Estradiol repression of tumor necrosis factor-alpha transcription requires estrogen receptor activation function-2 and is enhanced by coactivators

Abstract

The tumor necrosis factor-alpha (TNF-alpha) promoter was used to explore the molecular mechanisms of estradiol (E(2))-dependent repression of gene transcription. E(2) inhibited basal activity and abolished TNF-alpha activation of the TNF-alpha promoter. The E(2)-inhibitory element was mapped to the -125 to -82 region of the TNF-alpha promoter, known as the TNF-responsive element (TNF-RE). An AP-1-like site in the TNF-RE is essential for repression activity. Estrogen receptor (ER) beta is more potent than ERalpha at repressing the -1044 TNF-alpha promoter and the TNF-RE upstream of the herpes simplex virus thymidine kinase promoter, but weaker at activating transcription through an estrogen response element. The activation function-2 (AF-2) surface in the ligand-binding domain is required for repression, because anti-estrogens and AF-2 mutations impair repression. The requirement of the AF-2 surface for repression is probably due to its capacity to recruit p160 coactivators or related coregulators, because overexpressing the coactivator glucocorticoid receptor interacting protein-1 enhances repression, whereas a glucocorticoid receptor interacting protein-1 mutant unable to interact with the AF-2 surface is ineffective. Furthermore, receptor interacting protein 140 prevents repression by ERbeta, probably by interacting with the AF-2 surface and blocking the binding of endogenous coactivators. These studies demonstrate that E(2)-mediated repression requires the AF-2 surface and the participation of coactivators or other coregulatory proteins.

Figure 1

The −125 to −82 region of the TNF-α promoter is required for E₂ repression. A deletion series of the human TNF-α promoter (3 μg) was transiently transfected individually into U937 cells with 1 μg of expression vector for human ERα (A) or ERβ485 (B). Cells were treated for 24 h with TNF-α (5 ng/ml) in the absence or presence of 10 nM E₂, and luciferase activity was measured. (C) ERβ is more potent than ERα at repressing TNF-α activation of the TNF-RE. U937 cells were transfected with 3 μg of TNF-RE TKLuc and 1 μg of human ERα, ERβ485, or ERβ530. Cells were treated for 24 h with TNF-α (5 ng/ml) in the presence of increasing concentrations of E₂, and luciferase activity was measured. (D) ERα is more effective than ERβ at activating an ERE. U937 cells were transfected with 3 μg of ERE TKLuc and 1 μg of human ERα, ERβ485, or ERβ530. Cells were maintained in the absence or presence of 10 nM E₂ for 24 h and then assayed for luciferase activity.

Figure 2

E₂ represses the collagenase promoter in U937 cells. (A) Cells were transfected with 3 μg of −1044 TNF-α Luc or the AP1-driven collagenase luciferase reporter (Δcoll73) plasmid (33) and 1 μg of human ERα or ERβ485. After transfection, the cells were treated for 24 h with TNF-α (5 ng/ml) in the presence of increasing concentrations of E₂, and luciferase activity was measured. (B) The ERα LBD contains the repression domain. U937 cells were transfected with 3 μg of TNF-RE TKLuc and 1 μg of human ERα, ERβ485, human progesterone receptor A (PRA), GAL-ERα A/B domain, GAL-ERα LBD, GAL-c-jun, or GAL-VP16. Cells were treated for 24 h with TNF-α (5 ng/ml) in the presence 10 nM E₂ or 100 nM progesterone and then assayed for luciferase activity. (C) ERα and ERβ do not inhibit GAL-VP16 activation of GALRE₅ Luc. Cells were transfected with 3 μg of GAL-responsive element-5 (RE₅) Luc (pG5-Luc, Promega), 1 μg of GAL-VP16, and 1 μg of either ERα or ERβ485. After 24 h treatment with 10 nM E₂, cell extracts were assayed for luciferase activity. (D) ERs do not bind to the TNF-RE. Electrophoretic mobility-shift assays were performed by using ³²P-labeled ERE or TNF-RE probes with 2 μl of in vitro-transcribed and -translated ERα or ERβ485. Binding was performed in the absence (−) or presence (+) of 10 nM E₂. U937 cell nuclear extracts (NE) were prepared (23) and incubated with ³²P-labeled TNF-RE in the absence or presence of 100 ng of unlabeled TNF-RE (last lane) as described in Materials and Methods.

Figure 3

Anti-estrogens do not inhibit TNF-α activation of the TNF-RE, but block E₂ repression of TNF-α activation. U937 cells were transfected with 3 μg of TNF-RE TKLuc and 1 μg of human ERα (A and C) or human ERβ485 (B and D). U937 cells were treated for 24 h with TNF-α (5 ng/ml) in the presence of increasing concentrations of E₂, raloxifene, tamoxifen, or ICI (A and B). In C and D, all cells were treated for 24 h with TNF-α (T) and 1 nM E₂ (T + E2) in the presence of increasing concentrations of raloxifene, ICI, or tamoxifen, except for the control (C) cells.

Figure 4

Mutations in the ERβ485 AF-2 surface impair repression of TNF-RE TKLuc. (A) U937 cells were transfected with 3 μg of TNF-RE TKLuc and 1 μg of wild-type ERβ485 or human ERβ485 AF-2 mutants (helix 3, K269A, and helix 12, E448K, based on ERβ485, which corresponds to K314A and E493K based on hERβ530, respectively). Cells were treated for 24 h with TNF-α (5 ng/ml) in the absence or presence of 10 nM E₂ and then assayed for luciferase activity. (B) Overexpressing GRIP1 enhances repression activity of ERβ485. Cells were cotransfected with 3 μg of TNF-RE TKLuc and 50 ng of ERβ485 and in the absence or presence of 5 μg of pSG5-GRIP1. All cells were treated for 24 h with TNF-α (5 ng/ml) in the absence or presence of 10 nM E₂. (C) Overexpressing GRIP1 restores repression activity of the helix 3 ERβ485 AF-2 mutant (K269A). Cells were transfected with 3 μg of TNF-RE TKLuc and 1 μg of ERβK269A in the presence of increasing amounts of pSG5–wild-type (WT) GRIP1 or pSG5-GRIP1 NR box II and III mutant (mut). All cells were treated for 24 h with TNF-α (5 ng/ml) in the presence of 10 nM E₂. The data are expressed as per cent repression of the TNF-α activation of the TNF-RE. (D) RIP140 blocks E₂ repression of the TNF-RE in the presence of wild-type ERβ485. Cells were transfected with 3 μg of TNF-RE TKLuc and 1 μg of ERβ485 in the presence of increasing amounts of an expression vector for RIP140 and 5 μg of pSG5-wild-type (WT) GRIP1 or pSG5-GRIP1 NR box II and III mutant. All cells were treated for 24 h with TNF-α (5 ng/ml) in the presence of 10 nM E₂ and then assayed for luciferase activity.