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From the * Department of Medicine, University of
Sydney, Sydney, New South Wales 2006, Australia; and
ANZAC Research Institute, Concord Hospital,
Concord, New South Wales 2139, Australia.
| Correspondence to: J. Singh, ANZAC Research Institute, Concord Hospital, Concord, NSW 2139, Australia (e-mail: jas{at}med.usyd.edu.au). |
| Received for publication December 20, 2001; accepted for publication March 22, 2002. |
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Abstract |
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Key words: Hpg, up-regulation, subtractive hybridization, differential expression, in situ hybridization
The choice of paradigm to identify androgen-regulated genes requires consideration, because genes that mediate androgen action are likely to be cell type specific and temporally regulated. Using individual cell lines was considered unsuitable because each cell type in the prostate is a potential target for androgen action. Although androgen-regulated genes from the prostate can be identified from mice undergoing androgen withdrawal, replacement, or both, these animals have had exposure to androgens until castration. As such, genes mediating androgen action at an early stage of prostate development may be missed. We therefore decided to utilize the hpg mouse (Cattanach et al, 1977) to identify androgen-regulated genes in the prostate. The hpg mouse has a deletion (>33.5 kilobases) in the gonadotropin-releasing hormone gene (Mason et al, 1986), resulting in a substantial decrease in blood gonadotropins and immature sex accessory organs, including the prostate. Using a subtractive hybridization approach between androgen-deficient and androgen-replaced prostate glands, we have identified a novel gene, AUMP (ie, androgen up-regulated gene in the mouse prostate) whose expression is up-regulated by androgens in the course of growth from rudimentary to mature prostate, and which codes for a palmitoyl protein thioesterase (PPT)-like protein.
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Materials and Methods |
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Tissue Preparation
Prostates from homozygous hpg mice with (n = 12) or without (n =
35) testosterone treatment and from non-hpg mice with (n = 20) or
without (n = 12) orchidectomy were excised using a micro-dissection
microscope, washed in cold phosphate-buffered saline (PBS) and snap-frozen in
liquid nitrogen before being transferred to a -70°C freezer. Testosterone
was administered as described previously
(Singh and Handelsman, 1996).
Briefly, a silastic tube (1.47 mm inner diameter and 1 cm in length) filled
with crystalline testosterone (Sigma, Sydney, NSW, Australia) was implanted in
homozygous hpg mice subdermally for 14 days before ventral prostates
were removed. Orchidectomy was performed on 8-week-old mice via scrotal
incision 14 days prior to prostate tissue harvesting. All procedures were
performed under anesthesia and were approved by the University of Sydney's
Animal Care and Ethics Committee.
RNA Isolation and Reverse Transcription
Pooled prostate glands (40-70 mg) were homogenized in 1 mL of TRI Reagent
(Sigma) using a pellet pestle (Kontes, Vineland, NJ). Total RNA was isolated
following the TRI Reagent protocol. RNA concentration was determined by
absorbance at 260 nm using a UV spectrophotometer (Pharmacia Biotech, Uppsala,
Sweden). The quality of total RNA was assessed by formaldehyde-agarose gel
electrophoresis. Poly(A)+ RNA was isolated from total RNA using
oligo(dT) attached magnetic beads (DYNAL, Oslo, Norway). Poly(A)+
RNA (250 ng) was reverse transcribed to first-strand complementary DNA (cDNA)
using 200 U/µL of Moloney murine leukemia virus reverse transcriptase in
the presence of a modified oligo(dT) primer provided by the SMART PCR cDNA
synthesis kit (Clontech, Palo Alto, Calif). The single-stranded cDNA was then
used for suppressive subtractive hybridization, PCR, and virtual Northern
analysis as described below.
Suppression Subtractive Hybridization
Double-stranded cDNA was generated using primers provided by the SMART PCR
cDNA synthesis kit (Clontech) after optimizing cycle numbers to ensure that
the cDNA remained in the exponential phase of amplification. The resultant
cDNA was subjected to column purification following the protocol provided by
the SMART PCR cDNA synthesis kit. Suppression subtractive hybridization (SSH)
was conducted as described previously
(Diatchenko et al, 1996) using
a PCR-Select cDNA subtraction kit (Clontech). The tester was cDNA derived from
testosterone-replaced hpg prostate, and the driver was from
hpg prostate. Both underwent RsaI digestion, but only the
tester cDNA was ligated with adaptors. After 2 rounds of hybridization between
excess driver and ligated tester cDNA, PCR amplification was carried out using
primers based on adaptor sequences.
Differential Screening
The PCR products were subcloned into pGEM T-Easy Vector (Promega, Sydney,
Australia), and the resultant constructs were used to transform
Escherichia coli. White colonies were randomly selected and cultured
for 4-5 hours at 37°C in 96-well microtiter plates containing 200 µL of
Luria Bertani medium supplemented with ampicillin (Sigma) (100 µg/mL). PCR
products of LB culture were denatured and dot-blotted onto 2 sets of identical
nylon membranes (Hybond, Amersham, Buckinghamshire, United Kingdom) and fixed
by UV irradiation (Stratagene, La Jolla, Calif). The 2 sets of membranes were
hybridized overnight at 72°C to radiolabeled hpg and the
testosterone-replaced hpg probe, respectively. The washing condition
included low (2x saline-sodium citrate [SSC] and 0.5% sodium dodecyl
sulfate [SDS]; 4 times for 20 minutes) and high (0.2x SSC and 0.5% SDS;
twice for 20 minutes) stringency washes at 68°C. The hybridization signals
on the membranes were detected with a PhosphorImager (Cyclone; Packard, Mount
Waverly, Australia) after overnight exposure. To decrease the number of false
positive clones being selected for plasmid purification and sequencing, all
positive clones identified were dot-blotted in duplicate on 2 sets of fresh
membranes, and underwent a second round of hybridization using the same probes
and conditions. Insert sequences of the truly differentially expressed clones
were determined by single direction sequencing.
Reverse Transcription and PCR
Total RNA (1 µg) extracted from various normal and hpg mouse
organs were treated for elimination of DNA with 1 unit of DNase I (Gibco BRL,
Melbourne, Australia) and reverse transcribed to cDNA using Superscript II
RNase H-reverse transcriptase (Gibco BRL). Both oligo(dT) and random hexamer
primers were included in final concentrations of 20 ng/mL and 1 ng/mL,
respectively. For PCR, the single-stranded cDNA was amplified using AUMP
forward 5'-GCGGGGACATTTGTTGGTAT-3' and reverse
5'-GGACAGAGAAAGAAAGCGGCTA-3' primers. PCR amplification was
performed using 0.4 mM primers, 1' PCR buffer (Perkin Elmer), 0.4 mM
dNTPs, and 0.02 U/mL of Taq DNA polymerase. Samples were denatured at
95°C for 1 minute, followed by 25 cycles of denaturation at 95°C for
15 seconds, annealing at 55°C for 30 seconds, and extension at 72°C
for 45 seconds. Mouse ß-actin primers (forward,
5'-CCTAAGGCCAACCGTGAA-3'; reverse,
5'-AACCGCTCGTTGCCAATA-3') were used to monitor equality of
loading.
Virtual and Classical Northern Analysis
Virtual Northern analysis was performed to examine the transcript size
using the SMART cDNA synthesis kit (Clontech). A total of 200 ng of
double-stranded cDNA obtained following 17-20 cycles of PCR was
electrophoresed on 1% agarose gel containing 10 mg/mL of ethidium bromide for
1 hour at 90 V and transferred onto a positively charged Hybond N nylon
membrane and covalently bound using a UV cross-linker. The probe was the PCR
product of AUMP. The PCR product was purified from a 1% low-melting-point
agarose gel with the GeneClean kit (BIO 101, Vista, Calif) and labeled with
[
-32P]dCTP. Labeled probe was added to a hybridization
solution (ExpressHyb, Clontech) to give a concentration of 1 x
106 cpm/mL and hybridized with the membrane at 68°C for 12-16
hours. The washing conditions were 2x SSC, 0.05% SDS (4 times for 20
minutes) followed by 0.1x SSC and 0.1% SDS (twice for 20 minutes) at
50°C. The same membrane after stripping was hybridized with mouse
ß-actin as a control for loading. For classical Northern analysis, 20
µg of total RNA from each of 8 normal mouse organs was electrophoresed for
2.5 hours at 55 V on a formaldehyde (pH 7) denaturing gel as described
previously (Margan et al,
2000). Denatured total RNA was transferred to a nylon membrane.
The probe, hybridization, and wash conditions were the same as those used for
virtual Northern analysis.
In Situ Hybridization
Frozen sections (8 µm) were cut using a cryostat (Shandon, Cheshire,
United Kingdom) mounted on charged SuperFrost (Menzel-Glaser, Germany) slides
and fixed in 4% paraformaldehyde in PBS (4°C) for 10 minutes followed by 3
washes in 1x PBS and one in 2x SSC at room temperature. The slides
were then placed in a solution of proteinase K (2 µg/mL) for 7 minutes at
37°C, then in 0.1 M glycine for 5 minutes at 37°C, followed by two
washes in 2x SSC at room temperature. Slides covered with
prehybridization solution (50% deionized formamide, 6x SSC, 5x
Denhardt solution, 0.1 mg/mL salmon sperm DNA (Gibco BRL), 10% dextran
sulfate, 5 mg/mL sodium pyrophosphate, 0.5% SDS, and 1 mM levamisole) were
incubated for 30 minutes at 55°C in an Omnislide thermal cycler (Hybaid,
Ashford, Middlesex, United Kingdom). All components of the prehybridization
solution were obtained from Sigma, Sydney, Australia, unless stated otherwise.
The hybridization solution, consisting of the prehybridization solution and
digoxigenin (Roche) (DIG)-labeled sense or antisense probe was then applied to
the slides, covered with parafilm to avoid drying, and incubated at 55°C
for 19 hours. After hybridization, slides were washed with 2x SSC,
0.2x SSC, and 0.1x SSC at 55°C for 15 minutes each. The last
rinse was in 0.1x SSC at room temperature. For detection of
hybridization signals, the slides were first blocked in 2% goat serum (Sigma)
in Tris-buffered saline (100 mM Tris pH 7.4 and 150 mM NaCl) containing 0.1%
Triton X-100 for 30 minutes followed by 30 minutes in 2% blocking solution
(Roche, Castle Hill, Australia). The slides were then incubated in a 1:250
dilution of anti-DIG antibody-alkaline phosphatase conjugate (Roche) in
blocking buffer for 2 hours at room temperature. After washing in
Tris-buffered saline, the hybridization signal was developed overnight in a
dark chamber using 4-nitroblue tetrazolium and 5-bromo-4-chloro-3-indoyl
phosphate (Roche) in Tris-buffered saline (100 mM Tris, 100 mM NaCl, and 50 mM
MgCl2 pH 9.5) at room temperature. The sense and antisense probes
were prepared by conducting an asymmetric PCR
(Paine et al, 1995;
Gibbins et al, 1999) for 50
cycles using 250 ng of template AUMP cDNA and 20 pmol of AUMP forward
(5'-CCTGAACCATCCCAAATGT-3') or reverse
(5'-AGAGAAAGAAAGCGGCTATGT-3') primers in the presence of 5 µL
of 10x DIG-labeled dUTP (Roche).
5'-Genomic DNA Walking
Genome walking was performed with the Mouse Genome Walker kit (Clontech)
following the manufacturer's protocol. Four mouse genomic DNA libraries were
provided with the kit. Each library was amplified using the adaptor primer and
AUMP-specific primer, 5'-GTTATTTGCCATGATTCTAGCTGT-3'. To improve
specificity, a nested PCR was performed using the nested adaptor primer and
AUMP-specific primer, 5'-AGGACCTAGTGACCTGGCATCA-3'. Nested PCR
products were subcloned into pGEM T-Easy Vector, and recombinant plasmids were
purified using the GeniePrep DNA Isolation kit (Ambion, Thebarton, Australia)
and sequenced unidirectionally.
Bioinformatics
Sequence homology searches, open reading frame (ORF) determination, and
isoelectric point (pI) calculation were performed using Bionavigator
(http://www.eBioinformatics.com).
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Results |
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To obtain the genomic DNA sequence upstream of the AUMP transcription start site, 5' genome walking was performed using AUMP gene-specific primers, and adaptor primers were used to ligate genomic DNA digests. A 457-bp genomic DNA fragment was obtained after 2 rounds of genome walking. No androgen response element was found within the region. The only steroid-binding site identified was for glucocorticoid receptor at position -430 (Figure 1B).
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Prediction of AUMP function
The longest ORF of AUMP was defined as 408 bp
(Figure 1A). The initiation
codon (ATG) is positioned at nt 94 and the stop codon at nt 499, encoding for
a protein of 135 amino acids (Figure
1C). The predicted protein has a molecular weight of 16 kd with a
pI of 7.4. Amino acids 48 to 119 were found to share approximately 65%
similarity and 35% identity to PPT of human (SwissProt P50897), rat (P45479),
mouse (O88531), and bovine (P45478) (Figure
2), as determined by the BlockSearcher program, with e values of
<0.015. A motif for protein-transport protein, SecY/Sec61- subunit
(P38379), was present at amino acid positions 38-78.
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Androgen Regulation of AUMP Expression
To evaluate the AUMP expression level in prostate under various androgen
conditions, RT-PCR was conducted on poly(A)+ RNA derived from
hpg, testosterone-replaced hpg, castrate non-hpg,
and intact non-hpg mouse prostates. AUMP expression was strong in
testosterone-replaced hpg and normal mouse prostate, but was absent
in untreated hpg as well as castrate mouse prostate
(Figure 3A).
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Virtual Northern assay was used to examine the AUMP transcript size under various androgen conditions. The reason for conducting virtual rather than a classical Northern assay was the insufficient amount of RNA generated from hpg mouse prostates. The weight of hpg prostate (which is about 8% of intact non-hpg mice) would necessitate using a large number of mice for prostate collection. As evident in Figure 3B, AUMP transcript size in testosterone-replaced hpg mouse prostate was the same as that in non-hpg normal prostates. Equally important was the finding that AUMP transcript size determined by virtual and classical Northern analysis was consistent (around 800 bp).
Tissue and Cellular Distribution of AUMP
The tissue expression profile of AUMP was studied by RT-PCR and classical
Northern analysis on total RNA derived from 9 organs (cerebral cortex,
cerebellum, heart, kidney, liver, lung, prostate, testis, and spleen) in
normal mice. Both analyses showed exclusive expression of AUMP in the prostate
(Figure 4A and B). A search of
the mouse expressed sequencing tag (EST) database revealed that no sequences
had a significant match with AUMP except those derived from male bladder.
Similarly, RT-PCR analysis of various hpg mouse organs showed no AUMP
signal in any of the 9 organs (data not shown). In situ hybridization was used
to characterize the cellular distribution of AUMP mRNA in mouse prostate. In
testosterone-replaced hpg and intact non-hpg mouse
prostates, a strong hybridization signal with AUMP antisense probe was
observed in the cytoplasm of luminal epithelial cells. Although it appears
that AUMP signal may also be present in basal cells, further studies using
specific basal cell markers would be required to confirm this. No AUMP signal
was detected in any cells of the stromal compartment of the prostate.
Hybridization signal with AUMP antisense probe was undetectable in
hpg and castrate mouse prostates
(Figure 5).
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Discussion |
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, which is a major component of the
endoplasmic reticulum (ER) translocation site, that promotes translocation as
well as integration of secretory proteins into the ER membrane
(Greenfield and High, 1999; Oliver et al, 1995). The mRNA sequence of AUMP consists of 805 nucleotides with an ORF of 408 bp. Alignment of AUMP sequences from 6 clones resulted in the assembly of the cDNA sequence of 805 bp. This was considered to be the full length for the following reasons. First, some AUMP clones showed the presence of a GGG sequence at the 5' end of mRNA that is generated by SMART technology. The addition of dG by reverse transcriptase occurs only after it has reached the 5' end of mRNA. Other AUMP clones contained both poly(A)+ signal and tail. Second, the assembled transcript was consistent with the size of the transcript determined by Northern analysis as described below. Last, during preparation of this manuscript, a full-length enriched male mouse bladder cDNA of 768 bp was submitted to GenBank (accession number AK020534). Mouse bladder cDNA has 99% identity to AUMP cDNA except for the missing 35 bp at its 5' UTR.
AUMP is clearly up-regulated by androgens because its transcripts are present only in testosterone-replaced hpg or intact non-hpg mouse prostates, but they are absent in untreated hpg and castrate mouse prostates. In addition, this androgen-induced AUMP expression is highly selective. Among the 9 mouse organs examined, AUMP is expressed only in the prostate. Searching the mouse EST database revealed that the only cDNAs showing a significant match with AUMP were from the male mouse bladder cDNA library. Although it is likely that some cell lineages in the bladder may express AUMP, the possibility that these transcripts are of prostate origin, arising from contamination of the bladder with adjacent prostate tissue, cannot be excluded. A high level of expression restricted to the prostate gland indicates that AUMP may have important roles related to prostate physiology. Although it is clear that AUMP is an androgen target gene, it is unknown whether it is under direct or indirect regulation by androgens, because we were unable to locate any androgen response element within the 457 bp in the 5' flanking region of AUMP. Considering the potential of selectively driving gene expression to the androgen-sufficient prostate, further study is required to clone the promoter responsible for AUMP expression.
When studying gene expression in the prostate, it is important to consider
the changes in the proportion of different cell types following androgen
depletion or repletion. Morphometric studies have shown that androgen ablation
can result in a major reduction of glandular epithelial cells
(DeKlerk and Coffey, 1978), leading to an altered ratio of epithelial to stromal cells in the prostate
(English et al, 1985;
Montpetit et al, 1986; Isaacs et al, 1992). As a
result of the changes in cellular composition, any gene that is localized in
the epithelium may appear to be down-regulated after androgen ablation due to
a decrease in epithelial cell numbers and not due to a decrease in expression
level. Techniques such as in situ hybridization are therefore essential to
demonstrate alteration in the expression level of a gene. In the present
study, in situ hybridization has localized AUMP expression to the epithelial
cytoplasm of the luminal cells in androgen-sufficient mouse prostates. AUMP
expression was absent in androgen-depleted prostates even though epithelial
cells are maintained in hpg as well as 14-day castrated mice. Thus,
the lack of mRNA in androgen-depleted prostates was not due to a reduction in
epithelial cell numbers, but to an absence of AUMP expression. Our results,
therefore, demonstrate that AUMP gene expression in the prostate is both
epithelial-specific as well as androgen-regulated. Based on subcellular
location of PPT and SecY/Sec61-, it is likely that AUMP may be
localized to lysosome or ER.
The presence of a PPT-like gene in the prostate and its functional significance are unclear at present. PPT is a lysosomal enzyme (Verkruyse and Hofmann, 1996) that is involved in deacylation of palmitoylated proteins. It was first isolated from bovine brain (Camp and Hofmann, 1993) and subsequently shown to be present in several tissues in rats (Camp et al, 1994), mice (Salonen et al, 1998), and humans (Schriner et al, 1996). Palmitoylation is believed to promote membrane localization of proteins and to mediate protein-protein interactions at the plasma membrane (Camp and Hofmann, 1993). Deficiency of PPT has been shown to cause a neurodegenerative disease in infants (Vesa et al, 1995). Recent evidence indicates that it is developmentally regulated in rat brain (Suopanki et al, 1999) and may have a protective role against apoptosis in human neuroblastoma cells (Cho and Dawson, 2000). However, we have not come across any studies reporting androgen regulation of PPT. Induction of AUMP by testosterone and its down-regulation by castration suggests that it may have a role in suppression of apoptosis.
Searching the draft human genome database has so far revealed no matching human sequence. Because the overall ontogeny of rodent prostate is very similar to that of humans, further efforts will be made to obtain the human homologue of AUMP that will enable evaluation of its role in normal and diseased prostate.
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Acknowledgments |
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Footnotes |
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