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Originally published In Press as doi:10.1074/jbc.M609149200 on February 26, 2007

J. Biol. Chem., Vol. 282, Issue 16, 11776-11785, April 20, 2007
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Block of Nuclear Receptor Ubiquitination

A MECHANISM OF LIGAND-DEPENDENT CONTROL OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR {delta} ACTIVITY*Formula

Davide Genini and Carlo V. Catapano1

From the Laboratory of Experimental Oncology, Oncology Institute of Southern Switzerland, CH-6500 Bellinzona, Switzerland

Received for publication, September 26, 2006 , and in revised form, February 7, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxisome proliferator-activated receptor {delta} (PPAR{delta}) is a ligand-activated transcription factor involved in many physiological and pathological processes. PPAR{delta} is a promising therapeutic target for metabolic, chronic inflammatory, and neurodegenerative disorders. However, limited information is available about the mechanisms that control the activity of this nuclear receptor. Here, we examined the role of the ubiquitinproteasome system in PPAR{delta} turnover. The receptor was ubiquitinated and subject to rapid degradation by the 26 S proteasome. Unlike most nuclear receptors that are degraded upon ligand binding, PPAR{delta} ligands inhibited the ubiquitination of the receptor, thereby preventing its degradation. Ligand binding was required for inhibition of the ubiquitination since disruption of the ligand binding domain abolished the effect. Site-directed mutagenesis showed that the DNA binding domain was also required, indicating that ligands preferentially stabilized the DNA-bound receptor. In contrast, the activation function-2 domain and co-repressor binding site were not involved in ligand-induced stabilization. Block of ubiquitination by ligands may be an essential step to avoid rapid degradation of a receptor, like PPAR{delta}, with a very short half-life and sustain its transcriptional activity once it is engaged in transcriptional activation complexes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxisome proliferator-activated receptors (PPARs)2 belong to the superfamily of nuclear hormone receptors and act as ligand-activated transcription factors (1). PPARs are activated by a diverse group of lipophilic compounds, including long-chain fatty acids, prostaglandins, and leukotrienes (2). PPARs form heterodimers with the retinoic X receptor and bind DNA in correspondence to specific PPAR response elements (PPRE) located in the promoter of target genes (1). Unliganded receptors maintain the promoter in a repressive or inactive state (3). Ligand binding induces a conformational remodeling of the receptor, resulting in the release of co-repressor molecules and recruitment of co-activators necessary for transcriptional activation (3). The PPAR subfamily includes three isotypes, named {alpha}, {delta} (or beta), and {gamma}, that share extensive structural homology (1). Although all three isotypes act as lipid sensors and are involved in various aspects of lipid metabolism, they have distinct tissue distribution, ligand specificity, and functions (2, 4). PPAR{alpha} is involved in fatty acid metabolism, and high affinity ligands of this receptor, like fenofibrate and bezafibrate, are effective hypolipidemic drugs (5). PPAR{gamma} prevalently controls lipid and glucose metabolism, and PPAR{gamma} agonists, like rosiglitazone and pioglitazone, are widely used anti-diabetic drugs (6). PPAR{delta}, which is the less studied of the three isotypes, has been implicated in wound healing, inflammatory responses, and embryo implantation in addition to lipid metabolism (4, 7). PPAR{delta} is a potential therapeutic target for diseases such as atherosclerosis and other inflammatory, metabolic, and neurodegenerative disorders (7). PPAR{delta} has also been associated with cancer (8). PPAR{delta} is overexpressed in colon, endometrial, and head and neck cancers (9–12). PPAR{delta} agonists stimulate proliferation and survival of cancer cells in vitro (13–16) and promote tumor growth in mice (17–19). Consistent with a tumor promoting role, PPAR{delta} has been shown to increase the expression of anti-apoptotic genes, like ILK (integrin-linked kinase) and PDK1 (3-phosphoinositide-dependent kinase-1), and to activate the pro-survival Akt signaling pathway in keratinocytes (20). However, the involvement of this nuclear receptor in carcinogenesis is still controversial. Somatic knockdown of PPAR{delta} decreased growth of human colon cancer xenografts in mice (21), whereas in other studies the frequency of colon polyps was not affected or even increased in PPAR{delta} knock-out mice (22, 23) and upon treatment with PPAR{delta} ligands (24). Thus, this nuclear receptor is involved in many critical cell functions, including proliferation, survival, and differentiation. However, the mechanisms mediating its effects and the outcome in different cells and tissues remain unclear.

Although much is known about the mechanisms that regulate the activity of most nuclear receptors, very limited information is available regarding PPAR{delta}. At the transcriptional level, PPAR{delta} was shown to be a downstream target of the oncogenic Wnt/APC/beta-catenin pathway, showing for the first time a link with colon carcinogenesis (9). Activated oncogenic Ras stimulated PPAR{delta} expression in intestinal cells (25), whereas activator protein-1 (26) and C/EBP{alpha} (CCAAT/enhancer-binding protein) (27) controlled its expression in keratinocytes. Activity and expression of PPAR{delta} were inhibited by nonsteroidal anti-inflammatory drugs and cyclooxygenase-2 inhibitors in colon cancer cells, relating this nuclear receptor to the chemopreventive effects of this class of anti-inflammatory compounds (9, 14). Less is known about post-transcriptional mechanisms that control PPAR{delta} protein level and activity. A better understanding of the multiple mechanisms that modulate ligand-dependent and -independent activity might provide hints into the involvement of PPAR{delta} in human diseases and indicate ways to exploit its therapeutic potential.

In this study we examined the role of the ubiquitin-proteasome system (UPS) in basal and ligand-dependent turnover of PPAR{delta}. The UPS is the major cellular machinery responsible for degradation of proteins and determines the level of critical regulatory factors, including nuclear receptors and transcription factors (28–31). We found that PPAR{delta} was ubiquitinated and subject to rapid degradation by the 26 S proteasome. However, unlike most nuclear receptors that are degraded upon ligand binding, PPAR{delta} was stabilized by its ligands. PPAR{delta} agonists inhibited ubiquitination of the receptor with consequent block of its degradation. Thus, PPAR{delta} ligands have a dual effect on the receptor. Ligands induce conformational changes, allowing co-activator binding and promoter transactivation, and at the same time prevent ubiquitination of the receptor engaged in transcriptional activation complexes. Block of ubiquitination may be an essential step to avoid rapid degradation of a receptor with very short half-life, like PPAR{delta}, and sustain its transcriptional activity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines, Plasmids, and Chemicals—Human osteosarcoma U2OS cells and non-small cell lung cancer H358, H441, and A549 cells were purchased from American Type Culture Collection (LGC Promochem, Molsheim Cedex, F) and maintained in RPMI supplemented with 10% fetal bovine serum. In all experiments involving incubation with PPAR{delta} ligands, cells were grown in phenol red-free RPMI supplemented with 5% charcoal-stripped serum (HyClone, Logan, UT). Full-length human wild type PPAR{delta} (a gift of Bert Vogelstein, John Hopkins University, Baltimore, MD) was subcloned into pCMV (Stratagene, La Jolla, CA) and pcDNA3.1/His (Invitrogen) expression vectors. The C91A/C94A, F270A, and L432A/E435A PPAR{delta} mutants and the truncated form of PPAR{delta}-(1–299) were generated by site-directed mutagenesis of pcDNA3.1/His-PPAR{delta} using the QuikChange site-directed mutagenesis kit (Stratagene). The HA-Ub expression vector was kindly provided by Ron R. Kopito (Stanford University, Stanford, CA), and the PPREx3-tk-Luc reporter vector was a gift of Ronald M. Evans (Salk Institute, La Jolla, CA). Cells were transfected with expression and reporter vectors using Lipofectamine 2000 (Invitrogen). Cells stably expressing PPAR{delta} were obtained upon transfection with pCMV-PPAR{delta} vector and selection in the presence of 600 µg/ml G418 (Calbiochem). GW501516 (Alexis, Lausanne, CH), L165041 (Sigma), cPGI2 (carboprostacyclin; Biomol, Plymouth Meeting, PA), and prostaglandin E2 (Cayman Chemical, Ann Arbor, MI) were dissolved in Me2SO. Proteasome inhibitors PS341 and MG132 were dissolved in Me2SO. Puromycin dihydrochloride (Sigma) was prepared in water.

Reporter Assay—Cells (2 x 104 cells/well) were plated in 48-well plates and transfected with the PPREx3-tk-Luc, pRL-SV40 vectors and, when indicated, with PPAR{delta} expression vectors. Dual-luciferase reporter assay was performed according to manufacturer's instructions (Promega, Catalys AG, Wallisellen, CH) using a Turner luminometer (Turner Design, Sunnyvale, CA). Data were normalized for Renilla luciferase activity used as control for transfection efficiency.

PPAR{delta} Half-life—U2OS cells were grown in 100-mm dishes to 90% confluence. Cells were transfected with 3 µg of His-PPAR{delta} expression vector. The next day cells were plated into 6-well plates at a concentration of 3.5 x 105 cells/well. After overnight incubation with or without a PPAR{delta} ligand, cells were treated with 50 µM puromycin and harvested at the indicated times for Western blotting.

PPAR{delta} Ubiquitination—U2OS cells were transfected with His-PPAR{delta} and HA-Ub and incubated with proteasome inhibitors and/or PPAR{delta} ligands. After 24 h cells were lysed in a denaturing buffer consisting of 8 M urea, 0.1 M Na2HPO4/NaH2PO4, and 10 mM imidazole, pH 8.0. His-PPAR{delta} was pulled down using His-select nickel affinity gel (Sigma) starting with ~100 µg of protein from cell lysates. After multiple washes with urea buffer, proteins were eluted from the beads with the same buffer supplemented with 250 mM imidazole, pH 6.0. Aliquots of whole lysate and flow-through samples corresponding to 15 µg of proteins and an equivalent fraction of the eluates were loaded on gels and analyzed by Western blotting.

Western Blotting—Cells were lysed in a buffer containing 25 mM Tris-HCl, pH 7.4, 150 mM KCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors mixture (Roche Applied Science). To isolate nuclear and cytoplasmic fractions, cells were lysed in 10 mM Tris-HCl, pH 8.0, 7.5 mM ammonium sulfate, 1 mM EDTA, 0.025% Nonidet P-40, and 1 mM dithiothreitol as previously described (32). After incubation on ice for 5 min, sucrose (0.3 M final concentration) was added to the cell homogenate, and the cellular fractions were separated by centrifugation at 4000 x g for 10 min at 4 °C. Protein concentration was determined using a BCA assay (Pierce). Proteins were loaded on 10% polyacrylamide gels and analyzed by immunoblotting with antibodies against PPAR{delta} (H-74, Santa Cruz Biotechnology, Santa Cruz, CA), tubulin (DM1B, Calbiochem), His tag (H1029, Sigma), FLAG tag (M2, Sigma) and HA tag (Roche Applied Science). Horseradish peroxidase-conjugated secondary antibodies and the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences) were used for detection. Band intensity was assessed with the AlphaImager 3400 and AlphaEase Software (Alpha Innotech, San Leandro, CA).

RNA Isolation and Analysis—Cells (1 x 106 cells/flask) were incubated with PPAR{delta} ligands overnight and proteasome inhibitors for 4 h. For RNA interference studies, cells were transfected with 100 nM small interfering RNA (Ambion Ltd., Huntingdon UK) specific for PPAR{delta} (siPPAR{delta}) or the firefly luciferase gene (siGL3) using Lipofectamine 2000, incubated for 48 h, and then treated with ligands and/or proteasome inhibitors. Total RNA was isolated using Trizol (Invitrogen) and further purified with RNeasy MiniKit (Qiagen). RNA concentration was determined using a NanoDrop spectrophotometer. RT-PCR was performed using the SuperScript One-Step RT-PCR system (Invitrogen) and gene-specific primers for the adipose differentiation-related protein (ADRP), glyceraldehyde-3-phosphate dehydrogenase, and PPAR{delta}. PCR products were run on 2% agarose gels, stained with ethidium bromide, and visualized using the AlphaImager 3400. Band intensity was determined using the AlphaEase software. For quantitative real time RT-PCR, 1 µg of total RNA was reverse-transcribed using the SuperScript First-Strand Synthesis system (Invitrogen). Real time PCR was performed on a 7900HT Fast Real Time PCR System (Applied Biosystems, Foster City, CA) using primer sets for ADRP and beta-actin and Absolute SYBR Green ROX Mix (ABgene, Epsom, UK). Standard curves were generated for each primer set, and beta-actin RNA was used as control to quantify ADRP RNA. Sequences of primers and small interfering RNAs are available as supplemental Table S1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
PPAR{delta} Ligands Stabilize the Receptor—Ligand-dependent transactivation of nuclear receptors is often associated with proteasome-mediated degradation of the receptor (33, 34). Ligand-induced proteolysis serves as a common mechanism to terminate transcriptional activity of the ligand-activated receptors (33, 34). To determine whether PPAR{delta} was subject to a similar ligand-dependent control, human lung cancer cell lines expressing different levels of the receptor were incubated overnight with a PPAR{delta}-selective ligand. PPAR{delta} protein level increased after incubation with L165041 in cells expressing high (H441) and low (H358) levels of the receptor, whereas there was no effect in A549 cells in which PPAR{delta} was undetectable in the immunoblots both before and after ligand treatment (Fig. 1A). The effect of the ligand on the receptor levels correlated well with the ligand ability to transactivate the PPAR-responsive reporter in these cells (Fig. 1B). Ligand treatment increased reporter activity ~13- and 5-fold in H441 and H358 cells, respectively. The effect on A549 and U2OS cells was minimal (~2-fold), in agreement with the very low levels of PPAR{delta} in these cells.

To better differentiate between transcriptional and post-translational effects, in subsequent experiments we used U2OS cells expressing recombinant PPAR{delta} from a heterologous promoter. Increased levels of PPAR{delta} were seen when PPAR{delta}-expressing U2OS cells were treated with synthetic agonists (i.e. GW501516 and L165041) and a stable prostaglandin analogue (cPGI2 (carboprostacyclin)) that binds to PPAR{delta} (Fig. 1, C and D). On the other hand treatment of cells with prostaglandin E2, a prostaglandin that activates PPAR{delta} indirectly and does not bind to the receptor (19), did not have any effect on the receptor protein level, suggesting that direct binding to the receptor was required to increase its stability. A time course experiment in PPAR{delta}-expressing U2OS cells showed that the ligand acted rapidly (Fig. 1D). A substantial increase of PPAR{delta} (~2-fold) was seen already after 4 h, and the levels continued to increase (up to ~4-fold) after 24 h. The effect on the protein level was reversible, and PPAR{delta} returned to base-line levels within 4 h after the removal of the ligand (Fig. 1E). The effect seen in cells expressing PPAR{delta} from a heterologous promoter made it unlikely that the increased protein level was a consequence of enhanced transcription. In fact, PPAR{delta} transcript levels measured by RT-PCR were identical in PPAR{delta}-expressing U2OS cells incubated with or without ligand (supplemental Fig. S1).


Figure 1
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  		FIGURE 1.
Ligand-induced stabilization of PPAR{delta}. A, cells were incubated with 5 µM L165041 overnight. PPAR{delta} protein was detected in immunoblots using an anti-PPAR{delta} antibody. B, cells were transfected with PPREx3-tk-Luc and pRL-SV40 reporter vectors and incubated with or without 5 µM L165041 for 24 h before measuring luciferase reporter activity. Data are presented as -fold increase (mean ± S.D. of triplicate experiments) in reporter activity compared with untreated control (ctrl) cells. *, p ≤ 0.005 compared with control cells. C, U2OS cells were transfected with His-PPAR{delta} expression vector and incubated overnight with GW501516 (GW), prostaglandin (GP) I2, or prostaglandin E2 at the indicated concentrations. D, U2OS cells expressing His-PPAR{delta} were incubated with 5 µM L165041 and harvested at the indicated time after the addition of the ligand. E, U2OS cells expressing His-PPAR{delta} were incubated overnight with 5 µM L165041. The ligand was removed, and cells were washed twice in phosphate-buffered saline before continuing the incubation in ligand-free medium for the indicated time. In panels C–E, PPAR{delta} was detected using an anti-His antibody. Tubulin was used as control for sample loading.

 
The data described above suggested that ligands could affect PPAR{delta} levels by influencing protein turnover. To estimate PPAR{delta} half-life, we transiently transfected U2OS cells with a His-PPAR{delta} expression vector and monitored protein level by Western blotting after the addition of the protein synthesis inhibitor puromycin. His-tagged PPAR{delta} level was greatly reduced within 1 h of puromycin treatment (Fig. 2A). The estimated PPAR{delta} half-life under these conditions was ~30 min (Fig. 2C). Next, His-PPAR{delta} expressing U2OS cells were incubated with puromycin in the presence of L165041 to determine the effects of the ligand on the receptor half-life. Incubation with L165041 increased PPAR{delta} protein level and completely prevented the decline induced by puromycin (Fig. 2, B and C). Thus, the ligand increased the receptor protein level by extending its half-life considerably.


Figure 2
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  		FIGURE 2.
PPAR{delta} ligands increase receptor half-life. A, U2OS cells were transfected with His-PPAR{delta} expression vector and, after an overnight incubation, treated with 50 µM puromycin. Cell lysates were prepared at the indicated times and analyzed by immunoblotting. B, His-PPAR{delta}-expressing U2OS cells were incubated overnight with 1 µM L165041 and then treated with 50 µM puromycin in the presence of L165041. In both experiments PPAR{delta} was detected with an anti-His antibody, and tubulin was used as control for sample loading. C, densitometric assessment of PPAR{delta} level in the immunoblots shown in A and B. Data represent the percentage relative to the starting level of PPAR{delta} in the presence or absence of L165041 (time 0). ctrl, control.

 
PPAR{delta} Turnover Is Controlled by the Ubiquitin-Proteasome Pathway—The 26 S proteasome is the major cellular complex responsible for the degradation of proteins, including nuclear receptors and transcription factors (28–31). To determine whether proteasome-mediated degradation played a role in controlling the turnover of PPAR{delta}, H441 and H358 cells expressing, respectively, high and low levels of the receptor were treated with the proteasome inhibitor PS341. In both cell lines PPAR{delta} protein level increased, consistent with reduced degradation of the receptor in the presence of the proteasome inhibitor (Fig. 3A). Similar results were obtained by treating cells with another proteasome inhibitor, MG132 (data not shown). To further study the role of the 26 S proteasome in PPAR{delta} turnover, U2OS cells expressing recombinant His-PPAR{delta} were treated with PS341, and protein level was determined at different times by Western blotting. As shown in Fig. 3B, treatment with PS341 led to a significant accumulation of PPAR{delta} (~3-fold) already after 4 h of incubation. Next, U2OS cells were treated either with puromycin, PS341, or both compounds together (Fig. 3C). PPAR{delta} level decreased rapidly in the presence of puromycin, whereas treatment with puromycin and PS341 resulted in a higher level of the receptor, indicating that PPAR{delta} turnover was under the control of the proteasome.


Figure 3
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  		FIGURE 3.
PPAR{delta} turnover is controlled by the proteasome. A, H441 and H358 cells were treated with PS341 for 4 h. B, U2OS cells were transfected with His-PPAR{delta} expression vector and, after an overnight incubation, treated with the proteasome inhibitor PS341 (10 µM) for the indicated times. C, His-PPAR{delta} expressing U2OS cells were left untreated or treated with puromycin, puromycin and PS341, or PS341 alone for 4 h. In panels A–C, PPAR{delta} was detected using an anti-PPAR{delta} antibody. D, U2OS cells were transfected with HA-ubiquitin vector along with His-pcDNA or His-PPAR{delta} vectors and after 24 h were incubated with 10 µM PS341 for 4 h. His-tagged PPAR{delta} was pulled down with nickel affinity gel under denaturing conditions. PPAR{delta} was detected in cell lysate, flow-through, and eluate fractions using an anti-His antibody, whereas ubiquitinated proteins were detected with an anti-HA antibody.

 
A necessary step for targeting proteins to the proteasome is the covalent attachment of ubiquitin (Ub) chains catalyzed by Ub ligases (28). To determine whether PPAR{delta} was ubiquitinated, U2OS cells were transfected with His-PPAR{delta} and HA-Ub expression vectors and then incubated with a proteasome inhibitor for 4 h. His-tagged PPAR{delta} was pulled down using nickel affinity gel under denaturing conditions. This strategy avoided the use anti-PPAR{delta} antibodies that had revealed limited specificity in preliminary immunoprecipitation experiments. In addition, it used highly stringent conditions to minimize non-covalent interactions of the receptor with potential ubiquitinated protein partners. Whole cell lysate and flow-through samples were examined along with the eluates to control for quantitative recovery of His-PPAR{delta} throughout the procedure (Fig. 3D, lanes 1–4). The specificity of the pulldown was demonstrated using cells transfected with Histag empty vector (lanes 1, 3, and 5). In His-PPAR{delta}-expressing cells, high molecular weight protein species were detected with the anti-HA antibody indicating the presence of PPAR{delta} with covalently linked poly-Ub chains (lane 6). No such bands were detected in the eluate from empty vector-transfected cells (lane 5).

Proteasome activity is required for receptor turnover, and its inhibition generally leads to reduced transcriptional activity of nuclear receptors (34, 35). To determine whether a functional proteasome was required for PPAR{delta} transcriptional activity, U2OS cells stably expressing recombinant PPAR{delta} and H441 cells were transfected with a PPAR-responsive reporter and incubated with PS341 in the presence or absence of a PPAR{delta} ligand (Fig. 4A). GW501516 increased reporter activity ~ 6 and 12-fold in U2OS and H441 cells, respectively. PS341 induced a ~2–3-fold increase in the absence of the ligand in both cell lines. The combination of PS341 and ligand increased reporter activity by ~13 and 26-fold in U2OS and H441, respectively. Thus, inhibition of proteasome activity did not negatively affect ligand-dependent activation of PPAR{delta}. Ligands and proteasome inhibitors had a positive and apparently synergistic effect on the activity of both recombinant and endogenous PPAR{delta} in the reporter assays. To determine whether ligands and proteasome inhibitors had similar effects on transcription of endogenous PPAR{delta} target genes, H358 and H441 cells were treated as described above, and the level of the ADRP RNA was monitored by real time RT-PCR or conventional RT-PCR. The ADRP gene contains a PPRE, and its transcription is activated by PPAR{delta} agonists (36). In both cell lines PPAR{delta} ligand increased ADRP RNA levels (by ~10 and 25-fold in H358 and H441 cells, respectively). This effect was enhanced in the presence of the proteasome inhibitor (up to ~25 and 100-fold in H358 and H441, respectively) (Fig. 4B). Neither the ligand nor proteasome inhibitor affected endogenous PPAR{delta} RNA levels in H358 and H441 cells, confirming that they acted post-transcriptionally, increasing the protein level of endogenous PPAR{delta} (Fig. 4C and data not shown). The specificity of these effects was further assessed by RNA interference. The increase induced by the PPAR{delta} ligand both alone and in combination with PS341 was attenuated in cells in which PPAR{delta} had been silenced (up to 75%) by a small interfering RNA against PPAR{delta} before the incubation with GW501516 and/or PS341 (Fig. 4C). Despite the evident reduction of PPAR{delta} RNA, ligand-dependent activation of ADRP transcription was reduced only partially by RNA interference because of the stabilizing effect of the ligand and proteasome inhibitor at the protein level. Taken together, these data indicate that inhibition of the proteasome did not reduce PPAR{delta} activity but, along with PPAR{delta} ligands, led to the accumulation of transcriptionally competent receptor. To determine whether the accumulated protein had also the proper subcellular localization, PPAR{delta}-expressing U2OS cells were treated with PS341 or a selective ligand before isolation of the nuclear and cytoplasmic fractions (Fig. 4D). Although a small amount was found in the cytoplasm, PPAR{delta} was detected predominantly in the cell nucleus of both PS341 and ligand-treated cells.


Figure 4
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  		FIGURE 4.
Proteasome inhibitors and PPAR{delta} ligands induce accumulation of transcriptionally competent PPAR{delta}. A, U2OS stably expressing PPAR{delta} and H441 cells were transfected with PPREx3-tk-Luc reporter and pRL-SV40 and incubated with or without 5µM GW501516 for 24 h. PS341 (10µM) was added during the last 4 h of incubation. Luciferase activity was measured with a Dual-Luciferase assay system using Renilla luciferase as a control for transfection efficiency. Data represent -fold increase (mean ± S.D. of triplicate experiments) relative to control cells. *, p ≤ 0.005 compared with control cells. B, H358 and H441 cells were treated with GW501516 and PS341 as described above. Total RNA was isolated, and the levels of ADRP and beta-actin were determined by quantitative real time RT-PCR. Data are expressed as ADRP/beta-actin ratios and normalized for the value in untreated control cells. *, p ≤ 0.01 C, H358 cells were transfected with 100 nM small interfering RNA against PPAR{delta} (siPPAR{delta}) or firefly luciferase (siGL3) and 48 h later treated with GW501516 and/or PS341 as indicated above. Total RNA was isolated, and ADRP, PPAR{delta}, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA was measured by RT-PCR. D, U2OS cells stably expressing FLAG-PPAR{delta} were incubated with or without PS341 (10 µM) for 4 h or L165041 (5 µM) overnight. Nuclear (n) and cytoplasmic (c) fractions were separated by sucrose gradient centrifugation. PPAR{delta} was detected using an anti-FLAG antibody.

 
Ligands Prevent Proteasomal Degradation of PPAR{delta} by Blocking Its Ubiquitination—Ligand-induced stabilization of PPAR{delta} might be the consequence of inhibition of proteasome-mediated degradation. To exclude the possibility that PPAR{delta} ligands might affect activity of the 26 S proteasome, U2OS cells expressing a Ub-enhanced green fluorescent protein fusion protein were treated with ligands or PS341 as a positive control and analyzed by flow cytometry. Although PS341 induced the expected accumulation of Ub-enhanced green fluorescent protein (EGFP), none of the PPAR{delta} ligands changed Ub-EGFP levels (supplemental Fig. S2), suggesting that the ligands did not inhibit the 26 S proteasome and might affect PPAR{delta} protein turnover by acting directly on the receptor.

To determine whether PPAR{delta} ligands modulated the ubiquitination or proteasomal degradation of the receptor, we treated U2OS cells transfected with HA-Ub and His-PPAR{delta} expression vectors with L165041 in the presence and absence of PS341. If the ligand blocked proteasomal degradation of the receptor, one would expect accumulation of ubiquitinated PPAR{delta}. On the contrary, a block of ubiquitination would lead to reduced amounts of ubiquitinated PPAR{delta} in the presence of ligand. As expected, L165041 and PS341 alone and in combination increased PPAR{delta} in the cell lysates (Fig. 5A, top panel). However, although PS341 led to the accumulation of ubiquitinated PPAR{delta}, treatment with the ligand both in the presence and absence of PS341 led to a reduction of the amount of ubiquitinated PPAR{delta} (Fig. 5A, bottom panel, lanes 2 and 4) compared with control and PS341-treated cells (lanes 1 and 3). Thus, the ligand selectively affected the ubiquitination of the receptor rather than its proteasomal degradation. The effect of the ligand on PPAR{delta} ubiquitination was reversible and depended on its continuous presence in the culture medium as seen before with the effect on PPAR{delta} protein level (Fig. 1E). Removal of the ligand restored the ability of the cells to ubiquitinate the receptor within 4 h, concomitant with the decrease of the receptor level (Fig. 5B). To confirm that ligand binding was required to prevent PPAR{delta} ubiquitination, a similar experiment was done with a truncated form of the receptor (PPAR{delta}-(1–299)), which missed most of the ligand binding domain. Disruption of this domain abolished the effects of the ligand on PPAR{delta} ubiquitination and stabilization (Fig. 5C). Thus, direct interaction of the ligand with the receptor was absolutely required for inhibition of PPAR{delta} ubiquitination.

Ligand-induced Stabilization of PPAR{delta} Involves the DNA Binding Domain of the Receptor—Like other nuclear receptors, PPAR{delta} has well defined structural and functional domains (1). To understand the role played by distinct domains in ligand-induced stabilization of the receptor, we introduced inactivating mutations in the DNA binding domain (C91A/C94A), in the AF-2 transactivating domain (L432A/E435A), and within the region (F270A) known to be responsible for co-repressor binding (Fig. 6A). Mutations in the DNA binding domain (C91A/C94A) were similar to those known to prevent binding of murine PPAR{delta} to DNA (37). The double mutant in the AF-2 domain (L432A/E435A) and the F270A mutation blocked the interaction of murine PPAR{delta} with coactivators (38) and corepressors (37), respectively. The ability of the mutated receptors to activate a PPAR-responsive reporter was tested in luciferase assays along with wild type PPAR{delta} (Fig. 6B). The DNA binding domain mutant (C91A/C94A) was less active than the wild type receptor in the reporter assay. The mutation in the putative co-repressor binding site (F270A) had a minimal effect on PPAR{delta}-induced transactivation. On the other hand, the AF-2 mutant (L432A/E435A) had greatly reduced transcriptional activity compared with the wild type receptor, confirming the critical role of this domain for ligand-dependent transactivation. In addition, the AF-2 mutant acted as a dominant negative, reducing ~-fold the activation of the reporter by endogenous PPAR{delta}.


Figure 5
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  		FIGURE 5.
Ligand binding prevents PPAR{delta} ubiquitination. A, U2OS cells expressing His-PPAR{delta} and HA-ubiquitin were incubated with or without L165041 (5 µM) overnight followed by PS341 (10 µM) for 4 h. B, His-PPAR{delta} U2OS cells were incubated with or without L165041 overnight. An aliquot of ligand-treated cells was washed twice in phosphate-buffered saline and incubated in ligand-free medium for an additional 4 h (recovery). C, U2OS cells were transfected with an expression vector encoding a truncated form of His-PPAR{delta}-(1–299) along with the HA-ubiquitin vector and incubated overnight with or without L165041. His-tagged PPAR{delta} was pulled down under denaturing conditions using nickel affinity gel. PPAR{delta} and ubiquitinated proteins were detected in cell lysates and eluates using anti-His and anti-HA antibodies, respectively.

 


Figure 6
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  		FIGURE 6.
Structural requirements for ligand-induced stabilization of PPAR{delta}. A, PPAR{delta} structural domains and positions of the mutations introduced in the wild type receptor. AF-1, activation function-1; DBD, DNA binding domain; Hinge, flexible hinge region; LBD, ligand binding domain; AF-2, activation function-2. B, U2OS cells were transfected with vectors encoding wild type PPAR{delta} or mutated receptors along with PPREx3-tk-Luc and pRL-SV40 reporter vectors. Cells were incubated overnight with or without L165041 (5 µM), and then luciferase activity was assessed as described in the legend for Fig. 1. Data represent -fold increase (mean ± S.D. of triplicate experiments) relative to empty vector-transfected and untreated cells. *, p ≤ 0.005 compared with control cells. ctrl, control. C, U2OS cells expressing wild type or mutated PPAR{delta} were incubated with L165041 overnight and analyzed by immunoblotting using an anti-His antibody. Pull-down experiments were performed to detect ubiquitinated His-PPAR{delta} as described in the legend for Fig. 3. D, PPAR{delta} levels in cells transfected with wild type or mutated PPAR{delta} and incubated with or without L165041 assessed by immunoblotting and densitometric analysis. Data (mean ± S.D. of triplicate experiments) represent the percentage of the band intensity relative to the corresponding untreated control.

 
The ability of ligands to stabilize wild type and mutated receptors was tested in transiently transfected U2OS cells. The L432A/E435A and F270A mutants were stabilized by the ligand to a level comparable with wild type PPAR{delta} (Fig. 6, C, top panel, D). The effect of the ligand was reduced with the C91A/C94A double mutant (Fig. 6, C and D). Ligand-induced stabilization of the L432A/E435A- and F270A-mutated receptors was associated with reduced ubiquitination as with the wild type receptor, indicating that mutations in the transactivation domain and co-repressor binding site did not affect the ability of the ligand to block ubiquitination and degradation of the receptor (Fig. 6C, bottom panel). In contrast, the ability of the ligand to affect receptor ubiquitination was considerably reduced in the case of the C91A/C94A mutant (Fig. 6C, bottom panel). This indicated that, although it was not necessary for ubiquitination and degradation of the receptor, DNA binding enhanced the effect of the ligand on receptor stability.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The nuclear hormone receptor superfamily includes at least 48 distinct members in the human and mouse genome (39). By controlling the activation state of several genes in response to specific stimuli, nuclear receptors exert fundamental functions in many physiological processes. The activity of nuclear receptors themselves needs to be tightly controlled at multiple levels to ensure rapid and timely ordered responses to the specific stimuli. Furthermore, distinct control mechanisms seem to have evolved for each nuclear receptor depending on its physiological function and ligand properties. In this study we examined basal and ligand-dependent turnover of PPAR{delta} and the role of the UPS in this process. PPAR{delta} is constitutively expressed at low levels in many tissues. However, a clear understanding of its functions in different cells and tissues is still missing. Understanding the mechanisms and pathways that control PPAR{delta} level and activity may provide insights into the diverse functions attributed to this nuclear receptor. Here we show that, like other nuclear receptors, PPAR{delta} protein turnover is under the control of the UPS. However, unlike most nuclear receptors that are degraded upon ligand binding, PPAR{delta} was more stable in the presence of ligands. PPAR{delta} ligands selectively inhibited ubiquitination of the receptor, thereby blocking its degradation by the proteasome. Ligand-induced stabilization may be an essential step to avoid rapid degradation of a receptor with very short half-life, like PPAR{delta}, and sustain its transcriptional activity once engaged in transcriptional activation complexes. Furthermore, our study reveals important differences between PPAR{delta} and the other PPAR isotypes as far as ligand-dependent control of receptor turnover, which may have implications for the diverse functions of PPARs in various cells and tissues.

The Ubiquitin-Proteasome System and PPAR{delta} Turnover—The UPS is responsible for degradation of many cellular proteins, including nuclear receptors and transcription factors (28–31, 33, 34). The first step in proteasome-mediated proteolysis is the covalent modification of proteins by the addition of Ub, a small 76-amino acid polypeptide (28, 31). Protein ubiquitination involves three classes of enzymes acting in a highly coordinated fashion: E1 Ub-activating enzymes, E2 Ub-conjugating enzymes, and E3 Ub ligases. E3 Ub ligases have the highest substrate specificity and represent the largest family of Ub-processing enzymes (28, 31). Ubiquitinated proteins are generally marked for rapid degradation by the proteasome (28). However, they can be rescued by the action of deubiquitinating enzymes that remove Ub chains before proteolytic attack (40). Deubiquitinating enzymes receive increasing attention since they can participate, along with E3 Ub ligases, in ubiquitination-deubiquitination cycles capable of rapidly modulating the level of target proteins (40). Ligand-induced degradation of nuclear receptors is probably operated by Ub ligases and proteasomal subunits present as integral components of transcription regulatory complexes and acting within the cell nucleus (29, 30, 33, 34). Proteasome-dependent proteolysis serves to ensure both nuclear receptor turnover and timely termination of the transcriptional response (34, 35). Accordingly, inhibition of proteasome activity generally leads to reduced transactivation by nuclear receptors (34, 35).

In this study we observed that PPAR{delta} was ubiquitinated and rapidly degraded by the 26 S proteasome. Brief incubation of cells expressing both endogenous and recombinant PPAR{delta} with proteasome inhibitors led to a rapid increase of the receptor. Upon proteasome inhibition most of the receptor was in the cell nucleus similar to control and ligand-treated cells. Moreover, it was transcriptionally competent as shown by luciferase reporter assays and direct assessment of the level of an endogenous target gene. PPAR{delta} behavior in this regard was quite different compared with other nuclear receptors, including the estrogen-, androgen-, thyroid hormone-, and retinoic acid receptors, whose transcriptional activity is reduced upon proteasome inhibition (34). Furthermore, although in the absence of ligand, PPAR{delta} had a very short half-life of ~30 min, the addition of ligand considerably increased its half-life. The effect of the ligands was rapid and specific for compounds able to bind and activate the receptor directly. PPAR{delta} protein level increased within 4 h after the addition of ligand to cells and remained high as long as the ligand was present in the culture medium. Removal of the ligand was followed by rapid reversal of the effect with return to the base-line level within 4 h. Also in this regard PPAR{delta} behavior was almost unique among nuclear receptors, which generally are negatively regulated by their own ligands (34). Only vitamin D3 receptor has been previously shown to be stabilized by the ligand with similar kinetics (41). PPAR{gamma} was degraded rather rapidly upon exposure to ligands (42), whereas PPAR{alpha} was stabilized in the presence of ligands but only transiently (43, 44). An increase of PPAR{alpha} protein level was seen within 3 h and was followed by rapid proteolysis upon continuous exposure to ligands (43).

Thus, the system in place for PPAR{delta} may be geared to prevent both accumulation of high levels and prolonged activation of the receptor. It is possible that overactivity of PPAR{delta} may be detrimental to cells, owing perhaps to the anti-apoptotic function and oncogenic potential associated with this nuclear receptor. The level of PPAR{delta} must be kept low and under constant control via UPS-dependent proteolysis. Only in the presence of high concentrations of the specific ligands PPAR{delta} would be stabilized and activated. Furthermore, the transcriptional response mediated by the ligand-bound receptor would persist only as long as the ligand is present. Under physiological conditions, termination of the transcriptional response would be ensured by the short half-life and low abundance of natural PPAR{delta} ligands, like prostaglandin I2. This hypothesis would be consistent with the observation that in processes such as wound healing, there is a sharp increase of PPAR{delta} level concomitant with increased production of ligands (45). Also in cancer, up-regulation of PPAR{delta} is apparently coordinated with up-regulation of cyclooxygenase-2 and consequent increased production of prostaglandin metabolites capable of stabilizing and activating PPAR{delta} (10, 17). In the absence of this coordinated increase of ligand and receptor levels, PPAR{delta} would not be able to exert its anti-apoptotic and growth-promoting functions.

Mechanism of Ligand-induced Stabilization of PPAR{delta}—Ligand-induced stabilization of PPAR{delta} was due to a selective block of receptor ubiquitination and not to interference with later steps of the proteolytic process, i.e. binding to and degradation by the proteasome. Like protein stabilization, reduced ubiquitination of PPAR{delta} depended on the continuous presence of the ligand and was rapidly reversed after ligand removal. Disruption of the ligand binding domain in PPAR{delta}-(1–299) abolished the effect of the ligand on PPAR{delta} ubiquitination and proteolysis, although the truncated form of the receptor was still ubiquitinated and degraded by the proteasome. This result supported the idea that ligand-induced stabilization of PPAR{delta} was due to the direct interaction of the ligand with the receptor. Ligand binding may induce conformational changes that, in addition to allow co-activator binding and transactivation, block the interaction of PPAR{delta} with Ub ligases or, alternatively, promote the activity of deubiquitinating enzymes. We are currently testing these hypotheses and attempting to identify the enzymes involved in ligand-dependent control of the PPAR{delta} ubiquitination. Ub-processing enzymes involved in this process may represent critical elements in the pathways regulating the activity of this nuclear receptor.

Using site-directed mutagenesis we investigated the role of distinct receptor domains in the ligand-dependent regulation of PPAR{delta} stability. This analysis also revealed additional differences between PPAR{delta} and the other PPAR isotypes as far as the mechanisms involved. Mutations in the DNA binding domain reduced the effect of the ligand on receptor ubiquitination and protein level. This indicated that the ligand acted preferentially on DNA-bound receptor molecules preventing their ubiquitination. Interestingly, mutations in DNA binding domain of PPAR{gamma} had no effect, and DNA binding of this receptor was not a pre-requisite for ligand-induced degradation (42). Furthermore, we show that the AF-2 domain and the co-repressor binding site were not required for ligand-induced changes in PPAR{delta} ubiquitination, indicating that the ligand effect was independent of co-activator and co-repressor binding. It is intriguing that the AF-2 domain of PPAR{delta} did not have any role in this process since for most nuclear receptors the trans-activation function is tightly linked to proteolytic degradation and mutations in the trans-activating domain are expected to affect both ubiquitination and proteolysis (34, 35). This is also in contrast with the established role of the AF-2 domain in ligand-dependent degradation of PPAR{gamma} (42). The AF-2 domain of PPAR{gamma} mediated ligand-induced degradation of the receptor, suggesting that conformational changes induced by the ligand favored the interaction with both co-activators and the UPS (42). Using a different approach, co-expression of the transcriptional co-activator CBP (cAMP-response element-binding protein (CREB)-binding protein) led to a decrease of PPAR{alpha} level in the presence of ligand, showing that the interaction with co-activators via the AF-2 domain promoted proteolysis of the PPAR{alpha} isotype (46). Thus, in the case of PPAR{alpha}, the initial stabilization induced by ligands is probably followed by recruitment of co-activators along with other co-factors that trigger proteolysis of the receptor (43, 46). In contrast, our data show that transactivation and receptor ubiquitination are physically and functionally separated in the case of PPAR{delta}. It is probably the absence of a physical link between these processes that permits independent regulation of the transactivating function and ubiquitination of PPAR{delta} upon ligand binding.


Figure 7
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  		FIGURE 7.
Model of ligand-dependent regulation of PPAR{delta} activity. In the absence of ligand, PPAR{delta} is bound to DNA as a heterodimer with retinoid X receptor forming a complex with co-repressor molecules and thereby functioning as a transcriptional repressor. PPAR{delta} can be ubiquitinated by ubiquitin ligases (Ub ligase) probably residing within the transcription regulatory complex, and its turnover is regulated by proteasome-dependent proteolysis. PPAR{delta}-selective ligands induce a conformational remodeling of the DNA-bound receptor that allows binding of co-activator molecules and displacement of co-repressors. Ligand-induced remodeling and block of ubiquitin ligases lead to increased stability of DNA-bound PPAR{delta} and promote transcriptional activation. Alternatively, deubiquitinating enzymes (DUB) may be activated upon ligand binding, resulting in reduced ubiquitination of the receptor. Deubiquitination of PPAR{delta} may also assist in switching between the distinct states of the receptor, favoring dissociation of co-repressor complexes or assembly of co-activator complexes.

 
Taken together, our data support a model of ligand-dependent regulation of PPAR{delta} activity that is in part different from what has been proposed for PPAR{gamma} and -{alpha} (Fig. 7). Under normal conditions, both unbound and DNA-bound PPAR{delta} may undergo constitutive ubiquitination and degradation to maintain low levels of the receptor in the absence of ligands. UPS-mediated proteolysis of the unliganded receptor may serve to control overall receptor level and, particularly, ligand-independent functions. In the absence of ligands, PPAR{delta} can regulate transcription by forming complexes with transcriptional repressors, like SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) (37) and Bcl-6 (36). We propose that upon ligand binding the DNA-bound receptor is protected from proteasome-dependent proteolysis by selective inhibition of receptor ubiquitination. This can be accomplished by blocking Ub ligases or, alternatively, activating deubiquitinating enzymes. The increased half-life of the DNA-bound receptor in the presence of ligand would allow time for transactivation of target genes to occur. Therefore, it is important that transactivation is functionally dissociated from ubiquitination of the receptor. In fact, this separation allows ligand binding, possibly via a conformational change independent of the AF2 domain and co-activator binding, to block ubiquitination of DNA-bound PPAR{delta}. This condition may be essential to prevent degradation of the DNA-bound receptor and elicit a proper transcriptional response. Receptor ubiquitination may also have effects independent of the proteolytic pathway and may assist in switching between the repressive and activated state of the receptor, perhaps favoring either dissociation or assembly of co-repressor and co-activator complexes.


   FOOTNOTES
 
* This work was supported in part by the Ticino Foundation for Cancer Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1 and S2. Back

1 To whom correspondence should be addressed: Laboratory of Experimental Oncology, Oncology Institute of Southern Switzerland, Via Vela 6, Bellinzona CH-6500 Switzerland. Tel.: 41-91-820-0365; Fax: 41-91-820-0397; E-mail: carlo.catapano{at}irb.unisi.ch.

2 The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator-activated receptor response elements; ADRP, adipose differentiation-related protein; AF, activation function; CMV, cytomegalovirus; Ub, ubiquitin; UPS, ubiquitin-proteasome system; HA, hemagglutinin; RT, reverse transcription. Back


   ACKNOWLEDGMENTS
 
We thank Drs. B. Vogelstein, R. R. Kopito and R. M. Evans for generously providing expression and reporter plasmids and Drs. C. Realini, G. M. Carbone and A. Rinaldi for advice and critical review of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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