cells were cotransfected with the indicated expression vector (0.3 µg/well). Cells were lysed and assayed for luciferase activity after 24 h. Data were normalized to cotransfected Renilla luciferase, and data shown are expressed as fold induction over basal reporter. Results represent the mean ± SEM of data from at least three independent experiments each performed in triplicate.
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Both Isoforms of Human GATA-6 Are Present in Adrenal and Can Activate SULT2A1 Gene Transcription
It has been reported previously that both the human and mouse genomes have two isoforms of GATA-6, which utilize two distinct promoters and initiation codons (25). Both isoforms are present throughout the tissues where GATA-6 is known to be normally present in the fetal and adult mouse, including extensive expression in the heart (25, 42). In the human, both the long and short isoforms of GATA-6 were found to be more highly expressed in ovarian theca cells of women affected by polycystic ovarian syndrome than in theca cells of normal controls (43). Analysis of the adrenal protein levels of each isoform indicates that both the long and short proteins are being produced, although the long form again seems to be the predominant isoform in the adrenal (Fig. 8A
). The converse is true in the adrenal carcinoma cell line H295R where the short isoform is the predominant protein expressed. Transient transfection analyses were performed in nonsteroidogenic HEK293 cells with expression vectors encoding either the long form (MALT), the short form (MYQ), or a mutated form that can only encode the long isoforms (M147L). As illustrated in Fig. 8B
, whereas basal expression of GATA-6 is marginally higher with the mutated long-only construct, no significant differences are observed between the three constructs when SF1 is included in the transfection.

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Fig. 8. The Presence of GATA-6 Isoforms Varies in Adrenal But Does Not Affect SULT2A1 Gene Transcription
A, Western analysis of GATA-6 long- and short-isoform protein in H295R and human adult adrenal (HAA) nuclear extracts. In vitro prepared short (GATA-6 Short) and long and short (GATA-6 Long/Short) proteins were included as positive controls as well as a mock in vitro protein preparation as a negative control. B, Comparison of the effects of GATA-6 isoforms on the transcriptional activity of SULT2A1 reporter gene activity with and without SF1. HEK293 cells were transfected with luciferase reporter constructs containing the SULT2A1 promoter construct at a concentration of 1 µg/well. Cells were cotransfected with the indicated GATA-6 isoform expression plasmid (0.1 µg/well) with (open bars) or without (black bars) SF1 (0.1 µg/well) or the empty expression plasmid pcDNA 3.1 zeo. Cells were lysed and assayed for luciferase activity after 24 h. Data were normalized to cotransfected Renilla luciferase, and data shown are expressed as fold induction over basal reporter. Results represent the mean ± SEM of data from at least three independent experiments each performed in triplicate.
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SF1 and GATA-6 Specifically Bind to Their Respective cis-Elements
To determine whether SF1 interacts directly with either the 85 or 65 cis-element, 32P-labeled oligonucleotides containing sequence to correspond with these specific elements were prepared and used in EMSAs (Fig. 9A
). Both elements bound in vitro prepared SF1 as well as proteins in nuclear extracts from adult adrenal tissue, although the 85 site bound with greater efficiency than the 65 site. A single specific band was inhibited by the addition of 100-fold excess unlabeled homologous oligonucleotide, indicating that the complexes formed represented specific binding. In addition, in vitro synthesized GATA-6 protein was tested with both oligonucleotide sets targeting the SF1 response element, and no binding was observed (data not shown).

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Fig. 9. Binding of SF1 and GATA-6 to Putative Response Elements on the SULT2A1 Gene Promoter by EMSA
A, EMSA of SF1 cis-elements. EMSA was performed using 32P-labeled oligonucleotide probes containing the 85 or 65 consensus sequences of SULT2A1. Radiolabeled probe alone (FP; free probe) is shown in lane 1. Lanes 2 and 3 (85 probe) and lanes 6 and 7 (65 probe) correspond to labeled probe incubated with nuclear extracts of protein from human adult adrenal tissue alone and with 100-fold molar excess nonradiolabeled self-competitor DNA, respectively. Lanes 4 and 5 (85 probe) and lanes 8 and 9 (65 probe) consist of the same experimental conditions as lanes 2, 3, 6, and 7 except with in vitro-translated SF1 as the protein source. In all cases a protein-DNA complex was formed that was able to be inhibited by the addition of 100-fold molar excess unlabeled oligonucleotide probe, although stronger binding is observed with the probe targeting the 85 region. B, EMSA of GATA cis-elements. EMSA was peformed using 32P-labeled oligonucleotide probes containing the 190/195 GATA consensus sequence of SULT2A1. Radiolabeled probe alone (FP; free probe) is shown in lane 1. Lanes 24 correspond to labeled probe incubated with in vitro-translated GATA-6 short protein (GATA Short), incubated with GATA-6 short and antibody targeting GATA-6 (GATA Short + Ab), and incubated with nonradiolabeled self-competitor DNA in a 100-fold molar excess (GATA Short + 100x), respectively. Lanes 57 and lanes 810 consist of the same experimental conditions as lanes 24 except with in vitro-translated GATA-6 long and short protein (GATA L/S) and human adult adrenal nuclear extract (HAA) as the protein source, respectively. In all cases a protein-DNA complex was formed that was able to be retarded by the addition of antibody targeting GATA-6 and inhibited by the addition of 100-fold molar excess unlabeled oligonucleotide probe.
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Further EMSA studies were performed with 32P-labeled oligonucleotides corresponding to the 190/195 cis-element for GATA. This element was able to bind in vitro synthesized proteins corresponding to both the long/short and short-only isoforms of GATA-6 as well as from nuclear extract derived from a human adult adrenal (Fig. 9B
). Complex formation was retarded, causing a supershift, when an antibody targeting GATA-6 was added to the reaction mixture. Inhibition of complex formation occurred when 100-fold excess unlabeled homologous oligonucleotide was added, indicating that the complexes formed again represented specific binding. In vitro prepared SF1 was also incubated with oligonucleotides targeted to the 190/195 GATA site with no specific binding observed (data not shown). Further studies were performed with mutated oligonucleotides targeting the GATA response element in which addition of 100-fold excess unlabeled mutant oligonucleotide was unable to block complex formation (data not shown).
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DISCUSSION
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The human steroid sulfotransferase gene, SULT2A1, is responsible for adrenal conversion of the C19 steroid hormone DHEA to DHEA-S as well as for the sulfonation of bile acids in the liver. Production of DHEA-S in the human adrenal relies on three steroid-metabolizing enzymes, which include CYP11A, CYP17, and the steroid sulfotransferase SULT2A1 (10). DHEA and DHEA-S fill a unique niche in adult and fetal life through their peripheral conversion to more potent steroids such as estradiol and testosterone. The best example for use of DHEA-S as a precursor steroid occurs in the human fetus where in late gestation up to 200 mg of steroids/d, approximately 60% of which is DHEA-S, are produced and subsequently used by the placenta as precursors for estrogen biosynthesis (44, 45). Although sulfonation of most molecules including hepatic bile acids generally increases solubility and aids in excretion from the system (26), sulfonation of DHEA to DHEA-S in both fetus and adult creates a more stable molecule, which allows DHEA-S to remain in circulation, thus providing a pool of substrate for placental estrogen production (46, 47, 48). It has been reported that approximately 30% of androgens in men and greater than 90% of estrogens in postmenopausal women are thought to be synthesized in peripheral tissues from DHEA-S (49), illustrating the importance of these compounds as androgen precursors.
Of the enzymes necessary for DHEA-S biosynthesis, CYP11A and CYP17 are common to other metabolic pathways as well. Both genes are required for production of cortisol in the human adrenal cortex (50, 51), and therefore a great deal more is known about their transcriptional regulation. The CYP17 gene, like most adrenal P450 steroidogenic enzymes, requires the transcription factor SF1 for both basal and cAMP-mediated expression (52, 53). SF1 is critical for adrenal development and is found in all steroidogenic tissues, including the adrenal and gonads (14). In addition, it has recently been reported in several studies that adrenal CYP17 requires the GATA-6 transcription factor for gene transcription (22, 54). However, this factor does not act through the GATA response element on the promoter but instead requires an interaction with Sp1 at the Sp1 binding site (54). Unlike the steroidogenic P450 enzymes, regulation of the human adrenal SULT2A1 gene has not been well characterized. Hornsby and Aldern (55) examined cultured fetal adrenal cells and noted that ACTH treatment increased the activity of the SULT2A1 enzyme. Further studies by McAllister and Hornsby (56) illustrated that forskolin also induced enzyme activity whereas the protein kinase C activator 12-O-tetradecanoyl phorbol 13-acetate inhibited sulfotransferase activity in cultured fetal adrenal cells. Additionally, fetal adrenal studies by Parker et al. (57, 58) demonstrated that sulfotransferase mRNA levels were increased in a dose-dependent manner by ACTH and that this increase was blunted by cytokines such as TGFß and TNF
.
SULT2A1 is present in high amounts in human liver hepatocytes where it functions to sulfonate a wide range of secondary bile acids (4, 29, 30). Due to their detergent-like qualities, secondary bile acids are hepatotoxic, and sulfonation of these compounds reduces toxicity and increases solubility and subsequent renal excretion. Although there is a wealth of data examining regulation of SULT2A1 in the rat hepatocyte (34, 59, 60, 61, 62), sequence comparisons reveal little similarity between rat and human SULT2A1 gene-regulatory regions. Both positive and negative regulation of hepatic SULT2A1 transcription has been studied in the rat in relation to its effects on bile acid biosynthesis. Rat SULT2A1 transcription is positively regulated by the secondary bile acid chenodeoxycholic acid via the farnesoid X receptor (NR1H4) (62) and negatively regulated indirectly via the androgen receptor (61). Humans do not appear to exhibit sex-dependent differences in the expression of SULT2A1 in the liver, and this may be due to the divergence seen between the rat and human 5'-flanking region of this gene, which may also help to explain the lack of expression in the rat adrenal gland. This lack of expression in rodent adrenals as well as other common laboratory models has led to a paucity of information regarding adrenal SULT2A1 regulation.
The present study demonstrates that SF1 and GATA-6 are required for the regulation of SULT2A1. Although there are a number of putative SF1 response elements present in the regulatory region of SULT2A1, it is the 85 site and, to a lesser extent, the 65 site that induce transcription of SULT2A1 gene expression in the adrenal. Deletion analysis also identified a region in the promoter, from 228 to 117, which, when lost, resulted in an approximate 50% decrease in SF1-mediated transcriptional activation, although no SF1 response elements were present in that region. However, a perfect consensus GATA response element, at 190/195, was present in that region which was able to bind and directly activate SULT2A1 gene transcription along with SF1. Mutating either the SF1 or GATA cis-acting element in the promoter completely negated the effect of that respective factor on SULT2A1 reporter gene activation. However, each factor is able to activate promoter activity above basal levels in the absence of the other, indicating that SF1 and GATA-6 act independently, although maximal stimulation is achieved when both factors are present and all response elements are intact.
GATA-6 is a member of a highly conserved family of six trans-acting nuclear regulatory proteins characterized by the consensus sequence (response element) to which they bind, (A/T)GATA(A/G) (16, 38). Based on detailed embryonic studies of several GATA family knockout mice, these transcription factors have been further subgrouped based on their roles in embryonic development. The hematopoetic group consists of GATA 1, -2, and -3 whereas the cardiac group is comprised of GATA 4, -5, and -6 (16, 38). Each GATA family member has distinct spatial and developmental expression patterns such that there is little overlap between factors. The closely related factors GATA-4 and GATA-6 are both found in the human gonads. Both factors are also found in the fetal adrenal cortex although GATA-6, but not GATA-4, has been localized to the adult adrenal cortex by our laboratory as well as others (21, 39). Several studies have indicated that GATA-4 positively regulates expression of genes involved in steroidogenesis in the ovary and testis such as steroidogenic acute regulatory protein and Müllerian inhibiting substance. GATA-4 has been shown to positively regulate transcription of Müllerian inhibiting substance gene in Sertoli cells of the testes, and this effect has been further augmented by SF1 (23). Further, the increased induction by the combination of SF1 and GATA-4 can be ablated by DAX-1 (24). Our findings, that GATA-6 works in concert with SF1 on SULT2A1 gene transcription, are the first to demonstrate a regulatory mechanism for the human adrenal enzyme SULT2A1.
Human GATA-6 consists of two transcript isoforms transcribed from two distinct promoters (25). The long isoform of GATA-6 (MALT) encodes a protein of 595 amino acids whereas the short isoform (MYQ) encodes a protein of 449 amino acids. In this study we examined the protein levels of each GATA-6 isoform in the human adrenal as well as the ability of each isoform to activate SULT2A1 reporter gene activity alone and in combination with SF1. Western analysis indicated that, in normal human adrenal, the long form was preferentially expressed whereas in the adrenal carcinoma cell line H295R the short isoform was the major isoform present. These results are similar to a published report that illustrated higher levels of the short isoform in theca cells from polycystic ovarian syndrome patients as compared with normal theca cells (43), indicating a possible link to steroidogenic pathology when higher levels of short-form protein are present. Transactivation studies in a nonsteroidogenic cell model were in agreement with a previous study in that basal activation of SULT2A1 was highest with the long form of GATA-6 (25). However, our results indicate there was no significant difference in the effects of either isoform on SULT2A1 transcription when SF1 was cotransfected.
Although the human SULT2A1 enzyme was purified and characterized in 1980 (63), lack of an appropriate animal model has stymied research regarding its regulation in humans. The role of orphan nuclear receptors as trans-acting regulators of transcription has been the key to understanding the mechanisms controlling regulation of the SULT2A1 gene. The results presented here demonstrate an adrenal-specific regulation of the SULT2A1 gene by the orphan nuclear receptor SF1 and the zinc-finger protein GATA-6.
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MATERIALS AND METHODS
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RNA Extraction, cDNA Synthesis, and Real-Time RT-PCR
Normal human adult adrenals and liver were obtained through the Cooperative Human Tissue Network (Philadelphia, PA). The use of these tissues was approved by the Institutional Review Board of the University of Texas Southwestern Medical Center (Dallas, TX). Total RNA was extracted from tissues (64), and purity and integrity of the RNA were checked both spectroscopically and by gel electrophoresis. Deoxyribonuclease I (2 µg) (Ambion, Inc., Austin, TX)-treated total RNA was reverse transcribed using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) following the manufacturers recommendations and stored at 80 C. Primers for the amplification of the target sequences were based on published sequences for the human LRH, SF1, SULT2A1, GATA-4, and GATA-6. The primer sequences used were: LRH (GenBank accession no. NM_003822) forward: 5'-TTACCGACAAGTGGTACATGGAA-3' (exon 6); reverse: 5'-CGGCTTTGTGATGCTATTATGGA-3' (exon 7) that gave an 89-bp fragment; SF1 (NM_004959) forward: 5'-GGAGTTGTCTGCCTCAAGTTCA-3' (exon 6); reverse: 5'-CGTCTTTCACCAGGATGTGGTT-3' (exon 7) that gave an 80-bp fragment; SULT2A1 (NM_003167) forward: 5'-TCGTGATAAGGGATGAAGATGTAATAA-3' (exon 1); reverse: 5'-TGCATCAGGCAGAGAATCTCA-3' (exon 2) that gave a 84-bp fragment; GATA-4 (NM_002052) forward: 5'-CGGAGGGCGAGCCTGTGT-3'; reverse: 5'-CCGCATTGCAAGAGGCCTGGG-3' that gave a 75-bp fragment; and GATA-6 (NM_005257) forward: 5'-GCGCGTGCCTTCATCAC-3'; reverse: 5'-TCTGCGCCATAAGGTGGTAG-3' that gave a 76-bp fragment.
PCRs were performed using the ABI Prism 7000 Sequence Detection System (Applied Biosystems) with a total volume of 30 µl per reaction following the reaction parameters recommended by the manufacturer. The 30 µl total volume consisted of SYBR Green Universal PCR Master Mix (2x) (Applied Biosystems), 50 nM of each primer, and 5 µl of each first-strand cDNA sample. Standard curves were prepared using human SULT2A1, SF1, LRH, GATA-4, and GATA-6 vectors. Negative controls contained water instead of first-strand cDNA. Samples were normalized on the basis of 18S ribosomal RNA content (µg). The quantification of the 18S in each sample was performed using a TaqMan Ribosomal RNA Reagent Kit (Applied Biosystems) following the manufacturers recommendations.
Cell Culture and Transfection Assays
HEK293 cells were routinely cultured in DMEM/Ham F12 medium (Life Technologies, Carlsbad, CA) supplemented with 5% NuSerum (Collaborative Biom, Bedford, MA) and antibiotics. Transfection assays were performed with HEK293 cells seeded at 200,000 cells per well in 12-well dishes using Fugene 6 (Roche, Indianapolis, IN) to transfect 1 µg of reporter plasmid and the indicated amounts of expression vectors for 6 h. To ensure constant amounts of DNA for each transfection, pcDNA 3.1 zeo empty vector was added to each well as needed. To normalize luciferase activity, cells were cotransfected with 50 ng/well of Renilla plasmid (Promega Corp., Madison, WI). Cells were assayed 24 h after recovery for activity using the Dual Luciferase assay system (Promega).
H295R human adrenocortical tumor cells were cultured in DMEM/Hams F12 medium supplemented with 10% Cosmic Calf serum (Hyclone Laboratories, Inc., Logan, UT) and antibiotics. For transfection experiments, cells were subcultured onto 12-well dishes at a density of 400,000 cells per well. Transfections were carried out using the transfection reagent Transfast (Promega) according to manufacturers directions for 6 h. To normalize luciferase activity, cells were cotransfected with 50 ng/well of Renilla plasmid (Promega). After recovery of the cells for 2024 h, cells were lysed and assayed for activity using the Dual Luciferase assay system (Promega).
Preparation of Reporter Constructs and Expression Vectors
The 5'-flanking DNA from the human SULT2A1 gene was created by PCR from human adrenal genomic DNA using a forward primer derived from sequence upstream of exon 1(5'-GCCAACTGATCTGTGTATAGTCCTTTA-3') and a reverse primer derived from sequence in exon 1 that is 5' to the translational start site (5'-GTGGTGTGAGGGTTTCAACTGTAG-3') (GenBank accession no. L36191). The resulting PCR product of approximately 1259 bp was T/A cloned into the pCRII-TOPO vector (Invitrogen, Carlsbad, CA). The insert was then digested and inserted upstream of the firefly luciferase gene in the reporter vector pGL3 Basic (Promega) using the restriction sites SacI and XhoI. The primers listed in Table 1
were used in conjunction with a vector-specific primer to create the SULT2A1 promoter deletion constructs 1063, 535, 228, 190, and 117 from the full-length construct of 1259 bp. The deletion construct SULT2A1 332 was created using a HindIII restriction site in the full-length promoter construct. Human SF1, DAX, SHP, and GATA-6 were inserted into the eukaryotic expression vector pcDNA 3.1 zeo (Invitrogen) as previously described (22).
Mutations to putative SF1 binding sites in the SULT2A1 promoter were created using the QuikChange XL Site Directed Mutagenesis kit (Stratagene, La Jolla, CA) following manufacturers recommendations. Primer sets used for the mutations can be found in Table 1
.
The construct pMALT, encoding the full-length human GATA-6, was a gift from Dr. Christopher Gove (Kings College London, UK) (25). Expression plasmids hG6-MALT and hG6-MYQ have been described previously (65). To generate hG6-M147L encoding only the long, but not the short, isoform of GATA-6, a PCR was carried out using pMALT-ORF as template and the following primer pair: forward, 5'-GCTGAGCCCCTTCGCACCCGAGCAGCCGGAGGAGCTCTACCAGACCCTCGCCGCTC-3'; and reverse, 5'-GCCATGGGCGGGCTGGGAGAGTAGGGGAAG-3' to introduce site-directed mutagenesis at the second transcriptional start site (ATG
CTC) such that methionine 147 of the long isoform was replaced by leucine. Leucine was chosen because both methionine and leucine are nonpolar and generally interchangeable without causing conformational changes (66). The resultant PCR product was then digested with BlpI and ligated to BlpI-restricted hG6-MALT to yield hG6-M147L.
Immunohistochemistry
Immunohistochemical analysis was performed on serial sections of adrenal tissue employing the streptavidin-biotin amplification method using a Histofine Kit (Nichirei, Tokyo, Japan). Antibodies used include SULT2A1 (67), SF1 (kindly provided by Dr. K. Morohashi, National Institute for Basic Biology, Okazaki, Japan (68), and GATA-6 (rabbit polyclonal, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Antigen retrieval was performed by heating the slides in a microwave oven for 15 min in citric acid buffer (2 mM citric acid and 9 mM trisodium citrate dehydrate, pH 6.0). The dilutions of the primary antibodies used in this study were: SULT2A1 (1:1000), SF1 (1:1000), and GATA-6 (1:600). The antigen-antibody complex was visualized with 3.3'-diaminobenzidine solution (1 mM 3.3'-diaminobenzidine; 50 mM Tris-HCl buffer, pH 7.6; and 0.006% H2O2), and counterstained with hematoxylin. For negative controls (data not shown), normal rabbit or mouse IgG was used instead of primary antibodies, and no specific immunoreactivity was detected in these sections. Histological identification of three zones of the human adrenal cortex was based on previously published criteria (69).
Western Blot Analysis
Nuclear extracts from H295R cells were prepared as previously described (70). Nuclear extracts from frozen adrenal tissues were prepared by first homogenizing tissue in Tris-buffered saline (2 mM Tris; 13.7 mM NaCl, pH 7.6) and resuspending the pellet in 400 µl ice-cold buffer A (10 mM HEPES, pH 7.9; 10 mM KCl; 0.1 mM EDTA; 0.1 mM EGTA; 1 mM dithiothreitol; and 0.5 mM phenylmethylsulfonylfluoride). Cells were allowed to swell on ice 15 min, and then 25 µl of a 10% Nonidet P-40 solution were added. The sample was then vortexed and centrifuged at 3000 x g for 30 sec. The pellet was then resuspended in 50 µl ice-cold buffer C (20 mM HEPES, pH 7.9; 0.4 M NaCl; 1 mM EDTA; 1 mM EGTA; 1 mM dithiothreitol; and 1 mM phenylmethylsulfonylfluoride). The solution was then rocked vigorously at 4 C for 15 min and protein content was measured. Human GATA-6 MYQ and M147L proteins were prepared using an in vitro transcription/translation system (Promega). PAGE was carried out using NuPAGE 412% Bis-Tris gels (Invitrogen) in 3[N-morpholino]propane sulfonic acid buffer. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes for 75 min at 30 V. After transfer, the membranes were blocked for 2 h at room temperature with a 5% milk solution and incubated overnight at 4 C with GATA-6 antibody (1:2000) obtained from Santa Cruz Biotechnology, Inc. Membranes were washed with Tris-buffered saline-Tween (2 mM Tris; 13.7 mM NaCl, pH 7.6; 0.5% Tween-20) before incubation with horseradish peroxidase-conjugated secondary antibody. Membranes were washed with Tris-saline-Tween (2 mM Tris; 30 mM NaCl, pH 7.4; 0.05% Tween-20), and immunoreactive bands were visualized using the ECL plus Western Blotting Detection System (Amersham Biosciences, Buckinghamshire, UK).
EMSA
Nuclear extracts from human adult adrenal tissue were prepared as described above. EMSAs were performed as previously described (70) using the primers listed in Table 1
for mutation primers, but without the mutations.
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FOOTNOTES
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This work was supported by National Institutes of Health Grants T32-HD07190 (to B.R.C.), HD-06274 (to J.F.S.), HD11149 (to W.E.R.), DK069950 (to W.E.R.), and DK43140 (to W.E.R.).
First Published Online September 23, 2004
Abbreviations: CYP11A, Cholesterol side-chain cleavage; CYP17, 17
-hydroxylase/17,20 lyase; DAX, dosage-sensitive sex reversal-adrenal hypoplasia congenital critical region on the X chromosome, gene 1; DHEA, dehydroepiandrosterone; DHEA-S, dehydroepiandrosterone sulfate; HEK, human embryonic kidney; LRH, liver receptor homolog; SF1, steroidogenic factor 1; SULT, sulfotransferase.
Received for publication August 29, 2003.
Accepted for publication September 14, 2004.
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