The orphan nuclear receptor SHP inhibits hepatocyte nuclear factor 4 and retinoid X receptor transactivation: two mechanisms for repression

Abstract

The orphan nuclear hormone receptor SHP interacts with a number of other nuclear hormone receptors and inhibits their transcriptional activity. Several mechanisms have been suggested to account for this inhibition. Here we show that SHP inhibits transactivation by the orphan receptor hepatocyte nuclear factor 4 (HNF-4) and the retinoid X receptor (RXR) by at least two mechanisms. SHP interacts with the same HNF-4 surface recognized by transcriptional coactivators and competes with them for binding in vivo. The minimal SHP sequences previously found to be required for interaction with other receptors are sufficient for interaction with HNF-4, although deletion results indicate that additional C-terminal sequences are necessary for full binding and coactivator competition. These additional sequences include those associated with direct transcriptional repressor activity of SHP. SHP also competes with coactivators for binding to ligand-activated RXR, and based on the ligand-dependent interaction with other nuclear receptors, it is likely that coactivator competition is a general feature of SHP-mediated repression. The minimal receptor interaction domain of SHP is sufficient for full interaction with RXR, as previously described. This domain is also sufficient for full coactivator competition. Functionally, however, full inhibition of RXR transactivation requires the presence of the C-terminal repressor domain, with only weak inhibition associated with this receptor interaction domain. Overall, these results suggest that SHP represses nuclear hormone receptor-mediated transactivation via two separate steps: first by competition with coactivators and then by direct effects of its transcriptional repressor function.

FIG. 1

Repression of HNF-4-mediated transactivation by SHP. (A) Repression of direct HNF-4 transactivation. HepG2 cells were cotransfected with 3 μg of (BA1)₅CAT, 50 ng of HNF-4, 0.75 μg of CMVβ-Gal, and 50 ng to 1 μg of SHP plasmid. At 40 h posttransfection, the cells were harvested for CAT and β-galactosidase assay. The CAT values are the means of normalized three independent transfection experiments, each carried out in duplicate. (B) Repression of Gal4-HNF4 transactivation. HepG2 cells were cotransfected with 25 ng of Gal4-HNF4D2CD1 (Fig. 3A), 200 ng of TKGH, 200 ng of Gal4Tkluc reporter plasmid, and increasing amounts of SHP expression vector. CDM8 was used to maintain a constant amount of receptor expression vector, and total DNA was transfected. Cell extracts were prepared 48 h following transfection. Luciferase activities normalized with GH values are plotted as the mean ± standard deviation (n = 3).

FIG. 2

HNF-4 interacts with the receptor interaction domain of SHP. (A) Previously described SHP mutants (33) are diagrammed, fused to Gal4. INT and REP represent receptor interaction and direct repression domains, respectively. (B) SHP sequences required for SHP interaction in the mammalian two-hybrid assay. Fifty nanograms of each of the deletion versions of murine SHP fused to the Gal4 DNA binding domain was cotransfected into HepG2 cells with 50 ng of a vector expressing VP16 alone or VP16-HNF4D2. Normalized luciferase expression directed by the Gal4Tkluc reporter is indicated as the mean ± standard deviation (n = 3). WT, wild type.

FIG. 2

HNF-4 interacts with the receptor interaction domain of SHP. (A) Previously described SHP mutants (33) are diagrammed, fused to Gal4. INT and REP represent receptor interaction and direct repression domains, respectively. (B) SHP sequences required for SHP interaction in the mammalian two-hybrid assay. Fifty nanograms of each of the deletion versions of murine SHP fused to the Gal4 DNA binding domain was cotransfected into HepG2 cells with 50 ng of a vector expressing VP16 alone or VP16-HNF4D2. Normalized luciferase expression directed by the Gal4Tkluc reporter is indicated as the mean ± standard deviation (n = 3). WT, wild type.

FIG. 3

SHP interacts with the AF-2 surface of HNF-4. (A) Gal4-HNF4 constructs used in mammalian two-hybrid mapping are diagrammed. (B) HNF-4 sequences required for SHP interaction in the mammalian two-hybrid assay. Gal4 fusions (50 ng each) to the deletion or point mutant versions of HNF-4 were cotransfected with 50 ng of VP16 or VP16-SHP into HepG2 cells. Normalized luciferase expression from the Gal4Tkluc reporter is shown. (C) The AF-2 surface of HNF-4 is required for interaction with SHP in vitro. GST alone or a GST-SHP fusion protein were expressed in E. coli, bound to glutathione agarose, and incubated with the indicated Gal4-HNF4 fusion proteins, which were ³⁵S labeled by in vitro translation. Specifically bound proteins were eluted by standard procedures (2) and are compared to 20% of the total input (bottom gel). WT, wild type.

FIG. 3

SHP interacts with the AF-2 surface of HNF-4. (A) Gal4-HNF4 constructs used in mammalian two-hybrid mapping are diagrammed. (B) HNF-4 sequences required for SHP interaction in the mammalian two-hybrid assay. Gal4 fusions (50 ng each) to the deletion or point mutant versions of HNF-4 were cotransfected with 50 ng of VP16 or VP16-SHP into HepG2 cells. Normalized luciferase expression from the Gal4Tkluc reporter is shown. (C) The AF-2 surface of HNF-4 is required for interaction with SHP in vitro. GST alone or a GST-SHP fusion protein were expressed in E. coli, bound to glutathione agarose, and incubated with the indicated Gal4-HNF4 fusion proteins, which were ³⁵S labeled by in vitro translation. Specifically bound proteins were eluted by standard procedures (2) and are compared to 20% of the total input (bottom gel). WT, wild type.

FIG. 3

SHP interacts with the AF-2 surface of HNF-4. (A) Gal4-HNF4 constructs used in mammalian two-hybrid mapping are diagrammed. (B) HNF-4 sequences required for SHP interaction in the mammalian two-hybrid assay. Gal4 fusions (50 ng each) to the deletion or point mutant versions of HNF-4 were cotransfected with 50 ng of VP16 or VP16-SHP into HepG2 cells. Normalized luciferase expression from the Gal4Tkluc reporter is shown. (C) The AF-2 surface of HNF-4 is required for interaction with SHP in vitro. GST alone or a GST-SHP fusion protein were expressed in E. coli, bound to glutathione agarose, and incubated with the indicated Gal4-HNF4 fusion proteins, which were ³⁵S labeled by in vitro translation. Specifically bound proteins were eluted by standard procedures (2) and are compared to 20% of the total input (bottom gel). WT, wild type.

FIG. 4

Stimulation of Gal4-HNF4 transactivation by SRC-3. Deletion or point mutant versions of HNF-4 (Fig. 3) fused to the Gal4 DNA binding domain (25 ng) were cotransfected into HepG2 cells with 500 ng of SRC-3. At 48 h after transfection, cells were harvested for luciferase and GH assays. Normalized luciferase expression directed by the Gal4Tkluc reporter is indicated as the mean ± standard deviation (n = 3). WT, wild type.

FIG. 5

SHP competes specifically with SRC-3 for binding to HNF-4 in a mammalian two-hybrid assay. HepG2 cells were cotransfected with 50 ng (each) of Gal4SRC-3(RID), VP16 or VP16-HNF4, and the indicated amounts of VP16-SHP and VP16-SHPW160X. Normalized luciferase expression directed by the Gal4Tkluc reporter is indicated as the mean ± standard deviation (n = 3). The decreased luciferase expression in the presence of increasing amounts of the SHP proteins reflects decreased interaction between SRC-3 and HNF-4. Essentially identical results were observed with intact SHP.

FIG. 6

SHP interacts with the AF-2 surface of RXR. (A) Mammalian two-hybrid assay for interaction. Gal4SHP was transfected into HepG2 cells along with VP16RXR, VP16RXRΔ19C, or VP16 alone. Approximately 20 h after transfection, the cells were treated with 1 μM 9-cis-RA for 30 h. Normalized luciferase expression directed by the Gal4Tkluc reporter is indicated as the mean ± standard deviation (n = 3). (B) The AF-2 surface of RXR is required for interaction with SHP in vitro. GST alone or a GST-SHP fusion protein were expressed in E. coli, bound to glutathione agarose, and incubated with wild-type RXR or the C-terminal Δ19 deletion mutant, both of which were ³⁵S labeled by in vitro translation. Specifically bound proteins were eluted by standard procedures (2) and are compared to 20% of the total input (bottom gel).

FIG. 6

SHP interacts with the AF-2 surface of RXR. (A) Mammalian two-hybrid assay for interaction. Gal4SHP was transfected into HepG2 cells along with VP16RXR, VP16RXRΔ19C, or VP16 alone. Approximately 20 h after transfection, the cells were treated with 1 μM 9-cis-RA for 30 h. Normalized luciferase expression directed by the Gal4Tkluc reporter is indicated as the mean ± standard deviation (n = 3). (B) The AF-2 surface of RXR is required for interaction with SHP in vitro. GST alone or a GST-SHP fusion protein were expressed in E. coli, bound to glutathione agarose, and incubated with wild-type RXR or the C-terminal Δ19 deletion mutant, both of which were ³⁵S labeled by in vitro translation. Specifically bound proteins were eluted by standard procedures (2) and are compared to 20% of the total input (bottom gel).

FIG. 7

SHP competes specifically with SRC-3 for binding to activated RXR in a mammalian two-hybrid assay. HepG2 cells were cotransfected with 50 ng (each) of Gal4-SRC3, VP16 or a VP16 fusion to the RXR LBD [VP16-RXR(L)], and the indicated amounts of plasmids expressing VP16-SHP and VP16-SHPW160X. 9-cis-RA (1 μM) was added 20 h after transfection, and cells were further incubated for 30 h before harvest. Normalized luciferase expression is plotted as the mean ± standard deviation from three independent experiments. Essentially identical results were obtained in treatments with the specific RXR agonist LG1069. WT, wild type.

FIG. 8

SHP requires the repression domain for full inhibition. (A) A Gal4-RXR(L) vector (50 ng) was cotransfected into HepG2 cells along with an SHP or SHPW160X vector in the indicated ratio (Gal:SHP). At 20 h posttransfection, cells were treated with 1 μM 9-cis-RA or vehicle alone and incubated for 30 h. Normalized luciferase expression is plotted as the mean fold activation by 9-cis-RA ± standard deviation from three independent experiments. (B) Gal4-RXR(L) vector (25 ng) was cotransfected into HepG2 cells with 200 and 800 ng of SHP or SHPW160X expression vector as indicated by concentration ratio (Gal:SHP). Normalized luciferase activities are plotted as percent activation or repression. Percent activation for each combination is relative to the activation observed with Gal4-RXR(L) in the presence of 9-cis-RA and the absence of SHP. Percent repression is relative to basal expression for each combination in the absence of 9-cis-RA. In this experiment, 800 ng of SHP or SHPW160X did not affect luciferase expression in the presence of Gal4 alone. (C) A thymidine kinase luciferase reporter containing the RXR response element from the CRBPII promoter was cotransfected into HepG2 cells with 50 ng of CDMhRXRα and either the wild-type SHP or the SHPW160X expression vector in the indicated ratio (RXR:SHP). Normalized luciferase expression was determined in three independent experiments, and the fold response to 1 μM LG1069 is shown.

FIG. 8

SHP requires the repression domain for full inhibition. (A) A Gal4-RXR(L) vector (50 ng) was cotransfected into HepG2 cells along with an SHP or SHPW160X vector in the indicated ratio (Gal:SHP). At 20 h posttransfection, cells were treated with 1 μM 9-cis-RA or vehicle alone and incubated for 30 h. Normalized luciferase expression is plotted as the mean fold activation by 9-cis-RA ± standard deviation from three independent experiments. (B) Gal4-RXR(L) vector (25 ng) was cotransfected into HepG2 cells with 200 and 800 ng of SHP or SHPW160X expression vector as indicated by concentration ratio (Gal:SHP). Normalized luciferase activities are plotted as percent activation or repression. Percent activation for each combination is relative to the activation observed with Gal4-RXR(L) in the presence of 9-cis-RA and the absence of SHP. Percent repression is relative to basal expression for each combination in the absence of 9-cis-RA. In this experiment, 800 ng of SHP or SHPW160X did not affect luciferase expression in the presence of Gal4 alone. (C) A thymidine kinase luciferase reporter containing the RXR response element from the CRBPII promoter was cotransfected into HepG2 cells with 50 ng of CDMhRXRα and either the wild-type SHP or the SHPW160X expression vector in the indicated ratio (RXR:SHP). Normalized luciferase expression was determined in three independent experiments, and the fold response to 1 μM LG1069 is shown.

FIG. 8

SHP requires the repression domain for full inhibition. (A) A Gal4-RXR(L) vector (50 ng) was cotransfected into HepG2 cells along with an SHP or SHPW160X vector in the indicated ratio (Gal:SHP). At 20 h posttransfection, cells were treated with 1 μM 9-cis-RA or vehicle alone and incubated for 30 h. Normalized luciferase expression is plotted as the mean fold activation by 9-cis-RA ± standard deviation from three independent experiments. (B) Gal4-RXR(L) vector (25 ng) was cotransfected into HepG2 cells with 200 and 800 ng of SHP or SHPW160X expression vector as indicated by concentration ratio (Gal:SHP). Normalized luciferase activities are plotted as percent activation or repression. Percent activation for each combination is relative to the activation observed with Gal4-RXR(L) in the presence of 9-cis-RA and the absence of SHP. Percent repression is relative to basal expression for each combination in the absence of 9-cis-RA. In this experiment, 800 ng of SHP or SHPW160X did not affect luciferase expression in the presence of Gal4 alone. (C) A thymidine kinase luciferase reporter containing the RXR response element from the CRBPII promoter was cotransfected into HepG2 cells with 50 ng of CDMhRXRα and either the wild-type SHP or the SHPW160X expression vector in the indicated ratio (RXR:SHP). Normalized luciferase expression was determined in three independent experiments, and the fold response to 1 μM LG1069 is shown.

FIG. 9

Two-step repression by SHP. In the first step, SHP displaces coactivators by competing for binding to the receptor AF-2 surface. In the second step, the repressor function of the receptor-bound SHP further decreases expression via an unknown mechanism.