Table 1.
Gene functions overrepresented in ERβ2 expressing PC3 cells.
Table 2.
ERβ2 regulation of hypoxic genes in PC3 cells.
Fig 1.
Regulation of HIF-1α protein expression independent of mRNA regulation.
A. Western blot of extracts from control and two different 22Rv1 and PC3 clones expressing ERβ2 assayed with HIF-1α antibody. B. Relative mRNA expression of ERβ2 and HIF-1α in the ERβ2clones of the 22Rv1 cell line. The graph shows the data as fold change compared with the control (mean of three separate experiments (±s.e.m.) calculated using Student’s t-test, *p≤0.016). C. Relative mRNA expression of HIF-1α in the ERβ2 #2 clone of the 22Rv1 cell line. The graph shows the data as fold change compared with the control (mean of three separate experiments (±s.e.m.) calculated using Student’s t-test, **p≤0.006).
Fig 2.
HIG2 promoter is activated by ERβ2 expression.
A. Schematic outline of the HIG2 promoter construct where (X) is representing sequence of HIF-1α response elements (HRE). B. Increasing amount of transient transfected ERβ2 expression plasmid into PC3 cells successively activates the transiently transfected HIG2 promoter as shown by luciferase assay of HIG2-A-LUC. The graph shows the data as an increase in luminescence of cells transfected with increased concentration of ERβ2 (mean of three separate experiments (±s.e.m.) calculated using Student’s t-test, ***p≤0.0002). Comparison of concentrations—0μg to 1000 μg. C. Addition of expression plasmid for HIF-1α activates HIG2 promoter in PC3 cell lines. The graph shows the data as an increase in luminescence of cells transfected with HIF-1α (mean of three separate experiments (±s.e.m.) calculated using Student’s t-test, ****p≤0.0001).
Fig 3.
Twist1 promoter with intact HIF-1α response element is activated by ERβ2 expression while the promoter with mutated site is not activated.
A. Description of Twist1 promoter constructs used. B. LNCaP cells transfected with Twist1-promoter-Luciferase reporter and increasing concentration of ERβ2 expression plasmid. The graph shows the data as an increase in luminescence of cells transfected with increased concentration of ERβ2 (mean of three separate experiments (±s.e.m.) calculated using Student’s t-test, **p≤0.0034). C. Transfection of Twist1-promoter-Luciferase reporter with (Twist) or mutated (mTwist) HIF-1α response element together with expression plasmids for HIF-1α or ERβ2 in LNCaP cells. The graph shows the data as an increase in luminescence of cells transfected with increased concentration of ERβ2 (mean of three separate experiments (±s.e.m.) calculated using Student’s t-test, ****p≤0.0001, ###p≤0.002).
Fig 4.
Interaction of HIF-1α with ERβ2 in vitro.
A. His-tagged LBD-ERβ2 pull down with nickel agarose beads of in vitro translated HIF-1α compared to His-tagged LBD-ERα, His-tagged LBD-ERβ1, His-tagged LBD-ERβ2 and His-tagged ERβ2ΔCX TNT is lane with only coupled reticulocyte lysate. B. Expression of the biotin ligase BirA, HA-HIF-1α, and the N-terminal biotin consensus peptide fused receptor constructs B7TEV-ERα, B7TEV-ERβ1, B7TEV-ERβ2, B7TEV-ERβ5, and B7TEV-ERβΔCX in PC3 cells. Cell extracts were then subjected to pulldown with streptavidin magnetic beads and proteins separated by SDS-PAGE followed by detection with HIF-1α antibody.
Fig 5.
The oxygen destabilization domain is not required for interaction with ERβ2 and ERβ5.
A. PC3 cells transfected with plasmids expressing GFP, HIF-1α, HIF-1α ΔODD, BirA (biotin ligase), ERβ2 and ERβ5 with N-terminal biotinylation consensus. Complexes were pulled down with streptavidin magnetic beads. Pulled down HIF-1α and HIF-1α ΔODD was detected using anti-HIF-1α antibody. B. Transfection of 200 ng of FR-luciferase alone, with 500ng PM2-ERβ2 and 300ng of either VP16-N-term HIF-1α (amino acids 1–401), VP16- ODD HIF-1α (amino acids 401–603), or VP16-C-term HIF-1α (amino acids 603–826). Measurement of luciferase activity 24 h after transfection.
Fig 6.
The prolines P405 and P564 are not required for interaction with ERβ2 and ERβ5.
PC3 cells transfected with GFP, HIF-1α, HIF-1α P405/A-P564/A, BirA (biotin ligase), ERβ2 and ERβ5 with N-terminal biotinylation consensus. Complexes were pulled down with streptavidin magnetic beads.
Fig 7.
ChIP assay showing recruitment of ERβ2 to the HIF-1α binding site in the Twist1 and VEGF promoters.
In PC3 cells (A) and 22Rv1 cells (B) expressing doxycycline-regulated ERβ2 expression, this ERβ isoform binds to HRE (HIF-1α response element in Twist1 promoter) and VEGF promoter (HIF-1α response element in VEGF promoter) but not to pS2 (ERE) promoter. ChIP-qPCR results are shown with a non-specific IgG and a specific anti-M2-FLAG antibody immunoprecipitation. ERβ2 binding is enriched only in HIF-1α response element containing Twist1 and VEGF promoters but not in the pS2 promoter lacking HIF-1α response element. The graph shows the data as an increase in binding of FLAG to the Twist1 (pHRE) and VEGF promoter (mean of three separate experiments (±s.e.m.) calculated using Student’s t-test, *p≤0.028, ##p≤0.041 (PC3 cells) and **p≤0.0049, ##p≤0.0481 (22Rv1 cells).
Fig 8.
Schematic model of the interaction between ERβ2 and HIF-1α.
Proposed model of how ERβ2 stabilizes HIF-1α and is recruited to VEGF and Twist1 promoters.