Repression of Runx2 by androgen receptor (AR) in osteoblasts and prostate cancer cells: AR binds Runx2 and abrogates its recruitment to DNA

Abstract

Runx2 and androgen receptor (AR) are master transcription factors with pivotal roles in bone metabolism and prostate cancer (PCa). We dissected AR-mediated repression of Runx2 in dihydrotestosterone (DHT)-treated osteoblastic and PCa cells using reporter assays and endogenous Runx2 target genes. Repression required DHT, but not AR's transactivation function, and was associated with nuclear colocalization of the two proteins. Runx2 and AR coimmunoprecipitated and interacted directly in glutathione-S-transferase pull-down assays. Interaction was ionic in nature. Intact AR DNA-binding domain (DBD) was necessary and sufficient for both interaction with Runx2 and its repression. Runx2 sequences required for interaction were the C-terminal 132 amino acid residues together with the Runt DBD. Runx2 DNA binding was abrogated by endogenous AR in chromatin immunoprecipitation assays and by recombinant AR-DBD in gel shift assays. Furthermore, AR caused increased nuclear mobility of Runx2 as indicated by faster fluorescence recovery after photobleaching. Thus, AR binds Runx2 and abrogates its binding to DNA and possibly to other nuclear components. Clinical relevance of our results was suggested by an inverse correlation between expression of AR-responsive prostate-specific antigen and osteocalcin genes in PCa biopsies. Given the tumor suppressor properties of Runx2, its repression by AR may constitute a mechanism of hormone carcinogenesis. Attenuation of Runx2 by AR in osteoblasts may play a role in skeletal metabolism: the bone-sparing effect of androgens is attributable, in part, to keeping Runx2 activity in check and preventing high-turnover bone disease such as seen after castration and in transgenic mice overexpressing Runx2 in osteoblasts.

Figure 1

AR represses Runx2 independently of its transcriptional activation property. A, COS7 cells were transiently transfected in 96-well plates with the Runx2 reporter 6XOSE2-Luc (firefly luciferase) along with expression vectors for human Runx2, AR, and GR as indicated. Cells were treated for 24 h with DHT, DEX, or vehicle and subjected to luciferase assay. The dotted line indicates the basal level of luciferase activity in the absence of Runx2, which was not affected by any of the receptors or ligands when present individually. Inset, Western blot analysis of Runx2 and AR in corresponding cultures treated with DHT or vehicle. B, Two sets of COS7 cell cultures were transfected in parallel with either the AR reporter MMTV-Luc or the Runx2 reporter 6XOSE-Luc, along with 1 ng each of AR and Runx2 expression plasmids. Cells were treated with the indicated DHT concentrations for 24 h, followed by luciferase assay. DHT concentrations required for 50% Runx2 repression and 50% AR activation were intrapolated from the curves as shown by the dotted lines. C, Block diagram at the top depicts the AR-NTD, -DBD, and -LBD, as well as the AF1 domain within the NTD and the position of the A573D mutation within the DBD. The two zinc-finger motifs constituting the AR-DBD are indicated by Z1 and Z2. Bar graphs represent luciferase assays, in which the reporters 6XOSE2-Luc (left panel) and MMTV-Luc (right panel), were transfected along with expression plasmids for the WT AR (WT) or a mutant lacking the AF1 domain (ΔAF1). Inset in the right panel represents side-by-side immunoblot analysis of AR and ARΔAF1 using anti-AR antibody. Results in all panels were corrected for expression of the internal control CMV-Renilla luciferase and presented as mean relative light units (RLU) ± sem, with n = 4 dish replicates of a representative experiment, repeated at least three times. Veh, Vehicle.

Figure 2

DBD of AR mediates its interaction with Runx2. A, GST and the indicated AR-derived GST-fusion proteins were expressed and purified from E. coli, and 10 μg of each was subjected to SDS-PAGE and Coomassie blue staining. B, Baits shown in panel A were used to pull-down ³⁵S-labled murine Runx2 produced using reticulocyte lysates. SDS-PAGE and autoradiography show the exclusive interaction between GST-AR-DBD and Runx2. C, ³⁵S-labeled Runx2 was pulled down using GST and GST-AR-DBD as baits in the presence of the indicated millimolar concentrations of NaCl. D, Coomassie blue-stained SDS-PAGE of intact or the indicated fragments of AR-DBD fused to GST. E, The baits shown in D were used to pull-down ³⁵S-labeled Runx2. SDS-PAGE and autoradiography show that only the intact AR-DBD bound Runx2. F, Coomassie blue-stained SDS-PAGE showing WT and mutant AR-DBD used as GST-fusion baits to pull-down ³⁵S-labeled Runx2. G, GST pull-down assay using the baits shown in F and ³⁵S-labeled Runx2. AR-DBD^A573D did not bind Runx2. In each pull-down assay, 5% of the input (In) prey is shown for reference. H, Whole-cell extracts from COS7 cells coexpressing Flag-Runx2 with either AR or AR^A573D were subjected to co-IP assays using anti-Flag or nonspecific (IgG) antibodies followed by Western blot analysis with anti-AR and anti-Flag antibodies. I, Luciferase assay of COS7 cells transiently transfected in 96-well plates with the 6XOSE-Luc reporter and expression vectors for human Runx2 alone or with those encoding WT AR, AR^A573D, Flag-AR-DBD, or Flag-AR-NTD as indicated. Below the bar diagram are side-by-side Western blots showing comparable expression of AR^A573D and WT AR (N-20 antibody) and of AR-DBD and AR-NTD (α-Flag antibody). The dotted line indicates the background luciferase activity in the absence of Runx2 (mean ± sem, with n = 4 dish replicates of a representative experiment, repeated at least three times). Veh, Vehicle.

Figure 3

Colocalization and evidence for interaction between AR and Runx2 in living cells. A, COS7 cells were transfected with the indicated plasmid/s (top), immunostained and analyzed by confocal microscopy to visualize the AR (red) and/or Runx2 (green). B, Runx2-GFP fusion protein was expressed in COS7 cells alone or together with WT AR or AR^A573D. The cells were treated with DHT or vehicle and subjected to FRAP analysis. Curves represent fluorescence intensity relative to the respective prephotobleaching levels. veh, Vehicle.

Figure 4

AR inhibits Runx2 in PCa cells. A, PC3 cells were transfected with AR-encoding or control vectors, fixed, immunostained, and analyzed by confocal microscopy to visualize the AR (red) and/or the endogenous Runx2 (green). B, PC3 cells that had been stably transduced with lentiviruses encoding AR or GFP as control were transiently transfected with the MMTV-Luc or 6XOSE2-Luc reporters, followed by DHT treatment and luciferase assay (mean ± sem, n = 4). C, Western analyses of cell extracts as in panel B showing the levels of Runx2 and tubulin (loading control). D, PC3-AR cells were treated with DHT or vehicle and subjected to RT-qPCR analyses of the indicated AR (left) and Runx2 target genes (right). Data were corrected for glyceraldehydes-3-phosphate dehydrogenase mRNA, which itself did not significantly change in response to DHT. The highest mean value for each gene was set at 100 (mean ± sem; n = 3). E, Primary prostate cancer tumors resected from 23 untreated patients (red diamonds) and 17 patients undergoing AAT (black squares) were previously subjected to comprehensive gene expression analysis (61). The negative correlation between OC and PSA mRNA levels in these tumors is demonstrated by best-fit linear regression of the respective values from the untreated patients (dashed line) and those undergoing AAT (solid line). The calculated r values and the levels of statistical significance assigned by the Wilcoxon signed-rank test are depicted in the inset. Veh, Vehicle.

Figure 5

Differential intracellular localization of AR in osteoblastic cell lines correlates with the repression of Runx2. A, ROS 17/2.8 and SaOS-2 cells, which express both AR and Runx2, were transiently transfected with the 6XOSE2-Luc reporter and treated for 24 h with DHT or vehicle, followed by luciferase assay. B, Confocal micrographs of immunostained ROS 17/2.8 and SaOS-2 cells showing the intracellular distribution of AR (red) and Runx2 (green) after 24 h of treatment with DHT or vehicle. C, Differentiating MC3T3E-1 cells stably transfected with the 6XOSE2-Luc reporter were treated with DHT or vehicle commencing at confluence (d 0), and levels of the indicated mRNAs were measured on d 1 and d 9. Data were corrected for ribosomal protein L10A mRNA, which itself did not significantly change in response to DHT (mean ± sem; n = 3). D, d 1 and d 9 MC3T3E-1 cultures were treated for 24 h with DHT or vehicle, and subjected to confocal microscopy for visualization of AR (red) and Runx2 (green) after immunostaining with the respective antibodies. Staining with 4′,6-diamidino-2-phenylindole (DAPI) demarcates the cell nucleus. E, Co-IP assay of d 9 MC3T3-E1 cells that were treated for 24 h with DHT or vehicle. Immunoprecipitates were obtained using Runx2-specific or nonspecific IgG antibodies and were subjected, along with 5% of the input, to Western blot analysis for the detection of AR and Runx2. Data are representative of at least three independent experiments. IP, Immunoprecipitation; veh, vehicle.

Figure 6

Mapping of Runx2 sequences required for binding AR-DBD. A, Shown at the top is a block diagram of Runx2 with the glutamine alanine (QA), Runt, and PST domains. Depicted within the PST domain are the nuclear localization signal (NLS), activation domain (AD), and the overlapping NMTS and SMID. The black thick line just below the block diagram represents the minimal Runx2 sequences required for binding AR-DBD as defined in the present study. Thick gray lines below represent a series of ³⁵S-labeled Runx2 fragments used as preys in pull-down assays with GST or GST-AR-DBD as baits. B, Western blot analyses of immunoprecipitates prepared using AR-specific or nonspecific (IgG) antibodies and whole-cell extracts from COS7 cells coexpressing AR and Runx2’s bipartite interaction domain (aa 176–332 and 465–576). In, Input; IP, immunoprecipitation.

Figure 7

AR-DBD diminishes Runx2’s interaction with its OSE2 target in vitro and in vivo. A, EMSA was performed using ³²P-labeled OSE2 probe and MC3T3-E1 whole-cell extracts as the source of Runx2. Where indicated, the binding reaction also contained purified GST, GST-AR-DBD, GST-AR-DBD^A573D (see Fig. 2F), or Runx2 specific antibodies, either native (n) or denaturated by boiling (b). The relative amounts of the indicated proteins added to the binding reaction are depicted by + or +++ (see Materials and Methods). Arrow indicates Runx2/OSE2 complex. B, Differentiating d 9 MC3T3E-1 cultures were treated for 4 h with DHT or vehicle and subjected to ChIP assay using anti-Runx2 or nonspecific IgG antibodies. Occupancy was quantified by qPCR of an OC promoter fragment containing the OSE2 site or a fragment of the insulin promoter as control. veh, Vehicle.