Tissue-specific expression of the prostatic acid phosphatase promoter constructs

BBRC Biochemical and Biophysical Research Communications 311 (2003) 864–869 www.elsevier.com/locate/ybbrc

Tissue-specific expression of the prostatic acid phosphatase promoter constructs Jingdong Shan,a Katja Porvari,a Anne Kivinen,a Lila Patrikainen,a Maria Halmekyt€ o,b c a,* Juhani J€ anne, and Pirkko Vihko a

Biocenter Oulu and Research Center for Molecular Endocrinology, University of Oulu, P.O. Box 5000, FIN-90014 Oulu, Finland b Institute of Applied Biotechnology, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland c A.I.Virtanen Institute, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland Received 4 September 2003

Abstract Human prostatic acid phosphatase (hPAP) is intensely expressed in epithelial cells of the prostate gland following puberty. Its regulatory regions were analyzed in transgenic mice and cell line transfections, in order to clarify the mechanisms of tissue-specific gene expression. A construct containing the sequence of hPAP between the nucleotides )734 and +467 in front of the CAT reporter gene was significantly expressed in the prostate of transgenic mice, while the proximal promoter )734/+50 alone achieved low levels of CAT mRNA in all tissues analyzed. Five homologous sequences (A–E) for our previously identified prostatic GAAAATA TGATA DNA-binding site were found in the area. The competitive reactions in electrophoretic mobility shift assays suggested that the same nuclear factor binds to the GAAAATATGATA and the sites C and E. The importance of the intronic area +57/+467 on the androgen-activated expression in prostatic cells was shown by the reporter construct containing heterologous promoter. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Prostatic acid phosphatase gene; Prostate-specific transcription; DNA-binding sites of prostatic nuclear factor; Androgen effect; Transgenic mice; Reporter gene analyses

Androgens, which are known to underlie the development and function of the prostate, are believed to be important for prostate-specific gene expression via androgen receptors. The rat probasin (rPB) gene contains multiple androgen response elements (AREs) and an upstream enhancer, but the proximal promoter ()426/ +28) is able to restrict the gene expression to the prostate in transgenic mice [1]. We have identified a binding site of a transcription factor involved in prostate-specific and androgen receptor-dependent gene expression of rPB [2]. This binding site is located at )251/)240, and about 20 additional 50 -flanking nucleotides are needed for optimal cooperation with the androgen receptor. An identical binding site was found in the first intron of the human prostatic acid phosphatase (hPAP) gene (AC:

*

Corresponding author. Fax: +358-8-315-5631. E-mail addresses: pirkko.vihko@oulu.fi, [email protected].fi (P. Vihko). 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.10.071

X74961), which is known for its prostatic expression [3]. hPAP (EC 3.1.3.2) is considered a differentiation marker of prostatic epithelium, and its expression decreases during the progression of prostate cancer [4]. So far, very little is known about the molecular basis of the tissue-/cell-specific expression of hPAP and the significance of androgen action on it. Both up- and downregulation of the hPAP gene by androgens has been reported at the RNA level in cell culture models [5–9]. On the contrary, the regulatory regions of the hPAP gene so far analyzed in reporter gene assays have not shown a clear response to androgen treatment [10]. However, several putative AREs are present in the hPAP gene, and some of them are even in conserved positions in the respective rat gene [11,12], suggesting a regulatory role for the elements. In general, the presence of both activating and silencing elements has been reported in the promoter region of the hPAP gene [13,14]. Here, we have studied the transcriptional regulation of hPAP promoter constructs in transgenic mice and in

J. Shan et al. / Biochemical and Biophysical Research Communications 311 (2003) 864–869

transiently transfected cell lines in order to clarify the processes leading to prostate-specific gene expression.

Materials and methods Generation and identification of transgenic mice. Licenses to produce and study transgenic mice in this project were received from the boards for experimental animals at the Universities of Kuopio and Oulu, respectively. All animal experiments described here were conducted in accordance with the accepted standards of humane animal care. hPAP )734/+50 and hPAP )734/+467 promoter/CAT reporter gene constructs [10] were used in transgenic production. The hPAP promoter-driven reporter gene fragments were released from vector sequences by restriction digestion with HindIII/BamHI/BglI and separated in gel electrophoresis. The appropriate DNA fragments were isolated from the gel by binding to glass beads (Geneclean; Bio101, Vista, CA) followed by passage through a Sephadex G-50 column and 0.45 lm filter, and they were used to generate transgenic mice by the pronucleus microinjection technique [15]. Fertilized oocytes were obtained from superovulated BALBc
DBA/2 (CD2F1) females mated with CD2F1 males. The presence of the transgene in DNA from tail biopsies [16] was analyzed by PCR, using hPAP (50 -AGAAATGTCTGCTAACATGCTCTGTG AC-30 ) and CAT (50 -TCCGGATGAGCATTCATCAG-30 ) oligonucleotides as primers. The transgene copy numbers were quantified on slot blot hybridization of the DNA samples from the mice lines, using pCAT vector as a probe. The intensities of the slots were compared to the one (hPAP )734/+467 X) containing a single copy of the transgene according to Southern blot analysis. Analysis of transgene expression by RNA slot blotting. Total RNA was isolated from tissues of the adult transgenic and control (BALB/c) mice by the CsCl-gradient method [17]. A slot blot of RNA samples was prepared as previously described [18] and hybridized with 32 P-labeled pCAT DNA. Nick-translated glyceraldehyde-3-phosphate dehydrogenase cDNA (GAD, AC: X02231) was used as a control probe. Preparation of reporter constructs, cell culture, and transient transfection assays. The PCR-amplified fragment +57/+467 of the hPAP intron was cloned into the SalI and XbaI sites of the pBLCAT4 vector (Promega, Madison, WI) containing thymidine kinase (TK) promoter. The human prostatic (LNCaP), breast (T47D), and lung (A-549) carcinoma cell lines and the COS-1 monkey kidney cell line were maintained according to the instructions of the supplier (ATCC, Rockville, MD). The cells (0.2
106 ) were divided into 6-well plates 1 day or 3 days (LNCaP) before transfection. The cells were transfected as described by Shan et al. [10] with 1.5 lg of reporter construct, 0.25 lg of androgen receptor and 1.0 lg b-galactosidase expression plasmids, using FUGENE 6 (Roche, Indianapolis, IN). The relative CAT activities are shown by comparing the values to that of the pBLCAT4 vector in hormone-depleted medium. Statistical analyses were carried

out with ANOVA, and p < 0:05 was kept as the limit for statistical significance. Electrophoretic mobility shift assays. Several different doublestranded oligonucleotides used as a32 P-labeled probes or unlabeled competitors in the electrophoretic mobility shift assays (EMSAs) [2] are shown in Table 1. The nuclear extracts were isolated according to Dignam et al. [19]. The HeLa nuclear extract was purchased from Promega (Madison, WI).

Results Characterization of transgenic mice In our previous work [10], the hPAP )734/+50 construct was found to be functional in both prostatic and non-prostatic cells, whereas hPAP )734/+467 seemed to be more active in prostatic cells. Therefore, the respective constructs were considered to be useful for studies clarifying prostate-specific gene regulation. In this study, we obtained 8 and 12 founder mice for the )734/+50 and )734/+467 constructs of hPAP, respectively. Only two founders, )734/+50 VIII and )734/+467 III, were unable to transmit the transgene to their offspring due to infertility or possible defective incorporation of the transgene, respectively. The copy number of the transgene was variable in different mouse lines, being, e.g., 7 and 9 in the cases of )734/+50 I and )734/+467 IV, respectively. Expression patterns of transgenic mice lines Expression of the reporter gene in the transgenic lines was studied first by slot blot analysis using total RNA isolated from tissues of adult animals. Expression of the reporter was under the detection level in the lines )734/ +50 III, IV, and )734/+467 V. In the cases where CAT expression was detectable, the relative distribution of CAT mRNA in the tissues was similar in the different lines generated by the same construct (results not shown). Extensive tissue distribution analysis was performed for the lines )734/+50 I and )734/+467 IV, representing the highest expression levels of the reporter gene directed by the respective hPAP regulatory region (Fig. 1A). The results indicate that CAT expression seems to be low and

Table 1 Oligonucleotides used as probes or competitors in EMSAs Oligonucleotide/element

Gene and position

Sequencea

A B C D E F Pb GATA Sox-5

hPAP )588/)558 hPAP )267/)237 hPAP )160/)130 hPAP +211/+241 hPAP +239/+269 hPAP +1136/+1164 rPB )251/)235 Consensus Consensus

50 -TATCTAAAGAAAGATAAAAGTAAACTGGCT-30 50 -AAAAAACAAAGATAAAAGTAAACTGAAAACA-30 50 -TTAAATGGGGAAACTGTGATCTCTCTCAGCT-30 50 -GTTCCCAGCAAAGTCTGATAAGGCAAGCGTC-30 50 -GTCAGGTTTCATCTTATCCTTGGATTGTTTC-30 50 -TTTCTACAGAAAATATGATACCCATGTGC-30 50 -AGTTAAGAAAATATGATAGCATC-30 50 -CACTTGATAACAGAAAGTGATAACTCT-30 50 -CGAGCACTAAAACAATGCCCGGGGA-30

a

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The elements are underlined (50 –30 direction) or dashed (30 –50 direction).

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the different lobes of the prostate are the main tissues for the higher reporter expression in the case of )734/+467 IV mice. In this line, relatively high CAT levels were also detected in the seminal vesicles of males and in the lungs of both genders. Although the expression levels in the other )734/+467 lines were lower than that in line IV, the prostate, seminal vesicle, and lung were also the main tissues for CAT expression for the lines examined, of which XI and XII are presented as examples (Fig. 1B). This indicates that the significant amount of the marker mRNA seen particularly in the prostate is not due to the integration site of the transgene. Effect of the hPAP intron fragment on heterologous promoter activity The potential of the hPAP+57/+467 intron fragment to restrict the function of the TK promoter and the SV40 enhancer in the pBLCAT4 vector in prostatic cells was evaluated using transient transfections (Fig. 2).

Fig. 1. Expression of the CAT reporter gene directed by the regulatory areas of hPAP in transgenic mice. (A) Total RNA was isolated separately from the tissues of three adult transgenic mice (per line) and from the tissues of non-transgenic control mice for slot blot hybridization. The expression levels of CAT were normalized by those of GAD, and are represented as mean values with SE in arbitrary units. The background for the CAT probe was determined from the tissues of non-transgenic control mice and is shown as a mean expression value  SE of all the analyzed tissues. (B) Tissue distribution of CAT transcripts in additional transgenic hPAP mice lines. The values of CAT expression in arbitrary units (equal to the scale in (A)) are shown in a representative assays.

nearly constant in the different tissues of line )734/+50 I mice. Similar expression was also observed in the lines )734/+50 II and VI (results not shown). On the contrary,

Fig. 2. Transcriptional regulation of the TK promoter by the hPAP+57/+467 fragment in the prostatic and non-prostatic cell lines. Prostate (LNCaP), breast (T47D), lung (A-549), and kidney (COS-1) cells were transiently transfected by reporter constructs containing TK or TK with the intronic fragment of hPAP. The AR and b-galactosidase expression vectors were cotransfected and the cells were cultured with 10 nM R1881 synthetic androgen (+) or without androgens ()). CAT activities were measured from duplicate samples of cell extracts from three independent experiments. Mock-subtracted CAT values were normalized by protein content as well as with b-galactosidase activity. The relative mean activities  SE compared to the activity of the TK construct (pBLCAT4) without androgen in each cell line are shown.

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In LNCaP prostatic cancer cells without androgens, 13fold activation was detected in the transcription when the intron fragment was included in the construct. An even higher level of activation (53-fold) was observed for the TK-hPAP+57/+467 construct in T47D breast cancer cells without androgens. Contrariwise, the intron fragment had practically no effect on the transcriptional activity of the TK promoter in COS-1 kidney cells or A-549 lung cancer cells. Interestingly, the androgen addition further increased (2.2-fold) the expression of the TK-hPAP+57/+467 construct in prostatic cells, but decreased (3.3-fold) it in breast cells. However, the changes in relative CAT activities were not statistically significant in either case. The CAT activity of the pBLCAT4 vector was 542  180, 258  127, 938  132, and 636  87 pg/mg protein/U of b-galactosidase in LNCaP, T47D, COS-1, and A-549 cells without androgens, respectively. These results indicate that the +57/+467 region of the hPAP gene contains regulatory elements important for gene expression in both prostate and breast cells.

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and E, while 58% is the respective value for site B. The sites C (Fig. 4A) and E (Fig. 4B) were able to bind transcription factors in EMSA, but complex formation

Search for prostatic transcription factor-binding sites Our results from transgenic animals and transfection studies suggested that the hPAP )734/+467 regulatory area probably contains elements involved in prostatic gene expression. Comparison analyses revealed several sequences similar to the recently identified prostatic GAAAATATGATA-binding site [2] in the area. These sites are located at )580/)569, )257/)246, )151/)140, +218/+229, and +244/+255 and have been named A, B, C, D, and E, respectively (Fig. 3, Table 1). The binding site at +1144/+1155 of the hPAP gene, which is identical to the original one (named Pb here) found in the probasin gene, is marked here as F. This site is known to bind the prostatic transcription factor efficiently and shows 75% nucleotide identity with the sites A, C, D,

Fig. 3. Location of the GAAAATATGATA-related DNA-binding sites and potential androgen response elements in a schematic representation of the hPAP regulatory region. Homologous sequences to GAAAATATGATA (A–F) were searched using FINDPATTERNS (in http://www.seqweb.csc.fi) and are indicated by ovals with the nucleotide location underneath. The binding capacities of the elements for the prostatic protein in vitro are also marked (), +). AREs are indicated by squares with the sequence position above. The in vitro androgen receptor-binding capacities are indicated by + or ++ above AREs. Transcription start site is marked by an arrow.

Fig. 4. Interactions of nuclear proteins with the elements C and E in EMSAs. (A) Double-stranded oligonucleotide containing element C was 32 P-labeled and incubated with nuclear extracts (10 lg) prepared from prostatic (LNCaP, PC-3) and non-prostatic (COS-1 and HeLa) cells. The resulting complexes were analyzed on a native 4% polyacrylamide gel. Lane 1, free probe; lanes 2, 5, 8, and 11, nuclear extract and labeled probe; lanes 3, 6, 9, and 12, nuclear extract and labeled probe in the presence of a 100-fold molar excess of the unlabeled probe as a specific competitor (S); and lanes 4, 7, 10, and 13, nuclear extract and labeled probe in the presence of a 100-fold molar excess of nonspecific sequence (N). (B) The same experiment performed using the labeled element E. The prostatic complex is indicated by *, the nonspecific complexes by +, and the free probe by ).

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was not detected in the case of the sites A, B, and D (data not shown). The DNA–protein interactions at the sites C and E seem to be prostate-specific, since at least the nuclear extracts isolated from renal (COS-1) and uterine (HeLa) cells do not contain the factors needed for the complex. Nor was this complex formed when nuclear extracts from breast (T47D) and lung (A-549) cells were used in EMSA with site E as a probe (data not shown). A computer search (Genomatix MatInspector professional) also revealed perfect core sequences of GATA and Sox-5 in the oligonucleotide used for site E in EMSA. However, the DNA-binding sites for the respective transcription factors were not able to compete with site E in protein binding (data not shown), suggesting that distinct factors are involved in the prostatic complex. To find out if the sites C and E bind the same factor as the sites F and Pb, we performed competition reactions in EMSA (Fig. 5). These studies indicated that the sites C, F, and Pb are actually competitors in the complex formation between site E and prostatic proteins. Interestingly, another sequence-specific complex was

Fig. 5. Elements C, E, and F of hPAP bind the same prostatic factor as the GAAAATATGATA element (Pb) in the probasin gene. An EMSA was performed using the 32 P-labeled probe for element E and the LNCaP nuclear extract. Lane 1, free probe; lane 2, nuclear extract and labeled probe; and lanes 3–10, nuclear extract and labeled probe in the presence of a 100-fold molar excess of the indicated competitor (unlabeled oligonucleotides for the elements A–F and Pb, or non-specific sequence N). The prostatic complex is indicated by *, the sequencespecific complexes by +, and the free probe by ).

detected in this assay. The probable reason for this is the larger amount of probe DNA (due to the decreased radioactivity) added to these binding reactions compared to those shown in Fig. 4B. The sites C, F, and Pb are not competitors for this complex. Discussion The prostate-specificity and hormonal regulation of hPAP have been intensively studied, but the mechanisms underlying the events are poorly understood. We have shown here that the hPAP intron fragment +57/ +467 is able to increase the transcriptional activity of its own proximal promoter in the prostate of transgenic mice, but not to the same extent in transiently transfected cancer cells of the prostate. This suggests that the regulatory processes involved are not identical in mouse tissue and the human cell line and/or in normal and cancerous prostate cells. The higher selective activity of the hPAP )734/+467 construct in prostate compared to the nearly constant activity of hPAP )734/+50 in transgenic tissues is probably due to the presence of the regulatory elements involved in prostatic gene expression in the intron area and/or the possible interactions of the transcription factors binding along the whole DNA area. The GAAAATATGATA-like elements found in the regulatory areas of hPAP might have impact on tissue-specific expression of the gene as shown in the case of probasin [2]. The presence of elements significant for prostatic gene expression in the hPAP+57/ +467 area is supported by the result showing that the heterologous TK promoter was also activated in LNCaP cells by the intronic fragment. On the other hand, the TK promoter was induced by this fragment in the T47D breast cells, indicating the lack of elements restricting expression exclusively in prostate or suggesting the presence of similar regulatory mechanisms in breast and prostate cancer cells. Noteworthy are the facts that T47D cells express AR endogenously [20] and that the prostate-specific upstream enhancer of hPSA has some activity in these cells [21]. Recent evidence of similar regulatory events in breast and prostate tissues has indicated over-expression of the prostatic transcription factor PDEF in human breast tumors [22]. However, the opposite effects of androgens on transcription of the TK-constructs in the breast and prostate cells are interesting on the tissue-specific expression point of view.

Acknowledgments We are grateful to Ms. Airi Vesala, Ms. Mirja M€akel€ainen, Ms. Pirkko Ruokoj€arvi, Ms. Tuula Reponen, and Mr. Jukka Pulkkinen for technical assistance. This work was supported by the Academy of Finland (Project 50003).

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