nTrip6 acts as a coactivator for AP-1 and NF-κB. (A,B,D) HEK293 cells or A549 cells (D, right) were cotransfected with the luciferase reporter constructs indicated in the top left corner, together with expression vectors for Trip6^190–476, p65 (B, left), or c-Fos (B, right), and treated with TPA (Coll-Luc) or TNFα (NF-κB-Luc) in the presence or absence of dexamethasone (Dex, D). (C) HEK293 cells were cotransfected with GAL-Luc reporter construct and expression vectors for either GAL_DBD or GAL_DBD–Trip6^190–476. Normalized luciferase activities are plotted as fold induction (mean ± S.D. of one representative experiment performed in triplicates).

Occupancy of AP-1- and NF-κB-dependent promoters by nTrip6 and GR. Chromatin immunoprecipitation (ChIP) was performed in HeLa cells treated as in Figure 2B and D, using antibodies against c-Fos (A) or NF-κB p65 (B), and against Trip6 and GR, as well as an isotype control Ab. PCR was performed with primers that detect either the promoter or a coding region of collagenase I (CollI, A) or interleukin 8 (IL-8, B) genes, or the U6 snRNA promoter. (C) Chromatin double immunoprecipitation assay. A first ChIP was performed on HeLa cells treated as indicated using an anti-c-Fos Ab, and the immunoprecipitated material was eluted and subjected to a second ChIP using either an anti-Trip6 or an anti-GR Ab.

nTrip6 is required for glucocorticoid-mediated repression of collagenase I and interleukin 8 expression. HeLa cells were transfected with siRNAs as in Figure 1E and treated with either TPA (A) or TNFα (B), in the presence or absence of dexamethasone (Dex). Total RNA was prepared and subjected to Northern blotting using probes specific for collagenase I IL-8 or GAPDH. (C) HeLa cells were cotransfected with either the control siRNA or the siRNA #2 together with either an AP-1-driven reporter gene (TRE-Luc) and a c-Fos expression vector (left), or an NF-κB-driven reporter gene and a p65 expression vector, and treated with dexamethasone (dex) as indicated. Normalized luciferase activities are plotted as fold induction (mean ± S.D. of one representative experiment performed in triplicates).

Trip6 interacts with GR, c-Fos, and NF-κB p65. (A) GST pull-downs. ³⁵S-labeled, in vitro translated GR, c-Fos, and p65 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–D) CoIPs. (B,D) HeLa cells stably transfected with HA-Trip6 were treated with either TPA (B) or TNFα (D) in the presence or absence of dexamethasone (Dex) as indicated. Proteins from nuclear extract were immunoprecipitated using either a rat anti-HA or a rat isotype control Ab (Con). Input (1%) and immunoprecipitated proteins were subjected to Western blotting (WB) using a mouse anti-HA (B,D), an anti-GR (B), or an anti-p65 Ab (D). (C) Cells were metabolically labeled with [³⁵S]methionine and [³⁵S]cysteine, and treated as in B. Proteins immunoprecipitated with the anti-HA and the control Ab were eluted and subjected to a second IP with an anti-c-Fos Ab. Input (1%) and precipitated proteins were subjected to SDS-PAGE and autoradiography.

Identification of a new GR-interacting protein: Trip6. (A) Schematic representation of wild-type (wt) GR and GR mutants. GAL4_DBD–GR_mH12ΔAF1 served as a bait in the two-hybrid screen. (AF) Activation function; (DBD) DNA-binding domain; (LBD) ligand-binding domain. (B) Transactivation and transrepression potential of the GR mutants shown in A. (Top) For transactivation, Cos7 cells were cotransfected with MMTV-luciferase reporter, the plasmids indicated, and treated as labeled. Normalized luciferase activities are plotted as fold induction (mean ± S.D. of three independent experiments). (Bottom) For transrepression, Cos7 cells were cotransfected with –517/+63-CollagenaseI-Luciferase reporter, the plasmids indicated, and treated as labeled. Normalized luciferase activities (mean ± S.D. of three independent experiments) are plotted relative to the value obtained in TPA-induced/Dex-untreated cells transfected with the empty expression vector. (C) Two-hybrid interaction of GR and Trip6. Y190 yeasts were transformed with the indicated plasmids. The β-galactosidase activity was measured after overnight incubation with vehicle alone or triamcinolone acetonide (TAC). (D) Subcellular localization of Trip6. HeLa cells were treated with TPA and dexamethasone (Dex) as indicated, and whole-cell extracts (WCE), cytoplasmic extracts (CE), and nuclear extracts (NE) were subjected to Western blotting using an anti-Trip6, an anti-c-Fos, and an anti-GR Ab. (E) Down-modulation of Trip6 by siRNA. HeLa cells were transfected with either a control siRNA (Con), or two different siRNAs targeting different sequences in Trip6 mRNA (#1 and #2). Whole-cell lysates were subjected to Western blotting using an anti-Trip6 and an anti-GR Ab.

nTrip6 mediates GR tethering to the repressed promoter. (A) Schematic representation of Trip6LIM3m fused to GST. Trip6 harbors 3 LIM domains in its C-terminal patch, each consisting of two zinc fingers, as illustrated for the LIM domain 3 (LIM3). In Trip6LIM3m, the four coordinating cysteines in the two zinc fingers of LIM3 were mutated to alanines. (B) Mapping of Trip6 LIM domains interaction with GR, c-Fos, and NF-κB p65. ³⁵S-labeled, in vitro translated GR, c-Fos, and p65 were subjected to GST pull-down assays using GST fusion of individual Trip6 LIM domains, or combinations of the LIM domains 1 and 2, or 2 and 3. Input represents 10% of the in vitro translated material used in each assay. (C) Trip6LIM3m does not interact with GR, but still interacts with c-Fos and NF-κB p65. GST pull-down assays were performed as in B using GST or GST fusions of Trip6 or Trip6LIM3m. (D) Trip6LIM3m has a dominant negative effect on AP-1 and NF-κB repression by GR. HEK-293 (left) or A549 cells (right) were cotransfected with the indicated luciferase reporter construct and expression vectors. Cells were treated with TPA (left) or TNFα (right), in the presence or absence of dexamethasone (Dex). Normalized luciferase activities (to renilla luciferase) are plotted as fold induction (mean ± S.D. of one representative experiment performed in triplicates). (E) nTrip6 mediates the tethering of GR to the repressed collagenase I promoter. Chromatin immunoprecipitation (ChIP) was performed as in Figure 5 in HeLa cells transfected with either the control siRNA or the siRNA #2, and treated with TPA in the presence or absence of Dex as indicated. The relative promoter occupancy by c-Fos, nTrip6, and GR (normalized to the input) is indicated below the bands.