The mechanisms underlying the temporal and tissue-specific effects of androgens are not understood. Like all steroid hormones, androgens act by binding to a nuclear hormone receptor, enacting conformational changes that allow binding of the receptor to specific regulatory elements in AR-responsive genes. The ensuing cascade of transcriptional events, unique to each cell type, underlies the dramatic and diverse effects of androgen. Understanding the molecular programs regulated by androgens in critical target tissues may lead to improvements in treatment of AIS in children, benign prostatic hyperplasia and prostate cancer in adult men, and diverse other conditions in which androgens play a key role.

Unfortunately, few model systems are available for studying the effects of androgens in human tissues. There are no practical models of early development, when androgen exerts its most striking effects on the genitalia, body habitus and brain. Only a handful of immortalized cell lines express AR and respond to its ligands. All of these cell lines have been isolated from patients with advanced prostate carcinomas that have become refractory to androgen deprivation therapy [5,6,7]. In three of these cell lines, LNCaP, MDA PCa 2a and MDA PCa 2b, mutations in the ligand-binding domain of the AR confer broad steroid-binding affinity [8,9]. A fourth cell line, LAPC-4, bears a wild-type AR.

The significance analysis of microarrays (SAM) procedure was used to identify genes with statistically significant changes in expression after treatment with R1881. The SAM procedure accurately identifies transcripts with reproducible changes in gene expression and is more reliable than conventional means of analyzing microarrays [11]. Hierarchical clustering of the expression patterns of 567 transcripts representing 517 unique genes with statistically significant changes in expression in response to 1 nM R1881 in LNCaP is displayed in Figure Figure1a.1a. Expression levels changed more than twofold in 539 (95%) of the transcripts. The raw microarray data and full details including transcript identities can be found at [12]. Approximately one third of these transcripts represent uncharacterized and unnamed genes and were not previously known to be regulated by androgen. Of the named genes, many have not been previously recognized as responsive to androgen. Virtually all of the known androgen-regulated genes that were represented on the array and adequately measured responded as previously reported (Figure (Figure1b1b).

To further examine the expression changes induced in response to androgen, we conducted two additional sets of experiments. In the first, LNCaP cells were treated for 24 hours with dihydrotestosterone (DHT), the predominant androgen found in the prostate in vivo. A clear relationship was observed between increasing dose of DHT and increased levels of gene expression (Figure (Figure2a2a and see Additional data files). Furthermore, the pattern of expression changes observed in response to DHT largely paralleled those observed after treatment with R1881 (R² = 0.7705; Figure Figure2b).2b). We conducted a second set of experiments to examine the response of LNCaP cells to androgen withdrawal. After carrying cells in androgen-replete media with charcoal-stripped fetal bovine serum (FBS) (supplemented with 1 nM R1881), cells were passaged in media without androgen and harvested at 48 and 70 hours (Figure (Figure2a).2a). After 48 hours, the change in gene-expression pattern was largely reciprocal to that induced by androgen treatment and these effects increased by 70 hours. For the SAM-selected gene set, expression patterns after 70 hours of androgen deprivation were inversely correlated with those seen 50 hours after R1881 treatment (R² = 0.7119; Figure Figure2c2c).

We have previously observed that varied stimuli produce temporally complex changes in gene-expression patterns in yeast and mammalian cells in vitro [13,14,15]. In many cases, genes can be grouped by their expression patterns using hierarchical clustering, and these groups often comprise genes that are functionally related. To our surprise, genes in the SAM-generated dataset varied little in their expression between 8 and 72 hours after androgen treatment. In general, expression levels of genes induced or repressed by androgen treatment remained at a constant level between 8 and 72 hours. As the SAM procedure identified genes with significant changes in expression in androgen-treated cells compared to controls, we were concerned that genes had been excluded whose expression levels varied for only part of the time course. Analysis of the raw data by hierarchical clustering analysis alone again generally failed to identify genes whose expression varied over the time course. The lack of complexity in the temporal response to androgen in this dataset precludes useful grouping of genes based on differences in their responses by cluster analysis. Perhaps a more detailed investigation of expression changes within the first 8 hours after treatment will reveal temporal patterns of response of LNCaP to androgens and provide additional insights into androgen action.

We were curious to know whether the expression changes observed in LNCaP would also occur in other androgen-responsive human prostate cancer cell lines (MDA PCa 2a, MDA PCa 2b, and LAPC-4), or in LNCaP and LAPC-4 subclones carried in androgen-depleted conditions for extended periods. All cell lines were treated with 1 nM R1881 and at least one sample was collected between 20 and 70 hours after addition of R1881. RNA samples from treated and control cell lines were compared directly on the same microarray. Responses to androgen of the set of transcripts identified as androgen-responsive in the LNCaP experiments were examined in each of these cell lines. Many genes were modulated similarly in all of the cell lines, suggesting conservation of a specific androgen-responsive program, perhaps a vestige of the prostatic epithelial phenotype, between these cell lines (Figure (Figure3;3; see also [12] and Additional data files). For example, the transcript levels of 310 of these genes changed by 1.5-fold or more in MDA PCa 2a and 2b cells following 48 hours of exposure to R1881; transcripts for 262 genes changed by 1.5-fold or greater in single treatments of all four LNCaP androgen-deprived subclones; and transcripts for 81 genes changed by 1.5-fold or greater in the three LAPC-4 and LAPC-4 (androgen-deprived) array experiments.

There were a few noteworthy differences in expression profiles between the cell lines. When we focused our attention on transcripts whose relative abundance changed by more than 2.6-fold in at least two experiments ([12] and see Additional data files), one group of genes differed strikingly between the cell lines (Figure (Figure4).4). Most of the genes in this group have previously been found to be consistently expressed at significantly higher levels in proliferating cells [10,16]. Treatment with 1 nM R1881 promotes proliferation in the MDA PCa 2a and 2b cell lines, has no proliferative effect in the LNCaP and LAPC-4 cell lines, and represses proliferation in LNCaP cell lines carried for prolonged periods in conditions of androgen deprivation (data not shown, and see references in [17,18,19]). As seen in Figure Figure3,3, the proliferation cluster is relatively induced in the MDA PCa 2a and 2b cell lines, shows little change in LNCaP and LAPC-4, and is repressed in the androgen deprived LNCaP cell lines. Thus, transcript levels of the genes in this proliferation cluster parallel the responses of these cell lines to treatment with androgens.

The expression patterns induced by R1881 offer possible insights into the effects of androgens in prostate physiology. Many features of the androgen-induced expression program appear to be related to activation of the cell's capacity to produce seminal fluid. After puberty, the normal prostate produces a protein- and organic solute-rich fluid that contributes approximately 30-50% of the volume of the ejaculate. The prostatic secretions are poorly characterized, but are undoubtedly critical to fertility as the prostate is the only sex accessory gland present in nearly all mammals [20]. Androgen induces several classes of genes that contribute to the production and secretion of prostatic fluid.

Perhaps the most striking change caused by treatment with R1881 occurs in the class of genes that contribute to the synthesis and modification of secretory proteins. All androgen-responsive genes in this class show increased expression in response to androgen. We have previously identified putative secretory and membrane-bound proteins based on their synthesis on membrane-associated polysomes ([21] and M.D. and P.O.B., unpublished observations) and over 200 of these are among the androgen-responsive genes (see [12] and Additional data files). Included in this set are genes encoding proteins previously known to be secreted by the prostate into the seminal plasma, including prostate-specific antigen (PSA), sorbitol dehydrogenase, apolipoprotein D [22], and vascular endothelial growth factor (VEGF) [23]. An additional set consists of 30 androgen-responsive genes with roles in protein trafficking and secretory vesicle formation and in transport of secretory vesicles (Figure (Figure5a).5a). This set of genes includes several with roles in membrane budding, vesicular formation, and membrane fusion, and translocation of proteins across the membrane of the endoplasmic reticulum. Androgen treatment also induced a number of genes that participate in protein folding and glycosylation (Figure (Figure5b).5b). One of these, FK506-binding protein 5/FKBP51, a recently described androgen target [24], was the most highly induced gene (more than 20-fold) we observed in our experiments. FK506-binding protein 5/FKBP51 is a co-chaperone protein that has peptidylprolyl isomerase activity and interacts with the progesterone receptor [25]. Whether it functions in the processing of androgen-induced secretory proteins or in other pathways induced by androgens or perhaps even in cell-cell signaling is unclear, but the degree of induction suggests it may have an important role in prostatic physiology.

Prostatic fluid is also rich in small organic compounds that account for much of its osmotic pressure. For instance, prostatic secretions contain the highest levels of polyamines of all bodily fluids. The transcript encoding ornithine decar-boxylase (ODC), a known androgen target that catalyzes the rate-limiting step in polyamine biosynthesis [26], was induced coordinately with enzymes that catalyze downstream steps in polyamine synthetic pathway (spermine synthase, spermidine/spermine N1-acetyltransferase, and S-adenosylmethionine decarboxylase) (Figure (Figure5c).5c). Cholesterol and lipids are also found at high levels in semen and are major components of secretory vesicles referred to as prostasomes, which are thought to regulate the function of spermatozoa [27]. Note that several genes for proteins involved in cholesterol biosynthesis (HMG CoA reductase, IDI1, sterol C4 methyl oxidase) and fatty acid metabolism (ACAT2, DEGS, ACATN, FACL3, PEC1) were modulated by R1881. Choline, sorbitol, amino acids and citrate are also found in the prostatic secretions, although only a few genes (for sorbitol dehy-drogenase, phosphatidylcholine transfer protein, amino-acid transporter) related to these pathways appeared to respond directly to androgen. Citrate production is regulated by transport of aspartate into the mitochondria by aspartate amino-transferase, and indeed, the gene encoding this enzyme was induced by androgen in each of these cell lines [28]. Genes encoding several mitochondrial proteins were also induced in response to androgen, perhaps reflecting the increased production of citrate, or the increased demand for energy in these cells as they mobilize secretory pathways.

Zinc, calcium, phosphate, potassium, sodium, chloride and other ions are abundant in the ejaculate, although sodium and chloride are at levels below their serum concentrations. Several transporter molecules and ion-channel genes which may have roles in the active transfer of these ions into and out of the lumen of the prostate acini and secretory granules were modulated in response to R1881 (Figure (Figure5d).5d). Transport of sodium and chloride out of the lumen of the prostate acini is accompanied by the passive transfer of water and the prostatic secretions are thereby concentrated [20]. Transport of ions and small organic compounds into the acinar lumen will concentrate them in the seminal plasma. The ank gene, which codes for a transmembrane protein that secretes pyrophosphate into joint spaces [29], was strongly induced by R1881 exposure. Our data suggests that the pyrophosphate in the ejaculate maybe secreted into the prostatic fluid by the ANK protein.

The prostate undergoes dramatic morphologic changes as it acquires the ability to produce seminal fluid, and these changes can be observed at puberty or by stimulating the prostate with androgens after castration. The epithelial cells proliferate and change from stratified to cuboidal, while glands branch and acini expand. These morphologic changes follow complex reprogramming of the prostate cell that involves poorly understood alterations of the cytoskeleton and the extracellular matrix and changes in downstream signaling pathways [30,31]. Many genes encoding components of the extracellular matrix, proteins important in cell-cell interactions and cytoskeletal proteins were regulated in response to R1881, suggesting that they may participate in the morphologic changes induced by androgens (Figure 6a,6b). Many previous studies have pointed to a critical role for the cytoskeleton and extracellular matrix in transducing signals for growth, differentiation and secretion in normal and cancerous prostate cells [32]. Our data may therefore help point to one important feature of the transcriptional program that sets the stage for these signaling events.

We also found evidence of modulation of several genes encoding transcriptional regulatory and signaling proteins in response to androgen in the prostate cancer cell lines (Figure 6c,6d). It is possible that some of these pathways have important roles in reprogramming the normal prostate cell in response to androgen [33]. However, some of these signaling and transcriptional proteins may be modulated abnormally in these cancer-derived cell lines because of molecular genetic alterations acquired with transformation [34,35]. Yet other pathways may not be direct targets of AR activation but could be modulated in response to androgen-induced changes in the internal or external milieu of the cells. For instance, treatment of LNCaP with androgens has been shown to cause a burst of oxidative stress that may contribute to prostate carcinogenesis [19]. The observed increased expression of NFκB and several DNA repair and stress-response genes may be a secondary response to this oxidative stress rather than a direct response to AR signaling [36]. Further work will clearly be necessary to dissect the roles of these signaling pathways in the response of the prostate to AR activation.