Only Fos-Containing AP-1 Dimers Are Trans-Repressed by GR
A, CEFs were cotransfected with pRSV-GR together with either the 5xcoll-TRE-TATA-luciferase reporter gene and an expression vector for the c-Fos seeking c-Jun mutant (c-Jun-m0) or the 5xjun2-TATA-luciferase reporter gene and an expression vector for the ATF2 seeking c-Jun mutant (c-Jun-m1). Luciferase activities were determined after treatment with dexamethasone (Dex) or solvent and are plotted as fold induction (mean ± sd of five independent experiments). B and C, HeLa cells were transiently cotransfected with the −1977/−1858uPA-TATA-luciferase reporter gene and Ubi-Renilla, together with expression vectors for either c-Jun-m0 and c-Fos or c-Jun-m1 and ATF2 (B) or expression vectors for either the single-chain AP-1 c-Jun∼c-Fos (cJ∼cF) or the single-chain AP-1 c-Jun∼ATF2 (cJ∼A) (C). Cells were treated with dexamethasone (Dex) as indicated. Luciferase activities normalized to Renilla activities are plotted as fold induction (mean ± sd of one representative experiment performed in triplicate; *, P < 0.05). D, HeLa cells were transiently cotransfected with either the −517/+63-collagenaseI-luciferase reporter gene (Coll-Luc) or the 5xjun2-TATA-luciferase reporter gene, together with the indicated single-chain AP-1. Normalized luciferase activities were determined after treatment with dexamethasone (Dex) or solvent and are plotted as fold induction (mean ± sd of one representative experiment performed in triplicate).

UV-Induced uPA Expression Is Repressed by GR
A, NIH-3T3 cells were either treated with TPA or UV irradiated in the presence or absence of dexamethasone (Dex). Total RNA was prepared and subjected to Northern blotting using probes specific for uPA, collagenase I (Coll), or GAPDH. B, HeLa cells were cotransfected with the −1977/−1858uPA-TATA-luciferase reporter gene and Ubi-Renilla and either treated with TPA or UV irradiated in the presence or absence of dexamethasone (Dex). Luciferase activities normalized to Renilla activities are plotted as fold induction (mean ± sd of one representative experiment performed in triplicates).

The Induction of MKP-1 by GR Mediates the Repression of JNK-Activated c-Jun:ATF2
A, GR does not inhibit the JNK-mediated activation of c-Jun:ATF2 through a direct interaction with JNK. Cos7 cells were transfected with the −1977/−1858uPA-TATA-luciferase reporter gene (uPA-Luc), together with Ubi-Renilla, and with either empty vector or expression vectors for wild-type GR (GR_wt) or two GR mutants unable to interact with and inhibit JNK (GR_R576N and GRR_576D), in the presence or absence of ΔMEKK1. Cells were treated with dexamethasone (Dex) as indicated. Luciferase activities normalized to Renilla activities are plotted as fold induction (mean ± sd of one representative experiment performed in triplicate). B, The repression of JNK-mediated activation of c-Jun:ATF2 requires the transactivation function of GR. Cos7 cells were transfected with the −1977/−1858uPA-TATA-luciferase reporter gene (uPA-Luc), together with Ubi-Renilla, and either empty vector or expression vectors for wild-type GR (GR_wt) or two GR mutants unable to transactivate (GR_ΔAF1mH12 and GRR_D4X), in the presence or absence of ΔMEKK1. Cells were treated with dexamethasone (Dex) as indicated. Luciferase activities normalized to Renilla activities are plotted as fold induction (mean ± sd of one representative experiment performed in triplicate). C, GR-mediated induction of MKP-1 is associated with an inhibition of JNK and c-Jun phosphorylation. NIH-3T3 fibroblasts were treated with dexamethasone (Dex) for 3 h and then UV irradiated as indicated. MKP-1 expression was assessed 20 min later by Western blotting (*, nonspecific band). GR was used as a loading control. JNK and c-Jun phosphorylation (p-JNK and p-c-Jun) was assessed by Western blotting using phosphospecific antibodies. The membranes were stripped and reprobed with phosphorylation state-independent anti-JNK1 and anti-c-Jun antibodies. D, The induction of MKP-1 by GR mediates the repression of JNK-activated c-Jun:ATF2. Primary MEFs from MKP-1 knockout mice (MKP-1^−/−) or littermate wild-type controls (MKP-1^+/+) were transiently transfected with the −1977/−1858uPA-TATA-luciferase reporter gene (uPA-Luc), together with Ubi-Renilla, and with either empty vector or an expression vector for ΔMEKK1. Cells were treated with dexamethasone (Dex) as indicated. Luciferase activities normalized to Renilla activities are plotted as fold induction (mean ± sd of one representative experiment performed in triplicate; *, P < 0.05).

nTrip6 Is a Fos Selective Coactivator
A, nTrip6 specifically interacts in vitro with Fos family members. ³⁵S-labeled, in vitro-translated Fos family members (c-Fos, Fra1, and 2, FosB), Jun family members (c-Jun, JunB, and JunD), or ATF2 were subjected to GST pull-down assays using either GST or GST-Trip6^190–476. Input represents 10% of the in vitro-translated material used in each assay. B, nTrip6 does not interact with c-Jun:ATF2 in living cells. Cos7 cells were transfected with either the single-chain AP-1 c-Jun∼c-Fos fused to YC and Trip6^190–476 fused to YN or the single-chain AP-1 c-Jun∼ATF2 fused to YC and Trip6^190–476 fused to YN, together with a mCherry expression vector as a transfection control. Nuclei were counterstained with DRAQ5. The reconstituted YFP fluorescence was allowed to mature for 1 h at 30 C before live cell imaging. The YFP complementation was observed in 80–90% of the cells transfected with c-Jun∼c-Fos-YC and Trip6^190–476-YN. C, NIH-3T3 cells were transfected with either a control siRNA (Con) or a siRNA targeting Trip6, together with the −1977/−1858uPA-TATA-luciferase reporter gene and Ubi-Renilla. Cells were induced with either TPA or UV 48 h after transfection, and luciferase activities were determined 16 h later. Top, Luciferase activities normalized to Renilla activities are plotted as fold induction (mean ± sd of one representative experiment performed in triplicate); bottom, cell lysates subjected to Western blotting using anti-Trip6, anti-c-Fos, and anti-c-Jun antibodies.

nTrip6 and GR Are Not Tethered to c-Jun:ATF2-Occupied Promoter
A, Schematic representation of the c-Jun:c-Fos- and c-Jun:ATF2-regulated gene unit amplified in the clone 2U. B, Clone 2U and parental NIH-3T3 fibroblasts were subjected to DNA in situ hybridization using a fluorescently labeled cDNA probe complementary to the luciferase coding sequence. A single gene array is visible in 100% of the 2U cells. C, The uPA gene array is functional. 2U cells were treated with either solvent alone (C) or TPA or UV irradiated. Luciferase activities were determined 6 h later and are plotted as fold induction (mean ± sd of one representative experiment performed in triplicate). D, AP-1 is functionally recruited to the uPA gene array. 2U cells were transfected with GFP-Pol II, together with the empty vector (V), an expression vector for an HA-tagged c-Jun∼c-Fos single chain (HA-cJ∼cF), or an expression vector for an HA-tagged c-Jun∼ATF2 single chain (HA-cJ∼A). The localization of the AP-1 single-chain proteins was determined by immunofluorescence using an anti-HA antibody. The enrichment of the AP-1 proteins and of GFP-Pol II was observed in 70–80% of the cotransfected cells. E and F, nTrip6 and GR are not tethered to c-Jun:ATF2 occupied promoter. E, 2U cells were transfected with empty vector, an expression vector for c-Jun∼c-Fos single chain (cJ∼cF), or an expression vector for c-Jun∼ATF2 single chain (cJ∼A), together with an expression vector for GFP-Pol II, an expression vector for Trip6^190–476 fused to YFP (YFP-Trip6^190–476), or an expression vector for GR fused to GFP. Cells were treated with dexamethasone for 30 min and imaged by confocal microscopy. Nuclei of representative cells are shown. F, Clone 2U uPA gene array cells were cotransfected with either GFP-Pol II, Trip6^190–476 fused to YFP (YFP-Trip6^190–476), or GR fused to GFP, together with empty vector (V), HA-tagged c-Jun-m0 mutant (HA-cJm0, c-Fos seeking) and c-Fos, or HA-tagged c-Jun-m1 mutant (HA-cJm1, ATF2 seeking) and ATF2. Cells were treated with dexamethasone for 30 min (Dex). The localization of the c-Jun mutants was determined by immunofluorescence using an anti-HA antibody. Cells were imaged by confocal microscopy. Nuclei of representative cells are shown.

JNK-Stimulated c-Jun:ATF2 Activity Is Repressed by GR
A, c-Jun-deficient MEFs were transfected with the −1977/−1858uPA-TATA-luciferase reporter gene (uPA-Luc), together with Ubi-Renilla, and either empty vector (V) or an expression vector for c-Jun∼ATF2 single chain. Cells were UV irradiated in the presence or absence of dexamethasone (Dex). Top, Luciferase activities normalized to Renilla activities are plotted as fold induction (mean ± sd of one representative experiment performed in triplicate, *, P < 0.05); bottom, the phosphorylation of the c-Jun moiety of the c-Jun∼ATF2 single chain was assessed by Western blotting using a phosphospecific antibody. The membrane was stripped and reprobed with a phosphorylation state-independent anti-c-Jun antibody. B, HEK-293 cells with an integrated GAL4-responsive reporter gene (GalUAS-Luc) were transfected with empty vector (V) or an expression vector for a GAL4 DNA binding domain fusion of c-Jun lacking its dimerization domain (Gal-Jun_Δzip), together with either empty vector or an expression vector for ΔMEKK1, and treated with dexamethasone (Dex), as indicated. Top, Luciferase activities are plotted as fold induction (mean ± sd of one representative experiment performed in triplicate); bottom, the phosphorylation of the c-Jun moiety of Gal-Jun_Δzip was assessed by Western blotting using a phosphospecific antibody. The membrane was stripped and reprobed with a phosphorylation state-independent anti-c-Jun antibody.