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The Journal of Immunology, 2006, 177: 3737-3745.
Copyright © 2006 by The American Association of Immunologists, Inc.

Peroxisome Proliferator-Activated Receptor {gamma} Promotes Lymphocyte Survival through Its Actions on Cellular Metabolic Activities1

Seung-Hee Jo*, Chunyan Yang*, Qi Miao*, Michal Marzec{dagger}, Mariusz A. Wasik{dagger}, Pin Lu{ddagger} and Y. Lynn Wang2,*

* Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, New York, NY 10021; {dagger} Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; and {ddagger} Immunoregulation Laboratory, Hospital of Special Surgery, New York, NY 10021


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) is a metabolic regulator that plays an important role in sensitizing tissues to the action of insulin and in normalizing serum glucose and free fatty acids in type 2 diabetic patients. The receptor has also been implicated in the modulation of inflammatory responses, and ligands of PPAR{gamma} have been found to induce apoptosis in lymphocytes. However, apoptosis induction may not depend on the receptor, because high doses of PPAR{gamma} agonists are required for this process. Using cells containing or lacking PPAR{gamma}, we reported previously that PPAR{gamma} attenuates apoptosis induced by cytokine withdrawal in a murine lymphocytic cell line via a receptor-dependent mechanism. PPAR{gamma} exerts this effect by enhancing the ability of cells to maintain their mitochondrial membrane potential during cytokine deprivation. In this report, we demonstrate that activation of PPAR{gamma} also protects cells from serum starvation-induced apoptosis in human T lymphoma cell lines. Furthermore, we show that the survival effect of PPAR{gamma} is mediated through its actions on cellular metabolic activities. In cytokine-deprived cells, PPAR{gamma} attenuates the decline in ATP level and suppresses accumulation of reactive oxygen species (ROS). Moreover, PPAR{gamma} regulates ROS through its coordinated transcriptional control of proteins and enzymes involved in ROS scavenging, including uncoupling protein 2, catalase, and copper zinc superoxide dismutase. Our studies identify cell survival promotion as a novel activity of PPAR{gamma} and suggest that PPAR{gamma} may modulate cytokine withdrawal-induced activated T cell death.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Peroxisome proliferator-activated receptors (PPARs)3 are members of the nuclear hormone receptor superfamily. They are involved in diverse biological functions and pathological conditions. PPARs consist of three subtypes, {alpha}, beta({delta}), and {gamma}, which differ in tissue distribution and ligand requirement. Like other members of the nuclear hormone receptors, PPARs serve as transcription factors and require ligands for activation. Among the subtypes, PPAR{gamma} is of particular interest because it plays a role in a variety of human diseases, including diabetes, atherosclerosis, inflammation, and cancer (1, 2). Ligands of PPAR{gamma} include derivatives of fatty acid molecules such as prostaglandins. 15-Deoxy-{Delta}12,14-PGJ2 (15d-PGJ2), a derivative of PGD2, is in particular thought to be one of the physiological ligands of PPAR{gamma}. Thiazolidinediones (TZDs; glitazone drugs), pharmacological agents for the treatment of type 2 diabetes, are also potent agonists for PPAR{gamma}.

Human PPAR{gamma} was first cloned from the bone marrow (3), and the receptor is expressed in monocytes/macrophages as well as in B and T lymphocytes and bone marrow precursors (4). Recent studies have demonstrated that activation of PPAR{gamma} suppresses inflammatory reactions in macrophages and monocytes. PPAR{gamma} ligand treatment leads to down-regulation of inflammatory genes, including inducible NO synthase and gelatinase B in activated macrophages (5), and inhibition of the production of inflammatory cytokines, including TNF-{alpha}, IL-6, and IL-1beta in monocytes (6). Moreover, ligands of PPAR{gamma}, including 15d-PGJ2 and troglitazone, are also effective against inflammatory diseases in animal models, including rheumatoid arthritis, inflammatory bowel disease, and allergic encephalomyelitis (7, 8, 9).

PPAR{gamma} also seems to play an anti-inflammatory role in lymphocytes. We and others (10, 11, 12) have shown that PPAR{gamma} is up-regulated during the activation of mouse and human T cells. In mouse T cells, this up-regulation requires the presence of IL-4 (10). Several studies have shown that treatment of T cells with PPAR{gamma} ligands leads to inhibition of T cell proliferation and a decrease in the production of IL-2 and IFN-{gamma} (13, 14, 15). A couple of groups have found that PPAR{gamma} ligands induce apoptosis in T, B, and lymphoma cell lines and suggest that the induction of lymphocyte death by PPAR{gamma} may contribute to its anti-inflammatory effects (12, 16, 17). It is, however, unclear whether the proapoptotic effects of PPAR{gamma} agonists are mediated through PPAR{gamma}, because high concentrations of the drugs are required to produce this effect in cell cultures. Accumulating evidence indicates that PPAR{gamma} agonists, such as 15d-PGJ2, ciglitazone and troglitazone, modulate various cellular activities not involving PPAR{gamma} (18, 19, 20, 21, 22, 23).

Although high concentrations of 15d-PGJ2 and ciglitazone indeed induce T cell death, we have observed no effects of these drugs on cell survival at the low concentrations that are comparable to their KD for PPAR{gamma} (11). Moreover, we have found that low doses of PPAR{gamma} ligands promote survival in an IL-3-dependent mouse lymphocytic cell line in the absence of the cytokine/growth factor. Using stable cell lines expressing or lacking PPAR{gamma}, we have shown that the survival-enhancing effects depend on both the presence and activation of PPAR{gamma}. Furthermore, PPAR{gamma} promotes cell survival by enhancing the ability of cells to maintain their mitochondrial membrane potential.

Lymphocytes depend on cytokines for survival as well as for proliferation. Cytokine withdrawal occurs in vivo following the peak of immune responses and causes apoptotic death in activated T lymphocytes (24). Removal of T cells at the end of the immune responses is thought to be important for the prevention of autoimmunity and the maintenance of T cell homeostasis. At the cellular level, cytokine withdrawal induces a series of metabolic events that precede cell death, including reduced glucose uptake, decreased glycolytic activity, depolarization of mitochondrial membrane, loss of mitochondrial integrity, and release of cytochrome c; these events ultimately lead to activation of caspases and cell death (25). Well-known anti-apoptotic factors such as Akt and Bcl-xL promote lymphocyte survival through their regulation of cellular metabolism. Akt, activated by the CD28 costimulatory signal, promotes glucose uptake and glycolysis that provide fuel to maintain mitochondrial function (26). Bcl-xL, a member of BCL2 family, prevents cell death by promoting efficient mitochondrial ATP/ADP exchange (27). Because PPAR{gamma} is a metabolic regulator, we investigated whether its prosurvival effects are linked to its regulation of metabolism in the current studies. We found that PPAR{gamma} activation leads to higher ATP levels in cytokine-withdrawn cells. Moreover, ATP production by mitochondria is required for PPAR{gamma} to exert its prosurvival effect. We also demonstrated that activation of PPAR{gamma} results in reduced reactive oxygen species (ROS) accumulation in growth factor-deprived cells. Furthermore, PPAR{gamma} regulates the ROS levels through transcriptional regulation of several proteins and enzymes that control cellular ROS. Taken together, we describe cell survival promotion as a novel activity of PPAR{gamma} that is sustained by a metabolic profile that favors cell survival. Our data suggest that PPAR{gamma} may play a role in modulating cytokine withdrawal-induced T cell death.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines and culture

FL5.12 cell lines transfected with pcDNA3.1-hPPAR{gamma}1 were described previously (11). Cells were cultured at 37°C in RPMI 1640 supplemented with 10% FBS (Mediatech), 0.3 pg/ml rIL-3 (BD Biosciences), 10 mM HEPES buffer, 1 mg/ml geneticin (Calbiochem), penicillin/streptomycin, and 50 µM 2-ME (Sigma-Aldrich). The lymphoma cell lines Karpas 299 and SUP-M2 have been described previously (28, 29, 30) and were maintained at 37°C in RPMI 1640 supplemented with 10% FBS and penicillin/streptomycin.

IL-3 withdrawal, serum starvation, and cell viability determination

To perform IL-3 withdrawal, FL5.12 cells were washed three times in RPMI 1640, resuspended, and cultured in otherwise complete medium lacking rIL-3. To perform serum withdrawal, cells were washed three times with RPMI 1640, resuspended, and cultured in serum-free medium. Cell viability was determined by cellular exclusion of 2 µg/ml propidium iodide followed by flow cytometric analysis of 10,000 events as described previously (11).

RNA preparation, reverse transcription, and real-time PCR

Total RNA was isolated from cells using an RNeasy kit (Qiagen) according to the manufacturer’s instructions. The amounts of total RNA were quantified using spectrophotometric measurements. RNA was reverse transcribed into cDNA using a Promega reverse transcription system according to the manufacturer’s protocol. Real-time PCRs were conducted in an ABI PRISM 7000 sequence detection system (Applied Biosystems). cDNA made from 100 ng of total RNA was added to a 20 µl of 1x TaqMan universal master mix (Applied Biosystems). PCR was conducted at 50°C for 2 min and 95°C for 10 min followed by 50 cycles at 95°C for 15 s and 60°C for 60 s. Primers and probe were purchased from Applied Biosystems for detection of PPAR{gamma} (Homo sapiens 00234592_m1), catalase (Mus musculus 00437992_m1), copper-zinc-dependent superoxide dismutase (CuZnSOD) (Mus musculus 01344231-g1), p40phox (Mus musculus 00476300_m1), and p67phox (Mus musculus 00726636_s1); sequences were not provided by the manufacturer. Uncoupling protein (UCP) 2 was detected using SYBR Green method. Primer sequences were 5'-GAC CTC CCT TGC CAC TTC AC-3' (forward) and 5'-GCA TGG AGC GGC TCA GAA AG-3' (reverse). Real-time PCR results were analyzed with ABI PRISM 7000 SDS software. Auto thresholds and auto baselines determined by the software were applied to generate values of corresponding threshold cycles (Ct). Ct values of mouse genes were normalized to the Ct of mouse beta-actin that was purchased from ABI (Mus musculus 00607939_s1). Ct values of human PPAR{gamma} were normalized to human beta-actin that was purchased from Applied Biosystems (Homo sapiens 99999903_m1).

Western blot analyses

The analyses were conducted as described previously (11). Abs against PPAR{gamma}, UCP2, and CuZnSOD were purchased from Santa Cruz Biotechnology, Alpha Diagnostic International, and Nventa Biopharmaceuticals, respectively.

Measurements of cellular ATP levels

Intracellular ATP levels were determined using the ATP bioluminescence assay kit HS II (Roche) following the manufacturer’s instruction. Briefly, 25 µl of lysis reagent was added to 25 µl cells (1 x 105 cells/ml) and incubated for 5 min at room temperature, and 50 µl of luciferase reagent was then added. Luminescence was quantified using an MLX microtiter plate luminometer (Dynex Technologies).

Measurement of intracellular ROS

Intracellular ROS were detected with 5-(and 6-)carboxy-2', 7'-dichlorodihydrofluorescein diacetate (DCF; Molecular Probes). Cells (3 x 105) were resuspended in 500 µl of RPMI 1640 containing 5% FBS and loaded with 10 µM DCF for 30 min at 37°C. DCF fluorescence of 10,000 events was measured by flow cytometry.

RNA interference and nucleofection

small interfering RNA (siRNA) against UCP2 and scrambled dsRNA controls were purchased from Dharmacon in SMARTpool form. The delivery of nucleic acids into cells was performed using nucleofection technology with the Amaxa Biosystems nucleofector instrument. For transfection into PPAR{gamma}-expressing FL5.12 cells, a total of 3 µg of the siRNA pools was delivered into 2 x 106 cells suspended in 100 µl of solution V using the G16 nucleofection program (Amaxa Biosystems).

Assay for catalase activity

The activity of the enzyme was assayed in whole cell extracts by the addition of H2O2 for cell extraction and by monitoring the decrease of H2O2 absorbance at 240 nm (31).

Statistical analysis

A Student t test was used to perform the statistical analyses. The p values are indicated in each figure.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Low concentrations of several PPAR{gamma} ligands promote survival in cytokine-deprived mouse pro-B cells in a receptor-dependent fashion

FL5.12 cells, an IL-3-dependent mouse lymphocytic cell line, undergo apoptosis in 24–48 h following the cytokine withdrawal. Previously, we showed that activation of PPAR{gamma} with low doses of rosiglitazone, a highly specific agonist of the TZD class, attenuates apoptosis in IL-3-deprived FL5.12 cells (11). To ensure that the prosurvival effect we observed depends on PPAR{gamma} instead of a specific effect of rosiglitazone, we used GW7845, another PPAR{gamma} agonist with a different chemical structure. To facilitate the assessment of receptor-dependent effects, previously established FL5.12 cell lines with or without stable PPAR{gamma} expression (11) were used with different agonists, and the survival of cells was determined under the condition of IL-3 deprivation. As shown in Fig. 1A, at a low dose of 0.5 µM, GW7845 promoted cell survival as well as rosiglitazone did in the PPAR{gamma}-expressing cell line but not in the control cell line that lacks PPAR{gamma} expression. This result provided additional evidence that the prosurvival effect depends on the presence and activation of PPAR{gamma} and is not the effect of a particular drug.


Figure 1
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  		FIGURE 1. PPAR{gamma} attenuates apoptosis in FL5.12 cells. A, Survival of FL5.12 cells transfected with PPAR{gamma} or control vector. Cell survival was determined at 24 h after IL-3 withdrawal by propidium iodide exclusion with flow cytometric analysis. DMSO, 0.5 µM rosiglitazone (Rosi), or 0.5 µM GW7845 was added to the PPAR{gamma} or control cell line at the time of IL-3 withdrawal. Data shown are mean ± SE of two independent experiments. B, Survival of IL-3-deprived PPAR{gamma} clones and control cells in the presence of ciglitazone at the concentrations indicated. Western blotting analysis showing PPAR{gamma} levels in the three clones is included as an inset. Cell survival was determined at 24 h after IL-3 withdrawal. Data shown are mean ± SE of three independent experiments. C, Survival of IL-3 deprived PPAR{gamma} clones and control cells in the presence of 15d-PGJ2 at the concentrations indicated. Cell survival was determined at 24 h after IL-3 withdrawal. Data shown are mean ± SE of three independent experiments.

 
To provide further evidence that cell survival is promoted by low doses of ligands through a PPAR{gamma}-dependent pathway, whereas apoptosis happens at high doses through receptor independent mechanisms, wide dose ranges of two PPAR{gamma} ligands, ciglitazone and 15d-PGJ2, were examined in cells with or without PPAR{gamma}. The two ligands were selected because they have been used in the studies that found that PPAR{gamma} induces cell death (12, 16, 17). In addition, two PPAR{gamma} clones with different levels of the receptor were tested to see whether the levels of the protein influence the choices of survival vs apoptosis. Our results show that ciglitazone increased cell viability in the dose range of 10–100 µM and killed cells at higher concentrations regardless of the presence or absence of PPAR{gamma} and the levels of the PPAR{gamma} protein (Fig. 1B). Notably, the KD of ciglitazone for PPAR{gamma} is ~4 µM. Similarly, 15d-PGJ2 promoted survival at doses <5 µM and induced cell death at doses ≥10 µM (Fig. 1C). Together, these data demonstrate that ligands at different concentrations generate different effects; low concentrations of ligands promote the survival of cytokine-deprived lymphocytes in a PPAR{gamma}-dependent fashion, whereas high concentrations cause cell death independently of the receptor. Unidentified endogenous ligands of PPAR{gamma} might be present in the culture medium or cells, because PPAR{gamma} cell lines treated with vehicle had better survival rates than the vector control cell line thus treated, and the clone with higher level of PPAR{gamma} treated with vehicle had better survival rates than the clone with the lower level thereof.

PPAR{gamma} attenuates apoptosis in human lymphoma cells in a receptor-dependent fashion

We next analyzed the survival of human lymphoma cells that express different levels of PPAR{gamma}. Karpas 299 and SUP-M2 are two malignant T cell lines derived from human anaplastic large cell lymphoma. We found that the level of PPAR{gamma} mRNA expression in Karpas 299 cells was ~3,500 times higher than that of SUP-M2 (Fig. 2A). Western blot analysis confirmed that PPAR{gamma} was highly expressed in Karpas 299 cells and not detectable in SUP-M2 cells (Fig. 2B). Apoptosis of Karpas 299 and SUP-M2 cells was induced by serum starvation in the presence or absence of the PPAR{gamma} ligand rosiglitazone, and the survival of the cells was determined and compared. As shown in Fig. 2C, survival of the lymphoma cells was not affected by rosiglitazone treatment in the presence of serum. However, at 48 h after serum withdrawal, Karpas 299 cells treated with rosiglitazone survived at rates better than those of cells treated with drug vehicle. In contrast, the survival of SUP-M2 cells was unaffected by rosiglitazone treatment. Moreover, the prosurvival effect of rosiglitazone on Karpas 299 cells was dose-dependent and could be observed at a concentration as low as 0.5 µM (Fig. 2D). Taken together with our previous findings, these results demonstrate that PPAR{gamma} not only promotes the survival of IL-3 dependent cells upon cytokine withdrawal but also promotes the survival of lymphoma cells upon serum starvation.


Figure 2
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  		FIGURE 2. PPAR{gamma} attenuates apoptosis in Karpas 299 lymphoma cells. A, PPAR{gamma} mRNA expression in lymphoma cell lines. Levels of PPAR{gamma} mRNA were measured by real-time RT-PCR assay. Expression levels were calculated relative to the level of PPAR{gamma} in SUP-M2 cells that is arbitrarily defined as 1. B, Western blot analysis of PPAR{gamma} in lymphoma cell lines. PPAR{gamma} in whole cell extracts was analyzed. D, Rosiglitazone promotes survival in serum-deprived Karpas 299 but not in SUP-M2 cells. Cells were cultured with or without 10% serum for 48 h in the presence of 2 µM rosiglitazone or DMSO as indicated. Data shown are mean ± SE of three independent experiments. D, Prosurvival is dose-dependent on the concentration of rosiglitazone. Serum-deprived Karpas 299 cells were cultured in the presence of rosiglitazone at the concentrations indicated, and survival was determined at 48 h after serum withdrawal.

 
PPAR{gamma} promotes lymphocyte survival by stimulating ATP production

Because PPAR{gamma} is a metabolic regulator, we hypothesized that PPAR{gamma} increases cell survival through its actions on cellular metabolic activities. We have previously shown that PPAR{gamma} enhances the ability of cells to maintain their mitochondrial membrane potential (11). Next, we investigated whether an increased total cellular ATP level is coupled with better maintenance of mitochondrial potential. We compared ATP levels of two PPAR{gamma} cell lines and a vector control cell line treated with or without rosiglitazone. As shown in Fig. 3A, in the presence of IL-3, the cellular ATP levels were similar in the vector and in the PPAR{gamma}-expressing cell lines, and the levels were not affected by rosiglitazone. Upon IL-3 withdrawal, the ATP levels declined in all cell lines but to a much lesser degree in the PPAR{gamma} cell lines as compared with the control cell line. Furthermore, rosiglitazone increased the amount of cellular ATP in the two PPAR{gamma} cell lines. The degrees of ATP increase by PPAR{gamma} activation are consistent with the degrees of the prosurvival effect. In contrast, the addition of rosiglitazone to the control cells showed no effect on the ATP levels. Taken together, the presence and activation of PPAR{gamma} attenuate the decline in ATP upon IL-3 withdrawal that correlates well with better-maintained mitochondrial membrane potential and improved cell survival.


Figure 3
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  		FIGURE 3. PPAR{gamma} attenuates the decline in ATP levels upon IL-3 withdrawal. A, ATP levels of FL5.12 cells transfected with PPAR{gamma} or control vector. Two independent PPAR{gamma} cell lines (PPAR{gamma}-A and PPAR{gamma}-B) are shown. Cells were cultured with or without IL-3 for 15 h in the presence of 0.5 µM rosiglitazone (Rosi) or DMSO as indicated. Cells were then harvested, and total cellular ATP levels were determined in lysates from a million cells. Data shown are mean ± SE of three independent experiments. B, Prosurvival effect of PPAR{gamma} is abolished by mitochondrial inhibitors. Time courses of cell survival were performed in the absence of IL-3. At the time of IL-3 withdrawal, 5 µg/ml oligomycin was added to the culture 3 min before the addition of 5 µM FCCP (FCCP + Olig). GD, PPAR{gamma} cell line treated with DMSO; GR, PPAR{gamma} cell line treated with 0.5 µM rosiglitazone; VD, vector control line treated with DMSO; and VR, vector control line treated with 0.5 µM rosiglitazone. C, Prosurvival effect of PPAR{gamma} is abolished by 2DOG. Time courses of cell survival were performed in the absence of IL-3 and glucose. 2DOG was added at a concentration of 10 mM. D, Prosurvival effect of PPAR{gamma} is abolished by IAA. Time courses of cell survival were performed in the absence of IL-3 and the presence of 10 mM glucose. IAA was added at a concentration of 20 µM. E, PPAR{gamma} promotes survival by using methyl-pyruvate (mPyr). Time courses of cell survival were performed in the absence of IL-3 and glucose. One millimolar methyl pyruvate was supplied to the cell cultures. Error bars in B–E represent the average ± SE of three independent experiments.

 
Higher cellular ATP in the PPAR{gamma} cells relative to the control cells might result from either increased ATP production or decreased ATP consumption. To distinguish between these two possibilities, we analyzed survival of the PPAR{gamma} and vector control cells in the presence of two drugs that inhibit mitochondrial ATP production. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) is a derivative of cyanide; it is a proton ionophore that acts to collapse the mitochondrial membrane potential required for ATP synthesis. Oligomycin is an inhibitor of the F1-F0-ATPase, the complex V of the mitochondrial respiratory chain. Acting together, the two drugs prevent ATP production from the mitochondrial source (32, 33). As shown in Fig. 3B, the survival benefit of the PPAR{gamma} line was lost in the presence of FCCP and oligomycin because the cells treated with or without rosiglitazone showed the same survival rate as the vector control cells. These data demonstrate that ATP production is required for PPAR{gamma} to exert its prosurvival effect.

To further test this notion, cell survival was determined in the presence of glycolytic inhibitors to prevent the fueling of glycolytic substrates to mitochondria for ATP production. Under the routine culturing condition (see Material and Methods), FL5.12 cells rely on glucose in the medium as the main energy source for ATP production to sustain their survival, growth, and proliferation. Inhibition of glycolysis impairs cellular ATP synthesis that may affect the prosurvival effect by PPAR{gamma}. To inhibit glycolysis, we made use of 2-deoxyglucose (2DOG), a nonmetabolizable analog of glucose, and iodoacetic acid (IAA), an inhibitor of GAPDH and a key enzyme in the glycolytic pathway. When cells were cultured in IL-3-free medium containing 2DOG in place of glucose, no survival advantage was observed in the PPAR{gamma} cells treated with or without rosiglitazone as compared with the control (Fig. 3C). Similarly, the addition of IAA in the presence of glucose abolished the survival advantage of the PPAR{gamma} cells (Fig. 3D). When cells were supplied with methyl pyruvate, a cell-permeable form of pyruvate as an alternative energy source replacing glucose, rosiglitazone was able to promote cell survival in a PPAR{gamma}-dependent fashion (Fig. 3E). Taken together, PPAR{gamma} relies on a source of energy to promote survival. Combined with our previous data, these results suggest that PPAR{gamma} promotes survival by maintaining mitochondrial integrity through an energy source that functionally leads to increased ATP production.

PPAR{gamma} suppresses accumulation of ROS in growth factor-deprived cells

ROS are generated following application of many apoptotic stimuli (34), and mitochondria are the major sites of ROS production in most apoptotic systems (35, 36). It has been shown that an increase in ROS precedes mitochondrial depolarization during TNF-{alpha}-induced apoptosis and that ROS scavenger treatment delays apoptosis (34). Based on our finding that PPAR{gamma} attenuates cell death by maintaining mitochondrial homeostasis, we reasoned that PPAR{gamma} might reduce the amounts of ROS that are harmful to the mitochondria during IL-3 withdrawal. We measured the intracellular oxidants in the PPAR{gamma} and the vector control cell lines in the presence or absence of rosiglitazone. As shown in Fig. 4, in the presence of IL-3, the ROS levels in the two cell lines were low and comparable to each other (dashed traces). In IL-3-depleted cells ROS was elevated in the vector control cells, and rosiglitazone addition had no appreciable effect on the ROS level (Fig. 4A). In comparison, fewer PPAR{gamma}-expressing cells had elevated ROS (Fig. 4B, thin solid trace). Moreover, even fewer PPAR{gamma} transfected cells had increased ROS when treated with rosiglitazone (Fig. 4B, thick solid trace). These results demonstrate that better survival of the PPAR{gamma} cell line in the absence of IL-3 is accompanied by suppressed ROS increase in the cells.


Figure 4
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  		FIGURE 4. PPAR{gamma} suppresses ROS accumulation in growth factor-deprived cells. Cells were cultured with or without IL-3 for 15 h in the presence of 0.5 µM rosiglitazone (Rosi) or DMSO as indicated. Cells were then harvested and incubated with 10 µM DCF at 37°C for 30 min. Ten thousand events in live cell gates were analyzed with a FACSCalibur flow cytometer. Histograms from a typical experiment show fluorescence of 10,000 live cells treated as indicated. A, Vector control cells. B, PPAR{gamma}-transfected cells.

 
UCP2 is up-regulated by PPAR{gamma} and involved in the cell survival-promoting effect of PPAR{gamma}

Cycloheximide and actinomycin D blocked the ability of PPAR{gamma} to promote survival in IL-3-deprived cells, suggesting that this function of PPAR{gamma} requires new mRNA and protein synthesis (data not shown). Having established that the limiting of ROS increase is one of the mechanisms underlying the prosurvival effect of PPAR{gamma}, we pursued potential transcriptional targets of the receptor that may mediate its regulation of ROS. UCP2, unlike UCP1 and UCP3, is mainly involved in the limitation of free radical levels in cells rather than in physiological uncoupling and thermogenesis (37, 38). Recently, it has been shown that UCP2 protects neurons and cardiomyocytes from death by reducing ROS production in mitochondria (39, 40). Interestingly, UCP2 is subject to regulation by unsaturated fatty acids. Recent observations suggest that fatty acids induce expression of UCP2 and UCP3 by acting as ligands for PPARs (41). We therefore tested whether PPAR{gamma} limits ROS increase via UCP2. Levels of UCP2 mRNA were measured in PPAR{gamma}-plus and -minus cell lines deprived of IL-3. Using a real-time RT-PCR assay, we found that UCP2 mRNA was significantly higher in the PPAR{gamma}-expressing cells than the control cells following the cytokine withdrawal (Fig. 5A). The level of UCP2 was further increased in the PPAR{gamma} line treated with rosiglitazone, whereas the control cells showed no response to the treatment. This finding was confirmed at the protein level with Western blot analysis. As shown in Fig. 5B, although little UCP2 protein was detected in the vector control cells, it was expressed in the PPAR{gamma}-expressing cells and its expression was further up-regulated when cells were treated with rosiglitazone.


Figure 5
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  		FIGURE 5. UCP2 is up-regulated by PPAR{gamma} and is involved in the prosurvival effect of PPAR{gamma}. A and B, Cells treated with 0.5 µM rosiglitazone (Rosi) or DMSO were harvested at 15 h after IL-3 withdrawal. A, UCP2 mRNA was measured by real-time RT-PCR using the SYBR Green method. The level of UCP2 mRNA in DMSO-treated vector cells was arbitrarily set as 100%. Data shown are mean ± SE of three independent experiments. B, Western blot analysis of UCP2 protein in whole cell extracts. D, DMSO; R, rosiglitazone. C–E, The PPAR{gamma}-expressing cell line was transfected with UCP2 siRNA or scrambled dsRNA. Cells were withdrawn from IL-3 in the presence of 0.5 µM rosiglitazone. C, UCP2 mRNA was measured in siRNA- or control RNA-transfected cells by real-time RT-PCR at 24 h after IL-3 withdrawal. The level of UCP2 mRNA in control RNA-transfected cells was arbitrarily set as 100%. D, UCP2 protein levels were determined by Western blot analysis in whole cell lysates at 24 h after IL-3 withdrawal. ctl, control. E, Cell viability was determined after IL-3 withdrawal at the indicated time points.

 
To obtain more definitive proof that UCP2 mediates the activity of PPAR{gamma} on cell survival, we knocked down the UCP2 level with a pool of UCP2 siRNA in the PPAR{gamma}-expressing cell line. In Fig. 5, C and D, both real-time RT-PCR and Western blotting revealed that UCP2 level was reduced by UCP2 siRNA as compared with scrambled dsRNA control. We then determined the survival of cells transfected with UCP2 siRNA and those with control RNA under the condition of IL-3 withdrawal. As shown in Fig. 5E, cells transfected with UCP2 siRNA had a lower survival rate compared with the cells transfected with control RNA. Together, these results suggest that UCP2 is one of the proteins that mediate the effects of PPAR{gamma} on cell survival.

PPAR{gamma} suppresses ROS accumulation by coordinated control of several ROS regulators at the transcriptional level

Several other enzymes are involved in the regulation of cellular ROS levels. Hydrogen peroxide is scavenged by catalase, an ubiquitously distributed heme-containing enzyme that converts H2O2 to water and molecular O2. Recently, a PPAR response element (PPRE) has been identified in the promoter region of the rat catalase gene, and this PPRE is necessary for PPAR{gamma} ligand-induced increase of catalase mRNA in rat endothelial cells (31). CuZnSOD scavenges superoxide radicals by converting two of the radicals into H2O2 and molecular O2. Its gene expression and protein levels are increased by troglitazone and pioglitazone treatment of endothelial cells (42). On the other side, NADPH oxidase catalyzes the production of many species of ROS, including superoxide, hypochlorite, H2O2, hydroxyl radicals, and NO. Although most abundant in phagocytes, it is expressed in FL5.12 cells. Interestingly, the expression of some of the regulatory subunits of NADPH oxidase in endothelial cells is decreased by PPAR{gamma} ligand treatment (42).

We examined mRNA and protein expression or enzyme activity of catalase, CuZnSOD, and some regulatory subunits of NADPH oxidase in the cells. As shown in Fig. 6A, the mRNA level of catalase was significantly increased in the PPAR{gamma} cell line treated with rosiglitazone in the absence of IL-3. This finding was confirmed when total cellular activity of the enzyme was measured (Fig. 6B). In contrast, no increase in the mRNA level or enzymatic activity of catalase was observed in the vector control cell line. Similarly, mRNA and protein expression of the CuZnSOD was up-regulated by the activation of PPAR{gamma} in the PPAR{gamma} cell line but not in the vector control cell line upon IL-3 withdrawal (Fig. 6, C and D). In comparison with catalase and CuZnSOD, expression of the regulatory subunits of NADPH oxidase, p67phox and p40phox, was not regulated by PPAR{gamma} under the condition tested (data not shown). Taken together with our UCP2 data, these results demonstrate that PPAR{gamma} coordinately controls several protein and enzymes at the transcriptional level, leading to ROS limitation and increased survival.


Figure 6
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  		FIGURE 6. PPAR{gamma} coordinately controls several ROS-regulating enzymes. Cells treated with 0.5 µM rosiglitazone (Rosi) or DMSO were harvested at 15 h after IL-3 withdrawal. A, Catalase mRNA was measured by real-time RT-PCR using TaqMan technology. The amount of catalase mRNA in DMSO-treated vector cells was arbitrarily set as 100%. B, Catalase activity in whole cell extracts was measured at 15 h after IL-3 withdrawal. C, CuZnSOD mRNA was measured by real-time RT-PCR using TaqMan technology. The amount of CuZnSOD mRNA in DMSO-treated vector cells was arbitrarily set as 100%. Data shown in A–C are mean ± SE of three independent experiments. D, CuZnSOD protein levels were determined by Western blot analysis in whole cell lysates at 15 h after IL-3 withdrawal. D, DMSO; R, rosiglitazone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this report we described cell survival promotion as a novel activity of PPAR{gamma}. We have found the receptor is capable of enhancing cell survival not only in a murine lymphocytic cell line but also in a human lymphoma cell line that highly expresses PPAR{gamma}. Using cells containing or lacking PPAR{gamma} with or without activation by its ligands, we have demonstrated that this activity depends both on the presence and activation of PPAR{gamma} rather than certain ligands. To further understand how PPAR{gamma} promotes survival, we characterized the metabolic changes induced by PPAR{gamma}. Our data showed that PPAR{gamma} helps maintain mitochondrial membrane potential, attenuates decline in ATP, and suppresses accumulation of ROS in cells deprived of the cytokine, all in a PPAR{gamma}-dependent manner. Fig. 7 shows a schematic diagram based on the collective findings in this report combined with what has been reported in the literature (see below). To our knowledge, we are the first group to show that PPAR{gamma} promotes lymphocyte survival.


Figure 7
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  		FIGURE 7. Schematic diagram shows how PPAR{gamma} might increase cell survival in cytokine-deprived cells. The scheme was made based on findings in the current report and the literature (see Discussion). Dashed arrow indicates the effects may or may not be direct.

 
During growth factor withdrawal, cells have to maintain their metabolism to sustain survival. Akt, a potent survival factor and an oncogene, maintains mitochondrial membrane potential and promotes an increase in ATP levels in growth factor-deprived cells (43). Cell death inhibition by Akt requires a functional glycolytic pathway, whereas active mitochondrial oxidative phosphorylation is dispensable (44). Unlike Akt, oxidative phosphorylation is required for PPAR{gamma} to inhibit cell death. In the presence of FCCP and oligomycin, PPAR{gamma} loses its ability to increase cell survival, although ATP derived from glycolysis is still available to the cells (Fig. 3B). In the mean time, glycolysis is not needed for the PPAR{gamma} survival-promoting effect. Although glycolytic inhibitors abolished survival-promoting effects by PPAR{gamma}, methyl pyruvate restored cellular response to rosiglitazone (Fig. 3E). These data suggest that glycolysis per se is not required. Instead, the feeding of pyruvate, the end product of glycolysis, to the tricarboxylic acid cycle and the subsequent mitochondrial oxidative phosphorylation are the processes that are needed for PPAR{gamma} to exert its survival effect.

ROS are generated in apoptotic processes induced by various stimuli, including TNF-{alpha} (45), Fas (46), glucocorticoid hormones (47), radiation (48), and chemotherapeutic drugs (49), and mitochondria are major sites of ROS production during apoptosis (35, 36). It has been shown that an increase in ROS inhibits mitochondrial electron transport and leads to mitochondrial depolarization and caspase activation (34, 50). In line with these findings, we showed that cellular ROS increased following IL-3 withdrawal. The increase in ROS was suppressed by the presence and activation of PPAR{gamma} (Fig. 4), which correlates well with the receptor’s ability to maintain mitochondrial membrane potential (11) and increase ATP production (Fig. 3A). Further investigation revealed that a set of proteins and enzymes regulating ROS were coordinately controlled by PPAR{gamma}. PPAR{gamma} increased UCP2, catalase, and CuZnSOD at mRNA and protein levels, leading to reduced ROS accumulation, limited mitochondrial damage, and, eventually, enhanced cell survival. At this point, it is unclear whether these genes are direct targets of PPAR{gamma} transcriptional activity. Functional PPRE have been identified in the promoters of the rat catalase gene and CuZnSOD (31, 51), but are not present in the promoter of UCP2 (41). Further studies are needed to determine whether PPAR{gamma} is directly involved in the transcriptional regulation of these genes.

In this report, we showed that the survival-promoting effect of PPAR{gamma} depended on the expression of the receptor and was reproducible with three classes of agonists at low concentrations. We demonstrated that PPAR{gamma} not only promoted the survival of PPAR{gamma}-expressing FL5.12 cells under the condition of cytokine withdrawal, but also attenuated the death of Karpas 299 lymphoma cells induced by serum starvation. Differing from our study, two other groups observed apoptosis in lymphocytes and lymphoma cells treated with the PPAR{gamma} agonist 15d-PGJ2 and/or TZDs (12, 16, 17, 52). To induce apoptosis, micromolar concentrations of 15d-PGJ2 were used. However, levels of this compound in body fluids fall in the picomolar to low nanomolar range (53), raising the questions of whether the findings have physiological relevance and whether the apoptotic effect by high doses of PPAR{gamma} ligands is mediated through the receptor. In the current study, titration of 15d-PGJ2 and ciglitazone in wide dose ranges showed differential effects of these ligands at different concentrations, increasing cell survival at low doses and inducing cell death at high doses. Previously, we also observed primary T cell apoptosis with high doses of 15d-PGJ2 and ciglitazone. In addition, apoptosis was produced with these drugs in FL5.12 parental cells that express little PPAR{gamma} (11). Collectively, our data suggest that apoptosis could be a result of drug cytotoxicity at high concentrations. In keeping with the receptor-independent mechanism, a recent study has shown that apoptosis induced by a synthetic triterpenoid (PPAR{gamma} ligand) in malignant Mycosis fungoides T cells cannot be blocked by a PPAR{gamma} antagonist (54). Another study has shown that 15d-PGJ2-induced death of lymphoma and myeloma cells cannot be reproduced with troglitazone at achievable pharmacological concentrations (55). Receptor-independent mechanisms of PPAR{gamma} agonists have also been documented in granulocyte death (56) and Fas-mediated lymphocyte death (57).

In murine and human T lymphocytes, PPAR{gamma} is significantly up-regulated during T cell activation (10, 11, 12). However, the functional significance of this up-regulation needs to be further characterized. Survival signals elicited by cytokines play an important role in survival of activated T cells. Along with Fas-mediated death, cytokine withdrawal is thought to be an equally important process in causing the death of activated T cells following the peak of the immune responses (24). Interestingly, ROS have been shown to be involved in the regulation of activation-induced T cell apoptotic death (58). Given our findings that PPAR{gamma} promotes survival in cytokine- or serum-deprived cells through its regulation on ROS, it would be interesting to see whether up-regulation of PPAR{gamma} in primary T cells may modulate activation-induced T cell death and other aspects of T cell immune responses.

Lastly, high expression of PPAR{gamma} in lymphoma cell is documented here and elsewhere (17, 59). Our data raise the interesting question of whether high levels of PPAR{gamma} in lymphoma cells may confer upon them a survival advantage. Further studies will need to be performed to assess this possibility.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported by a National Heart, Lung, and Blood Institute Career Award K08-HL068850 (to Y.L.W.). Back

2 Address correspondence and reprint requests to Dr. Y. Lynn Wang, Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, 525 East 68th Street, Box 69, New York, NY 10021. E-mail address: lyw2001{at}med.cornell.edu Back

3 Abbreviations used in this paper: PPAR, peroxisome proliferator-activated receptor; Ct, threshold cycle; 2DOG, 2-deoxyglucose; 15d-PGJ2, 15-deoxy-{Delta}12,14-PGJ2; CuZnSOD, copper-zinc-dependent superoxide dismutase; siRNA, small interfering RNA; DCF, 5-(and 6-)carboxy-2',7'-dichlorodihydrofluorescein diacetate; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; IAA, iodoacetic acid; PPRE, PPAR response element; ROS, reactive oxygen species; TZD, thiazolidinedione; UCP, uncoupling protein. Back

Received for publication February 1, 2006. Accepted for publication June 23, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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