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. 2001 Feb 27;98(5):2323-8.
doi: 10.1073/pnas.051619898. Epub 2001 Feb 20.

Fatty acids and hypolipidemic drugs regulate peroxisome proliferator-activated receptors alpha - and gamma-mediated gene expression via liver fatty acid binding protein: a signaling path to the nucleus

Affiliations

Fatty acids and hypolipidemic drugs regulate peroxisome proliferator-activated receptors alpha - and gamma-mediated gene expression via liver fatty acid binding protein: a signaling path to the nucleus

C Wolfrum et al. Proc Natl Acad Sci U S A. .

Abstract

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a key regulator of lipid homeostasis in hepatocytes and target for fatty acids and hypolipidemic drugs. How these signaling molecules reach the nuclear receptor is not known; however, similarities in ligand specificity suggest the liver fatty acid binding protein (L-FABP) as a possible candidate. In localization studies using laser-scanning microscopy, we show that L-FABP and PPARalpha colocalize in the nucleus of mouse primary hepatocytes. Furthermore, we demonstrate by pull-down assay and immunocoprecipitation that L-FABP interacts directly with PPARalpha. In a cell biological approach with the aid of a mammalian two-hybrid system, we provide evidence that L-FABP interacts with PPARalpha and PPARgamma but not with PPARbeta and retinoid X receptor-alpha by protein-protein contacts. In addition, we demonstrate that the observed interaction of both proteins is independent of ligand binding. Final and quantitative proof for L-FABP mediation was obtained in transactivation assays upon incubation of transiently and stably transfected HepG2 cells with saturated, monounsaturated, and polyunsaturated fatty acids as well as with hypolipidemic drugs. With all ligands applied, we observed strict correlation of PPARalpha and PPARgamma transactivation with intracellular concentrations of L-FABP. This correlation constitutes a nucleus-directed signaling by fatty acids and hypolipidemic drugs where L-FABP acts as a cytosolic gateway for these PPARalpha and PPARgamma agonists. Thus, L-FABP and the respective PPARs could serve as targets for nutrients and drugs to affect expression of PPAR-sensitive genes.

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Figures

Figure 1
Figure 1
Transactivation of human PPARα in HepG2 cells. HepG2 cells were transfected with the expression vector for human PPARα, pSV-β-Gal, and the CAT-reporter gene vector under the control of ideal PPRE. (A) Cells were treated for 24 h with 100 μM fatty acid, except 50 μM for arachidonic acid and docosahexaenoic acid. As control, cells were either transfected with pCDNA3 instead of the expression vector for human PPARα (−PPARα) or treated with DMSO alone. (B) Concentration-dependent transactivation by linoleic acid (results are representative for all fatty acids tested). (C) Cells were treated for 24 h with phytanic acid (100 μM), bezafibrate (100 μM), ETYA (50 μM), and Wy14,643 (100 μM). β-Gal and CAT concentrations were determined by ELISAs; DMSO control was set as one. Each value represents the mean of six independent experiments ± SD.
Figure 2
Figure 2
Direct interaction of murine L-FABP and murine PPARα. (A) Pull-down assay. Murine 35S-labeled PPARα (lanes 1 and 3, positive control) was precipitated with murine L-FABP covalently bound to CH-activated Sepharose and centrifuged; no 35S-PPARα was found in the supernatant (lane 5). The wash with PBS is free of 35S-PPARα (lane 6); thereafter, bound 35S-PPARα was eluted with SDS-loading buffer (lane 7). To test specific binding, the precipitate with bound 35S-PPARα was washed with PBS/murine L-FABP solution and eluted as described above (lane 8). To check for unspecific binding, purple acid phosphatase covalently linked to Sepharose (lane 2) or unmodified Sepharose (lane 4) was incubated with 35S-PPARα. Protein fractions obtained were separated by SDS/PAGE (13.5%), and 35S-PPARα was visualized by autoradiography. (B) Immunocoprecipitation (Upper, stained for PPARα; Lower, stained for L-FABP). From nuclear lysates of mouse liver (lane 1, positive control), L-FABP–PPARα complex was precipitated with anti-murine L-FABP antibody immobilized on Sepharose. Neither L-FABP nor PPARα was found in the supernatant (lane 4). After washing the precipitate with PBS, L-FABP and PPARα were eluted with SDS-loading buffer (lane 5). For negative control, unmodified Sepharose was used (lane 2). Protein fractions obtained were separated by SDS/PAGE (13.5%), and bands were visualized after Western blotting and immunodecoration; protein size was determined by molecular mass marker (lane 3).
Figure 3
Figure 3
Two-hybrid interaction of L-FABP with nuclear receptors. Interaction was measured by using the mammalian two-hybrid assay in COS7 cells, using a fusion protein of the GAL4-DNA binding domain with PPARα, PPARβ, PPARγ, or RXRα and a fusion protein of the VP16 activation domain with L-FABP. Unspecific interaction was quantified by using a fusion protein of the GAL4-DNA binding domain with p53 or a fusion protein of the VP16 activation domain with the simian virus 40-T antigen. Positive and negative controls were used according to the supplier's manual. CAT and β-Gal expression was measured by ELISAs. Each column represents the mean of 5–8 independent experiments ± SD.
Figure 4
Figure 4
Transactivation of human PPARα depends on L-FABP concentration. A series of eight HepG2 cell clones, with different L-FABP contents after stable transfection with antisense L-FABP mRNA (17), were transfected with the expression vector for human PPARα, pSV-β-Gal, and the CAT-reporter gene vector under the control of ideal PPRE. Each data point represents the analysis of a single clone with ELISAs for the determination of β-Gal, CAT, and L-FABP concentrations. (A) Cells treated for 24 h with 200 μM stearic acid (●), 200 μM linoleic acid (▴), and 100 μM phytanic acid (■). (B) Cells treated for 24 h with 200 μM bezafibrate (●), 50 μM ETYA (▴), and 200 μM Wy14,643 (■). (C) Cells treated for 24 h with 100 μM linoleic acid (●), 100 μM ciglitazon (▴), and 100 μM Wy14,643 (■). DMSO control was set as one. Note the different scale of ordinates. Each data point represents the mean of six independent experiments ± SD. P < 0.001 for all graphs in A and B; P < 0.004 for all graphs in C.
Figure 5
Figure 5
Scheme for L-FABP action in PPAR-mediated gene regulation.

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