Rapid Signalling by Androgen Receptor in Prostate Cancer Cells

Heike Peterziel1, Sigrun Mink1, Annette Schonert1, Matthias Becker1, Helmut Klocker2 and Andrew CB Cato*,1

1Forschungszentrum Karlsruhe, Institut fuÈr Toxikologie und Genetik, Postfach 3640, D-76021 Karlsruhe, Germany; 2Department of Urology, University of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria

Androgens are important growth regulators in prostate interacts with c-Jun to inhibit the DNA binding cancer. Their known mode of action in target cells activity of this factor (Kallio et al., 1995) or with Ets requires binding to a cytoplasmic androgen receptor transcription factors to downregulate their activities followed by a nuclear translocation event and modulation (Schneikert et al., 1996). of the expression of speci®c genes. Here, we report The ability to regulate gene expression positively and another mode of action of this receptor. Treatment of negatively is not speci®c to the AR but is shared by androgen responsive prostate cancer cells with dihydro- other steroid receptors (Beato et al., 1995). In addition, testosterone leads to a rapid and reversible activation of a number of steroid hormones are reported to rapidly mitogen-activated protein kinases MAPKs (also called and reversibly activate important intermediates in extracellular signal-regulated kinases or Erks). Transient signal transduction pathways known to trigger cell transfection assays demonstrated that the androgen proliferation (Migliaccio et al., 1996, 1998; Endoh et receptor-mediated activation of MAP kinase results in al., 1997; Marcinkowska et al., 1997). For example enhanced activity of the transcription factor Elk-1. This estrogens activate two members in the family of c-src- action of the androgen receptor diers from its known related tyrosine kinases, c-src and c-yes as well as the transcriptional activity since it is rapid and insensitive to MAP kinases Erk-1 and -2 (Di Domenico et al., 1996). androgen antagonists such as hydroxy¯utamide or Progestins also activate MAP kinase, a reaction that casodex. Biochemical studies as well as analyses with has been correlated with an indirect association of the dominant negative mutants showed the involvement of progesterone receptor (PR) with c-src through the kinases such as MAPK/Erk kinase, phosphatidyl-inositol intermediary role of the estrogen receptor (ER) 3-kinase and protein kinase C in the androgen receptor- (Migliaccio et al., 1998). Interaction of the PR and mediated activation of MAP kinase. These results ER with c-src either directly or indirectly enhances the demonstrate a novel regulatory action of the androgen kinase activity of this protein which then activates receptor and prove that in addition to its known MAP kinase via the Ras-Raf-1 pathway (Castoria et transcriptional eects, it also uses non-conventional al., 1999). Other mechanisms have been proposed to means to modulate several cellular signalling processes. explain the ER-mediated activation of MAP kinase. In one report it was shown that it occurs via the release of Keywords: androgen receptor; anti-androgens; growth calcium from intracellular stores (Improta-Brears et al., factors; MAP kinase; rapid response 1999). Another study on the mechanism of ER- mediated activation of MAP kinase showed the involvement of Gaq and Gas proteins (Razandi et al., 1999). Thus the ability of ER to activate MAP Introduction kinase seems to involve several dierent signalling pathways. Androgens are the major regulators of prostate growth In this communication, we report that the AR also (Cunha et al., 1987). Their known eects are mediated activates MAP kinase. This occurs through an early by a cytoplasmic androgen receptor (AR) that is and a late response pathway in a cell-type dependent translocated into the nucleus upon hormone binding manner. We have in this report analysed the rapid to positively or negatively modulate the expression of response and shown with the use of kinase inhibitors speci®c genes (Beato et al., 1995). and dominant negative mutants that it is mechan- Transcriptional upregulation by the AR occurs istically dierent from that mediated by the ER and through homodimer binding of the receptor to distinct PR. The AR action requires in addition to MAPK/Erk DNA sequences (Cato et al., 1987; Ham et al., 1988; kinase, the phosphatidyl-inositol 3-kinase and protein Beato, 1989). This takes place alongside interaction of kinase C pathways. Unlike the eects of the ER and the receptor with basal transcription factors such as PR which are inhibited by hormone antagonists, the TFIIF and the TATA-box binding protein (McEwan AR response is insensitive to dierent antiandrogens. and Gustafsson, 1997) or with coactivators that We therefore report here a rapid cellular signalling potentiate its activity (Voegel et al., 1996; Yeh and response which appears to be speci®c for the AR. Chang, 1996). The AR also negatively regulates the expression of genes through interaction with transcription factors already bound to DNA. For example, it Results

DHT-mediated activation of Erk-1 and -2 *Correspondence: ACB Cato Received 12 February 1999; revised 15 June 1999; accepted 15 June Estrogen, progesterone and vitamin D3 have recently 1999 been reported to rapidly activate MAP kinase through Cellular signalling by androgen receptor H Peterziel et al 6323 a novel non-conventional mechanism (Migliaccio et al., (Figure 2a). These results show that the rapid DHT- 1996, 1998; Endoh et al., 1997; Marcinkowska et al., mediated activation of Erk-1 and -2 is strictly 1997). In this study we have asked whether androgens dependent on the presence of the AR. also mediate this response using two primary androgen responsive cells: human genital skin ®broblasts and prostatic stromal cells. Both cell lines were treated with the androgen dihydrotestosterone (DHT) and MAP kinase activity was determined by immunoblotting cellular extracts with antibodies that recognize active dually phosphorylated MAP kinases Erk-1 and -2. In the primary human genital skin ®broblasts, increased phosphorylation of Erk-1 and -2 was detected after 16 h of hormone treatment. This persisted several hours before ®nally returning to basal levels after 30 h (Figure 1a, compare lanes 6 ± 9 with lane 1; Phospho p44 and p42 column). During this time, AR level was increased possibly due to an androgen-mediated post-translational stabilization process but no change in the amount of non- phosphorylated Erk-1 and -2 was observed (Figure 1a). A rapid (10 ± 30 min) activation of Erk-1 and -2 was not observed in these primary human genital skin ®broblasts (Figure 1b) although a 10 min exposure to phorbol ester 12-O-tetradecanoyl 13- acetate (TPA) activated the MAP kinases (Figure 1b). The lack of rapid activation of Erk-1 and -2 by DHT was not a special feature of the primary human genital skin ®broblasts. Several cells into which we transiently transfected the AR also failed to show this response upon DHT treatment (results not shown). A dierent result was obtained in primary prostatic stromal cells. The basal level of Erk-1 and -2 phosphorylation was low (Figure 1c, lane 1) but this was rapidly and transiently increased (2 ± 60 min) by DHT treatment (Figure 1c, lanes 2 ± 5). Two further peaks of phosphorylated Erk-1 and -2 were observed at 8 ± 20 h and again at 30 h (Figure 1c, lanes 8 ± 12). The level of the AR and non- phosphorylated Erk-1 and -2 remained the same throughout the experiment. Early and late peaks of phosphorylation of Erk-1 and -2 following treatment with DHT were also observed in the androgen receptor negative human prostate PC3 cells into which we stably transfected the wild-type AR while only the early peak was detected in the androgen responsive human LNCaP prostate cancer cells Figure 1 Kinetics of DHT-mediated phosphorylation of Erk-1 (Figure 1d). The signi®cance of these cell-type and -2 in primary genital skin ®broblasts and in primary prostatic speci®c dierences is so far not clear. In subsequent stromal cells. Immunoblots were carried out with equal amounts studies we further characterized the rapid eect of of cellular proteins from primary genital skin ®broblasts after treatment with (a) 100 nM DHT for the indicated period of time DHT on MAP kinase activation in the prostate cells. or with (b) 100 nM DHT or 80 ng/ml TPA. (c) Immunoblots were also carried out with equal amounts of cellular proteins from human primary prostatic smooth muscle cells treated for the Ligand speci®city indicated period of time with 100 nM DHT. The ®lters were PC3 cells stably transfected with an empty or an AR- hybridized with antibodies against the human androgen receptor (AR), phosphorylated Erk-1 and -2 (phospho p42/p44) and non- expressing vector were treated for dierent lengths of phosphorylated Erk-1 and -2 (p42/p44). The sign (7) indicates time with DHT and MAP kinase activity was cells treated with vehicle alone (0.1% ethanol). (d) Kinetics of the determined. MAP kinase was immunoprecipitated phosphorylation of Erk-1 and -2 in PC3 ARwt or LNCaP cells. from the cellular extracts and subjected to an in vitro Upper panel: PC3 ARwt cells were treated with 100 nM DHT for the indicated periods of time (lanes 2 ± 6; 9 ± 13) or for 30 min kinase assay with the use of myelin basic protein as (lane 1) or 22 h (lane 8) with vehicle (0.1% ethanol) (7). As substrate. In these analyses, a transient activation of positive control the cells were also treated for 10 min with 80 ng/ MAP kinase was observed between 2 and 30 min in the ml TPA (lane 7). Lower panel: LNCaP cells were treated with AR-positive cells but no clear activation was detected 100 nM DHT for the indicated periods of time (lanes 2 ± 6) for 30 min with vehicle (0.1% ethanol, lane 1) and for 10 min with in cells transfected with the empty expression vector 80 ng/ml TPA (lane 7). Equal amounts of extracts from these cells (Figure 2a). As a control, both cell lines responded to were used for immunoblot analyses with an antibody speci®c for TPA treatment by an increased MAP kinase activity phosphorylated Erk-1 and -2 Cellular signalling by androgen receptor H Peterziel et al 6324 Concentrations of DHT as low as 0.1 nM suced for likely due to their inherent ability to activate Erk-1 and the activation of Erk-1 and -2 in both PC3 ARwt and -2 (Figure 2d). This response is mediated by the AR as LNCaP cells (Figure 2b, upper panel) and correlated well no activation was observed in PC3 cells transfected with the doses required for transactivation of a with an empty expression vector (results not shown). transiently transfected AR-responsive MMTV reporter The DHT or antiandrogen-mediated activation of Erk- construct in these cells (Figure 2b, lower panel). Note 1 and -2 is speci®c for this class of MAP kinase as that due to unequal transfection eciencies, the level of other MAP kinases such as p38 or JNK-1 and -2 were DHT-mediated activation of the MMTV construct in not activated although anisomycin which was used as a these two cell lines is strikingly dierent. control (Kortenjann and Shaw, 1995) activated these Dierences were observed in the androgen-mediated kinases (Figure 2c). transactivation and rapid activation of Erk-1 and -2. While transactivation is downregulated by the anti- Downstream eector molecules androgens hydroxy¯utamide and casodex (Cato et al., 1987; Berrevoets et al., 1993), DHT-mediated activa- Upon activation, Erk-1 and -2 become translocated tion of Erk-1 and -2 is insensitive to the action of these from the cytoplasm into the nucleus where they antagonists at equimolar or 100-fold molar excess enhance the activity of speci®c eector molecules such concentrations (100 nM and 10 mM) (Figure 2c). The as the transcription factor Elk-1 (Marias et al., 1993; lack of inhibitory action by the antiandrogens is most Gille et al., 1995a; Yang et al., 1998). We therefore

A C

Figure 2 Speci®city of androgen-receptor mediated activation of MAP kinase. (a) Dihydrotestosterone-induced activation of MAP kinase requires the presence of the androgen receptor. PC3 cells containing either an empty expression vector or stably expressing the wild-type androgen receptor (PC3 ARwt) were treated with 100 nM dihydrotestosterone, 80 ng/ml TPA or vehicle (0.1% ethanol) alone for the indicated period of time. Thereafter the cells were lysed and the extracts subjected to an in vitro kinase assay. The results of the in vitro phosphorylation are shown above. (b) Low concentrations of dihydrotestosterone suce for the phosphorylation of Erk-1 and -2 and transactivation. (Upper panel) PC3 cells stably transfected with the wild-type androgen receptor expression vector (lanes 1 ± 5) and LNCaP cells (lanes 6 ± 10) were treated for 30 min with the indicated concentration of DHT. Equal amounts of cellular extracts were used for an immunoblot with the phosopho-speci®c Erk-1 and -2 antibody. (Lower panel) The same cells were transiently cotransfected with pGL3 MMTV-luciferase construct and Renilla tk-luciferase and treated with the indicated concentrations of DHT for 36 h. The cells were then harvested and luciferase activity determined. The transfection eciency was normalized to the activity of the Renilla tk-luciferase used as an internal standard. The level of induction was calculated as luciferase activity in the presence of the dierent concentrations of DHT divided by the luciferase activity in the absence of hormone. The bars represent the average and standard deviation of the relative luciferase activities of three independent experiments. (c) Antiandrogens do not block androgen-induced phosphorylation of Erk1 and -2. Shown are phospho-Erk-1 and -2 immunoblots with equal amounts of cellular extracts of PC3 ARwt-cells treated with vehicle (0.1% ethanol, lane 1) or 100% nM DHT (lane 2) alone or simultaneously with 100 nM DHT and 100 nM or 10 mM of the antiandrogens hydroxy¯utamide (HydroxyF, lanes 3 and 4) or casodex (lanes 5 and 6). (d) Antiandrogens enhance the activity of a speci®c subtype of MAP kinase. PC3 ARwt- cells were treated with the indicated concentrations of DHT, hydroxy¯utamide (HydroxyF), casodex or vehicle (0.1% ethanol) alone (7) for 30 min. As controls the cells were also treated with anisomycin (5 mg/ml) or TPA (80 ng/ml) for 10 min. After treatment, the cells were harvested, lysed and equal amounts of protein was used for an immunoblot assay. The results show phosphorylated forms of the mitogen-activated protein kinases Erk-1 and -2 (p42/P44, upper panel), p38 (middle panel) and JNK1/2 (lower panel) Cellular signalling by androgen receptor H Peterziel et al 6325 investigated whether Elk-1 was activated by DHT (EGF), a known activator of the Erk-1 and -2 treatment. This was determined through transient signalling pathway (Ahn et al., 1991). In the presence transfection of a construct containing the DBD of of the Gal 4 DBD-Elk-1, the basal promoter activity in the yeast Gal 4 transcription factor fused to Elk-1 and the PC3 cells was slightly increased possibly due to an indicator gene carrying Gal 4 binding sites into PC3 residual growth factors in the culture medium that cells. activated the Elk-1 protein (Figure 3a). This activity In the presence of Gal 4 DBD, the reporter activity was further enhanced threefold by EGF but not by was low in the AR negative PC3 cells and was neither DHT (Figure 3a). In cells additionally cotransfected enhanced by DHT nor by the epidermal growth factor with the AR, the basal level was even higher possibly

Figure 3 Androgen receptor-mediated activation of Elk-1 and transient induction of c-fos expression in an androgen receptor positive prostate cancer cell line. (a) Androgen receptor-induced activation of Elk-1. PC3 ARwt cells or PC3 cells with empty expression vector were transiently cotransfected with Gal 4 DBD alone or fused to Elk-1 and a GAL4-luciferase construct. After transfection, the cells were treated with vehicle (0.1% ethanol) alone (7) or vehicle containing 100 nM DHT or with 5 ng/ml EGF for 6 h. The cells were harvested and luciferase assay performed with equal amounts of protein. The results were expressed relative to the activity of the luciferase construct in the AR-positive cells in the presence of the Gal 4 DBD-Elk-1 but in the absence of hormone. The bars represent the average and standard deviation of the relative luciferase activities of ®ve independent experiments. (b) Antiandrogens enhance Elk-1 activity in androgen receptor positive PC3 prostate cancer cells. PC3 cells transfected as above were treated with vehicle (0.1% ethanol) alone (7) or vehicle containing DHT, hydroxy¯utamide (HydroxyF), casodex or EGF for 6 h. The cells were then harvested and luciferase assay performed with equal amounts of protein. The results were expressed relative to unity which is the nominal value assigned to the activity of the indicator plasmid in the presence of DHT in cells expressing the wild-type androgen receptor. The bars represent the averages and standard deviations of the relative luciferase activities of three independent experiments. (c) Transient induction of c-Fos expression in human prostate LNCaP cells by DHT. LNCaP cells were cultured under conditions for the determination of enhanced MAP kinase activity and treated with DHT for the indicated periods of time. Poly(A)+ RNA was prepared and 10 mg were used for Northern blot analyses with radioactively labelled Fos- (Curran et al., 1982) and GAPDH-speci®c (Fort et al., 1985) DNA probes Cellular signalling by androgen receptor H Peterziel et al 6326 due to the eect of residual androgens activating the al., 1995), protein kinase C (PKC) inhibitor AR (Figure 3a). The promoter activity in this case was GF109203X (Toullect et al., 1991), downregulation of not only enhanced 3 ± 4-fold by EGF but also by DHT PKC by prolonged treatment with phorbol esters (Figure 3a). (Fabbro et al., 1986) or phosphatidyl-inositol 3 (PI3) To prove that this AR response is a non-classical kinase inhibitor wortmannin (Ward et al., 1996). All transactivation function, the antiandrogens casodex these inhibitors downregulated the activation of Erk-1 and hydroxy¯utamide were analysed for their eects and -2 by DHT (Figure 4, compare lanes 3 ± 6 with 2). on Elk-1 activity. These antiandrogens do not normally As positive controls, the inhibitors were tested on activate transactivation via the wild-type AR but in known signalling pathways to con®rm their speci®city. this assay they enhanced the transcriptional activity of Elk-1 albeit to a lesser extent than DHT (Figure 3b). This eect was dependent on the presence of the AR as the antiandrogens did not exert any in¯uence on Elk-1 A activity in cells that lack the AR (Figure 3b). As a control, EGF activated Elk-1 independent of the presence of the AR showing that its signalling action does not rely on the AR. As Elk-1 transcriptionally activates c-fos expression (Gille et al., 1995a; Treisman, 1996), we determined whether DHT can rapidly enhance the expression of this gene. In a Northern blot assay carried out with RNA from LNCaP cells, we showed that DHT transiently activates the expression of c-fos with the same kinetics as it activates Erk-1 and -2 (Figure 3c; compare Northern blot assay with Figure 1d, bottom panel, lanes 1 ± 6). DHT did not activate the c-fos gene in PC3 cells transfected either with the AR or with the empty expression vector (results not shown). This is probably due to an insensitivity of this gene locus in the PC3 cells.

Sensitivity to kinase inhibitors

To identify the chain of events that accompany the B DHT-mediated phosphorylation of Erk-1 and -2 and activation of Elk-1, we examined the eect of various kinase inhibitors on the response of the AR. We used the MAPK/ERK kinase inhibitor PD98059 (Dudley et

Figure 5 Dominant-negative Raf and PI3 kinase inhibit androgen-induced activation of Elk-1. (a) PC3 ARwt cells were transiently cotransfected with a construct containing Gal 4 DBD alone or fused to Elk-1 in the presence of either an empty Figure 4 MEK, protein kinase C and phosphatidyl inositol-3 expression vector or dominant-negative constructs of Raf-1 kinase are involved in dihydrotestosterone-induced phosphoryla- (cRafC4) or p85 (p85D) and the GAL 4 luciferase indicator tion of Erk-1 and -2. PC3 ARwt cells were treated with vehicle plasmid. After transfection, the cells were treated with vehicle (0.1% ethanol) alone (lane 1) for 30 min or with 100 nM DHT for (0.1% ethanol) alone (7) or vehicle containing 100 nM DHT for 30 min in the absence (lane 2) or presence of dierent inhibitors 6 h. The cells were harvested and luciferase assay performed with (lanes 3 ± 6). As controls, 5 ng/ml EGF (lanes 7 ± 10) or 80 ng/ml equal amounts of protein. The results are expressed relative to the TPA (lanes 11 ± 14) were used alone as inducers for 10 min or in activity of the indicator plasmid in the presence of the Gal 4 the presence of dierent inhibitors. The vehicle alone (0.1% DBD-Elk-1 expression vector and the empty expression but in the DMSO) (lanes 1, 2, 7 and 11) or vehicle plus inhibitors were absence of hormone. The bars represent the averages and added 15 min before addition of DHT, EGF or TPA. The standard deviation of the relative luciferase activities of four concentrations of the inhibitors used were 50 mM PD98059 (lanes independent experiments. (b) Dihydrotestosterone activates 3 and 8), 500 nM GF109203X (lanes 4, 9 and 12) and 100 nM phosphatidyl-inositol 3-kinase in androgen receptor positive wortmannin (lanes 6, 10 and 14). In the case of prolonged prostate cancer cells. PC3 ARwt cells were treated with vehicle treatment with TPA (lanes 5 and 13), the cells were incubated (0.1% ethanol) alone for 10 min, with vehicle containing 100 nM with this compound for 16 h prior to the addition of the inducers. DHT for 5 and 10 min or with 20 ng/ml IGF-I for 10 min. The After the various treatments, the cells were harvested and cellular cells were harvested, lysed and subjected to PI3 kinase assay and proteins were prepared and used for immunoblots with the anti- TCL chromatography. The arrow heads indicate the positions of phospho-Erk antibody and an antibody against non-phosphory- the origin of application of the reaction products and of lated Erk phosphatidyl-inositol 3 phosphate (PI-3P) Cellular signalling by androgen receptor H Peterziel et al 6327 The MEK inhibitor but not the inhibitor of PKC nor where dominant negative mutants inhibited not only wortmannin abrogated EGF induced phosphorylation the androgen response but also the basal activity of the of Erk-1 and -2 (Figure 4, compare lanes 8 ± 10 with 7). indicator gene. This suggests the involvement of a basal In further controls, phorbol ester (TPA)-induced activity for the manifestation of the DHT response. phosphorylation of Erk-1 and -2 was suppressed by In contrast to recent studies where it was shown that the PKC inhibitor or by prolonged treatment with the ERb isoform rapidly activates Erk-1 and -2 as well TPA but not by the PI3 kinase inhibitor (Figure 4, as c-Jun kinase (Razandi et al., 1999), in this present compare lanes 12 ± 14 with 11). These inhibitor studies work we can only show a rapid eect of the AR on suggest the involvement of the Raf-MEK, PI3 kinase Erk-1 and -2. Other MAP kinases such as p38 or JNK- and PKC pathways in the AR mediated activation of 1 and -2 were not activated by the AR. A further Erk-1 and -2. dierence in the rapid mode of action of the AR To further con®rm the involvement of these path- compared to the other steroid receptors is its ways, we carried out Elk-1 transactivation studies with insensitivity to antihormones. Antiestrogens and dominant negative constructs of Raf-1 and the p85 antiprogestins inhibit the ER- and PR-mediated subunit of PI3 kinase (Bruder et al., 1992; Brennan et activation of MAP kinase (Migliaccio et al., 1996; al., 1997). These constructs did not only abrogate the 1998; Di Domenico et al., 1996) whereas in our studies DHT eect but they also reduced the basal activity antiandrogens are unable to inhibit the AR response. which possibly arose as a result of residual hormone in We have identi®ed c-fos as one of the downstream the culture medium (Figure 5a). A more direct link targets of DHT-mediated activation of MAP kinase. In between these kinases and the DHT signalling pathway AR-positive LNCaP prostate cell line, activation of was established in the case of PI3 kinase in an in vitro Erk-1 and -2 by DHT was correlated with an increased kinase assay. In this study, PC3 ARwt cells, showed a expression of this gene. Several other downstream low but clear increase in PI3 kinase activity following targets may exist and one of them could be the AR treatment with DHT or with IGF-I as a control itself since a dominant negative Erk-2 construct (Figure 5b). No increase in PI3 kinase activity was downregulated ligand-dependent transactivation by induced by DHT in PC3 cells lacking the AR (results the receptor (Peterziel H and Cato ACB, unpub- not shown). lished). This may therefore provide a means whereby These results together demonstrate that the AR the AR potentiates its own transactivation potential mediated activation of Erk-1 and -2 requires the through activation of MAP kinase. activation of intermediary kinases that may either be Other investigators have reported rapid eects of downstream of the AR or whose activity may be androgens such as increased intracellular calcium level required in parallel for transfer of signals from the AR or increased levels of inositol trisphosphates and to the MAP kinases. diacylglycerol in osteoblasts and prostatic cells (Steinsapir et al., 1991; Lieberherr and Grosse, 1994). These eects are very rapid (5 and 60 s) compared to Discussion the 2 ± 60 min response we have described here for activation of MAP kinase. As estrogen-induced We have demonstrated in this work that activation of activation of MAP kinase is reported to involve the AR by the androgen DHT leads to phosphoryla- mobilization of intracellular calcium (Improta-Brears tion of Erk-1 and -2 via two pathways: rapid and late et al., 1999), it is likely that these very rapid eects in response pathways. We have analysed the rapid intracellular calcium concentrations seen with andro- response to determine how it compares with other gens may preceed the activation of MAP kinase rapid eects of steroid hormone receptors in the reported here in our work. This idea is being activation of MAP kinase recently reported. investigated at the moment. With the use of kinase inhibitors such as the MEK We have demonstrated that rapid activation of MAP inhibitor PD98059 and a dominant negative mutant of kinase by androgen occurs in cells of prostatic origin Raf-1, we succeeded in blocking the rapid activation of and not in genital skin ®broblasts. This suggests a MAP kinase by the AR demonstrating the involvement rather restrictive tissue or organ speci®city for this AR of the Raf-1-MEK pathway. This is in agreement with response. The function of this increased MAP kinase reports of ER and PR activating MAP kinase through activity is not clear at the moment but whatever it may the Ras-Raf-1-MEK pathway (Migliaccio et al., 1996, be, it is certainly not essential for life as mice and 1998). In addition, we have shown for the AR that PI3 humans lacking a functional androgen receptor will kinase and PKC also contribute to the DHT-mediated survive (He et al., 1991; Marcelli et al., 1991). It is activation of MAP kinase. These ®ndings were therefore likely that this increase in MAP kinase obtained by the use of kinase inhibitors and confirmed activity by the AR may be con®ned to particular in the case of PI3 kinase with a dominant negative patho-physiological situations at de®ned sites of mutant and an in vitro kinase assay. From these studies androgen action. we can conclude that the rapid response of the AR is It is interesting to note that the rapid action of mechanistically dierent from that reported for the androgen reported in osteoblasts (Lieberherr and other steroid receptors in that it requires additional Grosse, 1994) and what we have described here in signalling pathways. We also conclude that the AR is prostatic cells are insensitive to antiandrogens. Thus either upstream of Raf-1 and PI3 kinase or that these the current therapeutically available antiandrogens may signalling pathways are activated in parallel such that not be totally eective in abrogating all androgenic their activities together contribute to the AR-mediated activity in target cells. This notion is consistent with activation of MAP kinase. This latter idea comes from ®ndings that long-term blockade of the growth results of our DHT-mediated Elk-1 activation studies promoting eects of androgen has so far not been Cellular signalling by androgen receptor H Peterziel et al 6328 achieved with antiandrogens. Cessation of antiandro- pGL3 MMTV, a mouse mammary tumour virus luciferase gen therapy in certain cases even improve rather than indicator plasmid (Schneikert et al., 1996) and 1 mg Renilla worsen the symptoms of the patients (Scher and Kelly, tk-luciferase plasmid (Promega). After transfection, the cells 1993; Small and Carrol, 1994; Moul et al., 1995). The were treated with DHT or with vehicle (0.1% ethanol) alone rapid action of the AR that we have reported here may for 36 h, harvested, lysed and luciferase assay performed using the Promega dual-luciferase reporter assay system. therefore provide a system for the identi®cation of a new class of antiandrogens which together with the classical androgen antagonists may be eective in PI3 kinase assay completely abrogating the action of the AR. PC3 ARwt cells or PC3 cells stably transfected with an empty Taken together these ®ndings de®ne a rapid action vector were cultured for 2 days in RPMI supplemented with of AR that is mechanistically dierent from what has 3% CCS. Prior to the addition of inducers, the cells were so far been described for other steroid receptors. The starved for 3 h in RPMI supplemented with 0.5% CCS and cell type speci®city of this response plus the known role treated with hormone or growth factor. They were then lysed of the activated kinases in cell proliferation, makes it with 1 ml ice-cold buer (50 mM HEPES pH 7.5, 150 mM an interesting regulatory pathway that requires further NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2,4mM EGTA, 100 mM NaF, 2 mM Na VO ,10mM Na P O , attention in future analyses of AR action. 3 4 4 2 7 20 mM PMSF, 0.14 mg/ml aprotinin) and centrifuged (10 000 g for 20 min) at 48C. Six hundred microliters of buer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Materials and methods EGTA, 10% glycerol, 1.5 mM MgCl2,2mM Na3VO4) were added followed by 25 ml protein A-sepharose (Pharmacia Materials and plasmid constructs Biotech., 1 : 1 in washing buer) and 5 mg anti-phosphotyr- Phorbol ester (TPA) was purchased from Sigma Ltd. osine antibody (4G10, Upstate Biotechnology). Immunopre- (Deisenhofen, Germany) and human recombinant epidermal cipitation was performed overnight at 48C and the protein A- growth factor (EGF) from Bachem (Bubendorf, Switzerland). sepharose pelleted and washed three times with ice cold Kinase inhibitors PD98059, GF109203X, and Wortmannin washing buer before ®nally resuspending in 50 ml of reaction buer (60 mM Tris pH 7.5, 8 mM MgCl , 100 mM NaCl, were all purchased from Calbiochem (Bad Soden/Taunus, 2 32 Germany). The indicator gene consisting of GAL 4 DNA 0.5 mM ATP, 10 mCi [g- P]ATP) containing 200 mg of the binding sites controlling the expression of a luciferase gene substrate L-a-phosphatidylinositol (Sigma). The reaction was pG5.E4D38lux3, the expression vector containing the GAL 4 incubated for 10 min at room temperature and stopped by DNA binding domain (DBD) alone or linked to the the addition of 150 ml 1 N HCl and the lipids were extracted with 450 ml CHCl /CH OH (1 : 1). The lower phase was dried transcription factor Elk-1, the empty expression vector 3 3 overnight and the lipid pellet was resuspended in 20 ml CHCl containing the RSV promoter RSV-H20 and the RSV 3 promoter driving the expression of a dominant negative and subjected to a thin-layer chromatography (TLC) (Polygram SilG, Macherey-Nagel) in CHCl /CH OH/acet- Raf-1 sequence RSV-cRafC4 have all been described 3 3 one/acetic acid/H O (80 : 26 : 30 : 24 : 14). The radioactively (Oringa et al., 1990; Bruder et al., 1992; Gille et al., 2 1995b; Kamano et al., 1995). labelled reaction products were visualized by autoradiography.

Cell culture and stable transfections Immunoblotting Stromal prostatic cells were derived from pathologically con®ned non-malignant areas of prostatectomy specimens Prior to the MAP kinase assay, the cells were cultured in essentially as described by Zhang et al. (1997). Primary medium supplemented with 3% CCS for at least 2 days. At genital skin ®broblasts were cultured as described by Klocker 70 ± 80% con¯uence, they were serum-starved for 18 h in et al. (1992). PC3 and LNCaP human prostatic carcinoma medium containing 0.5% CCS and thereafter incubated in cell lines were cultured routinely in RPMI medium fresh medium containing vehicle alone (0.1% ethanol) or supplemented with 10% foetal calf serum (FCS) at 378Cin vehicle with hormone. They were then harvested, lysed and subjected to a 10% SDS polyacrylamide gel electrophoresis a5%CO2 atmosphere. All the culture media contained 100 units/ml penicillin and 100 mg/ml streptomycin. PC3 cells and electroblotted onto PVDF membranes. For detection of were stably transfected with wild-type AR cDNA cloned in the phosphorylated MAP kinases, the membranes were the plasmid vector pcDNA3 (Invitrogen) by the calcium incubated with antibody (New England Biolabs) prior to phosphate coprecipitation method (PC3 ARwt). Stable the addition of a secondary goat anti-rabbit antibody transfectants were selected in medium containing 600 mg/ml conjugated to horseradish peroxidase and detection by the G418. enhanced chemoluminescence method (ECL, Amersham). Hybridizations with anti-AR and anti-Erk antibodies were performed similarly. Transient transfection assays Unless otherwise stated, transient transfection was performed In vitro MAP kinase assay according to the calcium phosphate coprecipitation method. For transient transfection assay to determine Elk-1 activity, The cells were washed twice with PBS, lysed in 1 ml lysis the cells were cultured 2 h after transfection in RPMI buer (30 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, medium supplemented with 0.5% charcoal treated FCS 0.5% Triton X-100, 0.5% (w/v) sodium deoxycholate, 10 mM (CCS) and 4.5 mg/ml glucose for a period of 16 h. They sodium ¯uoride, 5 mM sodium orthovanadate, 10 mg/ml were then treated with hormones or with epidermal growth aprotinin, 10 mg/ml leupeptin, 1 mM PMSF) and the lysates factor (EGF) for an additional 6 h, rinsed twice with cold cleared by centrifugation (10 000 g, 15 min at 48C). For phosphate buered saline (PBS) and harvested in 400 ml ice- immunoprecipitation 30 ml protein A-sepharose and antibody cold lysis buer (0.1 M Tris-acetate pH 7.5, 2 mM EDTA, 1% against Erk1 (Santa Cruz) were added to the supernatants v/v Triton X-100). Cellular extracts were prepared and and rotated at 48C for 3 h. The antigen-antibody-complexes luciferase activity determined with equal amounts of were pelleted, washed three times with 1 ml lysis buer and protein. For transactivation studies by the AR, 0.56106 once with 1 ml 25 mM Tris pH 8.0. In vitro kinase assay was LNCaP or PC3 ARwt cells were transfected with 9 mgof carried out in a ®nal volume of 50 ml kinase buer (25 mM Cellular signalling by androgen receptor H Peterziel et al 6329

Tris pH 8.0, 20 mM MgCl2,2mM MnCl2,1mM DTT, 5 mg Acknowledgements myelin basic protein, 10 mCi [g-32P]ATP) for 30 min at 308C. We thank AO Brinkmann for anti-AR antibodies, P Shaw The reaction was stopped by addition of 50 ml sample buer for supplying us with the constructs for the Gal 4 DBD- (0.16 M Tris pH 6.8, 4% SDS, 20% glycerol, 4% mercap- Elk-1 transfection assays and P Brennan for the dominant toethanol, 0.02% bromo-phenol blue) and subjected to a negative p85 PI3 kinase construct. We also thank E 15% SDS polyacrylamide gel electrophoresis. Thereafter the Metzinger for her help with the PI3 kinase assay. We proteins were transferred onto a PVDF membrane and acknowledge with thanks the excellent technical assistance analysed by autoradiography. of J Stober and A Hesselschwerdt. This work was supported by grants from the German Science Foundation and the Deutsche Krebshilfe. RNA preparation and Northern blot analysis Poly(A)+ RNA preparation and Northern blots were carried out essentially as described by Heck et al. (1997).

References

Ahn NG, Seger R, Bratlien RL, Diltz CD, Tonks NK and Klocker H, Kaspar F, Eberle J, UÈ berreiter S, Radmayr C Krebs EG. (1991). J. Biol. Chem., 266, 4220 ± 4227. and Bartsch G. (1992). Am.J.Hum.Genet.,50, 1318 ± BeatoM,HerrlichPandSchuÈ tz G. (1995). Cell, 83, 851 ± 1327. 857. Kortenjann M and Shaw PE. (1995). Oncogene, 6, 99 ± 115. Beato M. (1989). Cell, 56, 335 ± 344. Lieberherr M and Grosse B. (1994). J. Biol. Chem., 269, Berrevoets CA, Veldscholte J and Mulder E. (1993). J. 7217 ± 7223. Steroid Biochem. Mol. Biol., 46, 731 ± 736. Marais R, Wynne J and Treisman R. (1993). Cell, 73, 381 ± Brennan P, Babbage JW, Burgering BMT, Groner B, Reif K 393. and Cantrell DA. (1997). Immunity, 7, 679 ± 689. Marcelli M, Zoppi S, Grino PB, Grin JE, Wilson JD and Bruder JT, Heidecker G and Rapp U. (1992). Genes Dev., 6, McPhaul MJ. (1991). J. Clin. Invest., 87, 1123 ± 1126. 545 ± 556. Marcinkowska E, Widelocha A and Radzikowski C. (1997). Castoria G, Barone MV, Di Domenico M, Bilancio A, Biochem. Biophys. Res. Commun., 241, 419 ± 426. Ametrano D, Migliaccio A and Auricchio F. (1999). McEwan IJ and Gustafsson J-AÊ . (1997). Proc. Natl. Acad. EMBO J., 18, 2500 ± 2510. Sci. USA, 94, 8485 ± 8490. Cato ACB, Henderson D and Ponta H. (1987). EMBO J., 6, Migliaccio A, Di Domenico M, Castoria G, de Falco A, 363 ± 368. Bontempo P, Nola E and Auricchio F. (1996). EMBO J., Cunha GR, Donjacour AA, Cooke PS, Mee S, Bigsby RM, 15, 1292 ± 1300. Higins SJ and Sugimura Y. (1987). Endocrinol. Rev., 8, Migliaccio A, Piccolo D, Castoria G, Di Domenico M, 338 ± 362. Bilancio A, Lombardi M, Gong W, Beato M and Curran T, Peters SG, van Beveren C, Teich NM and Verma Auricchio F. (1998). EMBO J., 17, 2008 ± 2018. IM. (1982). J. Virol., 44, 674 ± 682. Moul JW, Srivastava S and McLeod DG. (1995). Sem. Urol., Di Domenico M, Castoria G, Bilancio A, Migliaccio A and 13, 157 ± 163. Auricchio F. (1996). Cancer Res., 56, 4516 ± 4521. Oringa R, Gebel S, van Dam H, Timmers M, Smits A, Dudley DT, Pang L, Decker SJ, Bridges AJ and Saltiel AR. Zwart R, Stein B, Bos JL, Van der Eb A and Herrlich P. (1995). Proc. Natl. Acad. Sci. USA, 92, 7686 ± 7689. (1990). Cell, 62, 527 ± 538. Endoh H, Sasaki H, Maruyama K, Takeyama K-I, Waga I, RazandiM,PedramA,GreeneGLandLevinER.(1999). Shimizu T, Kato S and Kawashima H. (1997). Biochem. Mol. Endocrinol., 13, 307 ± 319. Biophys. Res. Commun., 235, 99 ± 102. Scher HI and Kelly WK. (1993). J. Clin. Oncol., 11, 1566 ± Fabbro D, Regazzi R, Costa SD, Borner C and Eppenberger 1572. U. (1986). Biochem. Biophys. Res. Commun., 135, 65 ± 73. Schneikert J, Peterziel H, Defossez P-A, Klocker H, de FortP,MartyL,PiechaczykM,elSabroutyS,DaniC, Launoit Y and Cato ACB. (1996). J. Biol. Chem., 271, Jeanteur P and Blanchard JM. (1985). Nucl. Acids Res., 23907 ± 23913. 13, 1431 ± 1442. Small EJ and Carrol PR. (1994). Urology, 43, 408 ± 410. Gille H, Kortenjann M, Thomae O, Moomaw C, Slaughter Steinsapir J, Socci R and Reinach P. (1991). Biochem. C, Cobb MH and Shaw PE. (1995a). EMBO J., 14, 951 ± Biophys. Res. Commun., 179, 90 ± 96. 962. Toullect D, Pianetti P, Coste H, Bellevergue P, Grand-Perret Gille H, Strahl T and Shaw PE. (1995b). Curr. Biol., 5, T, Ajakane M, Baudet V, Boissin P, Boursier E, Loriolle 1191 ± 1200. F, Duhamel L, Charon D and Kirilovsky J. (1991). J. Biol. HamJ,ThomsonA,NeedhamM,WebbPandParkerM. Chem., 266, 15771 ± 15781. (1988). Nucl. Acids Res., 16, 5263 ± 5277. Treisman R. (1996). Curr. Opin. Cell. Biol., 8, 205 ± 215. He WW, Kumar MV and Tindall DJ. (1991). Nucl. Acids Voegel JJ, Heine MJS, Zechel C, Chambon P and Res., 19, 2373 ± 2378. Gronemeyer H. (1996). EMBO J., 15, 3667 ± 3675. Heck S, Bender K, Kullmann M, GoÈ ttlicher M, Herrlich P Ward SG, June CH and Olive D. (1996). Immunol. Today, 17, and Cato ACB. (1997). EMBO J., 16, 4698 ± 4707. 187 ± 197. Improta-Brears T, Whorton AR, Codazzi F, York JD, Yang S-H, Yates PR, Whitmarsh AJ, Davis RJ and Meyer T and McDonnell DP. (1999). Proc. Natl. Acad. Sharrocks AD. (1998). Mol. Cell. Biol., 18, 710 ± 720. Sci. USA, 96, 4686 ± 4691. Yeh S and Chang C. (1996). Proc. Natl. Acad. Sci. USA, 93, Kallio PJ, Poukka H, Moilanen A, JaÈ nne OA and Palvimo 5517 ± 5521. JJ. (1995). Mol. Endocrinol., 9, 1017 ± 1028. Zhang J, Hess MW, Thurnher M, Hobisch A, Radmayr C, Kamano H, Burk B, Noben-Trauth K and Klempnauer K-H. Cronauer MV, Hittmair A, Culig Z, Bartsch G and (1995). Oncogene, 11, 2575 ± 2582. Klocker H. (1997). Prostate, 30, 117 ± 129.