![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Vol. 29, Issue 8, 1130-1135, August 2001
Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
The 5'-flanking region [1892 base pairs (bp)] of the rat aryl
sulfotransferase (SULT1A1) gene was cloned and the
cis-acting sequences involved in
glucocorticoid-inducible SULT1A1 gene transcription were characterized.
SULT1A1 promoter and 5'-flanking sequences lacked a TATA box and a
consensus glucocorticoid response element. Using a 5'-rapid
amplification of cDNA ends approach, four SULT1A1 transcription
start sites were identified. Transient transfection studies with
SULT1A1-5':luciferase reporter constructs in primary cultured rat
hepatocytes revealed that treatment with the potent glucocorticoid
dexamethasone (10
9-105 M) produced
concentration-dependent increases in luciferase activity in constructs
containing from 1892 to 119 bp of the SULT1A1 5'-flanking region.
Relative to the most upstream SULT1A1 transcription start site, the
minimal cis-acting sequences that were required for dexamethasone-inducible SULT1A1 expression were located between 
84
and 69 bp. Treatment of transfectants with a panel of steroids, including dexamethasone, triamcinolone acetonide, hydrocortisone, dihydrotestosterone, 17
-estradiol, and
pregnenolone-16
-carbonitrile, revealed that steroid-inducible
SULT1A1 gene expression was specific for glucocorticoid-class steroids.
Concentration-response studies, coupled with a robust inhibition of
glucocorticoid-inducible SULT1A1-5':luciferase reporter activity by
antiglucocorticoid/antiprogestin RU-486, recapitulated earlier findings
on endogenous SULT1A1 gene expression and implicated a major role for
the glucocorticoid receptor transcription factor in the regulation of
glucocorticoid-inducible SULT1A1 gene expression.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
The
aryl sulfotransferase (SULT1)1 enzyme family
plays a major role in xenobiotic metabolism. Sulfate conjugation
generally results in the formation of a polar end product that is
amenable to excretion and elimination from the body (detoxication). In rat liver, the presence of four classes of distinct SULT1 isoforms, including aryl/minoxidil sulfotransferase (SULT1A1) (Falany and Kerl,
1990
; Hirshey et al., 1992), a thyroid hormone/dopa-tyrosine sulfotransferase (SULT1B1) (Yamazoe et al., 1994; Sakakibara et al.,
1995; Araki et al., 1997), two estrogen sulfotransferases (SULT1E)
(Demyan et al., 1992; Falany et al., 1995; Rikke and Roy, 1996), and a
SULT1 enzyme that catalyzes the bioactivation of
N-hydroxy-2-acetylaminofluorene carcinogen (SULT1C1) (Nagata et al., 1993; Yamazoe et al., 1994), underscores the broad range of
substrate specificities that stamp this important subfamily of
conjugating enzymes.
SULT1A1 readily catalyzes the sulfation of simple phenols such as
1-naphthol and p-nitrophenol and also sulfates estrogens at
micromolar concentrations (Falany et al., 1994). Of all the SULT1
enzymes, SULT1A1 is particularly active in drug metabolism. SULT1A1 is
widely expressed in both hepatic and in metabolically active
extrahepatic tissues (Dunn and Klaassen, 1998; Dooley et al., 2000) and
catalyzes the sulfation of a number of common pharmaceutics such as
acetaminophen and minoxidil (Nagata et al., 1993; Yamazoe et al.,
1994). In most cases, sulfation by SULT1A1 is a detoxication reaction
for labile reactive intermediates (Larrey et al., 1986). However, the
sulfation of minoxidil by SULT1A1 is required for prodrug activation,
and represents an essential step in the conversion of minoxidil to its
physiologically active form (McCall et al., 1983; Meisheri et al.,
1993). Because alterations in SULT1A1 expression may have important
implications for drug metabolism and for hepatocellular toxicity during
drug therapy, it is critical to gain a clear understanding of the
molecular regulation of the SULT1A1 gene.
SULT1A1 is one of the "male dominant" SULT1 enzymes that undergoes
age-related changes in gene expression and is more abundantly expressed
in male relative to female rat liver (Liu and Klaassen, 1996a).
The basis for age- and gender-dependent changes in rat hepatic SULT1A1
gene expression is not yet known, but may involve pituitary factors
other than growth hormone (Liu and Klaassen, 1996a). Other
conditions that, like hypophysectomy, have pleiotropic effects on gene
expression, also affect rat hepatic SULT1A1 gene expression. For
example, the administration of CYP2B-inducing doses of phenobarbital to
rats significantly suppressed rat hepatic SULT1A1 mRNA expression
(Runge-Morris et al., 1998). Similarly, treatment of primary cultured
rat hepatocytes with CYP1A1-inducing concentrations of the
"environmental hormone"
2,3,7,8-tetrachlorodibenzo-p-dioxin produced substantial
decreases in SULT1A1 expression (Runge-Morris, 1998).
Glucocorticoids have been strongly implicated in the transcriptional
regulation of SULT1A1 gene expression, both in hepatic and
extra-hepatic tissues. For example, the treatment of cultured bovine
tracheobronchial epithelial cells with hydrocortisone produced concentration-dependent increases in SULT1A1 enzyme activity (Beckmann et al., 1994) and mRNA levels (Schauss et al., 1995). Similarly, the
administration of pharmacological doses of the potent glucocorticoid DEX to rats increased SULT1A1 mRNA levels in both male and female rat
liver (Liu and Klaassen, 1996b). Additional studies, which demonstrated glucocorticoid-mediated induction of SULT1A1 expression in
primary cultured rat hepatocytes, provided sound evidence that glucocorticoids regulate SULT1A1 gene expression directly at the level
of the hepatocyte and suggested a proximal role for the glucocorticoid
receptor transcription factor in the transcriptional control of SULT1A1
(Runge-Morris et al., 1996). The purpose of the present analysis was to
probe the molecular mechanisms that control glucocorticoid-inducible
SULT1A1 gene transcription. For these studies, the 5'-flanking region
of the rat SULT1A1 gene was cloned and cis-acting genomic
sequences that mediate glucocortioid-sensitive SULT1A1 gene expression
were evaluated in transient transfection studies conducted in primary
cultured rat hepatocytes.
| |
Experimental Procedures |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
Materials.
Steroids (DEX, triamcinolone acetonide, hydrocortisone,
dihydrotestosterone, -estradiol, and pregnenolone
16-carbonitrile) were purchased from Sigma Chemical Co. (St. Louis,
MO). Custom-synthesized oligonucleotides were purchased from Integrated
DNA Technologies, Inc. (Coralville, IA). The Rat GenomeWalker kit was
obtained from CLONTECH (Palo Alto, CA). The FirstChoice RLM-RACE
kit was obtained from Ambion (Austin, TX). All other supplies and
reagents were obtained from the sources previously described
(Runge-Morris et al., 1996; Kocarek et al., 1998).
Cloning of 5'-Flanking Region of SULT1A1 Gene.
An ~1.9-kb fragment of the 5'-flanking region of the rat SULT1A1 gene
was isolated using the PCR-based gene walking method (Rat GenomeWalker
kit). In a PerkinElmer Gene Amp PCR system 9700, two nested PCR
reactions were performed according to the manufacturer's instructions
with minor modifications. Five genomic GenomeWalker libraries were used
as templates, and two primers AP1 and AP2, corresponding to the adapter
sequences, were provided in the kit. Two gene-specific primers (GSP1
and GSP2) were selected based on the published rat aryl
sulfotransferase IV (SULT1A1) structural gene sequence (Khan et al.,
1993; GenBank accession no. L16241). GSP1 (5'-CACCTGATCCTGGGGTTCTGA-3',
+63 to +83) was paired with AP1 and used for primary PCR, while GSP2
(5'-CCTCCGGGTGCTCAGTGATA-3', +37 to +56) and AP2 were used for
secondary PCR. The annealing temperature was 70°C for primary PCR and
72°C for secondary PCR. An ~1.9-kb PCR product was obtained using
the DraI library, cloned into the pGEM-T Easy vector
(Promega, Madison, WI) and sequenced completely. The insert was then
transferred into pBluescript SK+ (Stratagene, La
Jolla, CA) at the NotI site, and finally ligated into the
firefly luciferase reporter plasmid pGL3-Basic (Promega) at the
SacI and XhoI sites.
Determination of SULT1A1 Transcription Start Site. The transcription start site of the SULT1A1 gene was identified using the 5'-RACE method and the First Choice RLM-RACE kit (Ambion), according to the manufacturer's instructions. Briefly, following treatment of total RNA from mature (age ~55 days) male Sprague-Dawley rat liver with calf intestinal alkaline phosphatase and tobacco acid pyrophosphatase, an RNA adapter was ligated to the 5' ends of the decapped mRNAs. The complementary DNA strands were then generated by reverse transcription of the adapter-containing RNAs, and fragments containing the 5' end of SULT1A1 mRNA were amplified by two consecutive PCR reactions. The first amplification was performed using the outer RNA adaptor primer provided with the kit and an outer SULT1A1 gene-specific primer (5'-GGAACCCCTGGACATTTGAACTCA-3'). The inner RNA adapter primer provided with the kit and the inner SULT1A1 gene-specific primer (5'-CGCGGCCACACTTCTCTAGCTTGCCACCCTGAT-3') were used for secondary PCR. The annealing temperature for both PCR reactions was 60°C. The PCR products were subcloned into the pGEM-T Easy vector, and the inserts contained in seven independent clones were sequenced.
Preparation of SULT1A1-5':Luciferase Reporter Constructs.
A fragment of the SULT1A1 5'-flanking region from 1892 to +56 was
inserted into the promoter-less luciferase reporter plasmid pGL3-Basic
as described above. Reporter plasmids containing nested deletions of
the SULT1A1 5'-flanking region were generated either by unique
restriction enzyme digestions or by PCR reactions. Specifically, constructs 745 and 449 were generated by restriction enzyme digestion with KpnI and NsiI, respectively,
followed by recircularization with T4 DNA ligase. Constructs 320,
119, 84, and 69 were generated by PCR. Using a 1982- to +56-bp
fragment of the SULT1A1 gene as template, a series of 5' primers were
designed to incorporate a SacI site for subcloning
(5'-GAGCTCAGGCGTGTGAATGCTCTG-3' for construct 320,
5'-GAGCTCTAACAACTCCGCCCCACT-3' for construct 119, 5'-GAGCTCTGTTTCTGGAGAACAGCCA-3' for construct 84,
5'-GAGCTCGCCAGTCCTAGCACTGTTT-3' for construct 69). The 3' primers
were designed with a XhoI site that was identical for all of
the constructs (5'-CTCGAGTCCGGGTGCTCAGTGATA-3'). These amplified
fragments were initially ligated into the pGEM-T Easy vector and then
cloned into the SacI and XhoI sites of
pGL3-Basic. The sequences of all constructs were verified by sequence
analysis (Center for Molecular Medicine and Genetics DNA Sequencing
Facility, Wayne State University, Detroit, MI).
Transient Transfections and Luciferase Assays.
The procedures for the isolation, primary culture, and transient
transfection of rat hepatocytes have been described previously (Kocarek
and Reddy, 1996; Runge-Morris et al., 1999). Briefly, hepatocytes
isolated from mature male Sprague-Dawley rats (220-300 g; Harlan,
Indianapolis, IN) were plated onto 12-well Vitrogen-coated plates
(3 × 105 hepatocytes/well) in Williams'
medium E supplemented with 0.25 U/ml insulin, 100 U/ml penicillin, 100 µg/ml streptomycin, and 107 M triamcinolone
acetonide. After ~21 h, the hepatocytes in each well were transfected
with 0.8 µg of reporter plasmid and cotransfected with 0.08 µg of
the pRL-TK plasmid (Promega) in 0.6 ml of Opti-MEM containing 5.5 µg
of Lipofectin reagent (Life Technologies, Inc., Grand Island,
NY). The pRL-TK plasmid, which expresses Renilla luciferase,
was used to normalize for variations in transfection efficiency among
samples. After 5 h of incubation, culture medium was replaced with
standard Williams' medium E without triamcinolone acetonide, and
hepatocytes were overlaid with 100 µg of Matrigel (Collaborative
Research Products, Bedford, MA) pipetted into the medium. At 48 h
after plating, hepatocytes were treated with either steroid or 0.1%
DMSO vehicle in fresh medium. After 24 h of treatment, hepatocytes
were harvested and lysed for luciferase activity measurement. The Dual
Luciferase Reporter Assay system (Promega) and a Dynex model MLX
luminometer were used for luciferase assays. Experimental data were
analyzed by the Student's t test using a two-tailed distribution and two-sample equal variance (GraphPad Software, San
Diego, CA).
| |
Results |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
Cloning and Sequence Analysis of 5'-Flanking Region of SULT1A1
Gene.
Although the coding sequence of the rat SULT1A1 gene (trivial name aryl
sulfotransferase IV) was previously reported (Khan et al., 1993), the
sequence of the SULT1A1 5'-flanking region and the location of the
transcription start site of this gene have not been established. To
explore the molecular mechanism of glucocorticoid-inducible SULT1A1
gene expression, an ~1.9-kb fragment of the rat SULT1A1 gene was
amplified by anchored PCR using oligonucleotides corresponding to
sequences located near the 5' end of the published SULT1A1 structural
gene sequence as gene-specific primers (Fig.
1). The fragment was subcloned into the
pGEM-T Easy vector, and sequence analysis confirmed that 27 bp of the
3' end of the PCR fragment were identical to the published 5' end of
the SULT1A1 structural gene (Khan et al., 1993).
|
) failed to identify
a consensus binding site for the glucocorticoid receptor (i.e., GRE).
The consensus GRE (GGTACA NNN TGT(T/C)CT, TRANSFAC matrix table) is a
variation of the pallindromic repeat of AGAACA, in which the two halves
are separated by 3 bp (i.e., AGAACA NNN TGTTCT). This motif is also
known as an IR3 for inverted repeat separated by 3 bp. We noted that,
located slightly upstream of the SULT1A1 transcription start site, at
bp 75 to 70 (see below for an explanation of this numbering
scheme), the perfect AGAACA hexanucleotide sequence was present (Fig.
1). Beginning 4-bp downstream from this half-site was a sequence
(AGTCCT) that differed from the GRE TGT(T/C)CT consensus
sequence by only one base (underlined), suggesting the possible
existence of an IR3 steroid receptor motif (Fig. 1). In addition, the
sequence TGTTTC, which also resembled a GRE half-site, was located just
upstream of the AGAACA half-site, suggesting the possible presence of
an ER3 (everted repeat) motif (Fig. 1). We also noted that, as has been
frequently reported for sulfotransferase genes (Her et al., 1995,
1998), the SULT1A1 gene did not contain a TATA box.
Determination of SULT1A1 Transcription Start Site. The transcription start site of the rat SULT1A1 gene was determined using a 5'-RACE approach. Seven independent clones were sequenced and aligned to the SULT1A1 gene sequence. The results suggested the existence of multiple transcription start sites within a 30-bp region (Fig. 1A). We have assigned the 5'-most site (30 bp upstream from the published 5' end of the rat structural gene) as bp +1 and all positions in the 5'-flanking region have been indexed relative to this reference transcription start site.
Functional Characterization of 5'-Flanking Region of SULT1A1
Gene.
To locate the region of the SULT1A1 gene that is essential for
conferring glucocorticoid-inducible promoter activity, a series of
deletion constructs of the 5'-flanking region were prepared and
subcloned into a promoter-less luciferase reporter plasmid (Fig. 1B).
Primary cultured rat hepatocytes were transiently transfected with each
of these luciferase reporter constructs and then treated for 24 h
with the potent glucocorticoid DEX (107 M). As
shown in Fig. 2, luciferase activity was
markedly increased in hepatocyte cultures that were transfected with
each of the SULT1A1 constructs containing from 119 to 1892 bp of
5'-flanking sequence. Relative to the benchmark 1892 construct, the
basal expression and magnitude of glucocorticoid-inducible luciferase activity were diminished in transfectants containing some of the more
truncated constructs, such as 119 and 69 (Fig. 2). These results
suggest that there may be cis-acting regulatory elements located between 1892 and 119 that are necessary for the
recapitulation of full glucocorticoid responsiveness (Fig. 2). Although
the glucocorticoid-inducible luciferase activity for construct 69 was
shown as significantly elevated relative to the control, this
statistical difference was not upheld in repeated studies. Therefore,
the nucleotide sequence between 119 to 69 appeared to be essential
for achieving glucocorticoid-mediated induction of SULT1A1 gene
transcription.
|
). This mode of gene regulation was revealed
by differences in the glucocorticoid concentration-response relationships that were observed when primary cultured rat hepatocytes were transfected with reporter constructs containing different fragments of the SULT2-40/41 5'-flanking region (Runge-Morris et al.,
1999). To examine the possibility that the regulation of SULT1A1 by
glucocorticoids is also complex, primary cultured rat hepatocytes that
were transiently transfected with either the full-length 1892
construct or with the minimal DEX-responsive 119 construct were
treated with DEX at concentrations ranging from
109 to 105 M. Consistent with our previous observations regarding DEX-mediated induction of SULT1A1 mRNA expression in primary cultured rat
hepatocytes (Runge-Morris et al., 1996), DEX treatment produced a
concentration-dependent induction of reporter gene expression in
primary cultured rat hepatocytes that were transfected with the
1892-bp SULT1A1-5':luciferase construct with a maximum increase of
~6- to 7-fold relative to DMSO control occurring at a DEX
concentration of 107 M (Fig.
3A). This same concentration-dependent
pattern of DEX-mediated induction was retained in primary cultured rat
hepatocytes transfected with the 119-bp SULT1A1 5'-luciferase
construct, although relative to the 1892 construct, the magnitude of
DEX-inducible luciferase activity in the shorter 119 fragment was
considerably reduced (Fig. 3B).
|
).
Therefore, to determine whether the same pattern of glucocorticoid
specificity held true for the activation of SULT1A1 gene transcription,
SULT1A1 transfectants were treated with a series of steroids (Fig.
4). In hepatocytes transfected with the
1892 SULT1A1 reporter construct, treatment with either DEX,
triamcinolone acetonide, or hydrocortisone increased luciferase activity 18-, 13-, and 7-fold relative to DMSO vehicle control, respectively (Fig. 4). In addition, we found that treatment with either
of the gonadal hormones dihydrotestosterone or 17-estradiol or with
the steroidal chemical pregnenolone 16-carbonitrile did not activate
reporter gene expression. Although a slight (~3-fold) increase in
luciferase activity was observed following dihydrotestosterone treatment in the experiment depicted in Fig. 4, this effect was not
reproduced in subsequent experiments. Similarly, in hepatocytes transfected with the 119 construct, DEX, triamcinolone acetonide, and
hydrocortisone treatment produced increases in reporter gene expression
of 9-, 8-, and 9-fold relative to controls, respectively, while
dihydrotestosterone, 17-estradiol, and pregnenolone
16-carbonitrile did not alter gene expression.
|
). In the current study, we examined the effect of RU-486 on
DEX-inducible luciferase activity in primary cultured rat hepatocytes
that were transiently transfected with three different
SULT1A1-5':luciferase reporter constructs (1892, 320, or
119). As shown in Fig. 5, cotreatment
of transfectants with 106 M RU-486 effectively
inhibited DEX (107 M)-mediated increases in
luciferase activity.
|
84 SULT1A1 reporter construct
(containing the putative IR3 and ER3 motifs) retained glucocorticoid
responsiveness, while the 69 construct (lacking the motifs) was
devoid of glucocorticoid-inducible luciferase activity (Fig.
6).
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
The genomic structure of the rat SULT1A1 gene coding region was
previously published and found to contain eight exons and seven
intervening introns (Khan et al., 1993). In the present study, we found
that the promoter region of the rat SULT1A1 does not contain a TATA
box. This is consistent with previous observations on sulfotransferase
gene structure. A canonical TATA box has been described for relatively
few of the cloned sulfotransferase genes (Her et al., 1995, 1998). Our
results, which indicate several alternative transcription start sites
in SULT1A1 promoter region, are also typical of other genes with
TATA-less promoters (Smale, 1997). They also reconcile with GenBank
information, which indicated that the 5'-untranslated region of the
mouse mSTp1 aryl sulfotransferase (SULT1A1) cDNA (GenBank accession no.
L02331) (Kong et al., 1993) extended farther upstream than did the
corresponding region of the rat SULT1A1 gene.
Rat "phenol sulfotransferase" (SULT1A1) enzyme activity has long
been observed to be positively regulated by glucocorticoids (Maus et
al., 1982). In corroboration of this perception, in vivo studies in
rats, using either SULT1A class-specific cDNA probes or SULT1A1
isoform-specific probes, confirm that treatment with DEX and other
glucocorticoids induces SULT1A1 enzyme activity and mRNA expression in
rat liver (Runge-Morris et al., 1996; Liu and Klaassen, 1996b).
Emerging evidence indicates that the induction of SULT1A1 gene
expression by glucocorticoids is biologically relevant. We found that
the administration of even moderate doses of triamcinolone acetonide to
rats in conjunction with minoxidil treatment, induced hepatic SULT1A1
gene expression and significantly amplified the hypotensive effects of
minoxidil (Duanmu et al., 2000), a drug that is dependent upon SULT1A1
activity for prodrug bioactivation (McCall et al., 1983; Meisheri et
al., 1993).
Foundation experiments in primary cultured rat hepatocytes have set the
stage for demonstration of the glucocorticoid receptor transcription
factor as a master regulator in the control of glucocorticoid-inducible SULT1A1 gene expression (Runge-Morris et al., 1996). Our previous work
showed that incubation of primary cultured rat hepatocytes with
concentrations of glucocorticoid that would be expected to bind to the
glucocorticoid receptor substantially induced SULT1A1 mRNA expression
(Runge-Morris et al., 1996). In addition, cotreatment with the
antiglucocorticoid/antiprogestin RU-486 inhibited DEX-inducible SULT1A1
expression in a manner that was consistent with
glucocorticoid-inducible tyrosine amino transferase gene expression
(Runge-Morris et al., 1996); a gene that is known to be regulated by
the classical glucocorticoid receptor transcription factor (Shinomiya
et al., 1984).
The results of the present series of transient transfection studies
provide the first direct evidence for the transcriptional regulation of
SULT1A1 by glucocorticoids and recapitulate our previous findings on
endogenous SULT1A1 gene regulation (Runge-Morris et al., 1996). As
illustrated in Fig. 2, luciferase activity in reporter constructs that
contained fragments of SULT1A1 5'-flanking sequences indicated that a
maximal induction of luciferase activity occurred with DEX
concentrations (107M) that would be expected to
saturate the glucocorticoid receptor. Deletional analyses established
that the SULT1A1 5'-flanking sequences located in proximity to the core
promoter contain sufficient glucocorticoid-sensitive sequences to
support the induction of gene transcription (Figs. 2 and 6). More
distal sequences appear to contain information that regulates the basal
level of SULT1A1 gene transcription and influences the magnitude of
DEX-inducible expression. In further support of prior studies on
endogenous SULT1A1 gene regulation in rat hepatocytes (Runge-Morris et
al., 1996), steroid induction of SULT1A1 transcriptional activity
appeared to be specific to steroids of the glucocorticoid class. In
addition, our results showed that DEX induction of luciferase activity
in transfectants containing any of three of the
glucocorticoid-sensitive SULT1A1-5':constructs tested (1892, 320,
or 119) was effectively inhibited by cotreatment with
antiglucocorticoid/antiprogestin RU-486 (Fig. 5).
The transient transfection data presented in this study provide
functional evidence in support of a classical glucocorticoid response
element located within 84 bp of the SULT1A1 5'-flanking sequence just
proximal to the core promoter. As shown in Fig. 6, the deletion or
disruption of either the IR3 or ER3 putative glucocorticoid response
sequence located in this gene domain, effectively ablated
glucocorticoid inducibility in SULT1A1 reporter constructs. Comparison
of the two candidate glucocorticoid response elements in SULT1A1
against available profiles of consensus and variant glucocorticoid
response elements indicated that the more proximal IR3 element (i.e.,
AGAACA GCC AGTCCT) had the structural features that are most consistent
with a classical GRE. The present investigation in primary cultured rat
hepatocytes provides convincing evidence in support of a primary role
for the glucocorticoid receptor transcription factor in the regulation
of glucocorticoid-inducible SULT1A1 gene expression. Future work will
focus on characterizing the binding and functional interactions between
the glucocorticoid transcription factor and the putative SULT1A1
glucocorticoid response element(s) that have been identified in this study.
| |
Footnotes |
|---|
Received February 20, 2001; accepted May 3, 2001.
This work was supported by National Institutes of Health Sciences Grants ES05823 (to M.R.M.) and HL50710 (to T.A.K.), and by services provided by the Cell Culture Facility Core and Imaging and Cytometry Facility Core of National Institute of Environmental Health Sciences Center Grant P30 ES06639.
Melissa Runge-Morris, M.D., Institute of Environmental Health Sciences, Wayne State University, 2727 Second Ave., Detroit, MI 48201. E-mail: m.runge-morris{at}wayne.edu
| |
Abbreviations |
|---|
Abbreviations used are: SULT1, aryl sulfotransferase; DEX, dexamethasone; PCR, polymerase chain reaction; GSP, gene-specific primer; RACE, rapid amplification of cDNA ends; bp, base pair; DMSO, dimethyl sulfoxide; GRE, glucocorticoid response element; ER3, everted repeat with three intervening bases; IR3, inverted repeat with three intervening bases.
| |
References |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
This article has been cited by other articles:
|
|
 |
|
  Y. Alnouti and C. D. Klaassen Regulation of Sulfotransferase Enzymes by Prototypical Microsomal Enzyme Inducers in Mice J. Pharmacol. Exp. Ther., February 1, 2008; 324(2): 612 - 621. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Pastorelli, D. Carpi, R. Campagna, L. Airoldi, R. Pohjanvirta, M. Viluksela, H. Hakansson, P. C. Boutros, I. D. Moffat, A. B. Okey, et al. Differential Expression Profiling of the Hepatic Proteome in a Rat Model of Dioxin Resistance: CORRELATION WITH GENOMIC AND TRANSCRIPTOMIC ANALYSES Mol. Cell. Proteomics, May 1, 2006; 5(5): 882 - 894. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-T. Yeh and G.-C. Yen Involvement of p38 MAPK and Nrf2 in phenolic acid-induced P-form phenol sulfotransferase expression in human hepatoma HepG2 cells Carcinogenesis, May 1, 2006; 27(5): 1008 - 1017. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. A. Nuber, S. Kriaucionis, T. C. Roloff, J. Guy, J. Selfridge, C. Steinhoff, R. Schulz, B. Lipkowitz, H. H. Ropers, M. C. Holmes, et al. Up-regulation of glucocorticoid-regulated genes in a mouse model of Rett syndrome Hum. Mol. Genet., August 1, 2005; 14(15): 2247 - 2256. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Hempel, H. Wang, E. L. LeCluyse, M. E. McManus, and M. Negishi The Human Sulfotransferase SULT1A1 Gene Is Regulated in a Synergistic Manner by Sp1 and GA Binding Protein Mol. Pharmacol., December 1, 2004; 66(6): 1690 - 1701. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. P. Hartley, X. Dai, Y. D. He, E. J. Carlini, B. Wang, S.-e. W. Huskey, R. G. Ulrich, T. H. Rushmore, R. Evers, and D. C. Evans Activators of the Rat Pregnane X Receptor Differentially Modulate Hepatic and Intestinal Gene Expression Mol. Pharmacol., May 1, 2004; 65(5): 1159 - 1171. [Abstract] [Full Text]
|
|
|
|
 |
|
 H.-L. Fang, S. Shenoy, Z. Duanmu, T. A. Kocarek, and M. Runge-Morris TRANSACTIVATION OF GLUCOCORTICOID-INDUCIBLE RAT ARYL SULFOTRANSFERASE (SULT1A1) GENE TRANSCRIPTION Drug Metab. Dispos., November 1, 2003; 31(11): 1378 - 1381. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Duanmu, D. Locke, J. Smigelski, W. Wu, M. S. Dahn, C. N. Falany, T. A. Kocarek, and M. Runge-Morris Effects of Dexamethasone on Aryl (SULT1A1)- and Hydroxysteroid (SULT2A1)-Sulfotransferase Gene Expression in Primary Cultured Human Hepatocytes Drug Metab. Dispos., September 1, 2002; 30(9): 997 - 1004. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
indicates the transcription start sites determined by 5'-RACE
analysis. The 5'-most transcription start site is designated +1. The
first base of the published gene sequence is denoted by an asterisk.
Sequences comprising the imperfect inverted and everted repeats are
underlined. B, schematic diagram of SULT1A1 5':luciferase deletion
constructs that were used in transient transfections. In each
construct, the arrow indicates the location of the transcription start
site.
