Transactivation by retinoid X receptor-peroxisome proliferator-activated receptor gamma (PPARgamma) heterodimers: intermolecular synergy requires only the PPARgamma hormone-dependent activation function - PubMed (original) (raw)
Transactivation by retinoid X receptor-peroxisome proliferator-activated receptor gamma (PPARgamma) heterodimers: intermolecular synergy requires only the PPARgamma hormone-dependent activation function
I G Schulman et al. Mol Cell Biol. 1998 Jun.
Abstract
The ability of DNA sequence-specific transcription factors to synergistically activate transcription is a common property of genes transcribed by RNA polymerase II. The present work characterizes a unique form of intermolecular transcriptional synergy between two members of the nuclear hormone receptor superfamily. Heterodimers formed between peroxisome proliferator-activated receptor gamma (PPARgamma), an adipocyte-enriched member of the superfamily required for adipogenesis, and retinoid X receptors (RXRs) can activate transcription in response to ligands specific for either subunit of the dimer. Simultaneous treatment with ligands specific for both PPARgamma and RXR has a synergistic effect on the transactivation of reporter genes and on adipocyte differentiation in cultured cells. Mutation of the PPARgamma hormone-dependent activation domain (named tauc or AF-2) inhibits the ability of RXR-PPARgamma heterodimers to respond to ligands specific for either subunit. In contrast, the ability of RXR- and PPARgamma-specific ligands to synergize does not require the hormone-dependent activation domain of RXR. The results of in vitro and in vivo experiments indicate that binding of ligands to RXR alters the conformation of the dimerization partner, PPARgamma, and modulates the activity of the heterodimer in a manner independent of the RXR hormone-dependent activation domain.
Figures
FIG. 1
Synergistic activation by RXR- and PPARγ-specific ligands. (A) NIH 3T3 cells were transfected with PPREx3-TK-LUC and expression constructs for mouse PPARγ and human RXRα. After transfection, cells were cultured in the absence (None) or presence of the RXR-specific ligand LG100268 (1.0 μM), the PPARγ-specific ligand BRL49653 (5 μM), or 1.0 μM LG100268 plus 5.0 μM BRL49653 for 36 h. Activity relative to that of the PPREx3-TK-LUC reporter alone is reported. Each number above a bar indicates the fold induction relative to the activity in the absence of ligand. Note the break in the y axis. Transfections were normalized by cotransfection with a β-galactosidase expression plasmid (see Materials and Methods). (B to E). Confluent 3T3 L1 preadipocytes were cultured for 7 days in the absence (B) or presence of 100 nM BRL49653 (C), 100 nM LG100268 (D), or 100 nM BRL49653 plus 100 nM LG100268 (E). After treatment, cells were stained with oil red O to visualize lipids.
FIG. 1
Synergistic activation by RXR- and PPARγ-specific ligands. (A) NIH 3T3 cells were transfected with PPREx3-TK-LUC and expression constructs for mouse PPARγ and human RXRα. After transfection, cells were cultured in the absence (None) or presence of the RXR-specific ligand LG100268 (1.0 μM), the PPARγ-specific ligand BRL49653 (5 μM), or 1.0 μM LG100268 plus 5.0 μM BRL49653 for 36 h. Activity relative to that of the PPREx3-TK-LUC reporter alone is reported. Each number above a bar indicates the fold induction relative to the activity in the absence of ligand. Note the break in the y axis. Transfections were normalized by cotransfection with a β-galactosidase expression plasmid (see Materials and Methods). (B to E). Confluent 3T3 L1 preadipocytes were cultured for 7 days in the absence (B) or presence of 100 nM BRL49653 (C), 100 nM LG100268 (D), or 100 nM BRL49653 plus 100 nM LG100268 (E). After treatment, cells were stained with oil red O to visualize lipids.
FIG. 2
RXR- and PPARγ-specific ligands synergistically promote interaction with CBP. (A) A fusion between the DNA binding domain (DBD) of GAL4 and the receptor-interacting domain of CBP (amino acids 1 to 171) was cotransfected into NIH 3T3 cells along with a construct expressing a VP16 activation domain-human RXRα ligand binding domain fusion protein (VP16-RXRLBD) and a construct expressing the LBD of mouse PPARγ (PPARγLBD). A luciferase reporter with four GAL4 binding sites (UASGx4-LUC) was also included. After transfection, cells were cultured in the absence (None) or presence of the RXR-specific ligand LG100268 (1.0 μM), the PPARγ-specific ligand BRL49653 (5.0 μM), or 1.0 μM LG100268 plus 5.0 μM BRL49653 for 36 h. Activity relative to GAL4-CBP (amino acids 1 to 171) alone is reported. Panels B and C are identical to panel A except that VP16-RXRLBD (B) or PPARγLBD (C) was omitted. Note the y axis in panel A differs from that in panels B and C. Panels D to F are identical to panels A to C except that GAL4-CBP (amino acids 1 to 171) was replaced with a fusion between the DNA binding domain of GAL4 and the central receptor-interacting domain of human SRC-1 (amino acids 381 to 891). Each number above a bar indicates the fold induction relative to the activity in the absence of ligand. Transfections were normalized by cotransfection with a β-galactosidase expression plasmid (see Materials and Methods).
FIG. 3
RXR and PPARγ prefer different coactivators. (A) In vitro-translated receptors were incubated with a 32P-labeled PPRE oligonucleotide and 10 μg of GST (lanes 2 to 5) or GST-CBP (amino acids 1 to 352) (lanes 6 to 17). DNA-protein complexes were resolved as described in Materials and Methods. As noted above the gel, 1.0 μM BRL49653 and/or LG100268 were included (+) or not included (−). Heterodimers formed with wild-type RXR and wild-type PPARγ (lanes 1 to 9), with wild-type RXR and mutant PPARγ L466A/L467A (lanes 10 to 13), and with mutant RXR M454A/L455A and wild-type PPARγ (lanes 14 to 17) are shown. A nonspecific band derived from the reticulocyte lysate is indicated by the asterisk. (B) Interaction with SRC-1 was carried as described in panel A using wild-type receptors. GST–SRC-1 (amino acids 381 to 891) (10 μg) was included in lanes 2 to 5. (C) The interaction of PPARγ and RXRα with CBP and SRC-1 was examined by far-Western blotting. A Coomassie blue-stained gel (Stain Gel) of immobilized GST fusion proteins and far-Western blots probed with 35S-labeled PPARγ or 35S-labeled RXR are shown. Molecular mass markers (lane 1), GST (lanes 2, 6, 10, 14, and 21), GST–SRC-1 (amino acids 381 to 891) (lanes 3, 7, 11, 15, and 19), GST-CBP (amino acids 1 to 352) (lanes 4, 8, 12, 16, and 20), and GST-RXR (lanes 5, 9, 13, 17, and 21) were used. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes, renatured, and incubated with 35S-labeled mPPARγ in the absence (−Ligand) or presence of 5 μM BRL49653 or with 35S-labeled RXR in the absence (−Ligand) or presence of 5 μM LG100268.
FIG. 3
RXR and PPARγ prefer different coactivators. (A) In vitro-translated receptors were incubated with a 32P-labeled PPRE oligonucleotide and 10 μg of GST (lanes 2 to 5) or GST-CBP (amino acids 1 to 352) (lanes 6 to 17). DNA-protein complexes were resolved as described in Materials and Methods. As noted above the gel, 1.0 μM BRL49653 and/or LG100268 were included (+) or not included (−). Heterodimers formed with wild-type RXR and wild-type PPARγ (lanes 1 to 9), with wild-type RXR and mutant PPARγ L466A/L467A (lanes 10 to 13), and with mutant RXR M454A/L455A and wild-type PPARγ (lanes 14 to 17) are shown. A nonspecific band derived from the reticulocyte lysate is indicated by the asterisk. (B) Interaction with SRC-1 was carried as described in panel A using wild-type receptors. GST–SRC-1 (amino acids 381 to 891) (10 μg) was included in lanes 2 to 5. (C) The interaction of PPARγ and RXRα with CBP and SRC-1 was examined by far-Western blotting. A Coomassie blue-stained gel (Stain Gel) of immobilized GST fusion proteins and far-Western blots probed with 35S-labeled PPARγ or 35S-labeled RXR are shown. Molecular mass markers (lane 1), GST (lanes 2, 6, 10, 14, and 21), GST–SRC-1 (amino acids 381 to 891) (lanes 3, 7, 11, 15, and 19), GST-CBP (amino acids 1 to 352) (lanes 4, 8, 12, 16, and 20), and GST-RXR (lanes 5, 9, 13, 17, and 21) were used. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes, renatured, and incubated with 35S-labeled mPPARγ in the absence (−Ligand) or presence of 5 μM BRL49653 or with 35S-labeled RXR in the absence (−Ligand) or presence of 5 μM LG100268.
FIG. 3
RXR and PPARγ prefer different coactivators. (A) In vitro-translated receptors were incubated with a 32P-labeled PPRE oligonucleotide and 10 μg of GST (lanes 2 to 5) or GST-CBP (amino acids 1 to 352) (lanes 6 to 17). DNA-protein complexes were resolved as described in Materials and Methods. As noted above the gel, 1.0 μM BRL49653 and/or LG100268 were included (+) or not included (−). Heterodimers formed with wild-type RXR and wild-type PPARγ (lanes 1 to 9), with wild-type RXR and mutant PPARγ L466A/L467A (lanes 10 to 13), and with mutant RXR M454A/L455A and wild-type PPARγ (lanes 14 to 17) are shown. A nonspecific band derived from the reticulocyte lysate is indicated by the asterisk. (B) Interaction with SRC-1 was carried as described in panel A using wild-type receptors. GST–SRC-1 (amino acids 381 to 891) (10 μg) was included in lanes 2 to 5. (C) The interaction of PPARγ and RXRα with CBP and SRC-1 was examined by far-Western blotting. A Coomassie blue-stained gel (Stain Gel) of immobilized GST fusion proteins and far-Western blots probed with 35S-labeled PPARγ or 35S-labeled RXR are shown. Molecular mass markers (lane 1), GST (lanes 2, 6, 10, 14, and 21), GST–SRC-1 (amino acids 381 to 891) (lanes 3, 7, 11, 15, and 19), GST-CBP (amino acids 1 to 352) (lanes 4, 8, 12, 16, and 20), and GST-RXR (lanes 5, 9, 13, 17, and 21) were used. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes, renatured, and incubated with 35S-labeled mPPARγ in the absence (−Ligand) or presence of 5 μM BRL49653 or with 35S-labeled RXR in the absence (−Ligand) or presence of 5 μM LG100268.
FIG. 4
Inactivation of the PPARγ τc/AF-2 domain inhibits the response to RXR- and PPARγ-specific ligands. (A) NIH 3T3 cells were transfected with a reporter containing three copies of the acyl-CoA oxidase PPRE cloned upstream of the thymidine kinase-luciferase (TK-LUC) reporter (PPREx3-TK-LUC) and expression constructs for mouse PPARγ and human RXRα. Panel B is identical to panel A except that a PPARγ τc/AF-2 domain double point mutant (L466A/L467A) was used in place of the wild-type PPARγ. After transfection, cells were cultured in the absence (None) or presence of the RXR-specific ligand LG100268 (1.0 μM), the PPARγ-specific ligand BRL49653 (5.0 μM), or 1.0 μM LG100268 plus 5.0 μM BRL49653 for 36 h. Activity relative to that of the PPREx3-TK-LUC reporter alone is reported. Note the break in the y axis. (C) A fusion between the DNA binding domain of GAL4 and the receptor-interacting domain of CBP (amino acids 1 to 171) was cotransfected into NIH 3T3 cells along with constructs expressing a VP16 activation domain-human RXRα ligand binding domain (VP16-RXRLBD) fusion protein and the LBD of mouse PPARγ (PPARγLBD). Panel D is identical to panel C except that a PPARγ τc/AF-2 domain double point mutant (L466A/L467A) was used in place of the wild-type PPARγ. After transfection, cells were cultured in the absence (None) or presence of 1.0 μM LG100268, 5.0 μM BRL49653, or 1.0 μM LG100268 plus 5.0 μM BRL49653 for 36 h. Activity relative to GAL4-CBP (amino acids 1 to 171) alone is reported. Note the break in the y axis. Each number above a bar indicates the fold induction relative to the activity in the absence of ligand. Transfections were normalized by cotransfection with a β-galactosidase expression plasmid (see Materials and Methods). Western blot experiments indicate that the PPARγ mutant is expressed at a level similar to the wild-type level (data not shown).
FIG. 5
Inactivation of the RXR τc/AF-2 domain still allows synergy with PPARγ-specific ligands. (A and B) NIH 3T3 cells were transfected with a reporter containing three copies of the acyl-CoA oxidase PPRE cloned upstream of the TK-LUC reporter (PPREx3-TK-LUC) and expression constructs for human RXRα (wild type) or the human RXRα τc/AF-2 domain mutant M454A/L455A. After transfection, cells were cultured in the absence (None) or presence of the RXR-specific ligand LG100268 (1.0 μM) for 36 h. Activity relative to that of the reporter alone is reported. Western blot experiments indicate that the RXR mutant is expressed at levels similar to the wild-type level (data not shown). C and D) NIH 3T3 cells were transfected with a reporter containing three copies of the acyl-CoA oxidase PPRE cloned upstream of the TK-LUC reporter (PPREx3-TK-LUC), an expression construct for mouse PPARγ, and expression constructs for human RXRα (wild type) (C) or the RXRα τc/AF-2 domain mutant M454A/L455A (D). After transfection, cells were cultured in the absence (None) or presence of 1.0 μM LG100268, 5.0 μM BRL49653, or 1.0 μM LG100268 plus 5.0 μM BRL49653 for 36 h. Activity relative to that of the PPREx3-luciferase reporter alone is reported. Note the break in the y axis. (E and F) A fusion between the DNA binding domain of GAL4 and the receptor-interacting domain of CBP (amino acids 1 to 171) was cotransfected into NIH 3T3 cells along with a construct expressing the LBD of mouse PPARγ (PPARγLBD) and constructs expressing VP16-RXRLBD (wild type) (E) or VP16-RXRLBD fusions with the M454A/L455A mutation (F). After transfection, cells were cultured in the absence (None) or presence of 1.0 μM LG100268, 5.0 μM BRL49653, or 1.0 μM LG100268 plus 5.0 μM BRL49653 for 36 h. Activity relative to that of GAL4-CBP (amino acids 1 to 171) alone is reported. Western blot experiments indicate that the RXR mutants are expressed at levels similar to the wild-type level (data not shown). Each numbers above a bar indicates the fold induction relative to the activity in the absence of ligand. Transfections were normalized by cotransfection with a β-galactosidase expression plasmid (see Materials and Methods).
FIG. 6
Ligand binding to RXR alters the protease sensitivity of PPARγ. In vitro-translated receptors were incubated with an oligonucleotide containing a single PPRE and receptor-specific ligands. After this initial incubation, trypsin was added for 20 min (lanes 3 to 6), the reaction was stopped, and the 35S-labeled peptides were visualized by autoradiography (see Materials and Methods). 35S-labeled PPARγ plus unlabeled RXRα (wild type) (A) 35S-labeled PPARγ plus unlabeled RXR M454A/L455A (τc/AF-2 domain mutant) (B), 35S-labeled PPARγ plus unlabeled RXR L436S (ligand binding mutant) (C), 35S-labeled PPARγ alone (D), and unlabeled PPARγ plus 35S-labeled RXRα (wild type) (E) are shown. All autoradiographs were exposed for 15 h. In each panel, 14C-labeled molecular mass markers (lane 1), undigested controls (lane 2), no ligand (lane 3), 1.0 μM LG100268 (RXR specific) (lane 4), 5.0 μM BRL49653 (PPARγ specific) (lane 5), and 1.0 μM LG100268 plus 5.0 μM BRL49653 (lane 6) were run.
FIG. 6
Ligand binding to RXR alters the protease sensitivity of PPARγ. In vitro-translated receptors were incubated with an oligonucleotide containing a single PPRE and receptor-specific ligands. After this initial incubation, trypsin was added for 20 min (lanes 3 to 6), the reaction was stopped, and the 35S-labeled peptides were visualized by autoradiography (see Materials and Methods). 35S-labeled PPARγ plus unlabeled RXRα (wild type) (A) 35S-labeled PPARγ plus unlabeled RXR M454A/L455A (τc/AF-2 domain mutant) (B), 35S-labeled PPARγ plus unlabeled RXR L436S (ligand binding mutant) (C), 35S-labeled PPARγ alone (D), and unlabeled PPARγ plus 35S-labeled RXRα (wild type) (E) are shown. All autoradiographs were exposed for 15 h. In each panel, 14C-labeled molecular mass markers (lane 1), undigested controls (lane 2), no ligand (lane 3), 1.0 μM LG100268 (RXR specific) (lane 4), 5.0 μM BRL49653 (PPARγ specific) (lane 5), and 1.0 μM LG100268 plus 5.0 μM BRL49653 (lane 6) were run.
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