From the Laboratory for Experimental Medicine and Endocrinology, Faculty of Medicine, Onderwijs en Navorsing, Gasthuisberg, Catholic University of Leuven, Herestraat 49, B-3000 Leuven, Belgium
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ABSTRACT |
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Differential display was used to identify novel
androgen-regulated genes in the human prostatic adenocarcinoma cell
line LNCaP. A 322-base pair cDNA fragment that was consistently
induced by the synthetic androgen R1881 revealed 100% homology with
the human phosphatidic acid phosphatase type 2a isozyme very recently
reported by Kai et al. (PAP-2a; Kai., M., Wada, I., Imai,
S.-i., Sakane, F., and Kanoh, H. (1997) J. Biol. Chem.
272, 24572-24578). The fragment was used to clone the corresponding
cDNA from a human prostate library. The deduced amino acid sequence
confirmed the identity with human PAP-2a. The inducibility of PAP-2a
mRNA by androgens was confirmed by Northern blot hybridization. The
effect was time- and dose-dependent with a maximal
stimulation (4-fold) after 24 h of treatment with
109 M R1881. The steroid specificity of
PAP-2a mRNA regulation was found to be in agreement with the
aberrant ligand specificity of the mutated androgen receptor in LNCaP
cells, supporting the involvement of the androgen receptor in the
induction process. Furthermore, low basal levels of PAP-2a mRNA and
absence of androgen inducibility were observed in the poorly
differentiated and androgen receptor-negative cell lines PC-3 and
DU-145. Induction of PAP-2a mRNA was not affected by the protein
synthesis inhibitor cycloheximide and was accompanied by a marked
increase in PAP-2 activity as measured by the conversion of
phosphatidic acid into diacylglycerol in membrane fractions of LNCaP.
Comparison of the expression of PAP-2a mRNA in 50 different
human tissues revealed ubiquitous expression. The highest levels,
however, were observed in the prostate. Since PAP-2 plays a pivotal
role in the control of signal transduction by lipid mediators such as
phosphatidate, lysophosphatidate, and ceramide-1-phosphate, the ability
of androgens to stimulate the expression and activity of this enzyme in
prostatic cells may provide an important opportunity for cross-talk
between signaling pathways involving lipid mediators and androgens.
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INTRODUCTION |
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The human prostate is a prototypical androgen-dependent organ. Androgens are required not only for its normal development and growth, but also for its structural and functional integrity (1, 2). The prostate is also a main site for androgen-related pathology. Benign prostatic hyperplasia is the most common proliferative disorder of any internal organ and prostate cancer is the most commonly diagnosed cancer in Western males and the second leading cause of male cancer death (3, 4). Androgens are undoubtedly involved in the pathogenesis of these diseases but their exact role remains elusive. In fact, the mechanisms by which androgens affect proliferation, differentiation, and function of the normal and diseased human prostate remain poorly understood.
To get a more complete picture of the effects of androgens on human prostatic epithelial cells and to select novel candidate genes for detailed studies on the molecular mechanisms underlying these effects, our laboratory is exploring different strategies. An antiserum directed against the proteins in prostatic fluid has been used successfully to screen a prostate cDNA expression library for novel androgen-regulated secreted proteins (5). In addition, a differential display (6) approach has been adapted to permit a broader search for androgen-regulated mRNAs in the prostatic adenocarcinoma cell line LNCaP.
LNCaP cells are the only human prostatic epithelial cells which are
routinely available and which remain androgen-responsive in
vitro (7). Not only their proliferation (8-10), but also the
expression of differentiated secretory function (5, 11-13), and the
control of processes such as lipid synthesis and accumulation (14)
remain androgen responsive. LNCaP cells display the remarkable property
that androgen regulation of cell proliferation follows a typical
bell-shaped dose-response curve with optimal stimulation at
1010 M R1881 (a synthetic androgen) and less
stimulation or even growth inhibition at higher concentrations (8-10,
15). Parameters of differentiated function, however, such as the
production of prostate-specific antigen follow a classical hyperbolic
dose-response curve with a maximal response at high concentrations of
androgens (10
8 M R1881). Using a differential
display polymerase chain reaction (DD-PCR)1 technique (6), we
try to identify genes that are differentially expressed at low and high
concentrations of androgens. To this end, we "display" and compare
the mRNA expression levels (by means of PCR fragments) in LNCaP
cells that are treated with R1881 concentrations that optimally
stimulate growth (10
10 M) or differentiated
function (10
8 M). Up to now 126 primer
combinations have been investigated resulting in the identification of
5 genes that are clearly regulated by androgens as confirmed by
Northern blot analysis. Two of these genes are stimulated and two are
inhibited at 10
8 M R1881. One follows the
bell-shaped dose-response curve of cell proliferation.2
Here we report the isolation of one of the androgen-stimulated genes and its identification as a phosphatidic acid phosphatase type 2 (PAP-2). The sequence of the cDNA cloned from a human prostate library is identical to that of the human PAP-2a isozyme reported during the preparation of this manuscript (16). Since PAP-2 plays an important role in the control of signal transduction by lipid mediators such as phosphatidate, lysophosphatidate, and ceramide 1-phosphate (16-18) these findings open the possibility for an important cross-talk between androgens and other cell-signaling pathways controlled by lipid mediators.
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EXPERIMENTAL PROCEDURES |
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Cell Culture and Androgen Treatment-- Human prostatic adenocarcinoma cell lines were purchased from the American Type Culture Collection (Rockville, MD) and maintained in a humidified atmosphere of 5% CO2 in air in RPMI 1640 medium (LNCaP) or in Dulbecco's modified Eagle's medium (PC-3 and DU-145) supplemented with 10% fetal calf serum (FCS), 3 mM L-glutamine, 100 µg/ml streptomycin, and 100 units/ml penicillin (all from Life Technologies Inc., Paisley, Scotland). To examine the effects of steroids, cells were cultured for 2 days in RPMI 1640 medium containing 5% FCS pretreated with dextran-coated charcoal (CT-FCS) as described previously (5). Natural steroids and dexamethasone were purchased from Sigma. R1881 (methyltrienolone) and mibolerone were obtained from NEN Life Science Products Inc. Steroids were dissolved in absolute ethanol. Control cultures received similar amounts of ethanol only. Final ethanol concentrations did not exceed 0.1% (v/v).
RNA Preparation-- For DD-PCR and Northern hybridization, total RNA was extracted from LNCaP cells using a modified guanidinium isothiocyanate/phenol extraction procedure (19). Contaminating DNA was removed by treatment with RNase-free DNase I (Boehringer Mannheim, Germany) in the presence of placental RNase Inhibitor (Promega, Madison, WI). The RNA was phenol/chloroform extracted and ethanol precipitated. Quality and quantity of the RNA were determined by measuring the absorbance at 260 and 280 nm and by Northern blot analysis. For dot-blot hybridizations, total RNA was prepared using a guanidine isothiocyanate/CsCl ultracentrifugation method as described previously (5).
Northern and Dot-blot Analysis--
Northern and dot-blot
hybridizations were carried out as described previously (5). A
radiolabeled PAP-2a cDNA probe was prepared as follows. The 322-bp
DD-PCR fragment (cloned into pGEM-T plasmid; Promega, Madison, WI) was
first amplified by PCR using pUC/M13 forward and reverse sequencing
primers (Pharmacia, Uppsala, Sweden). Approximately 20 ng of the PCR
product was used in a radiolabeling reaction mixture (total volume: 12 µl) containing 10 mM Tris-HCl (pH 8.3), 50 mM
KCl, 1.5 mM MgCl2, 15 µCi of
[-32P]dCTP (3000 Ci/mmol, Amersham International,
Buckinghamshire, UK), 50 µM each of dATP, dTTP, and dGTP,
0.4 µM 10-mer primer 5'-TACAACGAGG-3', 0.4 µM dT11GA primer, and 0.025 units/µl
Taq DNA polymerase. PCR cycling conditions were as described
for "Differential Display Analysis" (see below). A radiolabeled 18 S probe was prepared as described before (20). Hybridization signals
were quantified using PhosphorImager screens (Molecular Dynamics,
Sunnyvale, CA).
Differential Display Analysis--
The mRNA DD-PCR was
performed using a Differential Display Kit (Display Systems Tandill
Ltd., Los Angeles, CA) according to the manufacturer's instructions,
with minor modifications to the protocol. For reverse transcription,
150 ng of DNase I-treated total RNA, 2.5 µM primer
(dT11MN; M,N = A, C, or G; supplied with the kit), 20 µM dNTPs, and 300 units of Superscript II Reverse Transcriptase (Life Technologies, Gaithersburg, MD) was mixed in a
total volume of 32 µl and incubated at 42 °C for 1 h. The reaction was stopped by incubation at 95 °C for 5 min. PCR reactions were performed in a total volume of 20 µl containing 2.5 µM downstream primer (dT11MN), 0.5 µM arbitrary 10-mer primer, 2 µM dNTPs, 2 µCi of [-33P]dATP (Amersham Int.), 10 mM
Tris-HCl (pH 8.3), 50 mM KCl, 3 mM
MgCl2, 1.1 µl of cDNA solution, and 0.5 unit of
AmpliTaq DNA Polymerase (Perkin Elmer, Norwalk, CT). PCR reactions were
carried out in a GeneAmp PCR System 2400 thermal cycler (Perkin Elmer) with the following cycling parameters: one denaturation step of 3 min
at 94 °C, 40 cycles of 30 s at 94 °C, 1 min at 40 °C, 1 min at 72 °C, and one extension step of 7 min at 72 °C. All
DD-PCR reactions were performed at least twice under identical
conditions, and loaded side by side on a 6% sequencing gel. Gels were
dried and bands were visualized by autoradiography. cDNA fragments
of interest were excised from the dried gel, eluted in 100 µl of boiling water, ethanol precipitated, and reamplified. The
resulting PCR products were purified using a Qiaquick PCR
Purification Kit (Qiagen, Hilden, Germany) and were cloned into
the pGEM-T plasmid (Promega, Madison, WI).
DNA Sequence Analysis-- DNA sequencing of the cloned cDNA fragments was performed using Autoread sequencing kits (Pharmacia, Uppsala, Sweden) and an A.L.F. automated sequencer (Pharmacia). The sequences of both strands were determined. Sequences were analyzed using the GCG Wisconsin package provided through the services of the Belgian Embnet Node (BEN). Comparison of DNA homology with the EMBL and the GenBank data bases was performed using BLAST (21) and FASTA (22) routines.
cDNA Cloning of PAP-2a from a Human Prostatic cDNA Library-- An oligo(dT) and random primed cDNA library derived from prostatic tissue from a 25-year-old male (HL3111b; CLONTECH, Palo Alto, CA) was screened for cDNA clones encoding PAP-2a essentially as described earlier (5). Plaque lifts with a total of 6 × 105 recombinant plaques were hybridized with a radiolabeled PAP-2a cDNA probe (0.5 × 106 cpm/ml). Positive plaques were identified by autoradiography and plaque-purified through a second round of screening. Clones were characterized by restriction enzyme analysis. Appropriate restriction fragments were subcloned into pGEM7Zf(+) (Promega, Madison, WI) and sequenced.
Determination of PAP Activity--
Microsomal fractions were
prepared from LNCaP cells by resuspension of the cells in 10 mM Hepes-KOH (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 mM
dithiothreitol. After incubation for 10 min at 4 °C the cell
suspensions were homogenized using a Dounce homogenizer. The homogenate
was centrifuged at 10,000 × g at 4 °C for 20 min.
The supernatant was centrifuged for 30 min at 100,000 × g (4 °C). Protein concentrations of the pellet fractions
were measured by the BCA procedure (Pierce, Rockford, IL). For the
determination of PAP activity, 1.5 µg of protein of LNCaP cell
membranes were incubated at 37 °C for 20 min, in a solution
containing 100 µM [14C]1--dipalmitoyl
phosphatidic acid (NEN Life Science Products), 50 mM
Hepes/NaOH (pH 7.0), and 3 mM Triton X-100, in a final
volume of 100 µl as described by Hoër et al. (23).
Equal results were obtained using 32P-labeled phosphatidic
acid as substrate (23).
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RESULTS |
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Use of DD-PCR Analysis to Identify Androgen-regulated Genes in
LNCaP Cells: Identification of One of the Androgen-induced Genes as
PAP-2a--
To identify mRNAs that are regulated by androgens,
DD-PCR was performed on total RNA derived from LNCaP cells treated with control medium or R1881. The synthetic androgen R1881 was selected for
these studies to circumvent the rapid metabolization of natural androgens such as 5-dihydrotestosterone and testosterone reported in
LNCaP cells (24). Cells were treated with 10
10
M R1881, a concentration that optimally stimulates growth,
or with 10
8 M R1881, a concentration that
optimally promotes differentiation (15).
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cDNA Library Screening and Sequencing of Human PAP-2a cDNA-- To obtain the full-length cDNA, a human prostate cDNA library was screened with the radiolabeled 322-bp cDNA clone. Seven positive clones, varying in length between 1.0 and 1.5 kilobases were isolated and sequenced. This resulted in a sequence of 1798 bp covering the entire mRNA except for a small fragment at its 3'-noncoding end. The 5' sequence was confirmed to be complete by 5-' rapid amplification of cDNA ends polymerase chain reaction (Life Technologies) on LNCaP mRNA. The entire sequence is available from EMBL, GenBank, and DDBJ data base (accession number Y14436). It encompasses a 937-bp fragment sequenced by Kai et al. (16) departing from HepG2 cDNA and referred to by them as PAP-2a because of its homology with mouse PAP-2. This fragment which is 100% homologous with our sequence contains a 852-bp open reading frame for a PAP-2a protein with a calculated Mr of 32,155. The 322-bp DD-PCR cDNA fragment was found to be located at the 5'-end of this open reading frame (bp +102 to +423 departing from the ATG start codon).
Comparison of the entire protein sequence with sequences in the EMBL/GenBank data bases using BLAST (21) and FASTA (22) routines, revealed 83% homology at the protein level, between PAP-2a and both mouse (25) and rat3 PAP-2. Other genes showing significant homology include the mouse hydrogen peroxide-inducible protein HIC-53 (26), mouse Dri 42 (an endoplasmatic reticulum resident transmembrane protein; Ref. 27), and its recently reported human counterpart PAP-2b (16), a human gene product with unknown function (clone 23748 mRNA)4 and the Drosophila germ cell guidance factor Wunen (28) (data not shown).Androgen Regulation of PAP-2a Expression in LNCaP
Cells--
Dose-response curves with R1881 confirmed that PAP-2a
mRNA induction follows a classical hyperbolic dose-response curve.
Maximal stimulation (approximately 4-fold) was observed from
109 M R1881 on (Fig.
2A). The time course of PAP-2a
mRNA induction revealed some effect as early as 4-8 h after
treatment. Maximal stimulation was obtained after 16-24 h. Thereafter
the level of mRNA tended to decrease slightly (Fig.
2B).
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PAP-2a mRNA Expression in the Prostate as Compared with Other Human Tissues-- Northern and dot-blot hybridization was used to analyze the expression of PAP-2a mRNA in different human tissues. Northern hybridization yielded identical data to those reported by Kai et al. (16) (not shown), including very high expression in the prostate. A broader screening was performed using dot-blot analysis (Fig. 5). Again expression turned out to be most pronounced in the prostate. Other tissues with high expression were: aorta > bladder > uterus > kidney > mammary gland > spinal cord > small intestine > adrenal gland > thyroid gland.
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Effects of Androgens on PAP-2 Activity--
To investigate whether
the observed effects of androgens on PAP-2a mRNA levels were
accompanied by changes in PAP activity, microsomal fractions were
prepared from LNCaP cells after stimulation with 1010
M R1881 and 10
8 M R1881. PAP-2
activity was determined by measuring the conversion of phosphatidic
acid into diacylglycerol (23). These results showed a 2.7-fold increase
of PAP-2 activity in microsomal fractions after induction with
10
10 M R1881, and a 5.4-fold increase with
10
8 M R1881 (Fig.
6). PAP-2 activity was independent of
Mg2+, inhibited by propranolol (2-fold decrease at 10 mM), and insensitive to N-ethylmaleimide (data
not shown). These characteristics are in accordance with those reported
for PAP-2 in other species (17, 32, 33) and for PAP-2a in the human
(16).
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DISCUSSION |
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It is universally accepted that androgens play a pivotal role in the control of growth and differentiation in the normal and diseased human prostate (1-4). Nonetheless, up to now only a limited number of androgen-regulated genes have been identified and investigated in any detail and most of these genes encode prostate-secreted proteins (5). In the present paper we have used a differential display technique (6) to identify novel androgen-regulated genes that are functionally relevant and/or can be used for studies on the molecular mechanisms governing androgen action in the human prostate.
A 322-bp DD-PCR cDNA fragment that was consistently induced by
108 M R1881 in the androgen-responsive
adenocarcinoma line LNCaP, revealed a 76% homology to mouse PAP-2 (25)
and proved 100% homologous to the human counterpart of this cDNA
reported during the preparation of this article (16). Screening of a
human prostate cDNA library with the mentioned fragment yielded 7 positive clones allowing sequencing of a 1798-bp fragment containing a
852-bp open reading frame, the entire 5'-upstream noncoding sequence, and most of the 3'-noncoding region of the human equivalent of PAP-2.
This sequence is 100% homologous with the 937-bp fragment derived from
HepG2 cDNA which has been shown to encode the human PAP-2a isozyme.
Accordingly we will conveniently denote the prostate-derived sequence
also as PAP-2a.
The human PAP-2a cDNA encodes a protein with a deduced size of 32 kDa and with a marked homology (83%) with the corresponding mouse (25) and rat3 proteins (16). Moreover, an NH2-terminal sequence of 25 amino acids and an internal sequence of 9 amino acids previously determined from porcine thymus PAP-2 (25, 34) are found unchanged in the human enzyme confirming a very high degree of conservation. A less pronounced but intriguing homology should be mentioned with three other gene products. (a) The originally identified 322-bp fragment is highly homologous to a region in HIC-53, a gene induced by hydrogen peroxide in a mouse osteoblastic cell line (26). The partial cDNA clone reported for HIC-53 is 95% identical with mouse PAP-2, but HIC-53 is supposed to use an initiation codon 5'-upstream of that of PAP-2 and the encoded protein would lack the 64 carboxyl-terminal amino acids of the latter enzyme. At the present time it is unknown whether the discrepant reading frame of HIC-53 is the result of a sequencing or a cloning artifact, alternative splicing, or the existence of highly similar but distinct genes. (b) Sixty percent homology was noted with Dri42 (27), a protein located in the endoplasmic reticulum and up-regulated during development and differentiation of rat intestinal epithelial cells. The human equivalent of Dri42 was recently sequenced and was also shown to display phosphatidic acid phosphatase activity (16). It has 47% homology with PAP-2a and was therefore denoted PAP-2b. Both isozymes display different patterns of substrate utilization and transcriptional regulation. (c) Finally, recent studies point to an important homology between PAP-2a and a transcript of the Drosophila gene Wunen (28). Interestingly, this gene encodes a membrane protein involved in guiding the germ cells to the gonads during embryonic development.
PAP-2a is obviously a ubiquitously expressed gene. Both Northern blot data (data not shown; Ref. 16), however, and the dot-blot data shown in Fig. 5 suggest that there are important differences in expression between tissues. The highest level of expression is observed in the prostate. At the present time the reason for this high expression in the prostate remains elusive. Preliminary experiments did not reveal marked differences in the expression of PAP-2a in biopsies derived from hyperplastic prostates (n = 29) and prostate cancers (n = 38). The levels of PAP-2a mRNA observed in these biopsies were comparable to those measured in LNCaP cells, prostatic adenocarcinoma cells with a high degree of differentiated function. Interestingly, much lower levels of expression were observed in the poorly differentiated prostate tumor lines PC-3 (30) and DU-145 (31). The latter finding is reminiscent of the decreased specific activity of PAP-2 observed in rat fibroblasts after transformation with ras or a non-receptor tyrosine kinase (35). In the latter model the decreased activity of PAP-2 has been related to higher levels of phosphatidate and an increased rate of cell division. It may be worthwhile to mention that for the above described HIC-53 gene markedly decreased expression has also been observed after transformation of the osteoblastic cell line MC3T3-E1 with ras (26).
Up to now little is known on factors regulating PAP-2a gene expression. In the studies of Kai et al. (16), PAP-2a in HeLa cells was shown not to respond to H2O2 and to epidermal growth factor, whereas PAP-2b was stimulated by epidermal growth factor. In this context it is of interest that androgens induce a 4-fold increase in the steady state level of PAP-2a mRNA in LNCaP cells. This increase in mRNA is accompanied by a comparable increase in PAP activity in membrane fractions of androgen-treated LNCaP cells. The measured enzyme activity displays the characteristics of PAP-2 (17, 33). Unlike PAP-1, it does not require Mg2+ for activity and it is not inhibited by the sulfhydryl reagent N-ethylmaleimide (33). Although the exact mechanism(s) by which androgens affect PAP-2a gene expression remain to be investigated, the data presented strongly suggest that the observed effects are mediated by the androgen receptor. (a) Androgens stimulate PAP-2a mRNA accumulation in androgen receptor-positive LNCaP cells but not in androgen receptor-negative PC-3 and DU-145 cells. (b) The steroid specificity of the response reflects the aberrant ligand specificity of the mutated androgen receptor in LNCaP cells (29). Stimulation is observed not only with natural and synthetic androgens but also with progesterone and estradiol whereas glucocorticoids are inactive. (c) Time dependence and dose-response of the effects of androgens on the expression of the PAP-2a gene are similar to the courses of other androgen-regulated genes in LNCaP such as prostate-specific antigen (5), diazepam-binding inhibitor/acyl-CoA binding protein (5), and fatty acid synthase (36). Further studies will be required to determine whether androgen regulation of the PAP-2a gene involves a direct interaction of the androgen receptor with a responsive element in the gene as demonstrated for prostate-specific antigen. The observation that, exactly as for prostate-specific antigen (5), androgen-induction of PAP-2a is not prevented by cycloheximide (an inhibitor of protein synthesis) is compatible with the contention that androgen-induced regulatory proteins are not essential for the induction process.
At the present time we can only speculate on the functional significance of the androgenic regulation of PAP-2a in LNCaP cells. By dephosphorylating phosphatidic acid, PAP can obviously supply diacylglycerol for glycerolipid synthesis (37, 38). As recently demonstrated in our laboratory, androgens provoke a marked accumulation of lipid droplets in LNCaP cells (14). These droplets contain cholesteryl esters and triacylglycerol and their accumulation is, at least in part, the result of the coordinate activation of several enzymes involved in cholesterol and fatty acid synthesis. This process is accompanied by a limited (approximately two-fold) increase in phospholipid synthesis. It is conceivable that the observed increase in PAP-2 might somehow be related to these marked changes in lipid metabolism. Alternatively, however, many recent data point to the possibility that PAP-2 plays a pivotal role in the control of signal transduction by lipid mediators (16, 17, 39). Phosphatidate has potent mitogenic effects in several cell lines and affects a variety of other cellular functions (40). PAP-2 can terminate signaling by this bioactive lipid, but in doing so it converts phosphatidate in diacylglycerol, another lipid second messenger that activates the protein kinase C cascade (41). In this way PAP-2 can act in a coordinate manner with the phospholipase D pathway, where phosphatidate is generated by the agonist-induced hydrolysis of the major membrane phospholipid phosphatidylcholine (42) to sustain a prolonged activation of protein kinase C. Furthermore, there is considerable evidence from studies on PAP-2 in other species (18, 23, 25, 35, 43) and PAP-2a in the human (16) that this enzyme is a multifunctional phosphohydrolase that can dephosphorylate not only phosphatidate but also lysophosphatidate and ceramide 1-phosphate. Since all these substrates as well as their dephosphorylated reaction products are potent lipid mediators, it is evident that PAP-2 occupies a central position in this signaling cascade. Taking these data into account it is tempting to speculate that the ability of androgens to stimulate PAP-2a provides LNCaP cells with an important opportunity for cross-talk between the signaling pathway for steroids and that for locally produced lipid mediators. The ability of androgens to stimulate phosphoinositide metabolism as described previously by Wilding et al. (44) may provide an additional and complementary way of cross-talk.
In conclusion, we have identified one of the androgen-induced genes in LNCaP cells as PAP-2a, the human equivalent of mouse and rat PAP-2. Evidence is provided that this gene is abundantly expressed in the prostate and that, at least in androgen-responsive tumor cells, its expression and activity is enhanced markedly by androgens via a pathway involving the androgen receptor. It is speculated that the ability of androgens to stimulate PAP-2 provides LNCaP cells with an important opportunity for cross-talk between signaling pathways involving androgens and lipid mediators.
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ACKNOWLEDGEMENTS |
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The excellent technical assistance of F. Vanderhoydonc and J. Rosseels is kindly acknowledged. We thank Prof. L. Baert and Dr. A. Elgamal for help in collecting prostate biopsies, and Prof. P. Van Veldhoven and Prof. M. Bollen for helpful advice for the determination of PAP-2 activity.
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FOOTNOTES |
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* This work was supported by grants from the Geconcerteerde Onderzoeksactie van de Vlaamse Gemeenschap, Grant 3.0048.94 from the Fund for Scientific Research, Flanders (Belgium) (Fonds voor Wetenschappelijk Ondersoek-Vlaanderen), grants from the Interuniversity Poles of Attraction Program, Belgian State, Prime Minister's Office, Federal Office for Scientific, Technical and Cultural Affairs, and the Vereniging voor Kankerbestrijding.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence reported in this paper has been submitted to EMBL, GenBank, and DDBJ data bases with accession number Y14436.
Senior Research Assistant of the Fund for Scientific
Research-Flanders (Belgium) (F. W. O.).
§ To whom correspondence should be addressed: LEGENDO, Onderwijs en Navorsing, Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. Tel.: 32-16-34-59-70; Fax: 32-16-34-59-34; E-mail: guido.verhoeven{at}med.kuleuven.ac.be.
1 The abbreviations used are: DD-PCR, differential display polymerase chain reaction; CT-FCS, charcoal-treated fetal calf serum; FCS, fetal calf serum; PAP-2, phosphatidic acid phosphatase type 2; bp, base pair(s).
2 W. Ulrix, J. V. Surinnen, W. Heyns, and G. Verhoeven, unpublished results.
3 D. W. Waggoner and D. N. Brindley, EMBL/GenBank accession number U90556.
4 W. Yu and R. A. Gibbs, EMBL/GenBank accession number U79294.
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REFERENCES |
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