Rapid Signalling by Androgen Receptor in Prostate Cancer Cells

Oncogene (1999) 18, 6322 ± 6329 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc 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).

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