A functional peroxisome proliferator-activated receptor-gamma ligand-binding domain is not required for adipogenesis

Abstract

The nuclear hormone receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) is the central regulator of adipogenesis. Although it is the target for several drugs that function as agonist activators, a high affinity endogenous ligand for this receptor that is involved in regulating adipogenesis has yet to be identified. Here, we investigated the requirement for ligand activation of PPARgamma in fat cell differentiation, taking advantage of a natural mutant of this receptor that does not bind or become activated by any known natural or synthetic ligand. When ectopically expressed in PPARgamma-null fibroblasts, this Q286P allele was able to strongly promote morphological adipogenesis, without any significant difference compared with wild-type PPARgamma. In addition, no significant differences were found in the expression of several adipogenic genes between the wild-type and Q286P mutant alleles. To extend our studies to an in vivo setting, we performed subcutaneous injections of PPARgamma-expressing fibroblasts into nude mice. We found that both wild-type and Q286P mutant-expressing fibroblasts were able to generate fat pads in the mice. These results suggest that the binding and activation of PPARgamma by agonist ligands may not be required for adipogenesis under physiological conditions.

FIGURE 1.

The PPARγ Q286P mutant is resistant to ligand activation. A, shown is the crystal structure of the apo-PPARγ ligand-binding domain, indicating the locations of relevant mutations. Q286P is shown in yellow, and E499Q is shown in blue. Helix 12, containing the AF-2 domain, is shown in red. The figure was generated by the PyMOL program (DeLano Scientific) using data from Ref. . B, 293 cells were transfected with PPARγ mutant expression plasmids along with a DR-1-luciferase reporter plasmid. The transfected cells were treated for 24 h with the indicated concentrations of the different known PPARγ-activating ligands and then harvested and assayed for luciferase activity. Each bar represents the mean ± S.E. of three separate cell preparations. Data are expressed as relative activity based on non-PPARγ-expressing and non-agonist-treated cells and are normalized to cotransfected renillin expression. 15-deoxy-PGJ₂, 15-deoxy-Δ^12,14-prostaglandin J₂; 15-S-HETE, 15-S-hydroxyeicosatetraenoic acid; azPAF, 1-O-hexadecyl-2-azelaoylphosphatidylcholine.

FIGURE 2.

The PPARγ Q286P mutant is fully adipogenic. PPARγ-null fibroblasts were infected with retrovirus expressing 1) no ectopic protein, 2) wild-type PPARγ, 3) PPARγ Q286P, or 4) PPARγ E499Q. All ectopic PPARγ constructs were dual-tagged at the N terminus with FLAG and HA epitopes. The cells were induced to undergo adipogenic differentiation with the dexamethasone/isobutylmethylxanthine/insulin hormone mixture (described under “Experimental Procedures”). A, shown is an immunoblot for PPARγ. Whole cell extracts were prepared from retrovirally infected cells as well as from fully differentiated adipocytes. Following SDS-PAGE, the extracts were immunoblotted for PPARγ. B, following 6 days of differentiation, the cells were stained with Oil Red O to detect lipid accumulation. C, at consecutive days of differentiation, cells were harvested, and total RNA was extracted. The expression levels of the adipogenic markers indicated in the figure were measured by real-time PCR. Each point is the mean ± S.E. of three separate cell preparations. FAS, fatty acid synthase; LPL, lipoprotein lipase; SCD, stearoyl-CoA desaturase. D, PPARγ-null fibroblasts expressing ectopic PPARγ mutants were injected subcutaneously into the sternal regions of athymic nude mice. After 6 weeks, the skin covering the sternum was removed and examined for the presence of a subcutaneous fat pad. Skin cross-sections were stained with hematoxylin and eosin and examined for the presence of fat cells (indicated by a red bar).