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.