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Am J Physiol Lung Cell Mol Physiol 294: L1238-L1249, 2008. First published April 4, 2008; doi:10.1152/ajplung.00017.2008
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PPARβ/{delta} agonist stimulates human lung carcinoma cell growth through inhibition of PTEN expression: the involvement of PI3K and NF-{kappa}B signals

ShouWei Han,1 Jeffrey D. Ritzenthaler,1 Ying Zheng,1,2 and Jesse Roman1,3

1Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; 2Department of Obstetrics and Gynecology, West China 2nd University Hospital, Sichuan University, Chengdu, China; and 3Atlanta Veterans Affairs Medical Center, Atlanta, Georgia

Submitted 9 January 2008 ; accepted in final form 4 April 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Recent studies suggest that activation of peroxisome proliferator-activated receptor β/{delta} (PPARβ/{delta}) promotes cancer cell survival. We previously demonstrated that a selective PPARβ/{delta} agonist, GW501516, stimulated human non-small cell lung carcinoma (NSCLC) cell growth. Here, we explore the mechanisms responsible for this effect. We show that GW501516 decreased phosphate and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor known to decrease cell growth and induce apoptosis. Activation of PPARβ/{delta} and phosphatidylinositol 3-kinase (PI3K)/Akt signaling was associated with inhibition of PTEN. GW501516 increased NF-{kappa}B DNA binding activity and p65 protein expression through activation of PPARβ/{delta} and PI3K/Akt signals and enhanced the physical interactions between PPARβ/{delta} and p65 protein. Conversely, inhibition of PI3K and silencing of p65 by small RNA interference (siRNA) blocked the effect of GW501516 on PTEN expression and on NSCLC cell proliferation. GW501516 also inhibited IKB{alpha} protein expression. Silencing of IKB{alpha} enhanced the effect of GW501516 on PTEN protein expression and on cell proliferation. It also augmented the GW501516-induced complex formation of PPARβ/{delta} and p65 proteins. Overexpression of PTEN suppressed NSCLC cell growth and eliminated the effect of GW501516 on phosphorylation of Akt. Together, our observations suggest that GW501516 induces the proliferation of NSCLC cells by inhibiting the expression of PTEN through activation of PPARβ/{delta}, which stimulates PI3K/Akt and NF-{kappa}B signaling. Overexpression of PTEN overcomes this effect and unveils PPARβ/{delta} and PTEN as potential therapeutic targets in NSCLC.

nuclear receptor; tumor suppressor; kinase signals; transcription factor; tumor cells

LUNG CANCER REMAINS THE LEADING cause of cancer-related mortality in the United States and 30–40% of newly diagnosed patients with non-small cell lung cancer (NSCLC) present with unresectable stage III disease (15, 40). Despite recent advances in understanding the molecular biology of lung carcinoma and the introduction of multiple new chemotherapeutic agents for its treatment, its dismal 5-yr survival rate (<15%) has not changed substantially (15, 18).

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-dependent transcription factors. There are three major PPAR isoforms: {alpha}, β/{delta}, and {gamma}; each with distinct tissue and cellular distributions, different modes of expression, and diverse agonist binding properties (37). In contrast to PPAR{alpha} and PPAR{gamma}, the consequences of PPARβ/{delta} activation are not well known (32). PPARβ/{delta} is expressed throughout the body in most tissues, such as brain, colon, skeletal muscle, and skin (6), and it is likely to play roles in cell proliferation, differentiation and survival, lipid metabolism, and development (23, 33). Activation of PPARβ/{delta} has also been shown to increase the growth of human cancers including colon, breast, and prostate, among others (3, 25, 42). We recently demonstrated that the PPARβ/{delta} agonist GW501516 stimulated NSCLC cell growth through upregulation of the PGE2 receptor subtype EP4 and downregulation of phosphate and tensing homolog deleted on chromosome 10 (PTEN) (19), thereby unveiling a mechanism of action for its effects on lung carcinoma. Here, we further explore this mechanism. PTEN is a dual-specificity lipid and protein phosphate and a negative regulator of phosphatidylinositol 3-kinase (PI3K)-mediated signaling. It functions as a tumor suppressor directly antagonizing growth factor receptor and integrin-stimulated signaling, thus promoting cell cycle arrest and decreasing cell migration (34, 36). Interestingly, overexpression of PTEN inhibits cell adhesion, migration, and invasion, whereas loss of PTEN is often associated with advanced stage tumors, such as lung and prostate, among others (44, 52).

In this study, we explore the cellular mechanisms responsible for the downregulation of PTEN expression by a selective PPARβ/{delta} agonist, GW501516, in NSCLC cells. We show that GW501516 induces the proliferation of NSCLC cells via inhibition of PTEN expression through activation of PPARβ/{delta}. This was associated with the activation of the PI3K pathway and induction of transcription factor NF-{kappa}B. Finally, we show that overexpression of PTEN reversed the effects of GW501516.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Culture and chemicals. The NSCLC cell lines H1838 and H2106 were obtained from The American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 medium supplemented with 10% FBS, HEPES buffer, 50 IU/ml penicillin/streptomycin, and 1 µg amphotericin (complete medium) as previously described (20). GW501516 was purchased from EMD Biosciences (San Diego, CA); [methyl-3H]thymidine was purchased from Amersham Biosciences (Piscataway, NJ); {gamma}-32P-dATP was purchased from Perkin Elmer Life Sciences (Boston, MA); antibodies against p65 (C-20) and IKB{alpha} (C-21) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies against PTEN (#9552), Akt (#9272), and their phosphorylated forms (#9271), and the inhibitors of PI3K (LY-294002) and ERK (PD-98059) were purchased from Cell Signaling Technology (Beverly, MA); the PPARβ/{delta} polyclonal antibody (#101720) was purchased from Cayman Chemical (Ann Arbor, MI); the Gel Shift Assay System and the Dual-Luciferase Report Assay kit were obtained from Promega (Madison, WI); the LightCycler-FastStart DNA Master SYBR Green I kit and the 5' DNA Terminus Labeling System were purchased from Roche Molecular Biochemicals (Indianapolis, IN); RT-PCR kit components were obtained from Perkin Elmer (Foster City, CA); ammonium pyrrolidinedithiocarbamate (PDTC) and all other chemicals were purchased from Sigma Aldrich (St. Louis, MO) unless otherwise indicated.

Western blot analysis. The procedure was performed as previously described (21). Protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad). Equal amounts of protein from whole cell lysates were solubilized in 2x SDS-sample buffer and separated on 10% SDS polyacrylamide gels. Blots were incubated with antibodies raised against PPARβ/{delta} (1:2,000), PTEN, p65, IKB{alpha}, Akt, and their phosphorylated forms (1:1,000). The blots were washed and followed by incubation with a secondary goat antibody raised against rabbit IgG conjugated to horseradish peroxidase (1:2,000, Cell Signaling). The blots were washed, transferred to freshly made ECL solution (Amersham, Arlington, IL) for 1 min, and exposed to X-ray film. In controls, the antibodies were omitted or replaced with a control rabbit IgG.

Reverse transcriptase PCR. Total RNA was prepared from human lung carcinoma cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. To amplify 264-bp PTEN and 200-bp GAPDH cDNA fragments, the sequences of PCR primers (Sigma Genosys, Woodlands, TX) were: for PTEN sense (5'-TCTACTCCTCCAACTCAGGAC-3') and antisense (5'-CATTATCCGCACGCTCTATAC-3') and for GAPDH sense (5'-CCATGGAGAAGGCTGGGG-3') and antisense (5'-CAAAGTTGTCATGGATGACC-3') according to published data (20, 26). RT-PCR was carried out as previously described (20). The samples were first denatured at 95°C for 30 s, followed by 32 PCR cycles, each with temperature variations as follows: 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. The last cycle was followed by an additional extension incubation of 7 min at 72°C. Analysis of amplicons was accomplished on 1% agarose gel containing 0.2 µg/µl ethidium bromide and visualized under UV transiluminator.

Real-time RT-PCR. Real-time RT-PCR was performed as described previously (20). Briefly, the treatment and total RNA preparation were identical to those described for RT-PCR. All PCR reactions using LightCycler-FastStart DNA Master SYBR Green I kit were performed in the Cepheid Smart-Cycler real-time PCR cycler (Sunnyvale, CA). The cycling conditions were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 60°C for 10 s, and 72°C for 10 s. Final results, which were expressed as n-fold differences in PTEN gene expression relative to the GAPDH gene, were calculated using a formula based on a doubling of the product after each cycle (12). Experiments were performed in triplicate for each data point. For all experiments, controls without templates were included.

Immunoprecipitation assays. Protein lysates were prepared from NSCLC cells treated with LY-294002 and with GW501516 for 24 or 48 h by extraction in modified RIPA buffer [50 mM Tris (pH 7.4), 0.5% Nonidet P-40, 0.25% Na-deoxycholate, 125 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, and protease inhibitor cocktail] on ice for 1 h. Cells were sonicated for 10 s, lysates centrifuged at 12,000 g for 15 min at 4°C, and the supernatant was removed for immunoprecipitation (IP). Samples containing 200 µg of proteins were precleared for 30 min with 30 µl of protein A/G Plus-agarose (sc-2003, Santa Cruz Biotechnology) and incubated for 1 h at 4°C with the appropriate antibodies (anti-PPARβ/{delta}, anti-IKB{alpha}, or anti-p65) or normal IgG preabsorbed to protein A/G Plus-agarose. Immune complexes were collected after incubation overnight at 4°C and washed once with lysis buffer and three times with PBS. Protein was eluted by boiling in 25 µl of 2x SDS sample buffer [125 mM Tris (pH 6.8), 10% 2-mercaptoethanol, 4% SDS, 20% glycerol], and eluted proteins were analyzed by SDS-PAGE and Western blotting using the appropriate antibodies described above.

Short interfering RNA treatment. The PPARβ/{delta} short interfering RNA (siRNA; #sc-36305), p65 siRNA (#sc-29410), I{kappa}B{alpha} siRNA (#sc-29360), PTEN siRNA (#sc-29452), and control siRNA (#sc-37007) were purchased from Santa Cruz Biotechnology; Akt siRNA (#6211) was purchased from Cell Signaling Technology. For the transfection procedure, cells were grown to 70% confluence, and PPARβ/{delta}, p65, Akt, I{kappa}B{alpha}, and PTEN siRNAs and their control siRNAs were transfected using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. Briefly, Lipofectamine 2000 reagent was incubated with serum-free medium for 10 min. Subsequently, a mixture of respective siRNAs was added. After incubation for 15 min at room temperature, the mixture was diluted with medium and added to each well. The final concentration of PPARβ/{delta}, p65, Akt, I{kappa}B{alpha}, and PTEN siRNAs in each well was 100 nM. After culturing for 40 h, cells were washed and resuspended in new culture media. Afterwards, the cells were treated with GW501516 for an additional 24 h followed by Western blot and 3H-thymidine incorporation assay.

Transient transfection assays. The human PTEN promoter constructs (pGL3-PTEN2526/427) ligated to the luciferase reporter gene were a gift from Dr. Vuk Stambolic, Division of Signaling Biology, Ontario Cancer Institute, University of Toronto, and have been reported previously (41). Briefly, NSCLC cells were seeded at a density of 5 x 105 cells/well in six-well dishes and grown to 50–60% confluence. For each well, 2 µg of the above PTEN plasmid DNA constructs, with or without 0.2 µg of the internal control phRL-TK Synthetic Renilla Luciferase Reporter Vector, were cotransfected into the cells using FUGENE 6 lipofection reagent (Roche Molecular Biochemicals) as described in our earlier work (20). After 24 h of incubation, cells were treated with or without GW501516 (1 µM) for an additional 24 h. The preparation of cell extracts and measurement of luciferase activities were carried out using the Dual-Luciferase Reporter Kit according to recommendations by the manufacturer (Promega). The assays for firefly luciferase activity and Renilla luciferase activity were performed sequentially in a Labsystems Luminoskan Ascent luminometer equipped with dual injectors. Changes in firefly luciferase activity were calculated and plotted after normalization with changes in Renilla luciferase activity within the same sample.

Adenovirus-mediated transfer of the PTEN gene. The adenoviral vector encoding a human PTEN (Ad-PTEN) gene and a control vector (Ad-CMV) were a gift from Dr. Jonathan M. Kurie (Univ. of Texas, M. D. Anderson Cancer Center). The construction and production of Ad-PTEN have been described previously (43). The particle numbers for Ad-PTEN and Ad-CMV were 1.98 x 1012 and 1.76 x 1012 particles/ml, respectively. H1838 cells were infected with control PBS, Ad-CMV empty vectors, and Ad-PTEN (from 100 to 10,000 particles per cells each) in PBS in six-well plates at 37°C incubation for 1 h before adding mediums and cultured up to 72 h. Afterwards, the exogenous PTEN protein levels were determined by Western blot analysis. In separate experiments, cell counting assays and [methyl-3H]thymidine incorporation assays described below were performed to determine cell growth; Western blot analysis was performed to evaluate Akt, IKB{alpha}, and p65 proteins.

Cell counting assay. NSCLC cells (1 x 105) were infected with or without Ad-PTEN, Ad-CMV, or control PBS as described before for up to 5 days. Afterwards, the cells were harvested by trypsinization using trypsin/EDTA, and cell numbers were counted under the microscope after trypan blue staining starting from day 1 until day 5.

[Methyl-3H]thymidine incorporation assay. NSCLC cells (0.5 x 104) were infected with or without Ad-PTEN, Ad-CMV for up to 72 h, or transfected with p65, IKB{alpha}, and PTEN siRNAs for 40 h before incubation with 1 µCi/ml [methyl-3H]thymidine (Amersham, specific activity 250 Ci/mmol) in the presence or absence of GW501516 (1 µM) for an additional 48 h. The medium was removed and the attached cells were washed with 1x PBS. Afterwards, the attached cells were treated with ice-cold 6% trichloroacetic acid (TCA) at 4°C for 20 min and washed once with 6% TCA. The cells were then solubilized with 0.1 N NaOH and counted in a liquid scintillation counter in 4 ml of scintillation fluid.

Cell viability assay. NSCLC cells (104 cells/well) were transfected with p65 or PTEN siRNAs for 40 h, or infected with Ad-PTEN or Ad-CMV for up to 96 h before exposing the cells to GW501516 (1 µM) for an additional 48 h in 96-well plates. Afterwards, the numbers of viable cells in culture were determined using The CellTiter-Glo Luminescent Cell Viability Assay kit which is based on quantitation of ATP, an indicator of metabolically active cells, according to the manufacturer's instructions (Promega).

EMSA. Nuclear protein extracts from NSCLC cells treated with GW501516 were prepared for EMSA as described earlier (20). The protein content of the nuclear extract was determined using the Bradford protein assay kit (Bio-Rad). EMSA experiments were performed as described before (20). The double-stranded oligonucleotides for NF-{kappa}B were as follows: wild-type NF-{kappa}B (5'-AGT TGA GGG GAC TTT CCC AGG C-3'), mutant NF-{kappa}B (5'-AGT TGA GGG GAC TTT CCC AGG C-3'). Nuclear proteins (5 µg) from control and treated cells were incubated with 32P-labeled oligonucleotide probe in the presence or absence of antibody against p65 protein (2 µg/µl). For cold competition, a 100-fold excess of the respective unlabeled consensus oligonucleotide was added before adding probe. The same amount of mutated oligonucleotides was used as another control.

Statistical analysis. All experiments were repeated a minimum of three times. All data were expressed as means ± SD. The data presented in some figures are from a representative experiment, which was qualitatively similar in the replicate experiments. Statistical significance was determined with Student's t-test (2-tailed) comparison between two groups of data set. Asterisks shown in the figures indicate significant differences of experimental groups compared with the corresponding control condition (P < 0.05, see figure legends).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PPARβ/{delta} agonist inhibits PTEN expression in a dose- and time-dependent manner through activation of PPARβ/{delta} and PI3K signals via new protein synthesis. We previously showed that activation of PPARβ/{delta} stimulated NSCLC cell growth (19). Since PTEN acts as a tumor suppressor, we tested whether activation of PPARβ/{delta} affects PTEN expression. In H1838 NSCLC cells, GW501516, a selective PPARβ/{delta} agonist, significantly inhibited PTEN mRNA expression in a dose- and time-dependent manner as determined by RT-PCR (Fig. 1, A and B). This result was confirmed by real-time RT-PCR analysis (Fig. 1C). Similarly, GW501516 significantly inhibited PTEN protein expression in a dose- and time-dependent fashion (Fig. 1, D and E). Next, we tested the specificity of the agonist by examining whether blockade of PPARβ/{delta} activation could influence the effects of GW501516 on PTEN expression. We found that silencing of PPARβ/{delta} using siRNA duplexes abolished the effect of GW501516 on PTEN protein expression, whereas the control siRNA had no effect (Fig. 1F). Note that the PPARβ/{delta} siRNA blocked PPARβ/{delta} expression, whereas GW501516 induced PPARβ/{delta} protein levels (Fig. 1F). GW501516 had no effect on the expression of PPAR{gamma} protein, another PPAR isoform (not shown). We also showed that the effect of GW501516 on PTEN protein expression was eliminated in the presence of cycloheximide (2 µg/ml) suggesting that new protein synthesis was required for this process (Fig. 1G). Similar results were obtained with H2106 cells (not shown).


Figure 1
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  		Fig. 1. The peroxisome proliferator-activated receptor β/{delta} (PPARβ/{delta}) agonist GW501516 inhibits phosphate and tensin homolog deleted on chromosome 10 (PTEN) gene expression in a dose- and time-dependent manner through activation of PPARβ/{delta} and PI3K and involves new protein synthesis. A: total RNA was isolated from H1838 cells that were cultured with increasing concentrations of GW501516 for 24 h followed by RT-PCR for PTEN. B: total RNA was isolated from H1838 cells that were cultured with GW501516 (1 µM) for the indicated time period followed by RT-PCR for PTEN mRNA. C: total RNA was isolated from H1838 cells cultured in the presence or absence of GW501516 (1 µM) followed by real-time RT-PCR analysis. GAPDH served as internal control for normalization purposes. *Significant differences from control (P < 0.05). Con, untreated control cells. D: cellular protein was isolated from H1838 cells that were cultured with increasing concentrations of GW501516 for up to 24, followed by Western blot analysis with antibodies against PTEN. E: cellular protein was isolated from H1838 cells that were cultured with GW501516 (1 µM) for indicated period of time followed by Western blot analysis with antibodies against PTEN protein. F: cellular protein was isolated from H1838 cells cultured for 30 h in the presence or absence of control or PPARβ/{delta} siRNA (100 nM each) before exposing the cells to GW501516 (1 µM) for an additional 24 h, then subjected to Western blot analysis for PTEN. G: cellular protein was isolated from H1838 cells that were cultured with cycloheximide (2 µg/ml) for 2 h before exposure of the cells to GW501516 (1 µM) for 24 h followed by Western blot analysis with antibodies against PTEN protein. H: cellular protein was isolated from H1838 cells that were treated with PD-98059 (25 µM) for 2 h before exposure of the cells to GW501516 (1 µM) for an additional 24 h. Afterwards, Western blot analysis was performed using polyclonal antibodies against PTEN. I: cellular protein was isolated from H1838 cells that were treated with LY-294002 (25 µM) for 2 h before exposure of the cells to GW501516 (1 µM) for an additional 24 h. Afterwards, Western blot analysis was performed using polyclonal antibodies against PTEN. Actin served as internal control for normalization purposes. Con, DMSO solvent control cells.

 
We previously demonstrated that GW501516 activated the phosphorylation of PI3K and extracellular signal-regulated kinase (ERK) signaling pathways in NSCLC cells (19). Thus, we examined whether these pathways were involved in the effect of GW501516 on PTEN expression. We showed that, while blockade of ERK signals did not block the inhibitory effects of GW50161 on PTEN (Fig. 1H), the inhibition of PI3K, LY-294002, restored PTEN protein levels in the setting of GW501516 (Fig. 1I).

GW501516 inhibits PTEN promoter activity and increases NF-{kappa}B DNA binding activity and p65 protein expression. We next examined whether the effects of GW501516 on PTEN expression occur at the transcriptional level. We found that when exposed to GW501516, H1838 cells transfected with the full-length wild-type PTEN promoter luciferase reporter construct showed decreased promoter activity, and this was abrogated by PPARβ/{delta} siRNA (Fig. 2A) and by the PI3K inhibitor, LY-294002, although less efficiently (Fig. 2B). Note that the PPARβ/{delta} siRNA greatly reduced the production of PPARβ/{delta} protein, whereas the control siRNA had no effect (Fig. 2A, top). Also, no changes were observed in promoter activity in cells transfected with control empty vector in the setting of GW501516 stimulation (not shown). Similar results were obtained with H2106 cells (not shown).


Figure 2
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  		Fig. 2. GW501516 reduces PTEN promoter activity and increases NF-{kappa}B DNA binding activity and p65 protein expression. A: H1838 lung cancer cells (1 x 105 cells) were transfected with control or PPARβ/{delta} siRNA (100 nM each) for 40 h followed by cotransfection with a wild-type PTEN promoter reporter construct (pGL3-PTEN2526/427) ligated to a luciferase reporter gene along with an internal control phRL-TK Synthetic Renilla Luciferase Reporter Vector as described in MATERIALS AND METHODS for 24 h in the presence or absence of GW501516 (1 µM). The inset at the top shows the Western blot result for PPARβ/{delta} protein production. B: cells were transfected with a wild-type PTEN promoter for 24 h and then treated with LY-294002 (25 µM) for 1 h before exposure of the cells to GW501516 (1 µM) for an additional 24 h. The ratio of firefly luciferase to renilla luciferase activity was quantified as described in MATERIALS AND METHODS. The bars represent the means ± SD of at least 4 independent experiments for each condition. *Significant increase of activity compared with controls. **Significance of combination treatment compared with GW501516 alone (P < 0.05). Con, DMSO solvent control cells. C: oligonucleotides containing the NF-{kappa}B site were end labeled with {gamma}32P-ATP and incubated with nuclear extracts (5 µg) from H1838 cells treated with GW501516 (1 µM) for 24 h. For competition assays, a molar excess (x100) of consensus NF-{kappa}B oligonucleotide was added to the binding reaction. To confirm specificity of binding, we used oligonucleotides containing a mutated NF-{kappa}B (Mut NF-{kappa}B) that were end labeled with {gamma}32P-ATP. Antibodies specific for NF-{kappa}B subunits p65 or p50 were included in the incubation mixture. D and E: cellular protein was isolated from H1838 cells that were treated with varying concentrations of GW501516 (D) and for increasing periods of time (E). Afterwards, Western blot analysis was performed using polyclonal antibodies against p65. The bar graph at the bottom represents the means ± SD of p65/actin of at least 3 independent experiments. Con, DMSO solvent control cells.

 
The NF-{kappa}B family of transcription factors is important in regulating the expression of cellular genes involved in inflammation, cellular transformation, proliferation, and metastasis (2). The PTEN promoter contains NF-{kappa}B DNA binding sites, and suppression of PTEN expression by NF-{kappa}B prevents apoptosis (46). Thus, we set out to test if the effects of GW501516 were mediated through NF-{kappa}B. First, we showed that GW501516 increased DNA binding activity of NF-{kappa}B via gel mobility shift assays (EMSA) (Fig. 2C). Addition of an anti-p65 antibody resulted in supershift of this binding complex, whereas an anti-p50 antibody had no effect (Fig. 2C). Second, we found that GW501516 increased the expression of p65 protein in a dose- and time-dependent fashion (Fig. 2, D and E). GW501516 had no effect on NF-{kappa}B subunit p50 protein levels (not shown). Similar results were obtained with H2106 cells (not shown).

The effect of GW501516 on p65 protein expression is dependent on activation of PPARβ/{delta} and PI3K signaling. Having demonstrated that GW501516 stimulates NF-{kappa}B signaling through DNA binding of p65 complexes of NF-{kappa}B, we then investigated whether this process involved protein-protein interactions between PPARβ/{delta} and p65. We showed that anti-PPARβ/{delta} efficiently immunoprecipitated a single PPARβ/{delta} protein, whereas IgG showed no immunoprecipitated product (Fig. 3A). Next, coimmunoprecipitation experiments found that PPARβ/{delta} coimmunoprecipitated with p65, and vice versa, and this was greatly enhanced by GW501516. Note that the control IgG had no immunoprecipitated effect (Fig. 3B). Furthermore, to investigate if PPARβ/{delta}-p65 interactions also lead to enhanced NF-{kappa}B binding activity, cells silenced for p65 were exposed to GW501516. We showed that the stimulatory effect of GW501516 on NF-{kappa}B DNA binding activity was abrogated in cells with knockdown of the p65 gene (Fig. 3C). We next examined if the effect of GW501616 on p65 protein expression was mediated through activation of PPARβ/{delta}. We showed that PPARβ/{delta} siRNA eliminated the effect of GW501516 on p65 protein expression, whereas the control siRNA had no effect. This suggested that activation of PPARβ/{delta} was required in this process (Fig. 3D).


Figure 3
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  		Fig. 3. Activation of PPARβ/{delta} and PI3K is required for the effect of GW501516 on p65 protein. A: cell lysates were immunoprecipitated with either IgG or anti-PPARβ/{delta}. Immunoprecipitates were separated by 10% SDS-PAGE Tris-glycine buffer, transferred onto nitrocellulose, and Western blot analysis was carried out with anti-PPARβ/{delta} antibody. B: cells were treated with GW501516 (1 µM) for 24 h, and cell lysates were immunoprecipitated with either IgG or anti-PPARβ/{delta}. Immunoprecipitates were separated by 10% SDS-PAGE Tris-glycine buffer, transferred onto nitrocellulose, and Western blot analysis was carried out with either anti-PPARβ/{delta} or anti-p65 antibodies. C: oligonucleotides containing the NF-{kappa}B site were end labeled with {gamma}32P-ATP and incubated with nuclear extracts (5 µg) from H1838 cells transfected with control or p65 siRNA (100 nM each) for 40 h before exposure of the cells to GW501516 (1 µM) for an additional 24 h. D: cellular protein was isolated from H1838 cells cultured for 30 h in the presence or absence of control or PPARβ/{delta} siRNA (100 nM each) before exposure of the cells to GW501516 (1 µM) for an additional 24 h and then subjected to Western blot analysis for p65. E: oligonucleotides containing the NF-{kappa}B site were end labeled with {gamma}32P-ATP and incubated with nuclear extracts (5 µg) from H1838 cells treated with LY-294002 (25 µM) for 2 h before exposure of the cells to GW501516 (1 µM) for an additional 24 h. For competition assays, a molar excess (x100) of consensus NF-{kappa}B oligonucleotides (Cold NF-{kappa}B) was added to the binding reaction. To confirm specificity of binding, we used a molar excess (x100) of oligonucleotides containing a mutated NF-{kappa}B (Mut NF-{kappa}B). F: cellular protein was isolated from H1838 cells that were treated with LY-294002 (25 µM) for 2 h before exposure of the cells to GW501516 (1 µM) for an additional 24 h. Afterwards, Western blot analysis was performed using polyclonal antibodies against p65. The bar graph at the bottom represents the means ± SD of p65/actin of at least 3 independent experiments. Con, DMSO solvent control cells. G: cellular protein was isolated from H1838 cells transfected with control or Akt siRNA (100 nM each) for 40 h before exposure of the cell to GW501516 (1 µM) for an additional 24 h. Afterwards, Western blot analysis was performed using polyclonal antibodies against total Akt and p65. Actin served as internal control for normalization purposes. H: cellular protein was isolated from H1838 cells treated with PD-98059 (25 µM) for 2 h before exposing the cells to GW501516 (1 µM) for an additional 24 h. Afterwards, Western blot analysis was performed using polyclonal antibodies against p65. Actin served as internal control for normalization purposes. I: cells were treated with LY-294002 (25 µM) for 1 h before exposure of the cells to GW501516 (1 µM) for up to 24 h, and cell lysates were immunoprecipitated with either IgG, anti-PPARβ/{delta}. Immunoprecipitates were separated by 10% SDS-PAGE Tris-glycine buffer, transferred onto nitrocellulose, and Western blot analysis was carried out with either anti-PPARβ/{delta} or anti-p65 antibodies. Con, DMSO solvent control cells.

 
GW501516 has been shown to activate PI3K signaling (19). Here, we demonstrated that an inhibitor of PI3K, LY-294002, diminished the effect of GW501516 on NF-{kappa}B binding activity and on p65 protein expression (Fig. 3, E and F). Consistent with a role for PI3K/Akt signaling, we showed inhibition of p65 expression with Akt siRNA, whereas control siRNA had no effect (Fig. 3G). In contrast, the inhibitor of ERK, PD-98059, had little effect (Fig. 3H). In addition, immunoprecipitation experiments showed that LY-294002 diminished the formation of PPARβ/{delta} and p65 proteins complexes (Fig. 3I).

The role of p65 and IKB{alpha} in mediating the effect of GW501516 on PTEN expression and on human lung carcinoma cell growth. To further determine the role of NF-{kappa}B components in mediating the effect of GW501516 on PTEN expression and on cell growth, we next assessed whether blockade of NF-{kappa}B abolished the effects of GW501516 on PTEN. NSCLC cells transfected with p65 siRNA showed inhibition of p65 protein expression, whereas the control siRNA had no effect (Fig. 4A). The p65 siRNA abrogated the effects of GW501516 on PTEN protein expression (Fig. 4A). A chemical NF-{kappa}B inhibitor, PDTC, also abolished the effect of GW501516 on PTEN expression (Fig. 4B). Similarly, silencing of p65 abolished the effect of GW501516 on PTEN promoter activity (Fig. 4C). In addition, the effect of GW501516 on cell growth was abated in the presence of p65 siRNA as determined by [3H]thymidine incorporation assay (Fig. 4D, bottom) and by cell viability assay (not shown). Note that the p65 siRNA blocked the p65 protein production, whereas the control siRNA had no effect (Fig. 4, C and D, top). Similar results were obtained with H2106 cells (not shown).


Figure 4
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  		Fig. 4. The role of p65 and IKB{alpha} in mediating the effect of GW501516 on PTEN expression and on human lung carcinoma cell growth. A: H1838 cells (0.5 x 104 cells/well) were transfected with p65 siRNA or control siRNA (100 nM each) for 30 h before exposure of the cells to GW501516 for an additional 24 h and then subjected to Western blot analysis for PTEN. Con, DMSO solvent control cells. B: cellular protein was isolated from H1838 cells that were treated with ammonium pyrrolidinedithiocarbamate (PDTC; 100 µM) for 2 h before exposure of the cells to GW501516 (1 µM) for an additional 24 h. Afterwards, Western blot analysis was performed using polyclonal antibodies against PTEN. Con, untreated control cells. C: H1838 lung cancer cells (1 x 105 cells) were transfected with control or p65 siRNA (100 nM each) for 40 h followed by cotransfection with a wild-type PTEN promoter reporter construct (pGL3-PTEN2526/427) ligated to a luciferase reporter gene along with an internal control phRL-TK Synthetic Renilla Luciferase Reporter Vector as described in MATERIALS AND METHODS for 24 h in the presence or absence of GW501516 (1 µM). The inset at the top showed the Western blot result for p65 protein production. The ratio of firefly luciferase to renilla luciferase activity was quantified as described in MATERIALS AND METHODS. The bars represent the means ± SD of at least 4 independent experiments for each condition. *Significant decrease of activity compared with controls. **Significance of combination treatment compared with GW501516 alone (P < 0.05). Con, DMSO solvent control cells. D: H1838 cells (0.5 x 104 cells/well) were treated with either solvent control or transfected with control siRNA or p65 siRNA (100 nM) for 30 h, followed by GW501516 (1 µM), and incubated with 1 µCi/ml [methyl-3H]thymidine for an additional 24 h. Afterwards, cell proliferation was determined. The inset at the top showed the Western blot result for p65 protein production. *Significant increase as compared to control. **Significance of combination treatment as compared to GW501516 alone.

 
We also tested if GW501516 affected the induction of NF-{kappa}B through effects on IKB{alpha}/IKBβ, one of the inhibitory protein complexes of the NF-{kappa}B family. GW501516 inhibited IKB{alpha} protein expression in a dose- and time-dependent manner (Fig. 5, A and B). However, GW501516 had no effect on IKBβ protein (not shown). As expected, IKB{alpha} siRNA enhanced the effect of GW501516 on PTEN protein expression (Fig. 5C), whereas knockdown of IKBβ had no effect (not shown). Silencing of IKB{alpha} also enhanced GW501516-induced cell proliferation (Fig. 5D, bottom). Note that the IKB{alpha} siRNA blocked IKB{alpha} protein production, whereas the control siRNA had no effect (Fig. 5D, top). Interestingly, GW501516 also reduced IKB{alpha} binding to p65 protein presumably because of lower IKB{alpha} levels (Fig. 5E). In contrast, silencing of IKB{alpha} augmented the GW501516-induced PPARβ/{delta}-p65 complex formation (Fig. 5F), whereas it had no effect on expression of either PPARβ/{delta} or p65 protein (not shown).


Figure 5
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  		Fig. 5. The role of IKB{alpha} in mediating the effect of GW501516 on PTEN, p65 expression, and on cell proliferation. A and B: effects of GW501516 on IKB{alpha} protein expression. Cellular protein was isolated from H1838 cells that were treated with varying concentrations of GW501516 (A) and for increasing periods of time (B). Afterwards, Western blot analysis was performed using polyclonal antibodies against IKB{alpha}. C: H1838 cells (0.5 x 104 cells/well) were transfected with IKB{alpha} siRNA or control siRNA (100 nM each) for 30 h before exposing the cells to GW501516 for an additional 24 h and then subjected to Western blot analysis for PTEN protein. D: H1838 cells (0.5 x 104 cells/well) were treated with either solvent control or transfected with control siRNA or IKB{alpha} siRNA (100 nM) for 30 h, followed by GW501516 (1 µM) treatment, and then incubated with 1 µCi/ml [methyl-3H]thymidine for an additional 24 h. Afterwards, cell proliferation was determined. The inset at the top showed the Western blot result for IKB{alpha} protein production. E: cells were treated with GW501516 (1 µM) for 24 h, and cell lysates were immunoprecipitated with either IgG or anti-IKB{alpha}. Immunoprecipitates were separated by 10% SDS-PAGE Tris-glycine buffer, transferred onto nitrocellulose, and Western blot was carried out with either anti-IKB{alpha} or anti-p65 antibody. F: H1838 cells (0.5 x 104 cells/well) were transfected with IKB{alpha} siRNA or control siRNA (100 nM each) for 30 h before exposure of the cells to GW501516 for an additional 24 h. Afterwards, cell lysates were immunoprecipitated with either IgG or anti-PPARβ/{delta}. Immunoprecipitates were separated by 10% SDS-PAGE in Tris-glycine buffer, transferred onto nitrocellulose, and Western blot was carried out with either anti-PPARβ/{delta} or anti-p65 antibody. Con, DMSO solvent control cell.

 
Overexpression of PTEN antagonizes the effect of GW501516 on phosphorylation of Akt and on NSCLC cell growth. Based on the above, we believe that activation of PPARβ/{delta} by GW501516 promotes tumor cell proliferation via inhibition of PTEN. To further assess the contribution of PTEN in mediating the effects of GW501516, we infected NSCLC cells with an adenoviral vector encoding the human PTEN (Ad-PTEN) gene. We showed that infection with increasing numbers of viral particles per cell (vp/cell) of Ad-PTEN resulted in increased expression of PTEN protein in a time-dependent manner (Fig. 6, A and B). Ad-PTEN significantly inhibited NSCLC cell growth compared with cells treated with empty vector Ad-CMV or PBS (Fig. 6C). [3H]thymidine incorporation experiments further confirmed that Ad-PTEN diminished the effect of GW501516 on cell growth, whereas cells treated with PBS or transfected with Ad-CMV empty vector had no effect (Fig. 6D). Similar results were obtained with H2106 cells (not shown).


Figure 6
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  		Fig. 6. Overexpression of PTEN antagonizes the effect of GW501516 on phosphorylation of Akt and on non-small cell lung carcinoma (NSCLC) cell growth. A: H1838 cells infected with increased particle numbers of Ad-PTEN as indicated for up to 72 h, followed by Western blot analysis for PTEN protein expression. B: H1838 cells (1 x 104 cells/well) were infected with 5,000 particle numbers/per cell of Ad-PTEN for different periods of time as indicated, followed by Western blot analysis for PTEN protein expression. Both exogenous and endogenous PTEN expressions were observed. Actin served as internal control for normalization purposes. C: H1838 cells (1 x 104 cells/well) were infected with Ad-PTEN, Ad-CMV, or PBS control for up to 5 days. Afterwards, cell numbers were determined. D: H1838 cells (1 x 104 cells/well) were infected with or without Ad-PTEN or Ad-CMV [5,000 viral particles per cell (vp/cell)] for 48 h and incubated with GW501516 (1 µM) and 1 µCi/ml [methyl-3H]thymidine for an additional 24 h. Afterwards, DNA synthesis as reflected by cpm counts for thymidine incorporation was determined. E: H1838 cells were infected with or without Ad-PTEN or Ad-CMV (5,000 vp/cell) for 72 h before exposure of the cells to GW501516 (1 µM) for an additional 24 h followed by Western blot analysis with antibodies against total Akt and phosphorylated Akt (p-Akt). Actin served as internal control for normalization purposes. F: H1838 cells were infected with or without Ad-PTEN or Ad-CMV (5,000 vp/cell) for 72 h before exposure of the cells to GW501516 (1 µM) for an additional 24 h followed by Western blot analysis with antibodies against p65 protein. Actin served as internal control for normalization purposes. G: H1838 cells were infected with or without Ad-PTEN or Ad-CMV (5,000 vp/cell) for 72 h before exposure of the cells to GW501516 (1 µM) for an additional 24 h. Afterwards, cell lysates were immunoprecipitated with either IgG or anti-PPARβ/{delta}. Immunoprecipitates were separated by 10% SDS-PAGE in Tris-glycine buffer, transferred onto nitrocellulose, and Western blot was carried out with either anti-PPARβ/{delta} or anti-p65 antibody. H: H1838 cells were infected with or without Ad-PTEN or Ad-CMV (5,000 vp/cell) for 72 h before exposure of the cells to GW501516 (1 µM) for an additional 24 h followed by Western blot analysis with antibodies against IKB{alpha} protein. Actin served as internal control for normalization purposes. The bar graph at the bottom represents the means ± SD of IKB{alpha}/actin of at least 3 independent experiments. Con, DMSO solvent control cells. *Significant difference from control (Con). **Significance of combination treatment compared with GW501516 alone.

 
We then tested the effect of PTEN overexpression on signaling pathways implicated in control of tumor cell growth. As expected, we showed that Ad-PTEN reduced the basal phosphorylation of Akt. It also alleviated the phosphorylation of Akt induced by GW501516 (Fig. 6E). PBS (not shown) and the Ad-CMV empty vector had no effect (Fig. 6E). We also showed that Ad-PTEN attenuated the effect of GW501516 on expression of p65 protein, whereas Ad-CMV had no effect (Fig. 6F). In addition, cells transfected with Ad-PTEN diminished the physical interaction between PPARβ/{delta} and p65 proteins induced by GW501516, whereas cells transfected with Ad-CMV had no effect (Fig. 6G). In addition, we found that Ad-PTEN not only induced IKB{alpha}, but also blocked the inhibitory effect of GW501516 on expression of IKB{alpha} protein (Fig. 6H).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
PTEN, also named MMAC1 or TEP1, is a tumor suppressor gene located on human chromosome 10q23 (39) that has been found to be mutated in several human sporadic cancers such as those in breast, endometrium, ovaries, and prostate (14). PTEN has been shown to play a pivotal role in apoptosis, cell cycle arrest, and angiogenesis (29). It antagonizes the PI3K pathway by dephosphorylating the signaling lipid phosphatidylinositol 3,4,5-triphosphate. In human lung carcinoma cells, overexpression of PTEN results in cell growth arrest (28, 51) suggesting that PTEN plays an important role in controlling lung cancer cell survival.

In view of the above, we tested whether PPARβ/{delta} regulates PTEN. As a member of the nuclear hormone receptor subfamily of ligand-dependent transcription factor, PPARβ/{delta} has been shown to be involved in fatty acid oxidation in skeletal muscle, insulin sensitivity, cell differentiation, cell survival, and tumor growth (9, 42). GW501516, a selective agonist of PPARβ/{delta}, has been shown to activate PPARβ/{delta} resulting in increased growth of breast, colon, and prostate cancer cells, and of primary endothelial cells (42). This suggests that PPARβ/{delta} antagonists may be of therapeutic value in the treatment of these cancers.

We found that GW501516 inhibited the expression of PTEN, which was overcome by PPARβ/{delta} siRNA and by cyclohexamide, suggesting that activation of PPARβ/{delta} and new protein synthesis were needed for the regulation of PTEN expression by GW501516. To our knowledge, this is the first demonstration of a link between PTEN and PPARβ/{delta} in human lung cancer cells. In contrast, we have shown that GW501516 has no effect on PPAR{gamma}, another PPAR isoform (Han, unpublished data). This highlights the specificity of GW501516. As discussed above, PTEN activity normally serves to restrict growth and survival signals by limiting the activity of the PI3K pathway. The absence of functional PTEN in some cancer cells leads to constitutive activation of the PI3K pathway including Akt (36). GW501516 has been shown to activate PI3K in several cancer cells including lung (19, 47). We previously showed that silencing of PPARβ/{delta} blocked the stimulatory effect of GW501516 on phosphorylation of Akt (19). Here, we found that cyclohexamide had no effect on GW501616-induced phosphorylation of Akt, suggesting that activation of PI3K by GW501516 did not require new protein synthesis. These findings suggest that PPARβ/{delta} activates PI3K/Akt and indirectly inhibits PTEN. PTEN has been shown to be regulated at the level of gene transcription in different cell types (38, 46). Our findings further suggest that activation of PPARβ/{delta} and PI3K signaling are required for transcriptional inhibition of PTEN gene expression in cells treated with GW501516.

As demonstrated for PPAR{gamma}, PPARβ/{delta} has been shown to associate with some transcription factors; one of which is NF-{kappa}B (12, 22). The NF-{kappa}B family of transcription factors is ubiquitous in carcinogenesis. It has been linked to cellular transformation, proliferation, apoptosis suppression, invasion, angiogenesis, and metastasis (2, 13). PPAR{gamma} ligands appear to inhibit NF-{kappa}B and lead to inhibition of inflammation (11, 31), whereas activation of PPARβ/{delta} stimulates NF-{kappa}B in several cell systems such as in ours. For example, the PPARβ/{delta} agonist L-165041 increased NF-{kappa}B activity and matrix metalloproteinase-9 (MMP-9) production in response to TNF-{alpha} in primary keratinocytes (12). Another study showed that PPARβ/{delta} activation by GW501516 induced cyclooxygenase-2 gene expression and cell proliferation in human hepatocellular carcinoma cells through activation of p65 (NF-{kappa}B) (17). However, opposite results have been reported in certain non-tumor cell types (4, 27). We speculate that the role of NF-{kappa}B in mediating the effects of PPARβ/{delta} agonists differs between cancer cells and some non-tumor cells. Interestingly, the PTEN promoter contains NF-{kappa}B binding sites, and suppression of PTEN is critical for cell proliferation and anti-apoptosis by NF-{kappa}B (46). Whether NF-{kappa}B is a repressor of PTEN promoter activity needs to be explored further. Studies identifying possible regulatory elements within the PTEN promoter that are involved in GW501516-inhibited PTEN expression are also warranted. Data generated using an inhibitor of NF-{kappa}B and the silencing of p65 (this report) not only demonstrate the critical role for this transcription factor in the regulation of PTEN, but also exclude the possibility of any potential off-target effects of the NF-{kappa}B inhibitor. Overexpression of NF-{kappa}B-inducing kinase and p65 ameliorates PTEN expression (24). On the contrary, inducible expression of PTEN inhibits constitutive NF-{kappa}B gene activation (1) suggesting a reciprocal control of NF-{kappa}B and PTEN. Thus, NF-{kappa}B appears to be both upstream and downstream of PTEN. Recent studies have also unveiled an unappreciated tumor suppressor role for NF-{kappa}B, suggesting a possible dual role for this molecule (10). While further testing the relationship between PPARβ/{delta} and NF-{kappa}B, we found that the induction of PPARβ/{delta} by GW501516 induces the recruitment of the NF-{kappa}B subunit p65, and that the presence of p65, followed by PPARβ/{delta} and p65 protein interactions, serves to enhance NF-{kappa}B activity. Of note, the association of PPARβ/{delta} with p65 in response to GW501516 correlated with reduced expression of PTEN. Activation of PPARβ/{delta} has been shown to activate PI3K and ERK signals (12, 19). By blocking PI3K signals, we confirmed that this kinase signaling pathway is an upstream signal for NF-{kappa}B. In contrast, ERK plays no role in this process. The connection between the PI3K and NF-{kappa}B signals has been reported in other studies (5). For example, there is complete inhibition of epinephrine-induced transcription by NF-{kappa}B after pretreatment with the PI3K inhibitor LY-294002 (30). Also, LY-294002 inhibited Akt phosphorylation and NF-{kappa}B DNA-binding activity in acute myeloid leukemia cells (5).

We also examined the effects of GW501516 on IKB. IKB{alpha} binds to the p65 subunit of the NF-{kappa}B complex and prevents the nuclear translocation of NF-{kappa}B. Others found that reduction or degradation of IKB{alpha} facilitates NF-{kappa}B activation (7). As expected, siRNA approaches and immunoprecipitation data demonstrated that reduction of IKB{alpha} enhanced NF-{kappa}B in this study, and this might result in suppression of PTEN expression. However, silencing of IKB{alpha} had no effect on PTEN expression. Therefore, the direct role of IKB{alpha} in mediating the expression of PTEN needs to be confirmed.

Finally, we tested the true contribution of PTEN. Knockdown of PTEN enhanced the effect of GW501516 on cell growth, confirming the tumor suppressor role of PTEN. Conversely, others found that the adenovirus-mediated transfer of PTEN (Ad-PTEN) suppressed cell growth in colorectal cancer cells, but not in normal colorectal fibroblast cells (35). Overexpression of PTEN suppresses cell growth and sensitizes cancer cells to cell death by anticancer drugs through reduction of Akt activity. PTEN inhibited Wnt-1-induced mammary tumorigenesis in early neoplastic stages by blocking the Akt pathway and by reducing insulin-like growth factor receptor levels in mammary gland, pointing towards PTEN as a therapeutic target for the treatment of mammary cancer and presumably other types of cancer (50). In agreement with this, our data suggest that inhibition of NSCLC cell proliferation by PTEN is likely due to inhibition of Akt phosphorylation.

Our findings also suggest a negative feedback loop between PTEN and NF-{kappa}B. Similarly, inhibition of AP-1 and NF-{kappa}B by the tumor suppressor p53 in mouse epidermal Cl41 cells seems to be mediated via activation of PTEN (48). NF-{kappa}B activation was necessary and sufficient for inhibition of PTEN expression, whereas restoration of PTEN expression inhibited NF-{kappa}B transcriptional activity and augmented TNF-induced mouse embryo fibroblasts RelA 3T3 cell apoptosis (46). Overexpression of PTEN enhanced IKB{alpha} further demonstrating the role of PTEN in regulation of NF-{kappa}B activation.

It should be highlighted that our results implicating PPARβ/{delta} activation in the upregulation of lung carcinoma cell growth (19) contradict those reported elsewhere in which a decrease in proliferation was observed (16). That particular work was performed in another lung carcinoma cell line (A549) and with the use of L-165041, another PPARβ/{delta} agonist. Note that L-165041 has also been shown to act as an agonist to PPAR{gamma}, which is known to reduce tumor cell proliferation (49). Thus, it is uncertain whether the latter results were mediated by PPARβ/{delta} at the concentrations tested. Our report is also inconsistent with other studies showing that activation of PPARβ/{delta} increases or has no effect on PTEN in cardiac fibroblasts and in keratinocytes (8, 45). However, this discrepancy might be explained by differences in the cell systems studied, the cell culture conditions tested, or the PPARβ/{delta} agonists used. This needs to be explored further.

In summary, our findings demonstrate that the PPARβ/{delta} agonist GW501516 stimulates the proliferation of NSCLC cells by inhibiting the expression of PTEN through activation of PI3K and NF-{kappa}B (Fig. 7). These effects are dependent on PPARβ/{delta} activation and interactions between PPARβ/{delta} and p65. Blockade of NF-{kappa}B signals abrogated the effect of GW501516 on PTEN expression and on cell growth, suggesting a critical role of NF-{kappa}B in mediating the effects of GW501516. Through a negative feedback loop, overexpression of PTEN attenuated the effect of GW501516 on NF-{kappa}B and phosphorylation of Akt. This, in turn, suppressed NSCLC cell growth (Fig. 7). Thus, upregulation of PTEN may be a promising adjuvant therapeutic strategy for the treatment of patients with lung cancer.


Figure 7
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  		Fig. 7. Schematic representation of signal pathways triggered in NSCLC in response to PPARβ/{delta} agonist. By activating the PPARβ/{delta}-RXR{alpha} receptor heterodimer, the PPARβ/{delta} agonist, GW501516, stimulates PI3K/Akt signaling, which promotes the expression of the NF-{kappa}B p65 protein. This, in turn, triggers the formation of a PPARβ/{delta}-p65 complex that acts to inhibit PTEN expression, and, consequently, induces cell proliferation. GW50516 also decreases IKB{alpha} protein expression, further increasing NF-{kappa}B. Overexpression of PTEN overcomes these effects by inhibiting the activation of Akt, reducing the expression of p65, and, ultimately, inhibiting cell growth.

 

   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by American Lung Association Grant RG-10215N (S. W. Han), by American Thoracic Society (ATS)/LUNGevity Foundation Partnership Grant LC-06-004 (S. W. Han), by Emory University Research Fund 2-55016 (S. W. Han), and by a Merit Review Grant from the Department of Veterans Affairs (J. Roman).


   ACKNOWLEDGMENTS
 
We are very grateful to Dr. Vuk Stambolic (Div. of Signaling Biology, Ontario Cancer Institute, Univ. of Toronto) for providing the human PTEN promoter construct and Dr. Jonathan M. Kurie (Univ. of Texas, M. D. Anderson Cancer Center) for supplying the Ad-PTEN and Ad-CMV.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. W. Han, Division of Pulmonary, Allergy, and Critical Care Medicine, Emory Univ. School of Medicine, Whitehead Bioresearch Bldg., 615 Michael St., Suite 205-M, Atlanta, GA 30322 (e-mail: shan2{at}emory.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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