From the
Mitochondrial aspartate aminotransferase (mAAT) is one of two
key enzymes in the pathway of citrate production in prostate.
Expression of mAAT is modulated by testosterone and prolactin in
prostate. We cloned the promoter and 5`-flanking region of the rat mAAT
gene and sequenced 2.0 kilobases of the DNA. This fragment contains the
5`-regulatory promoter region that lacks a TATA and a CCAAT box but is
G+C rich. The 5`-upstream flanking region contains sequences that
have high homology with the consensus glucocorticoid response
element/androgen response element (ARE) and a reported ARE sequence
that is different from the consensus sequence. Functional transcription
studies showed that a 481-base region containing the two ARE sequences
was sufficient for androgen-regulated gene expression. There are
multiple transcription start sites that are regulated by testosterone
in prostate. In liver, on the other hand, castration did not affect
transcription from any of the start sites. Therefore, these data
provide evidence that transcriptional regulation of the rat pmAAT gene
occurs through an ARE located in the 5`-region. In addition, not only
is gene expression modulated by testosterone, but the effect of
testosterone on transcription is cell specific.
An extraordinarily high concentration of citric acid is
characteristic of the prostate gland in many mammals. Our studies using
rat ventral prostate and pig prostate epithelial cells have shown that
aspartate via mitochondrial aspartate aminotransferase
(mAAT)
To further
investigate regulation of mAAT expression and the tissue-specific
effects of hormonal stimulation, we cloned the promoter and
5`-untranslated region of the rat mAAT gene. Sequence analysis revealed
that the general characteristics (absence of TATA box, G+C-rich
content, and the presence of Sp1 sites) of housekeeping gene promoters
were present. In addition, the gene contains multiple transcription
start sites that are regulated by testosterone in prostate but not in
liver. We speculate that the initiation of transcription from multiple
start sites contributes to the mechanism of testosterone modulation of
expression of mAAT and to the tissue-specific regulation of this
housekeeping gene. To identify functional androgen response elements,
transient expression experiments were performed using an androgen
receptor vector and reporter genes. The results provide evidence that
transcriptional regulation of the rat pmAAT gene occurs through an ARE
located in the 5`-region.
Before transient transfection, PC-3 cells were
plated at 1
The 5`-flanking region of the pmAAT gene was isolated by
screening a rat genomic library with a 500-bp fragment of the rat pmAAT
cDNA (Fig. 1). Fig. 2shows the sequence of the
HindIII fragments that comprise the first exon, part of the
first intron, and the 5`-regulatory region. The exon and 90 bp of the
5`-flanking sequence are 100% homologous to the 5`-region of the rat
pmAAT cDNA. A 200-bp region from -1 to -202 (+1 is A
of the ATG codon used for translation) is G+C rich (67% GC) and is
characteristic of the promoter in housekeeping genes. This region does
not contain a TATA-like element nor a CCAAT box in the G+C rich
area but does contain three putative Sp1 sites (Fig. 1). Two
sequences that are homologous to the ARE or to the ARE half-site are
located at -1460 and -1439. The sequence at location
-1439 (GGAAAAGACTGTTCT) differs from the consensus
(GGAACANNNTGTTCT) ARE
(13) by only one base in the left
half-sequence. The other sequence at location -1460
(ATCTTGTTCTGTAG) is very similar to the ARE-2 reported by Rennie et
al.(12) for the probasin gene. Thus, the sequences at
location -1439 and -1460 could participate in testosterone
modulation of pmAAT expression.
We have isolated the rat pmAAT gene from a
The sequence of the pmAAT gene is highly conserved
between the rat and mouse. In the region from -1 to -226,
the two sequences are 90% identical. The Sp1 binding sites at -9
and -88 in the rat gene are at the same positions in the mouse
gene. However, in both sequences there is a single base change between
the rat and mouse sequences (CCGCCC to CCACCC and GAGGCGTCGT to
GAGGTGTCGT). In each case, the change is a purine to purine or a
pyrimidine to pyrimidine change. In addition to these two Sp1 sites,
another sequence with high homology to the consensus Sp1 sequence
GCCCCGCCCA is located at -115. The sequence in the pmAAT gene is
GCCACGCCCA. The same sequence is present at the same location in the
mouse gene; however, Tsuzuki et al. (15) did not report this
sequence as an Sp1 site. Since the change here is purine to pyrimidine,
this sequence might not bind Sp1 with high affinity. Additional binding
studies are required to determine whether this sequence can function as
an Sp1 binding site.
Seven transcription start sites were identified
for the rat mAAT gene using primer extension analysis. Multiple start
sites have also been reported for other housekeeping genes lacking a
TATA box. Pave-Preux et al.(14) identified 5 start
sites for the rat cAAT gene, and Tsuzuki et al.(15) identified four sites for the mouse mAAT gene. Therefore,
our results are consistent with these other studies. In addition,
Schatt et al.(17) reported that the presence of
upstream enhancers in the absence of a TATA box resulted in initiation
from sites scattered around the promoter region. Consequently, multiple
transcription initiation sites may be a characteristic of genes that
lack a TATA in the promoter.
We previously reported that
testosterone stabilizes mAAT mRNA in rat ventral prostate and pig
prostate
(4) . This stabilization effect contributes to the
testosterone-stimulated increase in the steady-state level of pmAAT
mRNA. In addition, the kinetics of mRNA disappearance were not first
order and suggested the presence of two populations of pmAAT mRNA. In
the present study, castration and testosterone replacement resulted in
differential stimulation of transcription from these multiple start
sites in prostate. However, castration did not affect expression from
any start site in liver. Discrimination among start sites by
testosterone could account for a mixed population of pmAAT mRNAs.
Moreover, if the half-life of the various pmAAT mRNA species were
different due to structural differences, this might account for the
lack of first order kinetics that we observed in the disappearance of
pmAAT mRNA reported earlier. The intensity of the transcription start
sites closest to the translation start codon was greater than the
intensity of the most 5`-upstream start site. This difference in
intensity was greater in the liver than in the prostate. In addition,
the most 5`-upstream start site seemed to be affected by castration to
a greater extent than the more 3`-sites.
Utilization of multiple
transcription start sites has been reported for other genes. In some
cases, transcription initiation from different start sites is tissue
specific and may contribute to tissue-specific gene
expression
(18, 19, 20) . Our observations,
however, are different since we observed the same multiple start sites
in both prostate and liver in intact rats. In castrated rats, on the
other hand, the more distal transcription start sites are not used in
prostate but are used in liver. Thus, in this case it is the hormone
induction that is tissue specific. Glucocorticoid regulation of
multiple transcription start sites for the cAAT gene
(14) and
hypoglycemic modulation of transcriptional start sites in the
preproenkephalin
(19) gene have been reported. In the case of
cAAT, dexamethasone increased transcription from two of the five
transcription start sites 15-18-fold, while transcription from
the three other start sites was only increased 3-fold. In addition,
dexamethasone also induced transcription from three new start sites
that were below the level of detection in the controls. Here,
testosterone treatment of castrated rats resulted in induction of
transcription from four start sites that were below the level of
detection. Thus, if castration represents a condition in which there is
a basal level of gene expression in androgen-responsive tissues, then
these results suggest that the 5`-distal start sites might be the
testosterone-induced sites while the other sites are constitutive.
Multiple transcription initiation sites have been proposed as
characteristic of ``housekeeping'' genes that show little or
no tissue specificity. These results support the suggestion that
hormonal regulation of multiple transcription start site usage may
provide a mechanism to achieve tissue-specific hormonal responses in
so-called ``housekeeping'' genes.
Sequence analysis of the
2.0-kb DNA fragment was carried out to determine if conserved sequence
motifs might be involved in androgen induction of pmAAT expression.
Steroid regulation of gene expression involves binding of activated
steroid receptor to a specific DNA sequence motif (the steroid response
element) that enhances transcription of the gene. Among the most
studied and best characterized response elements is the GRE that
contains the consensus sequence GGTACAnnnTGTTCT. Several
studies
(12, 21, 22, 23, 24, 25) have revealed that the consensus GRE can also mediate
transcription activation by androgen. Studies with androgen-responsive
genes
(13, 26, 27) have revealed the presence of
an ARE sequence that has a high homology with the consensus GRE
sequence. Consequently, evidence is mounting to support the concept
that the GRE might be characterized as a GRE/ARE. However, studies with
the androgen-regulated probasin gene (12) revealed the presence of two
putative AREs. Both AREs are required for functional activity and are
unresponsive to glucocorticoid.
Two ARE-like sequences were
identified in the 1.7-kb 5`-region of the pmAAT gene. One sequence is
homologous to the consensus ARE sequence reported by Roche et
al.(13) , and the second sequence is similar to ARE-2 in
the probasin gene
(12) and contains the consensus GRE/ARE right
half-site. To determine whether the putative ARE sequences in the pmAAT
gene are functional, a 1.4-kb and a 481-bp fragment were tested in
transfection studies. The pmAAT-CAT constructs were cotransfected with
a rat androgen receptor expression vector. We tested functional
androgen receptor expression by determining mAAT activity in response
to DHT treatment in PC-3 cells. PC-3 cells have a significantly lower
mAAT activity level than LNCaP cells,
The pmAAT DNA
fragments were cloned into the CAT vector downstream of the SV40
promoter. CAT activity with the larger 1.4-kb construct was increased
3-fold by treatment with androgen with the maximum response seen at
10
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We express sincere appreciation to Dr. S. Biswas and
E. Biswas (University of Maryland at Baltimore) for valuable
suggestions and assistance. We are indebted to Dr. R. J. Matusik
(University of Manitoba) for the androgen receptor expression vector
and Dr. J. R. Mattingly (University of Missouri-Kansas City) for the
pmAAT cDNA.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
serves as the major source of
oxaloacetate for citrate synthesis in prostate. Therefore, mAAT is a
key enzyme in the pathway for citrate production by prostate epithelial
cells and contributes to the accumulation of citrate in the glandular
lumen. The expression of mAAT is regulated by testosterone and
prolactin in prostate. We have reported that prostate mAAT activity
(1) and biosynthesis
(2) are stimulated by testosterone.
In addition, we showed that the effects of testosterone
(3, 4) and prolactin
(5) on mAAT were at the level of gene
expression. mAAT is expressed in all tissues, however, it is
specifically regulated by testosterone in prostate epithelium but not
in other tissues
(1) . Therefore, although mAAT is expressed in
all cells, its hormonal regulation is cell specific.
Animals and Animal Treatment
Male
Wistar rats weighing 250-300 g were used as the source of
prostate and liver mRNA. The animals were maintained and sacrificed in
accordance with the National Institutes of Health guidelines. The
procedures for castration and sham operation were conducted as
previously described
(6) .
RNA Preparation
Total RNA was isolated
from rat prostate and liver using the guanidium thiocyanate extraction
method described by Chomczynski and Sacchi
(7) .
Poly(A) RNA was isolated by oligo(dT)-cellulose
chromatography
(8) .
DNA Used as Hybridization Probes
A
full-length rat precursor mAAT (pmAAT) cDNA cloned into the
EcoRI site of the BlueScript II KS phagemid
(9) was used. The full-length pmAAT cDNA was digested with
PvuII, and the three resulting fragments were separated by
agarose gel electrophoresis. A 560-bp EcoRI-PvuII
fragment containing the 5`-end of the pmAAT cDNA, a synthetic
oligonucleotide representing the first 22 bp of the signal peptide of
pmAAT, and the full-length cDNA were used as probes.
Isolation and Sequencing of the 5`-Region of the
pmAAT Gene
A SalI partially digested rat liver
genomic library in the DASH vector from Stratagene was screened by
the plaque hybridization technique
(8) . Plaques (8
10
) were screened according to the procedures recommended
by the manufacturer using the total pmAAT cDNA labeled by random
priming. Seven positive clones were isolated by three rounds of
screening, and DNA was prepared and characterized by restriction
endonuclease mapping. EcoRI fragments of the phage clone
inserts were screened by hybridization using the probes described
above. Four of these clones contained EcoRI fragments that
hybridized with all three probes and probably represent pseudogenes.
Only one of the three remaining clones contained an EcoRI
fragment that hybridized to the synthetic signal peptide
oligonucleotide probe. This 6.4-kb EcoRI fragment was digested
using HindIII. The four resulting HindIII fragments
were subcloned into BlueScript II KS
, and the two
5`-subclones were sequenced according to Sanger et
al.(10) .
Primer Extension Analysis
The primer
extension reaction was carried out according to a slight modification
of the method described by Modaressi et al.(11) . Two
18-base synthetic oligonucleotides (5`-GAAACCGAGGCGCGGGCTCC-3` and
5`-ACCGGAGTGCAGGAGGGC-3`) complementary to positions -80 to
-62 and +4 to +22, respectively, were 5`-end labeled
with [P]ATP (6000 Ci/mmol) and T4
polynucleotide kinase. The oligonucleotide at position -80 was
used to ensure that the start sites identified with the oligonucleotide
at position +4 included the most 5`-site. 4 µg of
poly(A)
RNA were mixed with 0.5 pmol of end-labeled
primer in 10 µl of a solution containing 0.4 M NaCl and 10
mM Pipes (pH 6.4) and incubated for 10 min at 75 °C. After
addition of 10 units of RNase inhibitor, the hybridization reaction was
carried out for 2 h at 56 °C. 20 µl of 5
reverse
transcriptase buffer (250 mM Tris-HCl, pH 8.3, 375 mM
KCl), 20 µl of 10 mM dithiothreitol, 20 µl of 10
mM dNTP mixture, and 10 µl of 1 mM MgCl
were added to the reaction solution. The final volume was
adjusted to 100 µl by addition of nuclease-free water. The reaction
was incubated at 42 °C for 2 h after adding 1200 units of Moloney
murine leukemia virus (MMLV) reverse transcriptase. The reaction was
stopped by addition of 5 µl of 0.5 M EDTA. The extended
DNA was extracted with phenol chloroform and ethanol precipitated. The
size of the extended DNA was analyzed by electrophoresis on a 7%
polyacrylamide sequencing gel. Aliquots of a sequencing reaction using
genomic DNA and the same oligonucleotides as primers were separated on
the same gel.
Construction of Transfection Vectors
The
6.4-kb EcoRI fragment containing the 5`-flanking region was
digested to remove the 5`-EcoRI-HindIII fragment
(Fig. 1). This fragment was then subcloned into the BlueScript II
SK phagemid. The resulting recombinant plasmid
(pj226H2-1) was digested with PstI and AccI. A
pmAAT-CAT chimeric gene (ARE(500)-CAT) was constructed by inserting the
481-bp PstI-AccI fragment of pj226H2-1 into the
PstI-AccI sites of the pCAT-promoter plasmid from
Promega. A second pmAAT-CAT chimeric gene (ARE(1400)-CAT) was
constructed by inserting the PstI-HindIII fragment of
pj226H2-1 into the PstI-HindIII sites of the
pCAT-promoter plasmid. The rat androgen receptor expression vector
(pSV40rAR) was a gift from Dr. Robert Matusik. Construction of this
vector has been previously described
(12) .
Figure 1:
5`-Region of
the rat mitochondrial aspartate aminotransferase gene. A restriction
map of the EcoRI fragment of the phage clone containing the
5`-region of the pmAAT gene is shown. The first exon is boxed;
A, AccI; B, BstEII; H,
HindIII; P, PstI; S, StuI;
V, AvaI.
Cell Culture and Transfection
The PC-3
cell line was obtained from the American Type Culture Collection. The
cells were cultured in Dulbecco's modified Eagle's medium
(DMEM) containing 4,500 mg/liter D-glucose, 25 mM
HEPES, 584 mg/liter L-glutamine, 44 mM
NaHCO, and 10% FBS. The medium was changed twice per week.
After transfection, the medium was replaced with DMEM containing 2%
charcoal-dextran-stripped FBS and either no added steroid or
10
-10
M
5
-DHT. The charcoal-dextran-stripped FBS was prepared as follows.
The charcoal-dextran mixture (0.125 g of activated charcoal, 0.125 g of
dextran T-70, 0.7 ml of 5 M NaCl, and 225 ml of
H
O) was vortexed for 5 min and then centrifuged for 10 min
at 1000
g. The pellet was transferred to 500 ml of
freshly thawed FBS, and the mixture was stirred with a magnetic stirrer
at room temperature for 30 min; the solution was then heat inactivated
at 56 °C for 30 min. The charcoal-dextran was removed from the FBS
by centrifugation at 3000
g for 10 min, and the
supernatant was filtered through a 0.22-µm pore filter. The
charcoal-dextran-stripped FBS was divided into aliquots and stored
-20 °C.
10
cells per 25-cm
culture
flask and grown in DMEM with 10% FBS until they reached 60-70%
confluence. 1-3 h prior to DNA addition, the medium was
replenished with fresh complete medium. The reporter vector and
androgen receptor vector were introduced into PC-3 cells by the calcium
phosphate-DNA coprecipitation method. A mixture of 50 µl of 2.5
M CaCl
, 10 µg of ARE(500)-CAT or ARE(1400)-CAT
reporter plasmid, 10 µg of pSV40rAR vector, and 20 µg of salmon
sperm DNA in a final volume of 500 µl was prepared. An equal volume
of 2
HBS (50 mM HEPES, 0.28 M NaCl, 1.6
mM Na
HPO
, pH 7.1) was slowly added to
the DNA mixture using a 1-ml pipette with vortexing. The mixture was
incubated 30-50 min at room temperature and added to the dishes.
After 6 h at 37 °C, the DNA-containing medium was removed, and the
cells were washed once with phosphate-buffered saline (2.7 mM
KCl, 1.5 mM KH
PO
, 137 mM
NaCl, and 16 mM Na
HPO
, pH 7.3),
followed by addition of 15% glycerol in HBS and incubated for 2 min at
room temperature. The cells were then washed twice with
phosphate-buffered saline and placed in DMEM supplemented with 2%
charcoal-dextran-stripped FBS with or without various concentrations of
DHT. The Ethanol vehicle was added to the medium of control groups. In
each transfection experiment, the treatment and control groups
contained three replicate flasks of transfected cells. The cells were
incubated for 48 h, after which they were harvested in TEN buffer (40
mM Tris-HCl, pH 7.5, 1 mM EDTA, and 15 mM
NaCl) and assayed for CAT activity.
CAT Activity Assay
The cell extracts were
prepared by three cycles of freezing and thawing. Each cycle consisted
of 5 min in an ethanol/dry ice bath and 5 min in a 37 °C water
bath. CAT activity was determined using the CAT enzyme assay system
from Promega. The reaction mixture was composed of 50 µl of
supernatant cell extract, 65 µl of 0.25 M Tris-HCl (pH
8.0), 5 µl of [C]chloramphenicol (25
µCi/ml, Amersham), and 5 µl of n-butyryl coenzyme A (5
mg/ml, Pharmacia Biotech Inc.). After incubation at 37 °C
overnight, the reaction was terminated by addition of 300 µl of
mixed xylenes and vigorous vortexing. The xylene organic phase was
extracted twice with 100 µl of TE buffer (10 mM Tris-HCl,
pH 7.5, and 1 mM EDTA). 200 µl of the final extracted
organic phase were counted in a liquid scintillation counter. CAT
activity is expressed as the mean ± S.E. of n-butyryl
[
C]chloramphenicol dpm for the three replicates
in each group.
Figure 2:
Sequence of the 5`-region of the rat
mitochondrial aspartate aminotransferase gene. Transcription start
sites are indicated by downarrows; Sp1 sites are
doubleunderlined; exon 1 (signal peptide) is
singleunderlined; putative AREs are boxed.
+1 corresponds to the A of the first ATG used for
translation. Asterisks are above bases that are different from
the cDNA sequence reported by Mattingly et al. (9). All bases
that are different are at the end of the cDNA and may be artifacts of
cloning the cDNA.
We determined the 5`-boundary of the
gene by identifying the transcription initiation sites using primer
extension analysis. The primers were labeled at the 5`-end, hybridized
with rat ventral prostate poly(A) RNA, and extended by
MMLV reverse transcriptase. Primer extension analysis showed the
presence of multiple transcription initiation sites. The sizes of the
extended products were determined by comparison with the sequencing
products of a DNA genomic fragment that was sequenced using the same
primer. Seven transcription initiation sites were identified
(Fig. 3). The presence of multiple transcription initiation sites
has been reported for other housekeeping genes including rat cAAT
(14) and mouse mAAT
(15) . Consequently, this may be a
general characteristic of genes that lack a TATA box in the promoter.
Figure 3:
Transcription initiation sites of rat
mitochondrial aspartate aminotransferase gene. Poly(A)
RNA (4 µg) extracted from rat ventral prostate was extended using
MMLV reverse transcriptase. The primer was complementary to the gene
sequence at +4 to +22 and was end labeled with
P. Arrows indicate locations of transcription
initiation sites. Lanes1-4 are sequencing
reactions of corresponding DNA (lanes are ACGT,
respectively).
Testosterone stimulates the transcription of the mAAT
gene
(4) . Therefore, we wanted to determine the affect of
testosterone on transcription from the multiple initiation sites.
Poly(A) RNA was extracted from the ventral prostate
and liver of sham-operated, castrated, and castrated +
testosterone-treated rats. Fig. 4shows the affect of castration
alone and with testosterone replacement on transcription from all of
the pmAAT start sites. Castration reduced transcription from all start
sites in prostate but did not affect any start site in liver.
Transcription from start sites at locations -112, -102,
-87, and -60 in ventral prostate were reduced below the
level of detection. Transcription from locations -25, -38,
and -42, on the other hand, were significantly decreased but were
clearly evident. Testosterone replacement restored transcription from
all sites to near the sham level.
Figure 4:
Effect of testosterone on transcription
from multiple transcription initiation sites. Poly(A)
RNA extracted from ventral prostate and liver of sham-operated
(S), castrated (C), or castrated plus
testosterone-treated (T) rats. RNA was extended using MMLV
reverse transcriptase and a
P end-labeled primer
complementary to the gene sequence at position +4 to +22.
Arrows indicate transcription initiation sites; PS,
prostate sham; PC, prostate castrate; LS, liver sham;
LC, liver castrate.
Transcription enhancer activity of
the 5`-flanking region of the rat mAAT gene was tested in PC-3 cells by
cotransfection with rat AR expression vector and with CAT reporter
plasmids containing a 481-bp PstI-AccI fragment or a
1400-bp PstI-HindIII fragment subcloned into the pCAT
promoter vector from Promega. To test the AR expression vector, PC-3
cells transfected with AR were treated with DHT, and the mAAT activity
was measured. Fig. 5shows that 10 nM DHT significantly
increased mAAT activity in AR-transfected cells. Fig. 6presents
the dose response of CAT activity to DHT in transiently transfected
PC-3 cells. The results demonstrate that DHT in the range of
10-10
M caused a
3-4-fold increase in CAT expression. PC-3 cells transfected with
the control pCAT promoter (no insert) vector did not respond to DHT at
a concentration of 10
M (data not shown).
Figure 5:
Effect of DHT on mAAT activity of PC-3
cells transfected with rat androgen receptor expression vector. PC-3
cells were transfected with pSV40rAR expression vector and incubated
with or without 10M DHT for 24 h. mAAT
activity of mitochondrial extracts was determined by activity staining
in a 1% agarose gel. Activity is expressed as relative absorbance units
determined by densitometer scanning. CONT.,
control.
Figure 6:
Androgen-dependent transcriptional
enhancer activities of 5`-flanking region fragments of the pmAAT gene.
DHT induction of CAT activity by pmAAT-CAT constructs in PC-3 cells is
shown. Fragments (1400 or 481 bp) containing putative AREs were cloned
upstream of the CAT reporter in the pCAT vector from Promega.
Nucleotide numbering corresponds to Fig. 1. Constructs were
cotransfected with pSV40rAR into PC-3 cells. Cells were then grown in
the absence or presence of the indicated concentrations of DHT.
Brokenline indicates deletion
construct.
phage
genomic library. The gene is 30 kb long and contains 11 exons. The
first exon is the signal peptide and is separated from the second exon
by a large (greater than 10 kb) intron. In this report, we describe a
2.0-kb fragment of the gene that contains the 5`-untranslated region,
the first exon, and part of the first intron. The sequence that we
determined for this fragment is 100% homologous with the reported
sequence for the rat pmAAT cDNA
(9) . The sequence of the
promoter region is G+C rich (67% from base -1 to -202)
and contains numerous CG or GC dinucleotide sequences. Similar CG
islands have been reported in the promoter region of the mouse cAAT
(16) and mAAT
(15) genes, the rat cAAT gene
(14) ,
and other mammalian housekeeping genes
(16) . The promoter does
not include a TATA box but does contain three putative Sp1 binding
sites. Although we did find four CCAAT boxes in the reverse
orientation, these were all far upstream of the G+C-rich area and
are probably not a part of the promoter. Therefore, we do not believe
that these CCAAT elements function in transcription regulation of the
rat pmAAT gene.
(
)
and since
they do not contain functional androgen receptor, they do not respond
to DHT treatment. However, after transfection with the androgen
expression vector, mAAT activity was inducible by androgen. In
addition, PC-3 cells, which do not accumulate citrate in the culture
medium, produced significant levels of medium citrate after
transfection (data not shown). Therefore, is it clear that the androgen
receptor was functional in the transfected cells.
M DHT. Deletion of this 1.4-kb fragment
showed that the 481-bp region containing the putative ARE region was
sufficient for androgen-responsive CAT activity. Furthermore,
10
M DHT caused CAT induction with the
481-bp construct, while this level of DHT was not effective with the
larger 1.4-kb construct. Thus, the functional transcription studies
demonstrated that DNA fragments containing the two ARE sequences were
capable of enhancing transcription of the SV40 promoter. Moreover,
sequence analysis of the 481-bp fragment revealed no other sequences in
this fragment with homology to any reported sequence for the GRE/ARE.
We conclude that the putative ARE sequences identified in this fragment
of the pmAAT gene function as an ARE. Moreover, these results are
consistent with cooperativity among AREs as has been described in other
genes regulated by androgen
(12, 28) . Mutation studies
must be carried out to determine whether both ARE sequences are
required. Most importantly the current studies continue to support our
proposal concerning the mechanism of testosterone regulation of citrate
production by prostate epithelial cells. These studies link the
important relationship of hormonal regulation of gene expression to a
major function (i.e. citrate production) of prostate as
described in our recent review
(29) .
/EMBL Data Bank with accession number(s) U21158.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.