Non-DNA binding, dominant-negative, human PPARgamma mutations cause lipodystrophic insulin resistance

Abstract

PPARgamma is essential for adipogenesis and metabolic homeostasis. We describe mutations in the DNA and ligand binding domains of human PPARgamma in lipodystrophic, severe insulin resistance. These receptor mutants lack DNA binding and transcriptional activity but can translocate to the nucleus, interact with PPARgamma coactivators and inhibit coexpressed wild-type receptor. Expression of PPARgamma target genes is markedly attenuated in mutation-containing versus receptor haploinsufficent primary cells, indicating that such dominant-negative inhibition operates in vivo. Our observations suggest that these mutants restrict wild-type PPARgamma action via a non-DNA binding, transcriptional interference mechanism, which may involve sequestration of functionally limiting coactivators.

Figure 1

Identification and characterization of loss-of-function mutations in human PPARγ A) Schematic representation of the three major domains of PPARγ, showing the locations of the five mutations (C114R, C131Y, C162W, FS315X, and R357X – PPARγ1 nomenclature) and the previously reported FSX mutation. NLS, nuclear localisation signal; RXR ID, retinoid X receptor interaction domain; AF2, activation function 2 domain. B) Family pedigrees showing genotypes (N, wild-type allele; M, mutant allele; NA, not available) and phenotypes (colored segments denote the presence of specific traits: green, type 2 diabetes mellitus/impaired glucose tolerance/hyperinsulinaemia; yellow, hypertriglyceridaemia; blue, hypertension; red, ischemic heart disease). Squares and circles represent male and female family members; slashed symbols denote deceased family members and arrows denote probands. C) PPARγ mutants are unable to mediate ligand-dependent transactivation. 293EBNA cells were transfected with 100 ng of wild-type (WT), mutant, or empty (pcDNA3) expression vectors, together with 500 ng of (PPARE)₃TKLUC reporter construct and 100 ng of Bos-β-gal internal control plasmid, and increasing concentrations of rosiglitazone. Results are expressed as a percentage of the maximum activation with WT PPARγ1 and represent the mean ± SEM of at least three independent experiments in triplicate. D) PPARγ mutants are unable to bind to DNA. EMSA with in vitro translated wild-type (WT) or mutant PPARγ1 (C114R, C131Y, C162W, FS315X, R357X, or FSX) and RXR proteins coincubated with oligonucleotide duplexes corresponding to various natural PPAREs. aP2, adipocyte protein 2; ACoABP, acyl coenzyme A binding protein; mCPT1, muscle carnitine palmitoyl transferase 1; LXRα, liver X receptor α; CAP, cbl-associated protein; LPL, lipoprotein lipase, ACoAOx, acyl coenzyme A oxidase; h, human; m, mouse; r, rat; RL, reticulocyte lysate. E) The C114R, C131Y, C162W, FS315X, and R357X mutants translocate to the nucleus whereas the FSX mutant remains cytoplasmic. 293EBNA cells were transfected as described. Top panels show DAPI-staining (blue) of nuclei, middle panels the cellular localisation of GFP-tagged receptors, and lower panels merged images. F) The DBD PPARγ mutants recruit SRC1 and TRAP220 coactivators, whereas the FS315X, R357X, and FSX truncation mutants do not interact. GST alone or WT and mutant GST-PPARγ fusion proteins were tested with ³⁵S-labeled in vitro translated SRC1 (upper panel) or TRAP220 (lower panel) in the absence or presence of rosiglitazone. Coomassie-stained gels confirmed comparable protein loading (data not shown). G) The LBD truncation mutants (FS315X, R357X) recruit PGC1α and PDIP1α coactivators, whereas the FSX mutant fails to interact. GST alone or WT and mutant GST-PPARγ fusion proteins were tested with ³⁵S-labeled in vitro translated human PGC1α and human PDIP1α in the absence of ligand. Coomassie-stained gels confirmed comparable protein loading (data not shown).

Figure 2

PPARγ mutants exhibit dominant-negative activity A) The C114R, C131Y, C162W, FS315X, and R357X PPARγ mutants inhibit transactivation by wild-type (WT) receptor, comparably to AF2, an artificial PPARγ mutant described previously, whereas the FSX mutant lacks dominant-negative activity (upper panel). 3T3-L1 cells were cotransfected with 33 ng of WT receptor plus an equal amount of either empty (pcDNA3) or WT or mutant expression vector, together with 265 ng of human aP2LUC reporter plasmid and 65 ng of the internal control plasmid Bos-β-gal. The dotted and dashed lines denote transcriptional activity of WT receptor in the absence and presence of ligand respectively. Results are expessed as fold induction relative to empty vector (pcDNA3 + pcDNA3) and represent the mean ± SEM of at least three independent experiments in triplicate. Expression of wild-type and mutant receptor proteins was confirmed by Western blotting (lower panel) and the positions of WT, C114R, C131Y, C162W, and AF2 PPARγ (open arrow) and FSX, FS315X and R357X truncation mutants (solid arrows) are indicated. B and C) Ligand-dependent regulation of PPARγ target genes in IDCs from subjects with PPARγ mutations. (B) Induction of the aP2 gene by rosiglitazone, measured by qPCR, is markedly impaired in IDCs derived from subjects with the C114R, C131Y, FS315X, and R357X mutations, compared to responses in cells from normal (WT), severely insulin resistant (IR) subjects without mutations in PPARγ and cells with the FSX, haploinsufficient, mutation. Results represent the mean ± SEM of more than three independent experiments in triplicate, except for cells with the FS315X mutation where a single representative experiment is shown. (C) Relative expression of several PPARγ target genes (5 downregulated and 5 upregulated) in WT and mutation-containing (FSX, C114R, R357X) IDCs, measured by qPCR using TLDA. Red indicates higher, and blue lower, levels of gene expression relative to rosiglitazone-treated (1000 nM) WT cells, whose responses are uniformly designated yellow. Fold changes in expression of each gene in rosiglitazone (RSG) versus vehicle (DMSO) treated WT cells are also listed. D and E) The R357X PPARγ mutant is expressed in IDCs. (D) PPARγ cDNA flanking the R357 codon was amplified by RT-PCR in IDCs from patient S5 and a control subject. Cac81 enzyme digestion of PCR products derived from the WT allele yields two fragments (161 and 74 bp), whereas abolition of this restriction site in the R357X mutant allele yields a larger 235 bp product. (E) Whole-cell lysates of WT and R357X mutant IDCs and 293EBNA cells transfected with R357X mutant were immunoprecipitated and Western blotted. The positions of WT PPARγ (open arrow), R357X (solid arrow), and nonspecific bands (solid arrowheads) are indicated.

Figure 3

Adenoviral-mediated expression of the C114R PPARγ mutant inhibits human preadipocyte differentiation Chub-S7 human preadipocyte cells were infected with comparable efficiency using recombinant adenoviruses expressing GFP, GFP-WT, or GFP-C114R mutant PPARγ and differentiated in the presence of rosiglitazone (100nM). A) Fully differentiated Chub-S7 cells fixed and stained with oil red O. B) aP2 expression quantitated by real-time qPCR at days 0, 3, 5, and 7 postdifferentiation with results representing the mean ± SEM of at least three independent experiments in triplicate. C) Western blotting of Chub-S7 cells at day 4 posttransduction with recombinant adenoviruses confirming comparable levels of total receptor expression. Nondifferentiated, (day 0) nontransduced, cell extracts (ND) are shown for comparison. The positions of PPARγ (open arrow) and nonspecific band (solid arrowhead) are indicated.