0013-7227/97/$03.00/0 Endocrinology Copyright © 1997 by The Endocrine Society
Vol. 138, No. 9 Printed in U.S.A.
Steroid-Involved Transcriptional Regulation of Human Genes Encoding Prostatic Acid Phosphatase, ProstateSpecific Antigen, and Prostate-Specific Glandular Kallikrein* JING-DONG SHAN, KATJA PORVARI, MINNA RUOKONEN, AULI KARHU, VIRPI LAUNONEN, PIRJO HEDBERG, JOUKO OIKARINEN, AND PIRKKO VIHKO Biocenter Oulu, World Health Organization Collaborating Center for Research in Human Reproduction and Department of Clinical Chemistry, University of Oulu, Oulu, Finland ABSTRACT We have compared the steroid regulation of human genes encoding prostatic acid phosphatase (hPAP), prostate-specific antigen (hPSA), and prostate-specific glandular kallikrein (hK2) at the level of transcription. Reporter constructs of hPAP promoter covering the region 2734/1467 were functional in both prostatic (LNCaP and PC-3) and nonprostatic (CV-1) cell lines in transient transfections. hPAP 2231/ 150 with eight identified transcription factor-binding sites showed the highest, and hPAP 2734/1467 showed the lowest transcriptional activity in CV-1 cells. The hPAP promoter could not be induced with androgen, glucocorticoid, or progesterone, contrary to the hPSA (2620/140) and hK2 (2493/127) promoters in PC-3 cells cotrans-
fected with the respective steroid receptor expression vector. Therefore, steroids cannot directly regulate hPAP gene expression via receptor binding to steroid response elements at 2178 and 1336, which have been shown to have androgen receptor-binding ability in vitro. Glucocorticoid was the most powerful activator of the hPSA construct at 10-nM steroid concentrations. On the contrary, glucocorticoid stimulation of the transcriptional activity of the hK2 construct was the weakest among the tested steroids. The results indicate that the steroid response elements in the proximal promoters of hPSA and hK2 genes are not androgen specific, offering the molecular basis for the expression of these genes outside the prostate in tissues containing steroid receptors. (Endocrinology 138: 3764 –3770, 1997)
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moter at 2400. This element is needed for the optimal androgen stimulation of hPSA transcription (10). Similarly to the hPSA and hK2 genes, both the hPAP promoter and the rat PAP promoter contain putative SRE at 2178 and 2174, respectively. These SREs in PAP genes have been shown to bind AR in vitro (11). The positional conservation of the SREs in these genes encoding prostatic proteins suggest that these elements might also be important for steroid regulation of PAP. Several investigations have shown that hPSA and hK2 gene expression is not strictly restricted to the prostate. Low levels of hPSA have been detected in the milk of lactating women (12), in several breast tumors (13), and in normal breast tissue induced with oral contraceptive (14). Furthermore, the expression of both hPSA and hK2 genes has been indicated by reverse transcription-PCR in the human endometrium (15). On the contrary, studies using monoclonal antibodies against hPAP have indicated the prostate specificity of this protein (16), and hPAP mRNA expression has not been reported outside the prostate (17). It is important to clarify further transcriptional regulation of the hPAP gene, because the probable prostate-specific promoter of this gene might have applications in the treatment of prostatic disorders in the future. The androgen-dependent prostatic carcinoma cell line LNCaP is able to produce the main secretory proteins of the prostate and is suitable for transcriptional regulation studies of the respective genes. Although the essential transcription factors needed for hPAP, hPSA, and hK2 gene expression are present in LNCaP cells, the disadvantages of these well dif-
HE HORMONAL regulation of human prostatic acid phosphatase (hPAP), one of the prostate cancer markers, is poorly understood. The effects of androgens on PAP expression have been intensively studied at protein and messenger RNA (mRNA) levels, but the precise mechanisms of androgen action remain unclear. Down-regulation of PAP by androgens has been reported in prostatic carcinoma cell line LNCaP (1, 2), whereas several studies support the opposite effect of the hormone (3, 4). Furthermore, the direction of androgen regulation of PAP has been suggested to be dependent on cell density (5) and other culture conditions, such as the amount of androgen (6) in LNCaP cells. Additionally, the longest transcript encoding rat PAP has been shown to be quite resistant to the hormonal status of the prostate, whereas two other mRNA species have been shown to be up-regulated by androgen (7). On the other hand, the androgen dependency of human prostate-specific antigen (hPSA) and human prostate-specific glandular kallikrein (hK2) has been clearly demonstrated. In these genes, the androgen effect is mediated via steroid response elements (SREs) at 2170 and 2160 in proximal promoters of hPSA and hK2, respectively (8, 9). In addition to SRE, another hormone response element with androgen receptor (AR)-binding ability has been recently identified in the hPSA proximal proReceived February 18, 1997. Address all correspondence and requests for reprints to: Dr. Katja Porvari, Kajaanintie 50, FIN-90220 Oulu, Finland. E-mail:
[email protected]. * This work was supported by the Research Council for Health of the Academy of Finland (Project No. 3314).
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ferentiated cells include instability, modest transfection efficiency, and poor ability to attach to the growth support (18). The androgen-independent prostatic cancer cell line PC-3 does not contain AR (19) and does not express detectable levels of hPAP mRNA in Northern blot analysis (17), nor are PC-3 cells able to secrete hPSA or hK2 (8). In this cell line the hPAP gene seems to be normal according to Southern blot analysis (20) and partial sequencing (our unpublished data). Transfection of the AR expression vector into PC-3 cells cannot trigger the expression of hPAP (20), and probably another transcription factor(s) essential for hPAP expression is missing from PC-3 cells. Here we have compared steroid regulation of the three prostatic proteins, hPAP, hPSA, and hK2, at the transcriptional level in prostatic (LNCaP and PC-3) and nonprostatic (CV-1) cell lines. The DNA-protein interactions in the proximal promoter of hPAP were also assessed, using nuclear extracts from LNCaP and PC-3 cells in deoxyribonuclease I (DNase I) footprint analysis, to expose the important factor(s) involved in hPAP gene transcription. Materials and Methods Chemicals and reagents The isotopes [a-35S]deoxy (d)-ATP (3000 Ci/mmol) and [a-32P]dCTP (3000 Ci/mmol) were obtained from Amersham Life Science (Little Chalfont, UK). The transfection reagent N-[(1-(2,3-dioleoyloxy)propyl)]N,N,-trimethyl-ammoniummethylsulfate (DOTAP), chloramphenicol acetyltransferase (CAT), and DNase I were products of Boehringer Mannheim (Mannheim, Germany). [3H]Acetyl coenzyme A, Econofluor, and synthetic androgen R1881 (17b-hydroxy-17a-methyl-estra-4,9,11trien-3-one) were purchased from DuPont-New England Nuclear (Boston, MA). Chloramphenicol and the synthetic glucocorticoid dexamethasone (1,4-pregnadiene-9a-fluoro-16a-methyl-11b,17a,21-triol-3, 20dione) were obtained from Sigma Chemical Co. (St. Lous, MO). Synthetic
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progesterone ORG2058 (16a-ethyl-21-hydroxy-19-nor-4-pregnene-3,20dione) was a gift from Organon (Oss, The Netherlands). Restriction endonucleases were obtained from New England Biolabs (Beverly, MA).
Preparation of reporter constructs Fragments of the hPAP (Fig. 1), hPSA, and hK2 gene were generated by PCR. Primers introducing HindIII (at the 59-end) and SalI (at the 39-end) restriction sites were used to amplify the hPAP fragments 2734/ 150, 2432/150, 2231/150, 2130/150, and 282/150. The hPAP fragment 21652/143 was amplified using oligonucleotides generating the PstI site at the 59-end and the XbaI site at the 39-end. Additional PCR products of the hPAP gene (157/1323 and 157/1467) contained SalI at the 59-end and XbaI at the 39-end. Correspondingly, SalI and XbaI sites were introduced into the 59- and 39-ends, respectively, of the hPSA (2620/140) and hK2 (2493/127) gene fragments. The fragments were cloned into the pCAT-Basic vector (Promega, Madison, WI). All inserts were verified by sequencing, using the T7 Sequencing Kit (Pharmacia Biotech, Uppsala, Sweden).
Cell culture and transient transfection assays The human prostatic carcinoma cell lines (LNCaP and PC-3) and the monkey kidney cell line CV-1 were obtained from American Type Culture Collection (Rockville, MD). The cells were maintained as recommended by the supplier. For transient transfections, 1 3 106 cells/ 100-mm dish were plated 24 h (PC-3 and CV-1) or 72 h (LNCaP) before transfection. The transfections were performed with DOTAP as recommended by the manufacturer. A total of 10 mg DNA were used for every transfection. The LNCaP cells were transfected with 8 mg reporter plasmid, 1 mg steroid receptor expression plasmid (hAR, hGR, or hPR, American Type Culture Collection), and 1 mg control plasmid (pCMVb, Clontech, Palo Alto, CA). Mock transfections were included in every series. The steroid response was verified by transfecting the mouse mammary tumor virus (MMTV) reporter construct (GMCAT, American Type Culture Collection). After 20 –24 h of transfection, the cell medium was replaced with medium containing charcoal-stripped FCS, and hormone (R1881, dexamethasone, or ORG2058) was added when indicated. The cells were collected and lysed after 48 h (PC-3 and CV-1) or 72 h (LNCaP) of incubation from the beginning of the transfection.
FIG. 1. Promoter fragments of the hPAP gene used in transient transfection assays. The locations of putative SREs are marked with in vitro AR-binding capacity (1/2, 1 or 11) (11) relative to the transcription start site (arrow at 11). In addition, TATA at 227, CAAT at 240, and the translation start site (ATG) at 151 are indicated. Deleted ATG is denoted by an open square.
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CAT measurements were performed using fluor diffusion assay (21, 22). Transfection efficiency was estimated by determining the b-galactosidase activity of the samples according to the method of Rosenthal (23). The protein content of the cell lysates was measured by the Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, CA). The CAT activities were normalized by the b-galactosidase activities and protein contents of the samples. The results are presented as relative CAT activities over the value of the pCAT-Basic vector without promoter in the absence of androgen. Statistical analyses of the differences between the means were performed with ANOVA. The means were regarded as statistically significant when P , 0.05.
Western blotting PC-3 cells were transfected with hPAP 2231/1467 and hPAP 2734/ 1467 constructs as described above. The cells were disrupted into 100 ml SDS buffer (62.5 mm Tris-HCl, 10% glycerol, 2% SDS, 5% 2b-mercaptoethanol, and 0.00125% bromophenol blue), and aliquots of the samples (40 ml) were run on 12% SDS-polyacrylamide gel together with mol wt markers. The proteins were transferred onto a polyvinylidene difluoride membrane. The CAT protein was detected using polyclonal CAT antibody (5 Prime-3 Prime, Boulder, CO), horseradish peroxidaselinked secondary antibody, and light-emitting substrate according to the ECL Western blotting protocol (Amersham Life Science).
RNA isolation and slot blotting CV-1 cells were transfected, as described above, with different CAT constructs (hPAP 2734/1467 and MMTV) and hAR expression plasmid. The cells were incubated for 3 h in the absence or presence of 10 nm R1881. Total RNA was isolated using TRIzol reagent (Life Technologies, Grand Island, NY) from the cells. Slot blot of DNase I-treated (20 U ribonuclease-free enzyme from Pharmacia; 10 min at 37 C) RNA samples (50 mg) was prepared as previously described (17) and hybridized with complementary DNA fragment encoding CAT (corresponding to nucleotides 2528 –2829 of pCAT-Basic). The probe was nick translated in the presence of [a-32P]dCTP. mRNA signals of the samples were quantified by densitometric analysis. The results are presented as relative mRNA values of CAT over the value of 1 for each construct in the absence of androgen.
Nuclear extracts Nuclear extracts were made from LNCaP, PC-3, and CV-1 cells as described by Dignam et al. (24). The nuclear extract prepared from HeLa
cells was obtained from Promega (HeLa nuclear extract in vitro transcription systems).
DNase I footprinting The hPAP fragment 2231/150 was cloned into the pCR II vector (Invitrogen, San Diego, CA). The plasmid construct was linearized by SpeI and EcoRV digestion. The 39-end was labeled by fill-in with [a-32P]dCTP. DNase I footprinting reactions were carried out by incubating the DNA probe with the nuclear extracts in the binding buffer (5 mg poly(dI-dC), 20% glycerol, 20 mm HEPES, 75 mm NaCl, and 2 mm dithiothreitol) for 30 min at room temperature. Control reactions were carried out in the presence of BSA (4 mg/reaction). The concentration of MgCl2 was adjusted to be 10 mm in the reaction. The samples were incubated for 1 min with freshly diluted DNase I (Boehringer Mannheim). The reactions were stopped by the addition of 140 ml stop solution (192 mm sodium acetate, 32 mm EDTA, and 0.14% SDS). The samples were proteinase K digested, further extracted with phenol-chloroform, and ethanol-precipitated, then electrophoresed on a 6% sequencing gel.
Results Promoter analyses
The activities of several promoter fragments of the hPAP gene linked to the pCAT reporter plasmid (Fig. 1) were determined in the androgen-dependent prostatic carcinoma cell line LNCaP, which is able to secrete hPAP, hPSA, and hK2 proteins. In the absence or presence of androgen, low CAT activities were produced, but all hPAP promoter constructs were functional in this prostatic cell line (results not shown). Some of the CAT constructs covering the entire region 2734/1467 of the hPAP gene and the AR expression vector were transiently transfected into an androgen-independent prostatic cell line PC-3 and a nonprostatic cell line CV-1 apart from the LNCaP cells (Fig. 2). The activities of the hPSA (2620/140) and hK2 (2493/127) promoter constructs were also analyzed in these three cell lines to allow comparison of the androgen-involved transcriptional regulation of the genes encoding prostatic kallikrein family proteins and hPAP. The promoter constructs of hPAP were
FIG. 2. Promoter activities of genes encoding hPAP, hPSA, and hK2 in prostatic and nonprostatic cells. LNCaP, PC-3, and CV-1 cells were transfected with the MMTV, hPAP, hPSA, and hK2 reporter constructs together with AR and b-galactosidase expression vectors as described in Materials and Methods. CAT activities with (1) or without (2) 10 nM R1881 were measured from duplicate samples of cell extracts from at least three independent experiments. Mock-subtracted CAT values were normalized for b-galactosidase activity and protein content. The data are presented as relative CAT activities over the value of the CAT expression vector without promoter in the absence of androgen. The relative CAT activities of the samples were averaged and are displayed with their SEs. Only 1/10th of the androgen-induced relative CAT value of MMTV is marked in the figure.
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functional in both prostatic and nonprostatic cells. Although hPAP constructs covered two SREs with in vitro AR-binding ability at 2178 and 1336 (11), androgen treatment had no effect on the activity of the proximal promoter of hPAP. The lack of androgen response was also evident in the case of the hPAP 21652/143 construct (results not shown), which covers two putative SREs at 21576 and 2178 with weak and strong in vitro AR-binding capacity (11), respectively. On the other hand, hPSA and hK2 constructs were also functional in all three cell lines, and androgens were able to stimulate the transcriptional activity of these promoters as previously described (8, 9) in PC-3 and CV-1 cells. Similar to previous reports (8, 25), androgen activation of hPSA and hK2 promoters could not be clearly demonstrated in LNCaP cells, although the control promoter (MMTV) showed a significant response to androgen treatment in this cell line. Among the hPAP constructs, the region 2231/150 gave the highest reporter activity in nonprostatic CV-1 cells; the mean values for relative CAT activity were 14.9 6 4.7 (mean 6 se; n 5 3) and 15.5 6 6.0 in the absence and presence of androgen, respectively. The corresponding values were 7.6 6 2.1/7.8 6 2.5 in LNCaP cells and 11.0 6 1.8/13.2 6 3.2 in PC-3 cells. The transcriptional activity of the hPAP promoter region from 2734 to 1467 was lowest at CAT values of 1.4 6 0.2 without androgen and 1.8 6 0.5 with androgen treatment in the nonprostatic cell line, whereas the corresponding values were 5.1 6 1.6/4.9 6 1.1 and 4.0 6 0.3/3.6 6 0.3 in LNCaP and PC-3 cells, respectively. The CAT expression vector without promoter represents the basal level of transcription with a relative CAT activity value 1. The CAT activity of this vector was 3.3 6 1.0 pg/mg protein without androgen and 4.1 6 1.2 pg/mg protein with androgen in LNCaP cells. The corresponding values were 24.9 6 5.8/21.1 6 3.6 pg/mg protein in PC-3 cells and 3.1 6 0.5/4.3 6 0.6 pg/mg protein in CV-1 cells. The activities without hormone vs. the activities with hormone were not statistically different in any of the cell lines. A long leader sequence in the hPAP 2734/1467 construct may cause the initiation of protein translation from an unexpected site, leading to the production of nonfunctional CAT protein and a misinterpretation of the possible androgen effect. One indication that constructs with this long leader are producing functional CAT enzyme is that the activities of the hPAP 2231/1467 and hPAP 2734/1467 constructs are significantly different (P , 0.001) when tested in PC-3 cells. The respective relative CAT values were 9.8 6 0.6 (n 5 3) and 4.0 6 0.3 (n 5 5) in the absence of androgen. Similar activities would be expected for these constructs if the CAT enzyme expressed from the transcripts with identical leader sequences were nonfunctional. However, we have also shown that hPAP 2231/1467 and hPAP 2734/ 1467 constructs produce immunologically detectable CAT enzyme (Fig. 3), confirming that these constructs are functioning as such and are usable for the detection of steroid effects. Furthermore, we also checked the androgen effect on the transcriptional regulation of the hPAP 2734/1467 and MMTV constructs at the mRNA level (Table 1). The relative amounts of CAT transcripts were similar in the absence and presence of androgen, supporting the original conclusion that androgen cannot induce the promoter activity of the
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FIG. 3. Immunological detection of CAT enzyme produced by hPAP 2231/1467 and hPAP 2734/1467 reporter constructs transfected into PC-3 cells. Polyclonal antibody against CAT enzyme was used in the immunostaining of the blotted SDS-polyacrylamide gel. Lane 1, hPAP 2734/1467; lane 2, hPAP 2231/1467. TABLE 1. Effect of androgen on CAT mRNA levels originating from the MMTV and hPAP2734/1467 reporter constructs transfected into CV-1 cells Relative amount of CAT mRNA Reporter construct
MMTV hPAP2734/1467
Without androgen
With androgen
1 1
3.1 0.8
Transiently transfected cells were incubated for 3 h in the absence or presence of 10 nM R1881 before RNA isolation. The results for a representative experiment are shown.
hPAP 2734/1467 construct. The induction of CAT mRNA from the MMTV construct was similar to that previously reported (26, 27). Because androgen failed to regulate the hPAP promoter, the effects of glucocorticoid and progesterone on transcriptional regulation of hPAP gene constructs covering the regulatory region from 2734 to 1467 were also tested by CAT assays in PC-3 cells cotransfected with respective steroid receptor expression vectors, but only minor changes in the CAT activities were observed, as shown in the case of the hPAP 2231/150 construct (Fig. 4), due to treatment with these steroids (10 or 100 nm), similar to the effects of androgen treatment. These results indicate that steroids cannot directly regulate hPAP gene expression via receptor binding to SREs at 2178 and 1336. On the contrary, the transcriptional activities of the promoters of the kallikrein family genes were induced significantly by glucocorticoid and progesterone as well as by androgen in PC-3 cells overexpressing the respective steroid receptors (Fig. 4). The effects of glucocorticoid and progesterone on the activity of the pCAT expression vector without promoter were similar to that of androgen (see values above). In the case of the hPSA promoter construct, androgen and progesterone acted as equal activators with relative CAT values of 37.7 6 5.6 and 39.7 6 2.6, respectively, in the presence of 10 nm hormone; the corresponding values were 58.1 6 4.8 and 57.7 6 7.4 in the presence of 100 nm hormone. Glucocorticoid, on the other hand, was the most powerful activator of hPSA gene transcription, raising the relative CAT value to 52.3 6 6.2 when the effects of 10 nm steroid were compared. On the contrary, the stimulus of glucocorticoid to the transcriptional activity
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FIG. 4. Steroid specificity of SREs at hPAP, hPSA, and hK2 proximal promoters. CAT assays were performed in PC-3 cells as described in Fig. 2. The relative CAT activities of duplicate samples from three independent experiments were averaged and are displayed with their SEs. For promoter activity in the presence of the steroid vs. promoter activity in the absence of the steroid: **, P , 0.01; and ***, P , 0.001.
of the hK2 construct was the weakest among the tested steroids, giving relative CAT values of 17.1 6 1.2 and 24.5 6 4.3 at 10- and 100-nm hormone concentrations, respectively. Comparable increases were obtained in hK2 promoter activity using androgen and progesterone treatments with relative CAT values of 39.9 6 11.6 and 42.2 6 6.2, respectively, in the presence of 10 nm hormone; the corresponding values were 51.0 6 11.3 and 40.1 6 6.2 in the presence of 100 nm hormone. These results indicate that the SREs in the proximal promoters of hPSA and hK2 genes are not androgen specific. Additionally, 10 nm glucocorticoid was about 3 times more effective as a transcriptional activator in the case of the hPSA gene construct than in the case of the hK2 gene construct. DNase I footprinting
The DNase I footprinting assay was used to study the protein and DNA interactions in the proximal promoter of the hPAP gene. Eight DNase I footprints (DFs) were found within the region from 2231 to 150: DF1 at 2206/2196, DF2 at 2192/2187, DF3 at 2163/2143, DF4 at 2130/294, DF5 at 293/281, DF6 at 268/237, DF7 at 234/221, and DF8 at 111/130 (Fig. 5). Two POU consensus sequences are covered by DF4. Furthermore, the CAAT box and TATA box are covered by DF6 and DF7, respectively. An E26 transformation specific (Ets)/serum response factor (SRF) consensus sequence is located in the area of DF8. The same binding pattern was observed in the hPAP promoter fragment when nuclear extracts of hPAP-expressing (LNCaP) or hPAP-nonexpressing (PC-3) prostatic cells were used. Nuclear extracts isolated from HeLa (Fig. 5) and CV-1 (results not shown) cells gave the same DFs as the prostatic nuclear extracts. Discussion
We have shown above that steroid-involved regulation of the hPAP gene at the level of transcription differs from that
of the hPSA and hK2 genes. Promoter constructs of hPAP covering the entire gene region of 2734/1467 were functional in both prostatic and nonprostatic cell lines, but these constructs could not be induced with androgen, glucocorticoid, or progesterone in transfection assays contrary to the hPSA and hK2 proximal promoters, nor was the androgen response evident in the case of the hPAP 21652/143 construct tested in LNCaP and PC-3 cells. In addition, several transcription factor-binding sites were identified in the regulatory region 2231/150 of the hPAP gene, but none of them seemed to be prostate specific. It has been proposed that the reported androgen regulation of hPAP (1–7) could be mediated via SREs present in the regulatory areas of the gene. We have previously shown that the SREs of the hPAP gene at 2178 and 1336 and the corresponding ones in the rat PAP gene are able to bind androgen receptor in vitro (11). Furthermore, putative SREs of the human and rat PAP gene at 21576 and 21612, respectively, have weak in vitro AR-binding capacities (11). Despite these facts, androgens were not able to increase or decrease the promoter activity of the hPAP constructs covering these elements. Therefore, androgen regulation of the hPAP gene has a mechanism different from that observed in the hPSA and hK2 genes. One possibility is that the distal parts of the hPAP gene contain additional SRE(s) that might cooperate with the SREs in the proximal areas and be essential for androgen regulation. There are several examples of genes where the androgen effect cannot be demonstrated maximally without the interaction of several SREs, as in the case of the probasin gene (28, 29). Another possibility is that the hPAP gene needs another transcription factor(s) along with AR to enhance the transcription and to define the prostatespecific expression. The response element for this factor(s) could be far upstream or somewhere else in the gene. Previous reports concerning the regulation of PAP have sug-
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FIG. 5. DNA-protein interactions in the proximal promoter of the hPAP gene. DF analysis using nuclear extracts from LNCaP, PC-3, and HeLa cells was carried out as described in Materials and Methods. BSA was used as a protein source in control reactions. DFs are indicated by lines, and nucleotide boundaries are beside the photograph.
gested both up- and down-regulation of gene expression by androgen at protein and mRNA levels (1–7). It has been shown that androgen is able to modify PAP expression and secretion, but it is not essential for these processes (3). This finding nicely summarizes the results of most of the PAP regulation studies available. In normal rat prostate, two of the transcripts encoding PAP were up-regulated by androgen, whereas one of the mRNA species was insensitive to the hormonal status of the gland (7). The androgen-sensitive transcripts were detectable even 4 days after castration. The above studies indicate that PAP gene expression in a normal prostate is a complicated process and is not regulated exclusively by androgen. It is also possible that the control of PAP expression is disturbed in prostate cancer cells and may have little linkage to the function of the AR (20). Anyway,
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none of the studies performed to date could prove or disprove the hypothesis that the existing androgen regulation of PAP expression appears at the level of transcription. Recently, an upstream enhancer demonstrating tissue specificity has been found in the hPSA gene between 25824 and 23738 (25). This enhancer contains three binding sites for prostate-specific factors together with a putative androgen response element. Our hPSA construct lacks the enhancer, and therefore, only low transcriptional activity was seen in the hPSA-secreting prostatic cell line LNCaP even in the presence of androgen. However, using a sensitive luciferase reporter system, androgen activation of the hPSA promoter area 2632/112 has been demonstrated in LNCaP cells (10). Despite the absence of the enhancer, our hPSA construct was androgen responsive in the androgen-independent prostatic carcinoma cell line PC-3 and in the nonprostatic cell line CV-1 cotransfected with the AR expression vector. These results suggest that PC-3 cells have probably lost some characteristics of prostatic cells that are still present in LNCaP cells, leading to differential transcription of the hPSA construct in the presence of androgen. The hK2 promoter construct studied here behaved almost identically with the hPSA promoter in transient transfection assays, and the mechanisms of androgen action seem to be similar in these genes. However, a difference was observed in the steroid specificity of the SREs in the kallikrein family genes. Glucocorticoid (10 nm) was the most potent transcriptional activator of the hPSA promoter area from 2620 to 140, but in the case of hK2 it caused the most modest increase in the promoter activity among the steroids tested here. The sequences of SREs at 2170 in the hPSA gene and at 2160 in the hK2 gene differ by only one nucleotide (A/GGAACAgcaAGTGCT), suggesting that the molecular basis for dissimilarity in steroid specificity of the SREs probably lies outside of this response element. The optimal androgen effect is obtained with the hK2 promoter construct covering the region from 21 up to 2323 (9), which suggests that no steroid response region similar to that of the hPSA gene at 2400 is present in the hK2 gene. Therefore, the cooperation between the steroid response regions of the hPSA proximal promoter (10) could represent the difference in glucocorticoid effect between the kallikrein family genes observed in this work. Recently, the promoter of prostatic protein, probasin, has been reported to contain SRE (59-GGTTCTtggAGTACT-39), in which the left subsequence excludes glucocorticoid receptor binding, offering a mechanism for differential in vivo effects between the steroids (30). In conclusion, the ability of glucocorticoid and progesterone to mimic the androgen effect via the SREs in the proximal promoters of hPSA and hK2 genes has been clearly demonstrated here. This phenomenon could explain the existence of hPSA and hK2 proteins in nonprostatic tissues containing steroid receptors, such as the breast. The DNA-protein interactions in the hPAP promoter region 2231/150 were similar regardless of whether nuclear extracts from prostatic cell lines or nonprostatic cells were used in the DF assays, supporting the result that this region is transcriptionally functional in both cell types. The regulatory area of hPAP tested here does not seem to contain a binding site(s) of factor(s) responsible for differential expression of the gene in the prostatic cell lines, leading to pro-
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duction of hPAP protein in detectable or nondetectable amounts in LNCaP and PC-3 cells, respectively. Because the hPAP promoter construct 2734/1467 was almost silent in nonprostatic cells, it will be interesting to see whether prostate-specific transcription factor-binding sites could be detected in the regions 2734/2231 and 150/1467. The Ets/ SRF-like footprint was interesting due to its location soon after the transcription start site. A similar element has been reported to be present in the gene of C3 (31), which is expressed in a prostate-specific manner. Additionally, a cluster of binding sites for POU family proteins was identified at DF4 in the hPAP gene. POU family proteins can act synergistically through several binding sites, and they can cooperate with the steroid receptor, as reported in the case of the PRL gene (32). However, the interaction of regulatory proteins and the precise factors responsible for the prostatespecific expression of the hPAP gene remain to be clarified. Acknowledgments We thank Ms. Marja-Riitta Hurnasti, Ms. Mirja Ma¨kela¨inen, Ms. Marja-Liisa Norrena, Ms. Pirkko Ruokoja¨rvi, and Ms. Airi Vesala for their expert technical assistance.
12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
References 1. Henttu P, Vihko P 1992 Steroids inversely affect the biosynthesis and secretion of human prostatic acid phosphatase and prostate-specific antigen in the LNCaP cell line. J Steroid Biochem Mol Biol 41:349 –360 2. Henttu P, Liao C, Vihko P 1992 Androgens up-regulate the human prostatespecific antigen messenger ribonucleic acid (mRNA), but down-regulate the prostatic acid phosphatase mRNA in the LNCaP cell line. Endocrinology 130:766 –772 3. Lin M, Garcia-Arenas R, Chao Y, Lai M, Patel P, Xia X 1993 Regulation of prostatic acid phosphatase expression and secretion by androgen in LNCaP human prostate carcinoma cells. Arch Biochem Biophys 300:384 –390 4. Banas B, Blaschke D, Fittler F, Ho¨rz W 1994 Analysis of the promoter of the human prostatic acid phosphatase gene. Biochim Biophys Acta 1217:188 –194 5. Lin M, Garcia-Arenas R, Kawachi M, Lin F 1993 Regulation of the expression of prostatic acid phosphatase in LNCaP human prostate carcinoma cells. Cell Mol Biol Res 39:739 –750 6. Langeler EG, van Uffelen CJ, Blankenstein MA, van Steenbrugge GJ, Mulder E 1993 Effect of culture conditions on androgen sensitivity of the human prostatic cancer cell line LNCaP. Prostate 23:213–223 7. Porvari K, Kurkela R, Kivinen A, Vihko P 1995 Differential androgen regulation of rat prostatic acid phosphatase transcripts. Biochem Biophys Res Commun 213:861– 868 8. Riegman PHJ, Vliestra RJ, van der Korput JAGM, Brinkman AO, Trapman J 1991 The promoter of the prostate-specific antigen gene contains a functional androgen responsive element. Mol Endocrinol 5:1921–1930 9. Murtha P, Tindall DC, Young CY 1993 Androgen induction of a human prostate-specific kallikrein, hKLK2:characterization of an androgen response element in the 59 promoter region of the gene. Biochemistry 32:6459 – 6464 10. Cleutjens KB, van Eekelen CC, van der Korput HA, Brinkmann AO, Trapman J 1996 Two androgen response regions cooperate in steroid hormone regulated activity of the prostate-specific antigen promoter. J Biol Chem 271:6379 – 6388 11. Virkkunen P, Hedberg P, Palvimo J, Birr E, Porvari K, Ruokonen M,
22. 23. 24. 25. 26. 27. 28.
29.
30. 31. 32.
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Taavitsainen P, Ja¨nne O, Vihko P 1994 Structural comparison of human and rat prostate-specific acid phosphatase genes and their promoters: identification of putative androgen response elements. Biochem Biophys Res Commun 202:49 –57 Yu H, Diamandis EP 1995 Prostate-specific antigen in milk of lactating women. Clin Chem 41:54 –58 Yu H, Diamandis EP, Sutherland DJA 1994 Immunoreactive prostate specific antigen levels in female and male breast tumors and its association with steroid hormone receptors and patient age. Clin Biochem 27:75–79 Yu H, Diamandis EP, Monne M, Croce CM 1995 Oral contraceptive-induced expression of prostate-specific antigen in the female breast. J Biol Chem 270:6615– 6618 Clements J, Mukhtar A 1994 Glandular kallikreins and prostate-specific antigen are expressed in the human endometrium. J Clin Endocrinol Metab 78:1536 –1539 Ho¨yhtya¨ M, Vihko P, Vuolas L, Tryggvason K, Vihko R 1987 High-affinity monoclonal antibodies specific for human prostatic acid phosphatase. Clin Chem 33:103–107 Solin T, Kontturi M, Pohlmann R, Vihko P 1990 Gene expression and prostate specificity of human prostatic acid phosphatase (PAP): evaluation by RNA blot analyses. Biochim Biophys Acta 1048:72–77 Ruokonen M, Shan JD, Hedberg P, Patrikainen L, Vihko P 1996 Transfecting well-differentiated prostatic cancer cell line LNCaP. Biochem Biophys Res Commun 218:794 –796 Brolin J, Skoog L, Ekman P 1992 Immunohistochemistry and biochemistry in detection of androgen, progesterone, and estrogen receptors in benign and malignant human prostatic tissue. Prostate 20:281–295 Garcia-Arenas R, Lin F-F, Lin D, Jin L-P, Shih CC-Y, Chang C, Lin M-F 1995 The expression of prostatic acid phosphatase is transcriptionally regulated in human prostate carcinoma cells. Mol Cell Endocrinol 111:29 –37 Neumann JR, Morency CA, Russian KO 1987 A novel rapid assay for chloramphenicol acetyltransferase gene expression. Biotechniques 5:444 – 447 Eastman A 1987 An improvement to the novel rapid assay for chloramphenicol acetyltransferase gene expression. Biotechniques 5:731 Rosenthal N 1987 Identification of regulatory elements of cloned genes with functional assays. Methods Enzymol 152:704 –720 Dignam JD, Lebovitz RM, Roeder RG 1983 Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 19:3921–3927 Schuur ER, Henderson GA, Kmetec LA, Miller JD, Lamparski HG, Henderson DR 1996 Prostate-specific antigen expression is regulated by an upstream enhancer. J Biol Chem 271:7043–7051 Darbre PD, Moriarty A, Curtis SA, King RJB 1985 Androgen regulates MMTV RNA in the short-term in S115 mouse mammary tumour cells. J Steroid Biochem 23:379 –384 Darbre PD, Page MJ, King RJB 1986 Steroid regulation of transfected genes in mouse mammary tumour cells. J Steroid Biochem 24:125–131 Rennie PS, Bruchovsky N, Leco KJ, Sheppard PC, McQueen SA, Cheng H, Snoek R, Hamel A, Bock ME, MacDonald BS, Nickel BE, Chang C, Liao S, Cattini PA, Matusik RJ 1993 Characterization of two cis-acting DNA elements involved in the androgen regulation of the probasin gene. Mol Endocrinol 7:23–36 Kasper S, Rennie PS, Bruchovsky N, Sheppard PC, Cheng H, Lin L, Shiu RPC, Snoek R, Matusik RJ 1994 Cooperative binding of androgen receptors to two DNA sequences is required for androgen induction of the probasin gene. J Biol Chem 269:31763–31769 Claessens F, Alen F, Devos A, Peeters B, Verhoeven G, Rombauts W 1996 The androgen-specific probasin response element 2 interacts differentially with androgen and glucocorticoid receptors. J Biol Chem 271:19013–19016 Celis L, Claessens F, Peeters B, Heyns W, Verhoeven G, Rombauts W 1993 Proteins interacting with an androgen-responsive unit in the C3(1) gene intron. Mol Cell Endocrinol 94:165–172 Nelson C, Albert VR, Elsholtz HP, Lu LI-W, Rosenfeld MG 1988 Activation of cell-specific expression of rat growth hormone and prolactin genes by a common transcription factor. Science 239:1400 –1405
