Although it is well established that androgens produce oxidative stress in prostate cells that plays a key role in the occurrence and progression of CaP, the exact molecular mechanism of androgen-induced oxidative stress generation in prostatic epithelia and prostate cancer cells is only recently being elucidated. We previously reported that AP-1 transcription factor JunD plays a key role in androgen induction of ROS (20
). Recently we reported that androgen significantly induces the expression and enzymatic activity of “spermidine/spermine acetyl transferase” (SSAT) (21
), a regulatory enzyme in the polyamine catabolic pathway that produces excess amount of ROS in polyamine-rich prostate cells. In this report, we show a relationship at the molecular level between these two components that establishes the mechanism of androgen-induced ROS generation in prostate cells.
Discovering the mechanism that regulates expression of SSAT is the focus of many studies (33
). Thus-far, binding sites of many important transcription factors in the SSAT
gene promoter have been identified (33
). Because the SSAT
gene promoter sequence lacks an AR binding site (ARE), the mechanism of androgen-induced SSAT expression is unclear. Here we show a direct binding of androgen-activated AR with JunD, and that an induction of SSAT by androgen occurs following an interaction of JunD with a specific sequence in the SSAT
promoter only in androgen treated LNCaP cells, probably due to the formation of an activated AR-JunD complex.
We previously showed that androgen-activated AR induces overexpression of transcription factor JunD as well as activates JunD binding to the AP-1 DNA-binding sequence in LNCaP cells (19
). Here we demonstrate that androgen treatment causes AR and JunD to co-precipitate as an immunocomplex from LNCaP cell lysate. Relatively more complex precipitates from the nuclear than from the cytoplasmic fraction (). Androgen treatment induces translocation of JunD into the nucleus in LNCaP cells () at the same time as AR translocates into the nucleus as shown by immunoprecipitation/western blot of nuclear extract () and also reported by other laboratories (31
). These observations suggest an interaction of activated AR with JunD in androgen-treated CaP cells that also causes functional activation of JunD.
More direct evidence of AR and JunD interaction was demonstrated using the Gaussia
luciferase reconstitution assay recently developed to study in situ
protein-protein interactions (30
). The significant reconstitution of Gaussia
luciferase activity only in androgen-treated Hep3B cells transfected with vectors expressing N-terminal and C-terminal fragments of Gaussia
luciferase enzyme linked to AR and JunD, respectively, provides clear and direct evidence of JunD interaction with androgen-activated AR in situ
(). While immunoprecipitation and co-localization of AR with another AP-1 family member, c-Jun, has been reported (31
), to the best of our knowledge this is the first direct demonstration of androgen-activated AR and JunD complex formation.
Since overexpression of JunD is necessary for the induction of ROS following androgen exposure (19
), presumably the AR-JunD complex regulates expression of genes involved in ROS production in LNCaP cells. The complex may bind via JunD to sequences containing binding sites for members of the AP1 family of transcription factors (TGAG/C
). These sequences may or may not contain any ARE sequence. Thus, many genes such as SSAT
that are not directly regulated by AR might be regulated by an AR-JunD complex.
By scanning the SSAT
gene promoter sequence in silico
, we identified six putative AP1 binding sites. Using our siJunD clone of the LNCaP cell line (20
), we demonstrated that in the absence of JunD, androgen-activated AR does not induce SSAT expression (). Thus we conclude that androgen activated AR requires
JunD for SSAT expression.
A direct binding of JunD to the SSAT
promoter sequence was demonstrated by ChIP assay (). By PCR analysis with primers designed to identify the SSAT
promoter, we obtained a PCR product that corresponds to a DNA fragment of the SSAT
promoter only in the chromatin fragment precipitated by JunD antibody and not in the chromatin fragment precipitated by AR antibody (). This suggests that under these conditions, where JunD directly binds to the −574bp to −651bp of the SSAT
promoter, there may not be a direct binding of AR to the SSAT
promoter. Elucidation of this mechanism also explains the delay in SSAT expression (72h) and ROS generation after androgen treatment as previously reported (21
Collectively, our data suggest that activated AR forms a complex with JunD that binds to an AP-1 DNA-binding sequence in the SSAT promoter to activate SSAT gene transcription resulting in overproduction of H2O2 in CaP cells, as shown schematically in . To the best of our knowledge, the data presented above provides for the first time a molecular mechanism of androgen-induced increase in SSAT activity and consequent ROS overproduction in CaP cells.
Schematic diagram showing a possible mechanism of androgen-induced increase in cellular ROS production in CaP cells through an AR-JunD complex
The demonstration of a mechanistic pathway of androgen-induced ROS production opens up a new avenue for development of drugs that specifically target steps in this ROS generating pathway in CaP cells and thus can be effective in therapy and prevention of CaP without major systemic toxicity.