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Department of Molecular Medicine (S.-T.P., K.D., G.N., A.F.-M.), Karolinska Hospital, Karolinska Institute, 171 76 Stockholm, Sweden; Division of Urology (S.-T.P.), Department of Surgery, Chang Gung Memorial Hospital, Tao Yuan 333, Taiwan; Department of Physiology (K.D.), Göteborg University, 405 30 Göteborg, Sweden; Division for Reproductive Endocrinology (X.X.W.) and Andrology Center (A.P.), Department of Woman and Child Health, Karolinska Hospital, Karolinska Institute, 171 76 Stockholm, Sweden
Address all correspondence and requests for reprints to: See-Tong Pang, Department of Molecular Medicine, Karolinska Institute, CMM L8:01, Karolinska Hospital, 171 76 Stockholm, Sweden. E-mail: jacob.pang{at}cmm.ki.se.
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Abstract |
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Introduction |
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Testosterone is the main circulating androgen secreted primarily by Leydig cell in testis, but it can also originate from the adrenal and can be formed by peripheral conversion of the adrenal steroid. Testosterone is mainly bound to serum proteins, and only a small fraction is dissolved freely in the serum. Once it enters prostate cells, about 90% is converted to dihydrotestosterone by the enzyme 5
-reductase. Dihydrotestosterone, which has 5-fold higher binding affinity for the androgen receptor (AR) than testosterone, can dissociate AR from heat-shock protein and phosphorylate the receptor. Subsequently, AR dimerizes and binds to androgen response elements in the promoter regions of target genes. Coactivators and corepressors interact with the AR complex and the general transcription apparatus to stimulate or inhibit target gene transcription (6). Using rat prostate as an animal model, many androgen-responsive genes have been identified by various screening methods, such as differential display and subtractive hybridization (7, 8). Identification of androgen-responsive genes in prostate provides a valuable way to understand androgen actions in the gland.
DNA microarrays can be used to study expression of thousands of genes simultaneously, and they are an important tool to advance biological discovery (9, 10, 11). In combination with cluster analysis, one can identify underlying gene expression patterns in complex data collected from multiple microarray experiments (12). Herein, we explored the utility of this method to study androgen actions in rat ventral prostate. Commonly two experimental models are used to identify androgen-responsive genes, namely, differentially analyze gene expression in prostate gland from either normal and castrated rats or castrated rats and castrated rats treated with androgen replacement. However, it has been shown that genes regulated in the ventral prostate gland during castration and testosterone replacement are not always reciprocally regulated (13). Thus, to understand the action of androgen in ventral prostate, we have analyzed gene expression both after castration and after testosterone replacement in a time-course manner using DNA microarray. In a screening attempt, we have identified 230 genes that were regulated in either condition; different gene expression patterns were seen among them.
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Materials and Methods |
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RNA preparation
Ventral prostates were harvested from normal rats, 1-, 4-, and 8-d castrated rats (C1, C4, and C8), and 8-d castrated rats treated with testosterone replacement for 1 and 4 d (T1 and T4). There were 5 animals in each group, except the group of 8-d castrated rats, which consisted of 10 animals. Total RNA was extracted using RNeasy Mini kit (QIAGEN, Chatsworth, CA) according to the manufacturer’s instructions, and quality was checked on denatured agarose gel. Total RNA was pooled from the same experiment samples, and then mRNA was purified using the oligo-dT cellulose kit (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the protocol supplied by the manufacturer.
Fabrication of cDNA chip
DNA chips were manufactured from a collection of about 3000 cDNA clones selected from the TIGR Rat GENE Index (www.tigr.org) and our own rat clones collection as described previously (14). Bacterial colonies were grown overnight in 1.5 ml Luria-Bertani medium in 96-well plates (Beckman, Fullerton, CA), and plasmid mini-preparations were followed by a PCR amplification of the inserts using vector-specific primer T3 (5'-AAT TAA CCC TCA CTA AAG GG-3') and T7 (5'-GTA ATA CGA CTC ACT ATA GGG C-3'). The amplified inserts, each produced by pooling two 100-µl PCRs, were purified by ethanol precipitation, resuspended in 40 µl 3x SSC, and purity checked on an agarose gel. Amino saline-coated slides (Corning, Inc., Corning, NY) were used as an adhesive surface for printing, using a GMS 417 arrayer (Affymetrix, Santa Clara, CA). The slides were post-processed as described before and stored in a dust-free dark box until hybridization.
Microarray hybridization
Hybridization of microarray was carried out essentially as described previously (14). Routinely, the same amount of mRNA, 2 µg, was used for the microarray experiments. mRNA derived from intact rat prostate was used as the control for the castration study, and 8-d castrated rat prostate was used as the control for the testosterone replacement study. Fluorescent-labeled cDNA was synthesized with oligo-dT primer (New England Biolabs, Inc., Beverly, MA) by reversed transcription reaction using Superscript II (Life Technologies, Inc., Rockville, MD) in the presence of labeled nucleotides (Cy3-uridine 5'triphosphate for control and Cy5-uridine 5'triphosphate for treatment mRNA; Amersham Pharmacia Biotech). Cy3 and Cy5-labeled cDNAs were pooled and purified using Microcon 30 (Millipore Corp., Bedford, MA). The final probe volume was adjusted to 15 µl with hybridization buffer containing 5x SSC, 0.2% SDS, 10 µg poly-A RNA, and 10 µg yeast tRNA. Probes were heated at 100 C for 2 min before application onto the array and were covered with a 22 x 22-mm cover slip (Grace Bio-Labs, Bend, OR). The chip was then placed in a hybridization chamber (Corning, Inc.) and kept at 65 C for 15–18 h. After hybridization, the chip was washed and dried before scanning with a GMS418 scanner (Affymetrix).
Data analysis
GenePix Pro software (Axon Instruments, Foster City, CA) was used for the analysis of the image. The signal of each spot was calculated as the average intensity of the spot minus background. Spots with intensity that was at least 1.4 times above the background were included in the study. Expression ratio calculated as Cy5/Cy3 signal was normalized using the LOWESS (Locally Weighted Scatter Plot Smoother) method in the SMA (Statistics for Microarray Analysis) package (15, 16). SMA is an add-on library written in the public domain statistical language R (17). The LOWESS algorithm performs a local fit to the data in an intensity-dependent manner. The intensity value for each spot is normalized on the basis of data distribution in the immediate neighborhood of the spot’s intensity. Genes with missing data in any of the studied time points were excluded. The significance of the expression ratios of both castration and testosterone replacement studies were estimated using the SAM software (Significance Analysis of Microarray) (18). SAM is a statistical technique for finding significantly regulated genes in a set of microarray experiments. For each gene (i) in the array, SAM computes the T-statistics (di), a score derived from the changes of gene expression in relation to the SD of repeated measurements for that gene. A threshold can be set on the basis of di to identify potentially significant changes in gene expression. The threshold can be adjusted on the basis of an associated false discovery rate (FDR) value: the percentage of genes expected to be wrongly identified as differentially expressed when a certain threshold (d value) is set. To each of the genes in the array, a q value was assigned. This value is similar to the familiar P value, and it measures the lowest FDR at which the gene is called significant. In this study, genes with a q value of more than 5% in either set of data were excluded from further analysis. To the statistically based criteria, we have added a further requirement based on the absolute changes in expression ratios. Only genes with average changes of 70% in any of the time points studied were listed as differentially expressed. This level of expression changes has been shown by us (14, 19) and others (20) to be reproducible when other direct methods, such as Northern blot, Rnase protection, and RT-PCR, are used to estimate gene expression. Although lower significant levels of changes in gene expression may have important biological consequences, insufficient information exists regarding its reproducibility by independent methodologies. The complete result of significantly regulated genes is available upon request.
Clustering analysis
Clustering analysis was performed essentially as described elsewhere (12). The fold ratios of gene expression for each of the experiments analyzed were scored and filtered as explained above. D-dimensional vectors (d = number of experiments included) were created for each of the N genes included from the data set selected as being regulated. The N, D-dimensional vectors were normalized to the unit sphere with the cluster program and used as input into hierarchical clustering algorithms. Gene clusters were viewed with TreeView software (12). Mean expression patterns were calculated from the normalized gene vectors in the clusters. All of the computer programs used are freely available at www.microarrays.org.
Real time quantitative RT-PCR
Six of the differentially regulated genes identified with cDNA microarray analysis were selected for validation analysis using LightCycler quantitative real-time PCR (Roche Diagnostics, Basel, Switzerland). The procedures were carried out essentially as described elsewhere with some modification (21). A total of 150 ng of mRNA, of pooled ventral prostate obtained from normal, 1-d and 4-d castrated, and testosterone replacement for 4-d animals were reverse transcribed using a First-Strand cDNA Synthesis Kit from Amersham Biosciences (Uppsala, Sweden) in an 11-µl reaction. After reverse transcription reaction, the resulting cDNA were brought up to a volume of 60 µl with diethyl pyrocarbonate-treated water. Gene-specific primers corresponding to the target genes on the microarray were used to generate amplicons, and these sequences were as follows: thioredoxin, 5'-TCC AAT GTG GTG TTC CTT GA-3' and 5'-TAG TGG CTT CGA GCT TTT CC-3'; peroxiredoxin 5, 5'-GAT CAA GGT GGG AGA CAC CA-3' and 5'-GCA GAT GGG TCT TGG AAC AG-3'; superoxide dismutase 2, 5'-GCT GGC TTG GCT TCA ATA AG-3' and 5'-AAT CCC CAG CAG TGG AAT AA-3'; selenoprotein 15 kDa, 5-ACG GGA ACA TTG CTG AAG A-3' and 5'-AAG TGA CAG ACG GAC AAC TGA-3'; vitamin D up-regulated protein 1 (VDUP 1), 5'-CCA GTG ACA GGG AAA AGG AG-3' and 5'-GGG GAA AGC CTC CAA AAG TA-3'; ezrin, 5'-ATC TTT GGC TTG GAG TGG A-3' and 5'-CGA TGG GCT TGA TGA CAA-3'. For each studied gene, a relative standard curve was constructed with serial dilution (1:1, 1:5, 1:25, and 1:125) using the cDNA sample that showed the higher fluorescent hybridization intensity in the array experiment. To measure the relative concentration of the gene expression, unknown cDNA samples were diluted to 1:10 to place its crossing point value approximately in the middle section of the standard curve. A crossing point is defined by the LightCycler software, using secondary-derivative-maximum measurement, as the intersection of the best-fit line through the log-linear region of the amplification curve. The real-time PCRs were performed in a 20-µl volume with 2 µl of respective cDNA sample, 0.4 µM primers, and 4 mM MgCl2. Nucleotides, Hot-Start Taq polymerase, reaction buffer, and SYBR Green I dye were supplied in the LightCycler DNA Master SYBR Green I Kit. The amplification program consisted of 1 cycle of 95 C for 10 min (hot start), followed by 45 cycles of 95 C for 15 sec, annealing temperature at 57 C for 10 sec, 72 C for 30 sec; fluorescent intensity was measured at a specific acquisition temperature for each gene. To confirm amplification specificity, a melting curve analysis (raising the sample temperature from 60–95 C with constant monitoring of fluorescence) was performed immediately after completion of PCR cycling, and products from each primer pair were checked with agarose gel electrophoresis. For each gene, two assays with duplicate reaction were performed. The level of individual mRNA measured in the real time RT-PCR was related to total RNA content measured by spectrophotometer.
Immunohistochemistry analysis
A 5-µm section was made from paraffin block of prostate tissue, and avidin-biotin-peroxidase immunohistochemical technique was used for the staining. Monoclonal mouse antibodies against human thioredoxin (M21) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); they have been tested to cross-react in rat protein. Monoclonal mouse antibodies against ezrin (3C12) were purchased from Lab Vision Corp. (Fremont, CA). Negative control was obtained by using nonimmune mouse serum as the primary antibody. The experimental procedure is briefly described as follows. The tissue sections were first dewaxed and rehydrated, and the antigen retrieval procedure was performed. Sections were pretreated with 0.01 M sodium citrate buffer (pH 6.0) for 10 min in a microwave oven at high power and allowed to cool for a further 20 min. After washing with buffer [0.1 M PBS (pH 7.5)], nonspecific endogenous peroxidase activity was blocked by treatment with 3% hydrogen peroxide (Merck, Darmstadt, Germany) in methanol for 10 min at room temperature (RT). The sections were then washed with PBS buffer for 10 min and exposed to a 30-min blocking using diluted normal horse serum (Vectastatin; Vector Laboratories, Inc., Burlingame, CA) in a humidified chamber at RT. The primary antibodies against thioredoxin and ezrin were diluted in normal horse serum (Vectastain, Vector Laboratories, Inc.), 1:25 and 1:500, respectively, before being applied to the sections and incubated at 4 C overnight. Second antibody, biotinylated horse antimouse (Vectastain, Vector Laboratories Inc.) was used and incubated with the sections for 60 min at RT. Thereafter, the tissue sections were incubated with horseradish peroxidase-avidin biotin complex (Vectastain Elite, Vector Laboratories, Inc.) for 60 min at RT. Sections were visualized after the application of 3,3'-diaminobenzidine in H202 (DAB kit, Vector Laboratories, Inc.) and counterstained with hematoxylin.
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Results |
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). We could identify clusters of genes that were only down-regulated or up-regulated by castration, whereas their expressions were not changed by testosterone replacement (Fig. 1, clusters A and D). We could also identify clusters of genes that were reciprocally regulated by castration and testosterone replacement (Fig. 1, clusters B and E). Finally, a cluster of genes that was only up-regulated by testosterone replacement was identified (Fig. 1, cluster C).
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), whereas 32 genes were regarded as androgen-repressed genes because they were up-regulated by castration and down-regulated by testosterone replacement (Table 2). Among them, ornithine carboxylase, S-adenosylmethionine decarboxylase (22), farnesyl pyrophosphate synthetase (8), glutathione peroxidase 1 (23), diazepam binding inhibitor (24), matrix Gla protein (25), and cathespin B (26) have been previously documented as androgen-responsive genes, and our results therefore confirm these findings. These results also provide an independent validation of our experimental approach to identifying androgen-responsive genes in rat ventral prostate.
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). There were 131 genes included in this category as shown at www.cmm.ki.se/users/Gnorstedt. In this category, we find genes that were previously described as secondary-responsive; for example, calmodulin and -prothymosin are known to be up-regulated only by castration and insensitive to testosterone replacement (13).
We assigned genes in both categories into different functional classes on the basis of their known biological or biochemical functions (Tables 1 and 2 and www.cmm.ki.se/users/Gnorstedt). The functions of these genes include regulation of cell cycle, DNA synthesis and transcription, cytoskeleton, extracellular matrix, cellular transport, intermediate metabolism, protein synthesis and folding, redox status, and signal transduction.
Validation of differential gene expression by quantitative real-time RT-PCR
We have used LightCycler real-time RT-PCR to measure the relative concentration of six genes obtained from the microarray analysis. The RT-PCR result of thioredoxin, peroxiredoxin 5, superoxide dismutase 2, selenoprotein 15 kDa, and ezrin confirmed the results obtained from the microarray; they were significantly down-regulated in castrated rat ventral prostate (Fig. 2), whereas vitamin D up-regulated protein 1 gene expression was significantly up-regulated in 4-d castrated animals.
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). The immunostaining of thioredoxin and ezrin were low in castrated animals, whereas a stronger signal was detected in testosterone replacement animals. The immunostaining of these two proteins therefore covaried with their gene expression at the RNA level. No staining was found in the negative control.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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With clustering analysis, we were able to tentatively distinguish the androgen-responsive from the secondary responsive genes. Changes in the latter category could be dependent on other nonandrogen factors from the testis (22, 23) or systemic effect related to castration or testosterone replacement. Many secondary-responsive genes possess biochemical function similar to the androgen-responsive ones. This may indicate that genes in both categories can contribute to different androgen-dependent processes in the prostate.
The many functions of the genes discovered here further support the important role of androgens in the regulation of prostatic epithelial cell functions, including growth and differentiation. It is of interest that a number of genes related to secretory function, such as integral membrane protein Tmp21-I (27), coat protein 
-COP (28), ADP-ribosylation factor 1 (29), signal peptidase complex 25-kDa subunit (30), and SEC61 -subunit (31) were regulated by androgen. We also identified several genes related to fatty acid metabolism (Table 1). This finding is consistent with reports that de novo lipogenesis occurs in prostate cancer cells and is stimulated by androgen (32). Because it is difficult to discuss all the findings in this paper, we will limit our discussion to a set of genes that may be related to the mechanism(s) of androgen withdrawal-induced prostate apoptosis.
Interestingly, we found that several antioxidant genes were modulated after castration in rat ventral prostate. Among them, thioredoxin (33), peroxiredoxin 5 (34), glutathione peroxidase 1 (35), superoxide dimutase 2 (36), and 15-kDa selenoprotein (Refs. 37 and 38 and Table 1) are genes functionally related to cellular redox regulation that were down-regulated after castration. In contrast, other redox-related genes such as glutathione reductase (Ref. 35 and Table 2), microsomal glutathione-S-transferase (39), and epoxide hydrolase (Ref. 40 and www.cmm.ki.se/users/Gnorstedt) were up-regulated after castration. Alteration of antioxidant proteins can cause oxidative stress to cells because the balance of reactive oxygen species and antioxidant system is lost (41). One interpretation of our data is that oxidative stress may be one of the initiation factors for androgen withdrawalinduced prostate apoptosis. Modulation of antioxidant molecules such as down-regulation of thioredoxin and superoxide dismutase 2 is also seen in dexamethasone-induced lymphocyte apoptosis (42, 43). In addition, it has been shown that up-regulation of glutathione-S-transferase Yb1 subunit, a family member of glutathione-S-transferase, is a secondary event to oxidative stress that occurred during steroid-mediated apoptosis (44). Glutathione-S-transferase Yb1 subunit is also known to be up-regulated in the regressing prostate of castrated rats (25), and antioxidant responsive element has been identified in its 5'-flanking region (45). Thus, upregulation of glutathione-S-transferase, glutathione reductase, and epoxide hydrolase in rat ventral prostate after castration could be an indication of oxidative stress experienced by the prostatic epithelial cells. Because androgen level decreases in both aging men and rodents (46, 47), it would be of interest to see whether reduction of antioxidant molecules presented here can play a role in increasing the oxidative stress in aging prostate (48). It is known that oxidative stress and loss of glutathione-S-transferase expression are risk factors for prostate cancer development (49, 50). Our observation may provide new clues to explain the paradoxical epidemiological finding that prostate cancer occurs during the period of life when testicular function is declining and levels of testosterone are falling.
Besides being a well known protein that is involved in cellular redox status, thioredoxin is also known as an antiapoptotic factor (51). Thioredoxin can directly block the apoptosis-signaling pathway activated by apoptosis signal regulating kinase 1 (ASK 1), a member of MAPK kinase kinase family (52). ASK 1 is expressed in many tissues, including prostate gland (53), and can be activated by oxidative stress after dissociation from its inhibitor thioredoxin (54). In this study we found that gene clone N27, also known as VDUP 1 gene (55, 56), a negative regulator of thioredoxin (57), was highly induced after castration. Speculatively, castration-reduced thioredoxin and -induced VDUP 1 expression could coordinately play a role in prostate apoptosis. Possibly, this may involve the regulation of ASK 1. It has been shown that ASK 1 can be activated by Fas death domain, and c-jun N-terminal kinase (JNK) is the downstream substrate of ASK 1 (54). Interestingly, activation of Fas and phosphorylation of JNK have been confirmed in rat ventral prostate after castration (58, 59). Moreover, we showed that Fas-activated serine/threonine kinase (60) and JNK substrate c-jun were up-regulated after castration. Taken together, although ASK 1 was not measured in our present study, results from our DNA chip experiments can be used to predict changes in pathways. Therefore, our data provide a clue that oxidative stress could contribute to the molecular mechanism of androgen withdrawal-induced prostate apoptosis, and ASK 1 may be involved in the apoptotic-signaling pathway. Further investigations are needed to confirm our current assumption.
In this study, we found the expression of ezrin was down-regulated after castration and up-regulated after androgen replacement. Ezrin is a membrane-cytoskeleton linker that can signal cell survival through the phosphatidylinositol 3-kinase/Akt pathway (PI3K/Akt) (61). Interestingly, because Akt can decrease the ASK 1 kinase activity stimulated by oxidative stress (62), one may speculate that down-regulation of ezrin can facilitate the apoptotic signaling conducted through the ASK 1 signaling pathway by compromising the phosphatidylinositol 3-kinase/Akt surviving pathway. Ezrin is also a member of the ezrin-radixin-moesin family of conserved protein in the band 4.1 superfamily, which has a positive role in the growth, morphologic changes, and invasion of cancer cells (63, 64, 65). Because we have shown that ezrin expression can be regulated by androgen, its role in prostatic growth and prostate cancer deserve further investigation.
In conclusion, we have demonstrated that DNA microarray is a valuable tool for investigating potential mechanism(s) of androgen action in prostate. Using this method, we have identified a large number of novel androgen-responsive genes and secondary-responsive genes regulated by castration and testosterone replacement in rat ventral prostate. These data provide unique opportunity for understanding the molecular events of androgen action in the prostate gland. Importantly, our findings have pointed out a possible molecular mechanism of androgen withdrawal-induced apoptosis in the prostate.
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Acknowledgments |
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Footnotes |
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Abbreviations: ASK 1, Apoptosis signal-regulating kinase 1; AR, androgen receptor; EST, expression sequence tag; FDR, false discovery rate; JNK, c-jun N-terminal kinase; RT, room temperature; VDUP 1, vitamin D up-regulated protein 1.
Received March 20, 2002.
Accepted for publication August 20, 2002.
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References |
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-COP, a coat subunit of non-clathrin-coated vesicles with homology to Sec21p. FEBS Lett 314:195–198[CrossRef][Medline]
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