Murine Pirh2 was reported to interact with the hAR N terminus in yeast and in vitro (1
). This interaction was tested in human cells; overexpressed hPIRH2-MYC and hAR were specifically coimmunoprecipitated from 293T cells (Fig. ). Endogenous hPIRH2 and hAR were also coimmunoprecipitated from LNCaP prostate cancer cells that had been cultured in steroid depleted medium (SDM). However, this interaction was further enhanced in the presence of synthetic androgen R1881 (Fig. ).
FIG. 1. hPIRH2 interacts with AR in human cells and increases AR-mediated transcription. (A) 293T cells were transfected with hPIRH2-MYC and either empty vector or full-length hAR (CMV-hAR) as shown. Immunoprecipitation was performed with AR C-19 antibody, and (more ...)
That R1881 enhanced hPIRH2-hAR interactions prompted assessment of hPIRH2 to influence AR transcriptional activity. AR-deficient 293T cells were transfected with a PSA promoter-driven AR-responsive luciferase reporter gene. In the presence of androgens, hPIRH2 cotransfection produced a dose-dependent enhancement of reporter gene activity to levels similar to those of the previously confirmed AR coactivator p300 (Fig. ). This did not occur in the absence of either cotransfected hAR (Fig. ) or androgens (not shown), and Western blotting demonstrated no significant alterations in AR protein levels upon hPIRH2 transfection (Fig. , lower panel). AR-positive LNCaP cells were used in similar reporter gene assays, and the hPIRH2 RING domain double mutant that lacks ubiquitin ligase activity (hPIRH2C145/8S) was included (25
). hPIRH2 cotransfection again led to increased reporter gene activity, whereas hPIRH2C145/8S expression did not (Fig. ), despite retaining hAR interactions (Fig. ). Again, AR levels were largely unaltered upon hPIRH2 transfection, and wild-type hPIRH2 was expressed at similar levels to mutant hPIRH2 (Fig. , lower panel). In addition, overexpression of increasing quantities of hPIRH2 did not alter AR steady-state protein levels in transfected 293T cells (Fig. ). These novel data are the first to suggest that hPIRH2 is an AR coregulator.
To confirm a transcriptional role for hPIRH2, recruitment of endogenous hPIRH2 and functionally related proteins to well-characterized androgen response elements (AREI and AREIII) within the PSA gene was examined in LNCaP cells (Fig. ). ChIP with hPIRH2 antibody at 20, 60, or 80 min after R1881 stimulation, combined with real-time PCR, demonstrated that hPIRH2 is recruited to both AREI and AREIII in response to androgens (Fig. ). The analysis revealed distinct hPIRH2 recruitment profiles to the two AREs. hPIRH2 was increasingly recruited to AREI at 20, 60, and 80 min, coinciding with AR and p300 recruitment (Fig. , left graph). Association of the AR coactivator TIP60 was not detected until 60 min after ligand exposure but was then largely lost after 80 min (Fig. , left graph). Examination of AREIII revealed initial loss of hPIRH2 and TIP60 upon ligand exposure (Fig. , right graph), whereas AR recruitment was robustly detected at this time (Fig. , right graph). After 60 min, hPIRH2 and TIP60 recruitment simultaneously peaked to levels above those prior to ligand exposure, and AR recruitment could still be detected (Fig. , right graph). At 80 min TIP60, hPIRH2 and AR began to dissociate (Fig. , right graph), demonstrating a cyclical, coordinated recruitment profile of hPIRH2 and TIP60 to AREIII. Further analyses showed that hPIRH2 and TIP60 were not recruited to non-ARE-containing regions between AREI and AREIII (not shown). p300 exhibited a noncyclical similar recruitment profile to AREIII and AREI, and in no scenario did hPIRH2 and p300 exhibit robust corecruitment, demonstrating a specific association between hPIRH2 and TIP60. In keeping with these data, hPIRH2-TIP60 protein-protein interactions have been previously described (30
To examine a possible mechanism by which hPIRH2 might increase AR-mediated transcription, hPIRH2 was assessed for its ability to overcome the effects of the potent AR corepressor and histone deacetylase, HDAC1 (11
). First, the effects of HDAC1 on the PSA reporter gene were determined in LNCaP cells. Prior transfection of HDAC1 siRNA led to a potent derepression of the subsequently transfected PSA reporter gene, whereas Western blotting showed HDAC1 knockdown in these cells (Fig. ). In agreement with previous findings (11
), reporter gene assays in LNCaP cells using the PSA-driven luciferase reporter gene demonstrated a strong repression of AR-mediated transcription by HDAC1, in the presence of synthetic androgens (Fig. ). Strikingly, cotransfection of wild-type hPIRH2 abrogated this HDAC1-mediated AR repression (Fig. ). Interestingly, introduction of the hPIRH2C145/8S mutant, which previously did not increase AR activity, did not substantially overcome the repressive effects of HDAC1 (Fig. ). To specifically test whether hPIRH2 enhances AR by HDAC1 downregulation, reporter gene assays were performed in LNCaP cells transfected with the HDAC1 siRNA. As shown in Fig. , HDAC1 depletion reduced the capacity for hPIRH2 to stimulate AR-mediated transcription. siRNA directed against luciferase acted as an internal control to confirm gene silencing was effective at the time of measuring reporter gene activity (Fig. ). Considering that hPIRH2 was able to overcome the repressive effects of HDAC1, protein-protein interaction between these coregulators was tested. Untransfected LNCaP cell lysates were subject to immunoprecipitation using the HDAC1 antibody or other control antisera. Only in the presence of HDAC1 antibody was hPIRH2 specifically recovered, demonstrating that endogenous HDAC1 and hPIRH2 complex in LNCaP cells (Fig. ). Because the hPIRH2C145/8S mutant could not overcome the repressive effects of HDAC1 on AR, hPIRH2C145/8S was compared against wild-type hPIRH2 for its ability to interact with HDAC1 in transfected 293T cells (Fig. ). Immunoprecipitation with HDAC1 antibody specifically recovered both wild-type hPIRH2 and hPIRH2C145/8S, showing that both interact with HDAC1. Immunoprecipitation of transfected cell lysate with an irrelevant antibody did not recover hPIRH2 (Fig. ). Having confirmed that hPIRH2 and HDAC1 can interact in human cells, their ability to co-occupy the androgen-responsive PSA promoter was next tested. In keeping with a role of the hPIRH2-HDAC1 interaction with modulation of AR-mediated transcription, ChIP with HDAC1 antibody, followed by re-ChIP with hPIRH2 antibody, revealed that the two factors were simultaneously present at AREI 80 min after R1881 stimulation (Fig. ). Re-ChIP performed with HDAC1 or nonspecific antibodies acted as controls (Fig. ).
FIG. 2. hPIRH2 can derepress HDAC1-mediated effects on AR and interacts with HDAC1. (A) LNCaP cells were transfected with the indicated siRNAs and then 24 h later with the PSA reporter gene. Cells were starved for 48 h, stimulated with R1881 or vehicle overnight, (more ...)
In order to delineate the mechanism by which hPIRH2 might derepress HDAC1, an alternative reporter gene assay was used that lacks AR and AREs. In this assay, hPIRH2 was tested for its ability to modulate the transcriptional activity of either the heterologous GAL4-DBD or a GAL4-DBD-HDAC1 chimera. 293T cells were transfected with plasmids encoding these factors and a GAL4-responsive luciferase reporter gene (Fig. ). As expected, the GAL4-DBD-HDAC1 chimera had substantially lower transcriptional activity than the GAL4-DBD (Fig. ). Transfection of hPIRH2 or the hPIRH2C145/8S mutant did not have a notable impact on the GAL4-DBD (Fig. ). However, in the presence of GAL4-DBD-HDAC1, hPIRH2 transfection led to a substantial increase in reporter gene expression, whereas transfection of the hPIRH2C145/8S mutant did not have a noticeable impact on reporter gene expression (Fig. ). The hPIRH2C145/8S mutant lacks ubiquitin ligase activity, which raised the possibility that hPIRH2 represses HDAC1 in a ubiquitin-dependent manner. To establish whether this might be the case, the previous reporter gene assay was repeated in the presence of overexpressed ubiquitin. As expected, transfection of hPIRH2 again led to derepression of HDAC1. However, cotransfection of ubiquitin slightly enhanced this effect (Fig. ). More notably, cotransfection of a mutant form of ubiquitin (7KR, in which all lysine residues are mutated to arginine residues) that is incapable of being incorporated into polyubiquitin chains almost completely abrogated the derepression of HDAC1 by hPIRH2 (Fig. ).
FIG. 3. hPIRH2 reduces the repressive activity of HDAC1 on transcription, causes HDAC1 ubiquitination, and reduces HDAC1 levels. (A) 293T cells were transfected with a GAL4-responsive luciferase reporter vector, either GAL4-DBD or HDAC1-DBD, and the indicated (more ...)
To examine the possibility that hPIRH2 might ubiquitinate HDAC1 or that hPIRH2 is somehow involved in regulation of HDAC1 stability, 293T cells were transfected with His-tagged HDAC1 and then subjected to nickel capture under denaturing conditions. Recovered material was probed with ubiquitin antibody to detect ubiquitin-conjugated HDAC1. In the presence of the proteasomal inhibitor MG-132, several slowly migrating HDAC1 species were detected that are likely to be ubiquitin-conjugated HDAC1. However, cotransfection of hPIRH2 produced a noticeable increase in high-molecular-weight ubiquitin-conjugated HDAC1, as detected with the ubiquitin antibody (Fig. ). When the purified material was probed with HDAC1 antibody, this confirmed that HDAC1 had been recovered, and an additional higher-molecular-weight HDAC1 species appeared upon cotransfection of hPIRH2. This additional HDAC1 species may represent ubiquitin-conjugated HDAC1 (Fig. , right panel). In an attempt to enhance the observed hPIRH2-mediated HDAC1 ubiquitination, His-tagged ubiquitin was overexpressed with hPIRH2 or Mdm2, which has previously been shown to ubiquitinate HDAC1 (12
). Nickel purified, ubiquitin-tagged proteins were analyzed by Western blotting with HDAC1 antibody (Fig. ). High-molecular-weight, ubiquitin-conjugated HDAC1 could be detected upon His-ubiquitin expression, and these species were enriched upon hPIRH2 transfection, suggesting that hPIRH2 does indeed ubiquitinate HDAC1 (Fig. ). Mdm2 transfection had a similar effect (Fig. ). Transfection of ubiquitin ligase-deficient hPIRH2C145/8S abolished HDAC1 ubiquitination, suggesting that the ubiquitin ligase activity of hPIRH2 is required for HDAC1 ubiquitination and that hPIRH2C145/8S may act as a dominant-negative form (Fig. , compare lanes 2 and 6). In order to determine whether hPIRH2 might regulate HDAC1 steady-state protein levels, hPIRH2 siRNA was transfected into LNCaP cells in the absence of proteasomal inhibition. Detection of hPIRH2 by Western blotting demonstrated hPIRH2 knockdown, while analysis of HDAC1 levels demonstrated that HDAC1 protein levels increased in response to hPIRH2 depletion (Fig. , right panels).
The data suggest that hPIRH2 may have an important role in AR-mediated transcription. The androgen-responsive LNCaP cell line is known to be at least partly dependent upon androgens for the proliferation and expression of androgen-responsive genes such as PSA. In order to examine whether hPIRH2 might be able to influence gene expression and proliferation in LNCaP cells, a gene-silencing approach was undertaken. Transfection of two siRNAs against hPIRH2 produced 21 to 58% decreases in PSA transcript levels compared to nonsilencing siRNA, as assessed by quantitative PCR (Fig. ). Western blotting confirmed hPIRH2 knockdown (Fig. , lower panel). In addition, hPIRH2 silencing resulted in a 20 to 27% decreased proliferation of LNCaP cells compared to nonsilencing transfectants (Fig. ).
FIG. 4. hPIRH2 controls PSA expression and LNCaP cell proliferation. (A) Nonsilencing siRNA (n/s) or siRNA against hPIRH2 (Si1 and Si2) was transfected into LNCaP cells. After 48 h, PSA and GAPDH transcript levels were measured by quantitative PCR. PSA levels (more ...)
Considering the involvement of hPIRH2 in prostate cancer cell proliferation, hPIRH2, like other AR coregulators, might be expected to be aberrantly expressed in prostate tumors. BL588 hPIRH2 antibody has previously been used for immunohistochemistry (9
). In LNCaP cells, this antibody produced a diffuse nuclear/cytoplasmic immunofluorescence staining pattern (Fig. ) as observed previously for GFP-tagged hPIRH2 (30
). To examine expression in human prostate tumors, paraffin-embedded prostate tissue samples, retrieved by transurethral resection, were assayed by immunohistochemistry. Samples were obtained from untreated patients at the time of diagnosis. Some patients did go on to develop AIPC posttreatment. hPIRH2 staining in tissues was detected in both cytoplasmic and nuclear compartments, and the intensity was categorized as negative (score of 0), weak (score of 1), medium (score of 2), or strong (score of 3). hPIRH2 expression in the benign sample group was weak or absent (mean staining score of 0.9, Fig. ). In lower-grade cancers (Gleason sum score of <7) staining was weak or medium (mean score of 1.4; Fig. ). In Gleason sum score 7 samples, hPIRH2 exhibited mainly medium expression, indicating that with a mean staining score of 2.1, hPIRH2 staining was significantly stronger in Gleason sum score 7 samples than in low-grade cancers (P
= 0.0078 [Kruskal-Wallis]) or benign tissue (P
= 0.0001 [Kruskal-Wallis]) (Fig. ). Strikingly, most high-grade tumors (above Gleason sum score 7) exhibited strong expression, while others exhibited medium staining (mean staining score of 2.79; Fig. ). hPIRH2 expression in these tumors was statistically significantly stronger than in Gleason sum score 7 tumors (P
= 0.0005 [Kruskal-Wallis]), low Gleason sum score tumors (P
= 0.0001, Kruskal-Wallis) or benign tissues (P
= 0.0001, Kruskal-Wallis). This is summarized in Fig. and Table .
FIG. 5. Expression patterns of hPIRH2. (A) LNCaP cells were fixed and subject to immunofluorescence with hPIRH2 BL588 antibody. (B) MCF-7 breast cancer cells were treated as in panel A. (C) Negative control immunofluorescence experiment with no primary antibody. (more ...)
PIRH2 expression in human prostate tumors
No correlation was observed between hPIRH2 immunostaining and PSA levels, patient survival, or patients that developed AIPC versus androgen-responsive cancers. Interestingly, a correlation was observed between hPIRH2 staining score and the presence of bone metastases (mean staining score of 2.76) or absence of bone metastases (mean staining score of 2.18) at diagnosis (P = 0.0004 [Mann-Whitney U test]) (Table ). The data show that hPIRH2 is overexpressed in prostate cancers with increasing Gleason sum scores, and strong expression correlates with the presence of bone metastases.