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. 2002 Jul;22(14):5114-27.
doi: 10.1128/MCB.22.14.5114-5127.2002.

Selective cooperation between fatty acid binding proteins and peroxisome proliferator-activated receptors in regulating transcription

Nguan-Soon Tan  1 , Natacha S Shaw, Nicolas Vinckenbosch, Peng Liu, Rubina Yasmin, Béatrice Desvergne, Walter Wahli, Noa Noy
Affiliations

Selective cooperation between fatty acid binding proteins and peroxisome proliferator-activated receptors in regulating transcription

Nguan-Soon Tan et al. Mol Cell Biol. 2002 Jul.

Erratum in

  • Mol Cell Biol 2002 Sep;22(17):6318

Abstract

Lipophilic compounds such as retinoic acid and long-chain fatty acids regulate gene transcription by activating nuclear receptors such as retinoic acid receptors (RARs) and peroxisome proliferator-activated receptors (PPARs). These compounds also bind in cells to members of the family of intracellular lipid binding proteins, which includes cellular retinoic acid-binding proteins (CRABPs) and fatty acid binding proteins (FABPs). We previously reported that CRABP-II enhances the transcriptional activity of RAR by directly targeting retinoic acid to the receptor. Here, potential functional cooperation between FABPs and PPARs in regulating the transcriptional activities of their common ligands was investigated. We show that adipocyte FABP and keratinocyte FABP (A-FABP and K-FABP, respectively) selectively enhance the activities of PPARgamma and PPARbeta, respectively, and that these FABPs massively relocate to the nucleus in response to selective ligands for the PPAR isotype which they activate. We show further that A-FABP and K-FABP interact directly with PPARgamma and PPARbeta and that they do so in a receptor- and ligand-selective manner. Finally, the data demonstrate that the presence of high levels of K-FABP in keratinocytes is essential for PPARbeta-mediated induction of differentiation of these cells. Taken together, the data establish that A-FABP and K-FABP govern the transcriptional activities of their ligands by targeting them to cognate PPARs in the nucleus, thereby enabling PPARs to exert their biological functions.

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Figures

FIG. 1.
FIG. 1.
H-FABP and A-FABP enhance the transcriptional activities of PPARα and PPARγ, respectively. Transactivation assays were carried out by using COS-7 cells as described in Materials and Methods. (A) PPARα was cotransfected into cells with either an empty vector or an expression vector for H-FABP. The ability of the receptor to activate expression of the luciferase reporter was examined in the absence of ligand or in the presence of either the naturally occurring pan-PPAR ligand linoleic acid (18:2) or the PPARα-selective ligand Wy-14643 (WY). (B) PPARγ was cotransfected into cells with either an empty vector or an expression vector for A-FABP. The transcriptional activity of the receptor was examined in the absence of ligand or in the presence of either linoleic acid (18:2) or the naturally occurring PPARγ ligand PGJ2. Luciferase activity was normalized to the activity of β-galactosidase.
FIG. 2.
FIG. 2.
A-FABP and K-FABP selectively enhance the transcriptional activities of PPARγ and PPARβ. Transactivation assays were carried out by using COS-7 cells as described in Materials and Methods. (A) Cells were cotransfected with PPARγ and either an empty vector (vec) or an expression vector for either K-FABP (K) or A-FABP (A). The transcriptional activity of PPARγ was examined in the absence or presence of the PPARγ-selective ligand troglitazone (TZD). (B) Cells were cotransfected with PPARβ and either an empty vector or an expression vector for either K-FABP or A-FABP. The transcriptional activity of PPARβ was examined in the absence or presence of the PPARβ-selective ligand L165041.
FIG. 3.
FIG. 3.
Determination of the ligand binding affinities of A-FABP and K-FABP. The Kd for the association of A-FABP (A) or K-FABP (B) with parinaric acid was measured by fluorescence titration. Protein (1 μM) was titrated with parinaric acid from a concentrated ethanolic solution. Fluorescence (λex = 303 nm; λem = 413 nm) was measured after each addition. Representative curves are shown. Titration curves were corrected for nonspecific increases in fluorescence due to addition of free parinaric acid. Corrected data (solid circles) were analyzed by fitting to an equation derived from binding theory to obtain the Kd and the number of ligand binding sites (solid lines through data points). The Kd for the association of troglitazone (C and D) and L165041 (E and F) with A-FABP (C and E) and K-FABP (D and F) were measured by competition fluorescence titrations. Protein was complexed with parinaric acid at a molar ratio corresponding to the measured number of binding sites. FABP-parinaric acid complexes were titrated with the appropriate ligand until a plateau was reached. Kd for the nonfluorescent ligands were calculated by using the EC50 of the competition curves and the measured Kd for parinaric acid.
FIG. 4.
FIG. 4.
A-FABP and K-FABP translocate into the nucleus in response to ligands for PPARγ and PPARβ, respectively. Expression vectors harboring either A-FABP (A through C) or K-FABP (D through F) tagged with GFP were transfected into COS-1 cells, and the subcellular locations of the proteins were examined by confocal microscopy. Images represent superpositions of the fluorescence and bright-field images of the same field. Ligands were added 3 to 4 h prior to imaging as follows: none (A and D), 1 μM troglitazone (PPARγ ligand) (B), 5 μM linolenic acid (C and F), and 1 μM L165041 (PPARβ ligand) (E).
FIG. 5.
FIG. 5.
K-FABP harboring an NES (NES-K-FABP) does not enhance the transcriptional activity of PPARβ. Transactivation assays were carried out using COS-1 cells as described in Materials and Methods. Cells were cotransfected with PPARβ and either an empty vector (vec) or an expression vector for either K-FABP (K) or NES-K-FABP (NES-K). The transcriptional activity of PPARβ was examined in the absence or presence of the PPARβ ligand L165041.
FIG. 6.
FIG. 6.
A-FABP and K-FABP interact with PPARγ and PPARβ selectively and in a ligand-dependent fashion. Coprecipitation assays were carried out by using bacterially expressed FABPs and 35S-labeled in vitro transcribed-translated PPARs as described in Materials and Methods. (A) GST-tagged A-FABP was immobilized on glutathione-agarose and incubated with 35S-labeled PPARγ in the absence or presence of the indicated concentration of the PPARγ ligand troglitazone (TZD). (B) Histidine-tagged A-FABP was immobilized on Ni2+-Sepharose and incubated either with 35S-labeled PPARγ in the absence or presence of troglitazone or with PPARβ in the absence or presence of either troglitazone or the PPARβ ligand L165041 (L). (C) GST or GST-tagged K-FABP was immobilized on glutathione-agarose and incubated with 35S-labeled PPARβ in the absence or presence of L165041.
FIG. 7.
FIG. 7.
A-FABP directly channels troglitazone to PPARγ-LBD, while K-FABP does not. The dependence of the rate of transfer of troglitazone (TZD) from FABP to PPARγ was examined. PPARγ-LBD was covalently labeled with a pyrene moiety. (A) The labeled protein (1 μM) was titrated with A-FABP precomplexed with troglitazone. Fluorescence (λex = 342 nm; λem = 377 nm) decreased upon titration until a plateau was reached at saturation. (B and C) To determine the rate constants for ligand transfer from FABP to PPARβ-LBD, pyrene-labeled PPARβ-LBD was mixed with A-FABP (B) or K-FABP (C) precomplexed with troglitazone. Mixing was accomplished using a stopped-flow apparatus. Final protein concentrations for the representative traces shown were 1 μM PPARβ-LBD and 5 μM FABP-ligand complexes. Traces were analyzed by fitting to a single first-order reaction (solid line through data points) to obtain the pseudo-first-order rate constant of the reaction. (D) t1/2 for transfer of troglitazone from A-FABP (solid circles) or K-FABP (open circles) to PPARβ-LBD as a function of the FABP/PPAR molar ratio.
FIG. 8.
FIG. 8.
K-FABP enhances the transcriptional activity of PPARβ only under limiting ligand concentrations. Transactivation assays were carried out by using COS-7 cells as described in Materials and Methods. Cells were cotransfected either with an empty vector or with an expression vector for K-FABP. The transcriptional activity of PPARβ was examined at various concentrations of the PPARβ-selective ligand L165041. (Inset) Fold activation observed upon cotransfection of K-FABP relative to fold activation in the absence of ectopic binding protein.
FIG. 9.
FIG. 9.
TNF-α-induced differentiation is inhibited or delayed in keratinocytes expressing a K-FABP antisense construct. Primary mouse prekeratinocytes were cultured, and their differentiation was induced by addition of TNF-α as described in Materials and Methods. (A) Expression of K-FABP in keratinocytes (control) or keratinocytes transfected with a vector harboring antisense (AS) K-FABP was examined by Western blotting using antibodies against K-FABP. Cells were transfected 12 h prior to treatment with TNF-α, and the expression level of K-FABP was monitored up to 48 h posttreatment. (B) Expression of the differentiation marker involucrin and the cell cycle protein cyclin A in primary keratinocytes and in keratinocytes expressing a K-FABP antisense construct was monitored by an RPA as described in Materials and Methods. (C) Relative expression levels of involucrin and cyclin A in TNF-α-treated primary keratinocytes or keratinocytes expressing a K-FABP antisense construct were measured by quantitation of the bands shown in Fig. 9B and normalization for the expression of L27.
FIG. 10.
FIG. 10.
Expression of a K-FABP antisense construct inhibits or delays TNF-α-induced differentiation of keratinocytes. Primary mouse prekeratinocytes were cultured as described in Materials and Methods and were visualized by direct bright-field microscopy. (A) Cultured prekeratinocytes; (B) keratinocytes 15 h post-TNF-α-treatment; (C) keratinocytes expressing a K-FABP antisense construct 15 h post-TNF-α-treatment.
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