A permissive retinoid X receptor/thyroid hormone receptor heterodimer allows stimulation of prolactin gene transcription by thyroid hormone and 9-cis-retinoic acid

Abstract

Heterodimers of the retinoid X receptor (RXR) with the thyroid hormone receptor (TR) are considered to be nonpermissive. It is believed that within these complexes RXR acts as a "silent partner." We demonstrate here that a permissive heterodimer mediates stimulation of prolactin expression by the thyroid hormone T3 and by 9-cis retinoic acid (9-cis-RA). A response element located in the prolactin distal enhancer mediates transactivation by both ligands in pituitary cells, and RXR recruits coactivators when bound to this element as a heterodimer with TR. Furthermore, transcription by the RXR agonist can be obtained in CV-1 cells only after overexpression of coactivators, and overexpression of corepressors inhibits the response in pituitary cells. Thus, cell type-specific differences in coregulator recruitment can determine the cellular response to both ligands. Coactivator recruitment by 9-cis-RA requires the ligand-dependent transactivation domains (AF-2) of both heterodimeric partners. Interestingly, the presence of the RXR ligand can overcome the deleterious effect of the AF-2 mutation E401Q on association with coactivators and transactivation. These results demonstrate an unexpected role for RXR in TR signaling and show that in particular cellular environments this receptor can act as a "nonsilent" partner of TR, allowing stimulation by RXR agonists.

FIG. 1.

T3 and 9-cis-RA stimulate PRL gene expression in rat pituitary GH4C1 cells. (A) Northern blot analyses were carried out with RNA from control cells and cells incubated for 48 h with 5 nM T3, 1 μM 9-cis-RA, or 100 nM estradiol (E2), as indicated. The blot was hybridized with a labeled cDNA probe for rat PRL. (Bottom) 18S rRNA. (B) GH4C1 cells were transfected with a PRL promoter construct containing the distal enhancer fused to the sequence from −422 to +34, and luciferase activity was determined after 48 h in untreated cells and in cells treated with increasing concentrations of T3 in the presence and absence of 1 μM 9-cis-RA. The data are expressed relative to those found in the untreated cells.

FIG. 2.

The PRL distal enhancer contains an RXR/TR binding site. (A) PRL luciferase reporter constructs containing the indicated combinations of the proximal promoter (−422/+34), the distal enhancer (−1530/−1831), or basal sequences (−36/+34) were transfected into GH4C1 cells, and reporter activity was determined after 48 h in control cells and in cells treated with 5 nM T3 or 1 μM 9-cis-RA. Results are expressed relative to the values obtained in the untreated cells transfected with the longest construct. The TRE is depicted as a shaded rectangle adjacent to the ERE. Black boxes, binding sites for GHF-1/Pit-1. (B) Mobility shift assays with in vitro-translated TRα, RXR, and RAR (1 μl) and an oligonucleotide encompassing the sequence from −1551 to −1573 of the PRL distal enhancer.

FIG. 3.

The PRL TRE mediates regulation by T3 and 9-cis-RA. The TRE was mutated in the PRL constructs indicated at the top. The wild-type (wt) and mutant plasmids were transfected into GH4C1 cells, and reporter activity was determined after 48 h of incubation with 5 nM T3, 1 μM 9-cis-RA, 10 μM forskolin (Fk), or 100 nM estradiol (E2). Results are expressed as factors by which induction exceeded that obtained in the corresponding control untreated cells.

FIG. 4.

The effect of 9-cis-RA is mediated by RXR. The PRL construct containing the distal enhancer was transfected into GH4C1 cells, and luciferase activity was measured after 48 h of incubation in the presence of 1 μM concentrations of the compounds indicated.

FIG. 5.

Expression of TR confers responsiveness to both T3 and 9-cis-RA in 235-1 cells. (A) The PRL construct was transfected into pituitary 235-1 cells together with 35 μg of an expression vector for TR or with the same amount of a noncoding vector. (B) The cells were cotransfected with the PRL promoter and either a wild-type receptor or the P363S mutant TR (20 μg). Reporter activity was measured after 48 h in the presence of 50 nM T3 and/or 1 μM 9-cis-RA, as indicated.

FIG. 6.

RXR acts as a silent partner for TR in CV-1 cells but not in GH4C1 or HeLa cells. (A) HeLa cells were transfected with plasmids in which PRL sequences from −1551 to −1573 (which contain the TRE) or −1551 to −1593 (which contain both the TRE and the ERE) were fused to a thymidine kinase (TK)-chloramphenicol acetyltransferase (CAT) reporter gene. The reporter gene was cotransfected with the expression vector for TR (2.5 μg) and/or that for RXR (0.5 μg) as indicated, and luciferase activity was determined after 48 h of incubation with T3 (200 nM) and 9-cis-RA (1 μM). (B) Stimulation of the −1551 to −1593 TK-CAT plasmid by the endogenous receptors in GH4C1 cells treated for 48 h with 5 nM T3 and 9-cis-RA (1 μM). (C) Reporter activity was determined in CV-1 cells transfected with the −1551 to −1593 TK-CAT plasmid and vectors for TR (200 ng) and RXR (50 ng). Cells were treated as for panel A.

FIG. 7.

RXR agonists cause coactivator recruitment by the RXR/TR heterodimer. (A) Gel retardation assays with the PRL TRE oligonucleotide and in vitro-translated TR and RXR. Assays were performed in the presence of the receptor-interacting domains of the p160 coactivators SRC-1 and ACTR fused to GST. In lanes without coactivators (lanes 1 to 4), the same amount of GST alone was added. As indicated, 9-cis-RA (1 μM) and T3 (1 and 100 nM) were present in the binding assays. Arrows, mobilities of the heterodimer and the complexes containing the heterodimer and the coactivator (CoA). (B) Gel retardation assays were performed with oligonucleotides conforming to either the PRL TRE or a consensus DR4. The binding of the p160 coactivator TIF-2 to RXR/TR was analyzed in the presence and absence of 1 μM 9-cis-RA. (C) Similar assays were performed with SRC-1 in the presence of 100 nM T3 or 1 μM 9-cis-RA, the rexinoid LG100268, or the RXR-selective antagonist LG101208. (D) Association of the RXR/TR heterodimer with wild-type His-tagged TIF-2 or with TIF-2 with mutations in the second (M2) or third (M3) LXXLL motif (450 ng). Assays were performed with the PRL TRE and in the presence of 20 nM T3 and 1 μM 9-cis-RA as indicated. (E) Gel retardation assays with the receptor-interacting domain of the corepressor SMRT fused to GST, the receptor heterodimer, and either the PRL TRE (top) or DR4 (bottom). Assays were performed with 9-cis-RA (1 μM) and/or T3 (20 nM). (F) PRL TRE was incubated with the corepressor in the presence of increasing amounts of GST-ACTR (200 to 600 ng) (top) or His-tagged TIF-2 (300 to 900 ng) (bottom). When indicated, 1 μM 9-cis-RA was present in the assays.

FIG. 8.

The TR AF-2 domain is required for coactivator recruitment by the RXR ligand. (A) Gel retardation assays with the receptor-interacting domains of TIF-2 (top) and DRIP205 (bottom) fused to GST and the PRL TRE oligonucleotide. As indicated, wild-type (wt) TR and RXR, receptors lacking helix 12 (ΔAF2), and the point mutant TR TR(E401Q) were used. (B) Assays were performed with TR(E401Q), native RXR, and wild-type (Wt) His-tagged TIF-2 or TIF-2 with a mutation in the second LXXLL box (M2). (C) The consensus DR4 oligonucleotide was incubated with GST-TIF-2 and either wild-type receptors or TR(E401Q). In all panels experiments were performed in the presence and absence of T3 (20 nM) and 9-cis-RA (1 μM) as indicated.

FIG. 9.

Transactivation by AF-2 mutant receptors. (A) Pituitary 235-1 cells were transfected with the PRL reporter, which contains the distal enhancer fused to proximal promoter sequences, and expression vectors (15 μg) for wild-type TR, TR(ΔAF-2), TR(E401Q). Luciferase activity was measured after treatment with 50 nM T3 and/or 1 μM 9-cis-RA for 48 h and is expressed as the factor by which induction exceeded the value for the untreated control within each group. (B) CV-1 cells were transfected with the PRL TRE fused to the heterologous thymidine kinase (TK) promoter and vectors for native TR (200 ng) and RXR (50 ng) or with the same amount of the specified AF-2 mutant receptors. Chloramphenicol acetyltransferase (CAT) activity was determined after treatment with T3 (5 nM) and/or 9-cis-RA (1 μM).

FIG. 10.

Expression of coregulators modulates responsiveness to 9-cis-RA. (A) CV-1 cell were transfected with the thymidine kinase (TK)-chloramphenicol acetyltransferase (CAT) construct containing the PRL TRE and expression vectors for TR (200 ng) and RXR (50 ng). Vectors (100 ng) for the coactivators ACTR, TIF-2, SRC-1, and DRIP205 or a noncoding vector (100 ng) was cotransfected with the reporter and the receptors, and CAT activity was determined after treatment with T3 (5 nM) and/or 9-cis-RA (1 μM) for 48 h. Data are expressed as the factors by which induction exceeded the value obtained for the corresponding control untreated cells. (B) Pituitary GH4C1 cells were cotransfected with the PRL promoter construct and 200 ng of expression vectors for SMRT or NcoR. Luciferase activity was determined after incubation with 5 nM T3 and/or 1 μM 9-cis-RA. In 235-1 cells the PRL reporter was cotransfected with 15 μg of TR and 500 ng of SMRT or NcoR.