ADD1/SREBP1 activates PPARgamma through the production of endogenous ligand

Abstract

Adipose differentiation is an important part of the energy homeostasis system of higher organisms. Recent data have suggested that this process is controlled by an interplay of transcription factors including PPARgamma, the C/EBPs, and ADD1/SREBP1. Although these factors interact functionally to initiate the program of differentiation, there are no data concerning specific mechanisms of interaction. We show here that the expression of ADD1/SREBP1 specifically increases the activity of PPARgamma but not other isoforms, PPARalpha, or PPARdelta. This activation occurs through the ligand-binding domain of PPARgamma when it is fused to the DNA-binding domain of Gal4. The stimulation of PPARgamma by ADD1/SREBP1 does not require coexpression in the same cells; supernatants from cultures that express ADD1/SREBP1 augment the transcriptional activity of PPARgamma. Finally, we demonstrate directly that cells expressing ADD1/SREBP1 produce and secrete lipid molecule(s) that bind directly to PPARgamma, displacing the binding of radioactive thiazolidinedione ligands. These data establish that ADD1/SREBP1 can control the production of endogenous ligand(s) for PPARgamma and suggest a mechanism for coordinating the actions of these adipogenic factors.

Figure 1

Expression of ADD1/SREBP1 selectively enhances the transcriptional activity of PPARγ. (A) NIH 3T3 cells were cotransfected with the 520-bp enhancer for aP2 gene (520-bp aP2 CAT) and indicated combinations of ADD1 with either PPARα/RXRα, PPARγ/RXRα, or PPARδ/RXRα expression vectors. Transfections were performed in duplicate and repeated at least three times. A representative experiment is shown above. All CAT enzyme activities were normalized to β-gal activity. (B) Quantitation of CAT enzyme activity of A was performed by PhosphorImager analysis (Molecular Dynamics).

Figure 2

A PPARγ ligand rescues the inhibition of adipogenesis by dominant-negative ADD1/SREBP1. 3T3-L1 preadipocytes were infected with retroviruses containing either empty vector, ADD1, or dominant-negative ADD1 (ADD1-DN) as previously described (23). 3T3-L1-vector, -ADD1, and -ADD1-DN cell lines were treated with differentiation inducers in the absence (DMI/-TZD) or presence (DMI/+TZD) of the TZD ligand pioglitazone (5 μM) for 48 hr at confluence and subsequently cultured for 5 days. Cells were fixed and stained with Oil Red O. (A) Macroscopic view of dishes. (B) Microscopic views of dishes shown in A at ×25 original magnification. (C) Total RNA (10 μg per lane) was isolated from each cell lines at confluence (−) and after 5 days of postconfluent culture with treatment of either DMI/-TZD or DMI/+TZD. RNA was separated by electrophoresis, blotted to nylon membrane, and hybridized with the indicated ³²P-labeled cDNAs.

Figure 3

Conditioned medium from cells transfected with ADD1/SREBP1 increases the transcriptional activity of PPARγ/RXRα through its ligand-binding domain (LBD). (A) Scheme of supernatant transfer assays. Section 1 shows a cotransfection analysis; section 2 illustrates transient transfection assays with conditioned medium. To collect conditioned medium, transfected cells were incubated with 0.5% BSA in DMEM, and supernatants were isolated after 24 hr. These supernatants were used for the sequential transfection experiments with a 520-bp aP2 CAT reporter in the absence or presence of PPARγ/RXRα. (B) As described above, conditioned medium was prepared by transient transfection of either empty expression vector or vectors expressing ADD1 or C/EBPα. Supernatants collected from different cells were incubated for 24 hr with cells transfected with the 520-bp aP2 CAT reporter and PPARγ/RXRα expression vectors. These transfections were performed in duplicate, and the above is a representative experiment. (C) Rat1-IR cells were transfected with luciferase reporter containing four copies of the UAS_G and either Gal4 DNA-binding domain (DBD), Gal4-PPARα LBD, or Gal4-PPARγ LBD expression vector. After transfection, cells were incubated with conditioned medium from cells transfected with either vector or ADD1. Normalized luciferase activity was determined and plotted as luciferase activity relative to Gal4 DBD expression vector.

Figure 4

ADD1/SREBP1 induces ligand activity for PPARγ. (A) Conditioned medium from cells transfected with either empty vector, ADD1, or C/EBPα was organically extracted as described in Materials and Methods. Fractions of each extract were diluted in ligand-binding buffer (100% denotes 5 μl of extract in methanol, 50% denotes 2.5 μl, etc.) and used in competition with [³H]BRL 49653 for binding to bacterial-produced PPARγ (see details in Materials and Methods). (B) Cotransfection of several alleles of ADD1 including ADD1, ADD1-R, and ADD1-DN with PPARγ/RXRα to determine transcriptional activity through PPREs with luciferase reporter (PPRE X3-TK-Luc). Luciferase activity was normalized with β-gal activity. (C) Ligand activity from cells transfected with either empty vector, ADD1, ADD1-R, or ADD1-DN. Conditions were the same as in A.