Down-regulation of PROS1 gene expression by 17beta-estradiol via estrogen receptor alpha (ERalpha)-Sp1 interaction recruiting receptor-interacting protein 140 and the corepressor-HDAC3 complex

Abstract

Pregnant women show a low level of protein S (PS) in plasma, which is known to be a risk for deep venous thrombosis. 17Beta-estradiol (E(2)), an estrogen that increases in concentration in the late stages of pregnancy, regulates the expression of various genes via the estrogen receptor (ER). Here, we investigated the molecular mechanisms behind the reduction in PS levels caused by E(2) in HepG2-ERalpha cells, which stably express ERalpha, and also the genomic ER signaling pathway, which modulates the ligand-dependent repression of the PSalpha gene (PROS1). We observed that E(2) repressed the production of mRNA and antigen of PS. A luciferase reporter assay revealed that E(2) down-regulated PROS1 promoter activity and that this E(2)-dependent repression disappeared upon the deletion or mutation of two adjacent GC-rich motifs in the promoter. An electrophoretic mobility shift assay and DNA pulldown assay revealed that the GC-rich motifs were associated with Sp1, Sp3, and ERalpha. In a chromatin immunoprecipitation assay, we found ERalpha-Sp protein-promoter interaction involved in the E(2)-dependent repression of PROS1 transcription. Furthermore, we demonstrated that E(2) treatment recruited RIP140 and the NCoR-SMRT-HDAC3 complex to the PROS1 promoter, which hypoacetylated chromatin. Taken together, this suggested that E(2) might repress PROS1 transcription depending upon ERalpha-Sp1 recruiting transcriptional repressors in HepG2-ERalpha cells and, consequently, that high levels of E(2) leading to reduced levels of plasma PS would be a risk for deep venous thrombosis in pregnant women.

FIGURE 1.

Down-regulation of PS mRNA and antigen in HepG2-ERα cells and human normal hepatocytes by17β-estradiol. A, analysis of whole cell extract from MCF7 cells used as a positive control (lane 1), the original HepG2 cells (lane 2), mock-transfected HepG2 cells (lane 3), and HepG2-ERα cells (lane 4) by Western blotting using anti-ERα antibody. Anti-β-actin antibody was used as a loading control. B and C, results of quantitative RT-PCR and an enzyme-linked immunosorbent assay to evaluate the effects of E₂ on the levels of PS mRNA in HepG2-ERα cells and its derived cells (B) and on the levels of PS antigen secreted from HepG2-ERα cells (C). After treatment of HepG2-ERα cells with E₂ for 48 h, total RNA and the culture medium were harvested and analyzed by quantitative RT-PCR and an enzyme-linked immunosorbent assay, respectively. Values are the mean ± S.D. for at least three independent experiments. *, p < 0.05 versus vehicle control. D, analysis of whole cell extract from MCF7 cells used as positive control (lane 1), the original HepG2 cells used as negative control (lane 2), and hNHep cells (lane 3) by Western blotting using anti-ERα antibody. Anti-β-actin antibody was used as a loading control. E, results of quantitative RT-PCR to evaluate the effects of E₂ in hNHep.

FIGURE 2.

Transient expression of PROS1 promoter-reporter gene constructs in HepG2-ERα cells with or without E₂ treatment. Luciferase activity of the HepG2-ERα cells transiently transfected with pPROS1/−4229 or its 5′-deleted constructs (A and B) and pPROS1/−175 or its mutant (C), with or without E₂ treatment, was measured. WT (pPROS1/−4229) or the full-length promoter construct with mutation (pPROS1/−4229Mut) was transiently transfected into HepG2-ERα cells or original HepG2 cells treated with or without E₂, and luciferase activities were measured (D). The luciferase activity levels of the respective constructs were expressed as relative values to that of the pGL3 Basic-derived empty vector without E₂ treatment. A, results for the constructs pPROS1/−4229 to pPROS1/−236. B, results for the constructs pPROS1/−236 to pPROS1/−137 with putative transcription factor binding sites. C, analysis of mutations in the GC-rich motifs in pPROS1/−175. Results for constructs pPROS1/−175 WT, Mut1 (−170A/−169A), Mut2 (−156A/−155A), and Mut3 (−170A/−169A/−156A/−155A) are shown. D, analysis of mutations in the GC-rich motifs in pPROS1/−4229. Results for WT (pPROS1/−4229) and mutated (pPROS1/−4229Mut) constructs are shown. Values are the mean ± S.D. for at least three independent experiments. *, p < 0.05 versus vehicle control.

FIGURE 3.

E₂-dependent PROS1 down-regulation mediated by ERα. A, luciferase (LUC) activity levels of cells transfected with pPROS1/−175, with or without E₂ treatment, expressed relative to that of the pGL3 Basic-derived empty vector without E₂ treatment. B, HepG2-ERα cells transfected with pPROS1/−175 and treated with 100 nm E₂, 10 μm ICI 182,780, or both. After 48 h, luciferase activities were measured, and results were expressed as relative values to that of the vehicle-treated cells. Values are the mean ± S.D. for at least three independent experiments. *, p < 0.05 versus vehicle control.

FIGURE 4.

Interaction of Sp and ERα proteins with the PROS1 promoter in vitro and importance of the GC-rich motifs. A, oligonucleotides used in EMSA and DNA pulldown analyses. GC-Mut contains mutations in both the distal and proximal GC-rich motifs. B, EMSA was performed with nuclear extracts from HepG2-ERα cells in the presence of the biotin-labeled DNA oligonucleotides GC-WT or GC-Mut. The biotin-labeled oligonucleotides were incubated alone or in combination with 5 μg of nuclear extract (N.E.) from E₂-treated HepG2-ERα cells in the presence of a 100-fold molar excess of specific unlabeled oligonucleotide (100X) or antibodies. Arrows indicate retarded (a, b, and c) and supershifted (SS) complexes. FP, free probe. C, DNA pulldown assays carried out by incubating biotin-labeled oligonucleotides containing WT or mutated GC-rich motifs with nuclear extract from HepG2-ERα cells with or without E₂ treatment. Specifically bound proteins were eluted and subjected to Western blotting using specific antibodies against Sp1, Sp3, and ERα, respectively. Similar results were obtained in multiple independent experiments. Veh, vehicle.

FIGURE 5.

Knockdown of Sp1 or Sp3 by RNA interference and its effects on E₂-dependent PROS1 repression. A, knockdown of Sp was determined by Western blotting. HepG2-ERα cells were transfected with iNS, iSp1, or iSp3, and whole cell lysate was analyzed by Western blotting as described under “Experimental Procedures.” The experiments were repeated at least three times, and similar results were obtained. β-Actin was used as a loading control (Cont.). B, RNA interference-luciferase reporter analysis of HepG2-ERα cells. siRNAs (50 nm) were transfected, and the next day pPROS1/−175 was transfected with or without E₂ treatment. Luciferase activity was expressed relative to that of the cells without E₂ treatment. C, quantitative RT-PCR analysis after siRNA transfection in HepG2-ERα cells. Cells were transfected with respective siRNAs for 4 h and treated with E₂ for 48 h. Values are the mean ± S.D. for at least three independent experiments. *, p < 0.05 versus vehicle control.

FIGURE 6.

ChIP and ChIP re-IP analyses of the E₂-responsive region or control regions of the PROS1 promoter and the effect of TSA on E₂-dependent PROS1 repression. A, ChIP analysis of the E₂-responsive region containing GC-rich motifs in the PROS1 promoter and of the control regions containing no GC-rich motif a or non-target GC-rich motif. Specific primers for detection of respective regions (Target GC-rich, −236 and −88; Untarget GC-rich, −3748 and −3574; No GC-rich, −4033 and −3875) are represented by thick arrows. B, ChIP assays for HepG2-ERα cells treated with E₂ or vehicle control performed using various antibodies. Similar results were obtained in multiple independent experiments. C, ChIP re-IP assays for HepG2-ERα cells treated with E₂ or vehicle control performed using anti-ERα antibody or anti-RIP140 antibody for primary immunoprecipitation (IP). Re-immunoprecipitation was performed with respective antibodies as described. D, HepG2-ERα cells were treated with E₂, TSA, or the two in combination, and total RNA was harvested and analyzed by quantitative RT-PCR. Values are the mean ± S.D. for at least three independent experiments. *, p < 0.05 versus vehicle control; **, p < 0.05 versus TSA(−) + E₂.

FIGURE 7.

A proposed model for down-regulation of PROS1 promoter by 17β-estradiol in HepG2-ERα cells. A, in HepG2-ERα cells without E₂ treatment, basal PROS1 expression was regulated by Sp1 and Sp3, probably together with other transcriptional coactivators. B, in HepG2-ERα cells with E₂ treatment, PROS1 expression was repressed by ERα-Sp1 interaction recruiting RIP140 and the NCoR-SMRT-HDAC3 corepressor complex sequentially, which consequently induced histone deacetylation. Ac, acetyl residue.