Down-regulated Peroxisome Proliferator-activated Receptor γ (PPARγ) in Lung Epithelial Cells Promotes a PPARγ Agonist-reversible Proinflammatory Phenotype in Chronic Obstructive Pulmonary Disease (COPD)*

Background: The mechanistic role of peroxisome proliferator-activated receptor γ (PPARγ) in chronic obstructive pulmonary disease (COPD) is poorly understood. Results: COPD and cigarette smoke exposure down-regulated PPARγ and produced inflammation that PPARγ agonists reversed through multiple pathways. Conclusion: PPARγ plays a pivotal role in COPD. Significance: PPARγ agonists may be the first effective treatment for COPD. Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory condition and a leading cause of death, with no available cure. We assessed the actions in pulmonary epithelial cells of peroxisome proliferator-activated receptor γ (PPARγ), a nuclear hormone receptor with anti-inflammatory effects, whose role in COPD is largely unknown. We found that PPARγ was down-regulated in lung tissue and epithelial cells of COPD patients, via both reduced expression and phosphorylation-mediated inhibition, whereas pro-inflammatory nuclear factor-κB (NF-κB) activity was increased. Cigarette smoking is the main risk factor for COPD, and exposing airway epithelial cells to cigarette smoke extract (CSE) likewise down-regulated PPARγ and activated NF-κB. CSE also down-regulated and post-translationally inhibited the glucocorticoid receptor (GR-α) and histone deacetylase 2 (HDAC2), a corepressor important for glucocorticoid action and whose down-regulation is thought to cause glucocorticoid insensitivity in COPD. Treating epithelial cells with synthetic (rosiglitazone) or endogenous (10-nitro-oleic acid) PPARγ agonists strongly up-regulated PPARγ expression and activity, suppressed CSE-induced production and secretion of inflammatory cytokines, and reversed its activation of NF-κB by inhibiting the IκB kinase pathway and by promoting direct inhibitory binding of PPARγ to NF-κB. In contrast, PPARγ knockdown via siRNA augmented CSE-induced chemokine release and decreases in HDAC activity, suggesting a potential anti-inflammatory role of endogenous PPARγ. The results imply that down-regulation of pulmonary epithelial PPARγ by cigarette smoke promotes inflammatory pathways and diminishes glucocorticoid responsiveness, thereby contributing to COPD pathogenesis, and further suggest that PPARγ agonists may be useful for COPD treatment.

cigarette smoke exposure and COPD have previously been poorly characterized. Known PPAR␥ agonists include the synthetic thiazolidinediones, used to treat type 2 diabetes, and various endogenous compounds. Physiologically relevant endogenous PPAR␥ agonists remain to be identified, but plausible candidates include nitrated fatty acids, which constitute one of the largest blood-borne pools of biologically active nitrogen compounds (9) and circulate in concentrations sufficient to activate PPAR␥ (10).
Here we assessed the anti-inflammatory potential of PPAR␥ in pulmonary epithelial cells of people with and without COPD and on smoke-induced epithelial responses. We also explored its mechanistic relationships with key transcription/signaling factors including the NF-B pathway, GR-␣, and HDAC2. We found that PPAR␥ expression and activity are down-regulated in human bronchial epithelial (HBE) cells from COPD patients and those exposed to cigarette smoke extract (CSE) in vitro, whereas proinflammatory pathways are up-regulated. Treating lung epithelial cells with either the thiazolidinedione rosiglitazone (Rosi) or the endogenous PPAR␥ agonist 10-nitro-oleic acid (OA-NO 2 ) reversed these CSE effects and the accompanying decreases in GR-␣ and HDAC2. PPAR␥ agonists also blocked CSE-induced inflammatory cytokine and chemokine production and ROS production by reversing the CSE-induced increase in NF-B activity through multiple PPAR␥-mediated mechanisms. Conversely, PPAR␥ knockdown augmented CSE responses. These findings raise the possibility that PPAR␥ agonists may be therapeutically useful for treating COPD, and furthermore, may reverse COPD patients' resistance to anti-inflammatory steroid therapy by restoring impaired HDAC2 activity.
Patient Lung Tissue Samples-Human lung tissues were obtained from excess pathologic tissue after lung transplantation and organ donation, under a protocol approved by the University of Pittsburgh Institutional Review Board. COPD lung tissues were obtained from explanted lungs of subjects with advanced COPD, and control lungs were donated lungs not suitable for transplantation from the Center for Organ Recovery and Education (CORE). Lung tissues were stored at Ϫ80°C until future usage.   each puff being of 2-s duration and 35-ml volume. The pH of CSE was adjusted to 7.4 and sterilized by filtration through a 0.22-m filter (EMD Millipore, Billerica, MA). The extract, defined as 100% CSE, was diluted to the indicated concentrations and used within 10 min of preparation. For control experiments, air was bubbled into 10 ml of medium, which was then treated as for CSE.
Measurement of Cytokine and Chemokine Levels in Culture Medium-Cell culture medium from different treatment groups was collected and stored at Ϫ80°C. Levels of TNF-␣, IL-6, and IL-8 were measured using ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.
Determination of Cellular ROS-H 2 O 2 production in cell culture media was determined using the Amplex Red hydrogen peroxide assay kit (Molecular Probes, Eugene, OR). Production of intracellular ROS in live cells was determined using the Cell Meter fluorimetric intracellular total ROS activity assay kit (AAT Bioquest, Sunnyvale, CA). Samples were mounted on glass slides with VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA). The slides were viewed by an Olympus FluoView FV1000 confocal microscope (Olympus, Center Valley, PA) using a 60 ϫ fluorescence lens along with FluoView confocal software (FV10-ASW v1.7, Olympus).
HDAC Activity and Transcription Factor DNA Binding Activity Assays-Nuclear proteins were extracted using a nuclear extraction kit (Active Motif, Carlsbad, CA), and their concentrations were determined using the BCA protein assay kit (Pierce). Nuclear extracts were used to quantify HDAC activity and DNA binding activity of PPAR␥, GR-␣, and the p65 subunit of NF-B using ELISA-based kits (56210, 40196, 45496, and 40096; Active Motif).
Immunoprecipitation-Nuclear extracts were prepared and were immunoprecipitated using the Dynabeads protein G immunoprecipitation kit (Invitrogen). Antibodies were bound to Dynabeads protein G, and Dynabeads-antibody complex was used to precipitate target proteins from the nuclear extracts. Unbound proteins were washed away, and complexes were eluted. All samples (20 g/lane) were separated by electrophoresis on SDS-polyacrylamide gels and transferred to PVDF membranes, and Western blotting was performed.
ChIP Assay-The ChIP assay was performed using the SimpleChIP enzymatic chromatin immunoprecipitation kit with magnetic beads (Cell Signaling Technology). Briefly, cellular chromatin was cross-linked with 1% formaldehyde for 10 min at room temperature, the cross-linking was stopped with 0.125 M glycine, and cells were washed twice with ice-cold PBS. Nuclei were pelleted and digested by micrococcal nuclease. Following sonication and centrifugation, equal amounts of sheared chromatin were incubated overnight at 4°C with antibodies, IgG as negative control, and RNA polymerase II as positive control (Cell Signaling Technology). Protein G magnetic beads were then added, and the chromatin was incubated with rotation for 2 h at 4°C. An aliquot of chromatin that was not incubated with any antibody was used as the input control sample. Antibody-bound protein-DNA complexes were eluted and subjected to real-time PCR as described under "RNA Isolation and Quantitative Real-time RT-PCR" with specific primers for TNF-␣, IL-6, IL-8, and ␣-satellite (supplemental Table 1).
RNA Isolation and Quantitative Real-time RT-PCR-RNA was isolated using the RNeasy mini kit (Qiagen), and cDNA was generated from 100 ng of total RNA using MultiScribe reverse transcriptase (Applied Biosystems, Foster City, CA) employing random and oligo(dT) primers. Real-time quantitative PCR was performed using 100 ng of cDNA with 2ϫ SYBR Green master mix (Applied Biosystems) and specific primers for the genes of interest (supplemental Table 1). These experiments were performed on an AB 7500 fast thermal cycler using a three-step protocol employing the melting curve method. The average of each gene cycle threshold (C t ) was determined for each experiment. Relative cDNA levels (2 Ϫ⌬⌬Ct ) for the genes of interest were determined using the comparative C t method, which generates the ⌬⌬C t as the difference between the gene of interest and the housekeeping genes ␤-actin and 9 S rRNA for each sample. Each averaged experimental gene expression sample was compared with the averaged control sample, which was set to 1.
Transfecting Small Interfering RNA into Normal HBE Cells-Normal HBE cells were incubated for 8 h with a liposome complex containing 100 nM of small interfering RNA (siRNA) targeted to PPAR␥ or scrambled control (Dharmacon, Lafayette, CO; supplemental Table 2) and Lipofectamine 2000 (Invitrogen) under serumand antibiotic-free conditions. After 8 h, fresh medium with 10% FBS was added, and the cells were incu- bated for a further 16 h. After a 24-h incubation, cells were treated with CSE as described.
Statistical Analysis-Data are presented as mean Ϯ S.D. Differences between groups were analyzed using an unpaired t test or analysis of variance followed by a Bonferroni's multiple comparison test using GraphPad Prism 5.03 (GraphPad Software, La Jolla, CA). A p Ͻ 0.05 was considered significant.

RESULTS
PPAR␥ Down-regulation in COPD Is Associated with Reduction of HDAC2 and Activation of NF-B-To assess the potential pathophysiological role of PPAR␥ in COPD, we tested whether PPAR␥ expression and function are altered in lung tissue samples of COPD patients and in airway epithelial cells. These cells are directly smoke-exposed in cigarette smokers and are pathogenic targets and mediators in COPD (12,13). We found that PPAR␥ protein (shown by Western blots) and DNA binding activity (Fig. 1A) were reduced in lung tissue and HBE cells of COPD patients when compared with those from individuals without COPD. This PPAR␥ down-regulation was associated with the previously reported (3) up-regulated expression and activity of the pro-inflammatory transcription factor NF-B and downregulation of the corepressor HDAC2 (Fig. 1B), a factor that participates in GR-␣-mediated anti-inflammatory activity. HBE cells from COPD patients also exhibited oxidative stress consistent with their proinflammatory state, as shown by immunostaining for ROS followed by confocal microscopy (Fig. 1C).

CSE Induces Inflammatory Responses and Oxidative Stress in Human Epithelial
Cells-Cigarette smoking, the major risk factor for COPD, produces lung inflammation and oxidative stress. To assess the mechanisms by which smoke down-regu- lates epithelial PPAR␥ and induces inflammation, we determined the time courses and concentration-response relationships of CSE-induced proinflammatory proteins, transcriptional mediators, and ROS in H292 human lung epithelial cells. Treating cells with varying concentrations of CSE for 6 h or with 10% CSE for various times up-regulated expression and release of the inflammatory cytokines TNF-␣ and IL-6 and the chemokine IL-8 (Fig.  2, A and B). Production of H 2 O 2 was also increased (Fig. 2C) as were intracellular ROS levels seen by immunofluorescence after a 6-h exposure to 10% CSE (Fig. 2D). CSE exposure likewise activated the proinflammatory transcription factor NF-B, seen via increased nuclear NF-B p65 levels (Fig. 2F) and DNA binding activity (Fig. 2G), and increased expressed protein levels of its transcriptional targets COX-2 and NADPH oxidase 4 (NOX4) (Fig. 2H).
We also measured changes in inhibitor of NF-B (IB), which down-regulates activity of NF-B by preventing its translocation to the nucleus, and in IB kinase (IKK), which drives ubiquitination and degradation of IB, thereby increasing NF-B activity (14). CSE treatment increased the levels of phosphorylated IB and phosphorylated (activated) IKK (Fig. 2E) and those of the activated form of mitogen-and stress-activated protein kinase 1 (MSK1), which phosphorylates IKK (Fig. 2F). MSK1 also promotes inflammatory gene transcription by phosphorylating NF-B itself, allowing it to recruit coactivators, and by phosphorylating histone H3, allowing it to induce chromatin loosening (15). The time courses and concentration-response relationships of all these CSE-induced responses were very similar, pointing to a broadly coordinated proinflammatory program in lung epithelial cells.
CSE Down-regulates PPAR␥, GR-␣, and HDAC2 while Enhancing Chromatin Acetylation-Based on the CSE-induced proinflammatory profile we saw in H292 cells and the suppression of PPAR␥ seen in HBE cells of COPD patients, we tested whether CSE influences PPAR␥ function in H292 cells. CSE induced time-and concentration-dependent decreases in nuclear levels (Fig. 3A, top panel) and DNA binding activity (Fig. 3B) of PPAR␥ that were accompanied by increased phosphorylation, an inhibitory post-translational modification (Fig.  3A, bottom panel). To test the idea that CSE-induced downregulation of GR-␣ might contribute to the ineffectiveness of glucocorticoids in COPD, we tested the effects of CSE on GR-␣. Supporting our hypothesis, exposing H292 cells to CSE downregulated both expression and activity of GR-␣ and also induced its lysine acetylation (Fig. 3, C and D), an inhibitory post-translational modification.
Transcription factors attract coactivators with histone acetyltransferase activity, which acetylates specific lysines in histones H3 and H4 and thereby loosens chromatin structure so as to allow RNA polymerase to bind and initiate transcription. GR-␣ suppresses proinflammatory gene expression in part by associating with NF-B and attracting the corepressor HDAC2, which inhibits transcriptional activation by deacetylating histones (16). We tested the influence of CSE on this system. CSE decreased the nuclear localization and activity of HDAC2 (Fig.  4, A and B) and thus increased the acetylation of histones H3 and H4, in a time-and concentration-dependent manner (Fig.  4C). CSE-induced suppression of HDAC activity reflected not only its down-regulated expression, but also induction of multiple concentration-and time-dependent post-translational modifications including serine phosphorylation, tyrosine nitrosylation, and cysteine alkylation by 4-hydroxy-2-nonenal, a specific marker of oxidative stress (Fig. 4D).

PPAR␥ Activation Reduces CSE-induced Inflammation and Oxidative Stress while Up-regulating GR-␣ and HDAC2-To
determine whether PPAR␥ activation can suppress the CSEinduced proinflammatory profile and resulting oxidative stress in epithelial cells, we treated H292 cells with either the synthetic agonist Rosi (1 M) or the endogenous agonist OA-NO 2 (100 nM) for the 30 min preceding a 6-h exposure to 10% CSE. Both PPAR␥ agonists reduced the CSE-induced increases in cytokine gene expression (Fig. 5A) and secretion (Fig. 5B) by ϳ80% and similarly reduced H 2 O 2 production (Fig. 5C) and intracellular ROS seen by immunostaining (Fig. 5D). Both agonists likewise markedly reduced the levels of NF-B and its target proteins COX-2 and NOX4, as well as the phosphorylated forms of MSK1, IKK, and IB (Fig. 5E). These data indicate that agonist-induced PPAR␥ activation inhibits the proinflammatory effects of CSE.
To determine the mechanisms by which PPAR␥ activation exerts its anti-inflammatory and antioxidant effects, we studied the effects of Rosi or OA-NO 2 on nuclear localization and activity of PPAR␥, GR-␣, and HDAC. Treatment with Rosi or OA-NO 2 more than reversed the decreases in nuclear PPAR␥ levels caused by CSE exposure, raising levels even above the air-vehicle treatment baseline (Fig. 6A). Both PPAR␥ agonists likewise up-regulated DNA binding activity of PPAR␥ and GR-␣ along with the deacetylase activity of HDAC2 (Fig. 6, C-E). These increases in activity were accompanied by decreases in PPAR␥ phosphorylation, GR-␣ acetylation, and the levels of HDAC2 phosphorylation, nitrosylation, and alkylation (Fig. 6B). The increased HDAC2 activity was associated with decreased acetylation of histones H3 and H4 (Fig. 6A). Taken together, these data demonstrate that PPAR␥ activation can reverse CSE-induced proinflammatory effects and ROS generation in epithelial cells and suggest that this is due both to direct anti-inflammatory effects of PPAR␥ itself and to restoration of the HDAC2 activity required by GR-␣ for effective antiinflammatory action.
Activation of PPAR␥ Modifies CSE-induced Protein-protein and Protein-DNA Associations-Transcription is regulated both by direct modulation of the activities of relevant transcrip-tion factors and their abilities to attract and bind coactivators or corepressors. Ligand-activated PPAR␥ inhibits NF-B by direct binding, among other mechanisms (17), while GR-␣ links NF-B to the corepressor HDAC2 and blocks its recruitment of the histone acetyltransferase coactivator p300 and the coactivator MSK1. Co-immunoprecipitation experiments showed that CSE exposure greatly increased the amounts of p300 and MSK1 associated with nuclear NF-B. These effects were largely reversed by treatment with either Rosi or OA-NO 2 (Fig.  7A). PPAR␥ activation likewise reversed the CSE-induced decrease in association between GR-␣ and NF-B, while NF-B was further inhibited by increased direct association with PPAR␥. Rosi or OA-NO 2 treatment also increased the association of HDAC2 and of PPAR␥ with GR-␣ (Fig. 7B). We confirmed by ChIP that the observed agonist-induced diminution of CSE-elevated nuclear localization of NF-B (Fig. 7C) and MSK1 (Fig. 7D) was associated with reduced binding to the promoter regions of the NF-B target genes TNF-␣, IL-6, and IL-8. This observation further implies that MSK1 was bound to NF-B.
PPAR␥ Knockdown Amplifies and Activation Reverses CSE Effects in HBE cells-To test whether endogenous PPAR␥ activity suppresses the CSE-induced proinflammatory and related transcriptional program in HBE cells, we used a gene silencing approach. Treating HBE cells with PPAR␥-directed siRNA abrogated PPAR␥ expression to the suppressed levels seen with CSE treatment (Fig. 8A), while agonist treatment up-regulated expression in whole-cell extracts above vehicle-treated baseline (Fig. 8B). Such PPAR␥ knockdown augmented CSE-induced alterations in HDAC and NF-B activity (Fig. 8C) and in CSEinduced stimulation of IL-8 release (Fig. 8D) versus those seen in wild-type cells. Thus, whereas PPAR␥ activation reversed the deleterious effects of CSE, reducing PPAR␥ greatly exaggerated them.
To assess the potential relevance of agonist-induced PPAR␥ activation to the effects of COPD we saw in HBE cells (Fig. 1), we tested the abilities of Rosi-and OA-NO 2 -induced PPAR␥ activation to inhibit CSE responses in HBE cells. As in H292 cells (Figs. 5-7), both agonists abrogated CSE-induced suppression of PPAR␥, GR-␣, and HDAC2 levels and its up-regulation of NF-B (Fig. 8E). These effects of Rosi and OA-NO 2 were accompanied by reduced NF-B binding to the promoter region of its target gene IL-8 (Fig. 8F) and decreased CSE-stimulated IL-8 release (Fig. 8G). These effects in human lung cells thus show that our results are generalizable beyond H292 cells and support their relevance for the smoke-exposed human airway in vivo.

DISCUSSION
Our studies lead to two central conclusions. First, PPAR␥ expression and DNA binding activity are down-regulated in lung tissue samples and bronchial epithelial cells from COPD patients. These observations are linked to down-regulation of GR-␣ and its associated corepressor HDAC2, whereas the transcription factor NF-B is up-regulated. The net effect of these findings is the enhanced airway inflammation typically observed in COPD. CSE treatment of human lung epithelial cells recapitulated the effects observed in samples taken directly from COPD patients, and these effects were exaggerated by PPAR␥ knockdown. Second, these deleterious effects of CSE were reversed by either of two structurally distinct PPAR␥ agonists, one synthetic, the other endogenous, that each upregulated expression and activity of the nuclear receptor. These agonists also largely or completely reversed CSE effects on other anti-inflammatory proteins, on the pro-inflammatory transcription factor NF-B, and on cytokine, chemokine, and ROS production. By demonstrating both PPAR␥ down-regulation in COPD and the ability of PPAR␥ activation to reverse all effects in a smoke-induced in vitro model of the disease, our findings thus support a crucial role of PPAR␥ down-regulation in pathogenesis of smoking-induced COPD.
Mechanistic studies supported multiple paths through which PPAR␥ activation, with consequent increases in its expression and activity, blocks the ability of cigarette smoke to up-regulate NF-B and thus induce inflammation. Co-immunoprecipitation demonstrated binding of PPAR␥ to NF-B, which can inhibit that transcription factor in at least two different ways (17,18). Furthermore, treatment with either PPAR␥ agonist reversed the CSE-induced phosphorylation of IKK, thus blocking degradation of IB and consequent nuclear localization of NF-B (14). This extends to airway epithelial cells and an additional PPAR␥ agonist previous observations that the thiazolidinedione pioglitazone blocks IKK activation through a PPAR␥-dependent mechanism in IL-1␤-stimulated vascular smooth muscle cells (19). The agonist-induced reduction in IKK phosphorylation we see presumably reflects the accompanying decrease in activating phosphorylation of MSK1. The ability of PPAR␥ to attack inflammation via multiple pathways thus enhances its attractiveness as a potential therapeutic target in COPD.
It is also unclear whether the PPAR␥ down-regulation and inhibition we observe directly reflect inflammatory signaling, are due to inflammation-associated oxidative stress, or both. Blanquicett et al. (20) have reported that H 2 O 2 induces a prolonged down-regulation of PPAR␥ mRNA expression in human vascular endothelial cells. The authors attributed this effect to oxidative stress activation of the transcription factor activator protein 1 (AP1). The inhibitory post-translational phosphorylation we also see appears to reflect a different mechanism, however, since it is driven by activation of mitogen-activated protein kinases (MAPKs). Previous studies have shown that such phosphorylation is mediated by members of the MAPK family (21)(22)(23)(24) and that it occurs specifically at Ser-84 (Ser-82 in the mouse) (21,22). There is redundancy in the pathways involved because PPAR␥ can be phosphorylated by either extracellular signal-related kinase (21)(22)(23) or c-Jun N-terminal kinase (21,24). Both are members of the MAPK family that can be activated by cigarette smoke (25).
The limited effectiveness of glucocorticoids represents a major therapeutic challenge in COPD. We demonstrated that CSE reduces nuclear localization and activity of both GR-␣ and the HDAC2 corepressor that it utilizes, with accompanying post-translational modifications of these proteins, and that these effects are reversed by PPAR␥ activation. This observation suggests that PPAR␥ activation may restore glucocorticoid sensitivity in lung epithelial cells and potentially in COPD patients, raising the possibility that joint administration of PPAR␥ and GR-␣ agonists would be therapeutically appropriate. PPAR␥ activation also increased its association with GR-␣, as has been observed by others (26). Whether this PPAR␥-GR-␣ binding is direct or indirect remains unclear, however. It has been suggested that it may represent binding to common coactivators (26).
A prior study found that PPAR␥ levels in lung tissues were up-regulated in patients with mild COPD, but in line with our present findings, were down-regulated in those with moderate or severe disease (27). The cell types involved were not identified. Because PPAR␥ levels were recently found to be unaltered in alveolar macrophages of COPD patients (28), our findings clearly point to lung epithelial cells as a key locus of PPAR␥ down-regulation and target for its potential therapeutic activation in COPD patients. Other previous investigations have shown that PPAR␥ activation can attenuate inflammation either in animal models of COPD or following CSE exposure in vitro, but have not addressed the signaling pathways we investigated. Both the thiazolidinedione pioglitazone (29) and the endogenous PPAR␥ agonist 15-deoxy-⌬ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) (30) have proven effective in the LPS-induced model of COPD, while both Rosi and pioglitazone were effective in a smoke-induced model (28). In vitro, Lee et al. (31) attributed the ability of Rosi to inhibit CSE-induced TNF-␣ and mucin production in H292 cells to up-regulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), with consequent down-regulation of the Akt signaling pathway. However, Rosi did not block CSE-induced cytokine production in a monocyte-macrophage cell line in which CSE disrupts the association between PPAR␥ and NF-B (32). Taken together with the previously mentioned study by Lea et al. (28), this suggests that the role of PPAR␥ in COPD may differ between macrophages and epithelial cells.
Our results show that down-regulation of epithelial cell PPAR␥ expression and activity plays an important role in cigarette smoke-induced inflammation and the pathophysiology of COPD (Fig. 9, top panel). Among its other effects, this downregulation decreases HDAC2 expression and activity, which contributes to increased acetylation of chromatin histones and a looser, unwound chromatin conformation. This conformational change facilitates binding of NF-B (p50 ϩ p65, expression and activity of which are up-regulated in COPD) and its p300 coactivator to the genes' promoter regions. Acetylation by p300 further loosens chromatic structure, allowing binding of RNA polymerase II and increased transcription of these genes. These changes are reversed by PPAR␥ activation (Fig. 9, bottom  panel). This leads to up-regulation of HDAC2 expression and activity, with removal of histone acetyl groups and rewinding of the chromatin. NF-B, now bound to PPAR␥, GR-␣, and HDAC2, no longer binds to the promoter regions of its target proteins. Furthermore, activated PPAR␥ itself attracts the coactivators that NF-B requires (33), limiting the activity of FIGURE 9. Schematic summary of how PPAR␥, GR-␣, and HDAC2 affect transcription of NF-B target genes in COPD. Top panel, cigarette smoke-induced inflammatory signaling, which leads to degradation and post-translational inhibitory modifications of PPAR␥, along with HDAC2 and GR-␣, results in histone acetylation and chromatin unwinding. This allows NF-B and its associated coactivators to bind to target promoters and stimulate transcription by RNA polymerase II (RNA Pol II). Bottom panel, activation of PPAR␥ up-regulates HDAC2 and GR-␣ expression in addition to expression of PPAR␥ itself. This results in removal of histone acetyl groups and condensation of chromatin structure, blocking NF-B and RNA polymerase II binding. NF-B activity is further reduced by binding to PPAR␥ and GR-␣ and, through GR-␣, to HDAC2, as well as by PPAR␥ competition for essential coactivators.
any remaining promoter-bound transcription factor. We also show that PPAR␥ and GR-␣ simultaneously bound to NF-B, as suggested by our finding that these two nuclear hormone receptors co-immunoprecipitate.
Our studies thus provide major new insights into the mechanisms by which COPD-induced down-regulation of PPAR␥ expression and inhibition of its activity contribute to the proinflammatory phenotype characteristic of this disease and the ways in which PPAR␥ agonists reverse these effects. They also illuminate the mechanisms underlying the glucocorticoid insensitivity seen in COPD and imply that PPAR␥ agonists could restore sensitivity. Taken together, these results support the possibility that PPAR␥ agonists might prove effective treatments for this common and deadly disease.