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The Journal of Immunology, 2008, 180, 7697 -7705
Copyright © 2008 by The American Association of Immunologists, Inc.
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*Cystic Fibrosis

Tissue Transglutaminase Activation Modulates Inflammation in Cystic Fibrosis via PPAR{gamma} Down-Regulation1

Luigi Maiuri2,*,{dagger}, Alessandro Luciani{dagger},{ddagger}, Ida Giardino§, Valeria Raia, Valeria R. Villella||, Maria D'Apolito{dagger}, Massimo Pettoello-Mantovani{dagger}, Stefano Guido||, Carolina Ciacci{ddagger}, Mariano Cimmino#, Olivier N. Cexus**, Marco Londei{dagger}{dagger},* and Sonia Quaratino2,**,{dagger}{dagger}

* European Institute for Cystic Fibrosis Research, San Raffaele Scientific Institute, Milan, Italy; {dagger} Institute of Pediatrics, University of Foggia, Foggia, Italy; {ddagger} Department of Experimental Medicine, University Federico II, Naples, Italy; § Department of Laboratory Medicine, University of Foggia, Foggia, Italy; Department of Pediatrics, University Federico II, Naples, Italy; || Department of Chemical Engineering, University Federico II, Naples, Italy; # Department of Otolaryngology, University Federico II, Naples, Italy; ** Cancer Research UK Oncology Unit, University of Southampton, Southampton, United Kingdom; {dagger}{dagger} Institute of Child Health, University College, London, United Kingdom; and * Novartis Pharma AG Translational Medicine, Basel, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cystic fibrosis (CF), the most common life-threatening inherited disease in Caucasians, is due to mutations in the CF transmembrane conductance regulator (CFTR) gene and is characterized by airways chronic inflammation and pulmonary infections. The inflammatory response is not secondary to the pulmonary infections. Indeed, several studies have shown an increased proinflammatory activity in the CF tissues, regardless of bacterial infections, because inflammation is similarly observed in CFTR-defective cell lines kept in sterile conditions. Despite recent studies that have indicated that CF airway epithelial cells can spontaneously initiate the inflammatory cascade, we still do not have a clear insight of the molecular mechanisms involved in this increased inflammatory response. In this study, to understand these mechanisms, we investigated ex vivo cultures of nasal polyp mucosal explants of CF patients and controls, CFTR-defective IB3-1 bronchial epithelial cells, C38 isogenic CFTR corrected, and 16HBE normal bronchial epithelial cell lines. We have shown that a defective CFTR induces a remarkable up-regulation of tissue transglutaminase (TG2) in both tissues and cell lines. The increased TG2 activity leads to functional sequestration of the anti-inflammatory peroxisome proliferator-activated receptor {gamma} and increase of the classic parameters of inflammation, such as TNF-{alpha}, tyrosine phosphorylation, and MAPKs. Specific inhibition of TG2 was able to reinstate normal levels of peroxisome proliferator-activated receptor-{gamma} and dampen down inflammation both in CF tissues and CFTR-defective cells. Our results highlight an unpredicted central role of TG2 in the mechanistic pathway of CF inflammation, also opening a possible new wave of therapies for sufferers of chronic inflammatory diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The breach of the innate immune system and a chronically infected and inflamed lung are the characteristic features of cystic fibrosis (CF)3 (1). It has been estimated that ~30,000 children and adults in the U.S. (70,000 worldwide) are affected by CF, with a frequency of ~1 in 2,500 livebirths (1).

The direct link between CF transmembrane conductance regulator (CFTR) dysfunction and airway infection has been shown. Defects in the CFTR result in an impaired clearance of lung pathogens, especially Pseudomonas aeruginosa (2, 3). Infections are then further exacerbated by the poor mucociliary clearance due to ciliary dysfunction and the hyperabsorption of water by the airways epithelium (4).

The mechanisms by which CFTR mutations might contribute to airways inflammation are, however, still elusive. Defects of the CFTR are also associated with a marked increase of proinflammatory cytokines, such as TNF-{alpha}, IL-6, IL-1β, IL-17 (5, 6), and the potent neutrophil chemoattractant and activator IL-8, which recruits large numbers of neutrophils into the airways (1). Chronic inflammation, however, is not merely the consequence of repetitive infections, because CFTR-defective cell lines spontaneously develop similar proinflammatory features when kept in vitro in sterile conditions (7, 8). Indeed, CFTR-defective cells have been reported to have an intrinsically proinflammatory phenotype, and despite different intracellular pathways that have been considered, none to date has been demonstrated to be associated (7, 8).

To underpin the molecular link between a defected CFTR and the excessive inflammatory responses typical of CF airways here we have studied nasal polyp mucosa from CF patients (9) as well as CFTR-defective bronchial epithelial cell lines (2, 10). The rationale to extend the study to the cell lines was to specifically target the role of the defective CFTR on peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) in sterile conditions, excluding the influence that recurrent infections might have on the epithelium and the generation of inflammation.

We have highlighted how a defected CFTR induces high levels of tissue transglutaminase (TG2) in the airways. TG2 has a pivotal role in the initiation of inflammation by sequestering PPAR{gamma}, a nuclear hormone receptor expressed in monocytes, macrophages, and epithelial cells that negatively regulates inflammatory gene expression by transrepressing inflammatory responses (11). This work also suggests that TG2 inhibition could become a therapeutic target to control inflammation in CF and possibly in other chronic inflammatory diseases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Human airway biopsies and ex vivo cultures

Nasal polyp explants from 10 CF patients carrying the common CFTR mutations ({Delta}F508/{Delta}F508, {Delta}F508/W1282X, {Delta}F508/N1303K, or {Delta}F508/G542X) and 10 non-CF patients with nonallergic idiopathic polyposis were cultured, for 4–24 h (9), with or without specific TG2 inhibitors 1,3-dymethyl-2-[(2-oxopropyl) thio] imidazolium (R283) (250 µM) (12) or halo-dihydroisoxazole-derivate transglutaminase inhibitor KCC009 (250 µM), reactive oxygen species (ROS) scavenger EUK 134 (50 µg/ml; Alexis Biochemical), N-acetyl-cysteine (NAC, 10 mM; Alexis Biochemical), PPAR{gamma} antagonist GW9662 (1 µM; Alexis Biochemical), or R283 for 24 h, followed by GW9662 (1 µM) for 4 h. Informed consent was obtained from all subjects, and the ethical committee of Regione Campania Health Authority approved the study.

Cell lines and cultures

IB3-1 (human CF bronchial epithelial cell line with the common {Delta}F508/W1282X CFTR mutation) and C38 (isogenic stably rescued with functional CFTR) cell lines (LGC Promochem) (2, 7, 10) were stimulated for 6 h with R283 (250 µM) or KCC009 (250 µM), ionomycin (1 µM; Calbiochem), BAPTA-AM (5 µM, Calbiochem), EUK 134 (50 µg/ml), rosiglitazone (10 µµ), NAC (10 mM), proteasome inhibitor MG132 (50 µM for 6 h; Calbiochem), or R283 for 24 h, followed by 6-h rosiglitazone. Normal human bronchial epithelial 16HBE cells were cultured, as previously described (8).

Quantitative RT-PCR

Quantitative RT-PCR was performed using iCycler iQ Multicolour Real-Time PCR Detector (Bio-Rad) with iQ TM SYBR Green supermix (Bio-Rad). A relative quantitative method was applied for TG2 mRNA (Qiagen; catalog QT00081277), normalized by the control GAPDH mRNA.

RNA interference

IB3-1 cells were transfected with 50 nM human TG2, human PPAR{gamma}, and scramble small interfering RNAs (siRNAs) duplex using Hiperfect Transfection Reagent (Qiagen) at 37°C for 72 h. The target sequence of TG2 siRNA was 5'-CCGCGTCGTGACCAACTACAA-3', and the whole-cell lysate was then analyzed for Western blot. The PPAR{gamma} siRNA is a pool of three sequences, as follows: CCAAGUAACUCUCCUCAAAtt, GAAUGUGAAGCCCAUUGAAtt, and CUACUGCAGGUGAUCAAGAtt. Transfected cells were then analyzed by confocal microscopy for expression using anti-PPAR{gamma} mAb clone E8.

Western blot

First Abs anti-phospho-p42/p44 MAPKs (Cell Signaling Technology), PPAR{gamma} (clone E8 sc-7273; Santa Cruz Biotechnology), TG2 (clone CUB7402; DakoCytomation), N{epsilon}({gamma}-L-glutamyl)-L-lysine isopeptide (clone 81DIC2; Covalab), and ubiquitin (1:100 clone FL-76, rabbit polyclonal IgG) were counterstained by a HRP-conjugated anti-IgG Ab (Amersham, General Healthcare). The amounts of proteins were determined by a Bio-Rad protein assay to ensure equal protein loading before Western blot analysis. Fifty micrograms of cell lysate were loaded in each lane.

Immunoprecipitation

Following cell lysis, 500 µg of proteins was incubated at 4°C overnight with anti-PPAR{gamma} Ab (clone E8) and then mixed with protein G-Sepharose beads for 2 h. The beads were washed three times with lysis buffer, and immunoprecipitates were resuspended in SDS loading buffer. Equal amounts of immunoprecipitate were loaded and analyzed for Western blot.

ROS detection

Cell lines were pulsed with 10 µM 5(and 6)-chloromethyl-2'7'-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA; Molecular Probes, Invitrogen), according to manufacturer suggestions, and analyzed with Wallac 1420 multilabel counter (PerkinElmer). Tissue sections (5 µm) of nasal mucosa and cells were incubated with 10 µM CM-H2DCFDA, according to manufacturer suggestions, and an LSM510 Zeiss confocal laser-scanning unit (Carl Zeiss, Germany) was used for detection.

In situ detection of TG2 activity and protein

TG2 protein and enzyme activity were detected and analyzed by confocal microscopy. Briefly, for detection of TG2 enzyme activity, live cells were preincubated with TG2 assay buffer (965 µl of 100 mM Tris/HCl (pH 7.4), 25 µl of 200 mM CaCl2) for 15 min and then with the same TG2 assay buffer added with 10 µl of 10 mM biotinylated monodansylcadaverine (bio-MDC; Molecular Probes) for 1 h at room temperature. The reaction was stopped with 25 mM EDTA for 5 min. The cells were then fixed in 4% paraformaldehyde for 10 min. The incorporation of labeled substrate was visualized by incubation with PE-conjugated streptavidin (DakoCytomation; 1:50) for 30 min. Control experiments included the omission of bio-MDC, as well as replacement of 200 mM CaCl2 with 200 mM EDTA. Blocking experiments were also conducted by incubating cells with the active site inhibitor R283 (250 µM) for 1 h before incubation with the substrates (bio-MDC or biotinylated peptides) and detection of TG2 enzymatic activity. Data were analyzed under fluorescence examination by LSM510 Zeiss confocal laser-scanning unit (Carl Zeiss).

The simultaneous detection of TG2 protein and activity was performed by incubating live cells with bio-MDC for 1 h at room temperature, according to the above described procedure, and then with anti-TG2 CUB 7402 mAb (NeoMarkers; 1:50) for 1 h at room temperature. This was followed by simultaneous incubation with PE-conjugated streptavidin (DakoCytomation; 1:50) and FITC-conjugated rabbit anti-mouse Ig F(ab')2 (DakoCytomation; 1:20) for 1 h.

The detection of TG2 activity and protein on tissue sections was performed, as reported in our previous paper (12).

Confocal microscopy

Cell lines were fixed in methanol and permeabilized with 0.5% Triton X-100 prior incubation with primary Abs. Frozen tissue sections were fixed in acetone for 10 min. Anti-phospho-p42/p44 (1:500; Cell Signaling Technology), anti-phosphotyrosine PY99 mAb (1:80, mouse IgG2b), anti-PPAR{gamma} (clone E8, sc-7273, 1:100, mouse IgG1; clone H100, sc-7196, 1:100, rabbit polyclonal IgG; Santa Cruz Biotechnology), anti-ubiquitin (1:100 clone FL-76, rabbit polyclonal IgG), anti-histone deacetylase 6 (HDAC6; 1:100 clone H300, rabbit polyclonal IgG), anti-p65 NF-{kappa}B (F-6; 1:100), and anti-ICAM-1 (15.2; 1:200) (Santa Cruz Biotechnology) Abs were used for first Ab. The Ag expression and distribution were visualized by indirect immunofluorescence, as previously described (12).

ELISA

Human TNF-{alpha} secretion was measured using the BD OptEIATM TNF-{alpha} ELISA kit II (BD Biosciences). Measurements were performed at least in triplicate. Values were normalized to 106 cells; results were expressed as mean ± SEMs.

Statistical analysis

All experiments were performed at least in triplicate. Data distribution was analyzed, and statistical differences were evaluated by using ANOVA Tukey-Kramer test by SPSS 12 software. A p value of <0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
PPAR{gamma} colocalizes in perinuclear aggresomes with ubiquitin and HDAC6 in CF epithelium

We initially determined whether PPAR{gamma}, a key inflammation regulator implicated in the prevention of several immunoinflammatory disorders (13), had any role in CF. PPAR{gamma}, produced by several cell types, including epithelial cells, regulates inflammation via the specific inhibition of proinflammatory cytokines, such as TNF-{alpha}, IL-6, IL-1β, and the NF-{kappa}B pathway, and by modulating oxidative stress (13, 14, 15).

Nasal polyp mucosal biopsies from CF patients and controls were ex vivo cultured, as previously reported, so that epithelial, myeloid, and lymphoid components can retain all the interactions with neighboring cells within their natural environment (9).

We found that PPAR{gamma} expression in nasal biopsies from CF patients and the CFTR-defective IB3-1 cell line was significantly reduced compared with controls and the isogenic C38 cell line with functional CFTR (Fig. 1, A and B). Remarkably, PPAR{gamma} was mainly limited to perinuclear localization in CF tissues and CFTR-defective IB3-1 cell line (Fig. 1, A and B).


Figure 1
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  		FIGURE 1. PPAR{gamma} expression in airway epithelial cells. Confocal images of nasal polyp mucosa from CF patients and controls (A) and IB3-1 and C38 cells (B): low PPAR{gamma} expression (green) (as detected by clone E8 Ab) with perinuclear aggregates in nasal CF epithelia and in IB3-1 cells; CyTRAK Orange (red), nuclear counterstaining. Yellow color indicates nuclear localization of PPAR{gamma}. C, Confocal images of IB3-1 cells: colocalization (yellow) of ubiquitin (red, top line) or HDAC6 (red, bottom line) in perinuclear aggregates of PPAR{gamma} (green). D, Left panel, Immunoblot detection of PPAR{gamma} in C38 and IB3-1 cells incubated with or without the proteasome inhibitor MG132. Right panel, Immunoprecipitated PPAR{gamma} species from whole-cell extracts of IB3-1 cells are immunoreactive for the anti-ubiquitin Ab. Immunoprecipitation (IP): anti-PPAR{gamma} Ab; immunoblot (IB): anti-ubiquitin Ab. E, Confocal images of IB3-1 cells: high PPAR{gamma} expression (green) in perinuclear aggregates upon MG132 incubation. F, IB3-1 cells after incubation with MG132: expression and colocalization of ubiquitin/PPAR{gamma} (yellow, left panel) or HDAC6/PPAR{gamma} (yellow, right panel) in aggresomes. A–C and E and F, Scale bar, 10 µm.

 
This perinuclear localization was suggestive of aggresomes, a specific cellular response against accumulation of ubiquitinated misfolded and/or aggregated proteins (16), which might also result from impaired proteasome function (16). Although aggresomes containing PPAR{gamma} have not been described before, aggresomes containing ubiquitinated misfolded {Delta}F508 CFTR molecules have been previously observed as result of overexpression of the defective CFTR (16). The aggresomes of CFTR also contain HDAC6, a microtubule-associated deacetylase that interacts with ubiquitin and stabilizes polyubiquitin chains (17). We have shown that in the CFTR-defective IB3-1 cell line, the perinuclear aggregates of PPAR{gamma} also colocalize with both HDAC6 and ubiquitin (Fig. 1C), thus demonstrating the aggresome nature of these aggregates. Because the presence of aggresomes and HDAC6 is usually the cellular response to eliminate cytoplasmic misfolded proteins (17), we have then investigated whether PPAR{gamma} was subjected to proteasome degradation. Treatment with the specific proteasome inhibitor MG132 (18) led to an increase in PPAR{gamma} protein levels (Fig. 1, D and E) and PPAR{gamma} ubiquitination (Fig. 1D) in CFTR-defective IB3-1 cells. Treatment with MG132 also led to an impressive perinuclear increase of HDAC6- and ubiquitin-PPAR{gamma} colocalization (Fig. 1F) in IB3-1 cells, suggesting a role for proteasome degradation.

Similar results were obtained using a polyclonal anti-PPAR{gamma} Ab with different epitope specificity (data not shown). To confirm the anti-PPAR{gamma} Ab specificity, IB3-1 cells were transfected with human PPAR{gamma} siRNA and then immunostained with anti-PPAR{gamma} mAb clone E8. We did not detect any PPAR{gamma} in the IB3-1 cell transfected with human PPAR{gamma} siRNA (data not shown).

TG2 is up-regulated in human CFTR-defective cells

Aggresomes are a prominent cytopathological feature of most neurodegenerative disorders, including Parkinson’s and Huntington’s diseases (19). In these pathologies, there is also an increase in TG2, responsible for the aggregation of {alpha}-synuclein in Parkinson’s (20, 21, 22, 23). TG2 is a pleiotropic enzyme expressed by many cell types, including epithelial cells, often up-regulated in many chronic inflammatory conditions (22, 23). Therefore, we explored whether TG2 levels or its enzymatic activity were up-regulated in CF tissues and CFTR-defective cell lines.

We observed a significant increase of TG2 protein and enzymatic activity in CF tissues as well as the IB3-1 cell line (Fig. 2, A–C). The increase of TG2 was demonstrated by immunoblot and confocal microscopy, whereas real-time PCR showed that TG2 transcript was significantly higher in IB3-1 than C38 cell lines (Fig. 2D). These results clearly indicate that CFTR-defective epithelial cells are characterized by an intrinsic TG2 functional increase.


Figure 2
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  		FIGURE 2. TG2 expression in airway epithelial cells. Confocal images identified expression of TG2 (green) and its activity (blue) in epithelial cells of nasal polyp mucosa from CF patients (A) and IB3-1 CF cells (B) as compared with controls. Merge indicates overlay of individual channels (cyan). Scale bar, 10 µm. C, Lysates of C38 and IB3-1 cells were immunoblotted with anti-TG2 Abs. D, Real-time PCR of TG2 mRNA in C38 and IB3-1 cells. TG2 transcript was significantly higher in IB3-1 than C38 cells (*, p < 0.05 vs C38 cells). Error bars represent SEMs.

 
TG2 induces cross-linking of PPAR{gamma} and mediates aggresome formation

We then tested the hypothesis that TG2 might induce posttranslational modifications (cross-linking) of PPAR{gamma} in CFTR-defective cells, because the QG and QXXP motifs in the PPAR{gamma} sequence could be recognized as specific sites for TG2 activity (22).

Double labeling immunofluorescence with anti-PPAR{gamma} Ab and an isopeptide cross-link specifically catalyzed by TG2 (22) demonstrated colocalization only in IB3-1 perinuclear aggregates (Fig. 3A). Furthermore, using an anti-PPAR{gamma} Ab, several high molecular mass bands ranging between 72 and 250 kDa were immunoprecipitated and detected by the anti-isopeptide Ab in the IB3-1 line (Fig. 3B). Normal 55-kDa PPAR{gamma} in IB3-1 cells was reduced (Fig. 3C), whereas C38 cell line presented only faint levels of the high molecular mass and a considerable amount of the normal 55-kDa PPAR{gamma} (Fig. 3, B and C). Exposure of the IB3-1 cells to the irreversible TG2-specific inhibitor R283 (a gift from M. Griffin, Aston University, Birmingham, U.K.) (12, 23, 24) tested at increasing concentrations 50–500 µM with similar results and induced a significant reduction of high molecular mass PPAR{gamma} (Fig. 3D) and a significant increase of the normal 55-kDa PPAR{gamma} in the IB3-1 cells (Fig. 3E). This indicated that the cross-linked PPAR{gamma} was due to increased TG2 activity. A similar result was achieved through TG2 gene silencing by siRNA (Fig. 3F). Exposure of both CF tissues and IB3-1 cells to R283 also resulted in a significant reduction of PPAR{gamma} aggregates, restoring a wider intracellular distribution of PPAR{gamma} (Fig. 3G).


Figure 3
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  		FIGURE 3. Effects of TG2 on PPAR{gamma} protein expression and localization. A, Confocal images of IB3-1 and C38 cells immunostained with PPAR{gamma} (green) and the anti-isopeptide Ab (red). Merge of individual channels shows colocalization (yellow) in perinuclear aggregates. B, Immunoprecipitated PPAR{gamma} species from whole-cell extracts of C38 and IB3-1 cells are immunoreactive for the anti-isopeptide cross-link Ab. Immunoprecipitation (IP): anti-PPAR{gamma} Ab; immunoblot (IB): anti-isopeptide Ab. C, Immunoblot of PPAR{gamma} in C38 and IB3-1 cells. D, Effect of R283 on PPAR{gamma} immunoreactivity for the anti-isopeptide Ab in IB3-1 cells. IP: anti-PPAR{gamma} Ab; IB: anti-isopeptide Ab. E, Immunoblot analysis of PPAR{gamma} in IB3-1 cells with or without R283. F, Immunoblot of PPAR{gamma} in IB3-1 cells: effect of TG2 siRNA. Scramble siRNA was used as a negative control. 72 kDa, TG2; 55 kDa, PPAR{gamma}. Confocal images of G, PPAR{gamma} (green) immunostaining of CF nasal mucosa and IB3-1 cells with or without R283, and H, PPAR{gamma} (green) in IB3-1 cells (left panel) and in C38 cells (right panel) after incubation with PPAR{gamma} agonist rosiglitazone (10 µM for 6 h) with or without prior incubation with R283. CyTRAK Orange (red) nuclear counterstaining. Yellow color indicates nuclear localization of PPAR{gamma}. A, G, and H, Scale bar, 10 µm.

 
In IB3-1 cells, PPAR{gamma} nuclear translocation was promoted only by R283, whereas rosiglitazone, a PPAR{gamma} agonist with potent anti-inflammatory activity used in clinic (25), induced only a marginal nuclear PPAR{gamma} increase (Fig. 3H). In contrast, rosiglitazone alone was able to induce an impressive PPAR{gamma} nuclear translocation in C38 cells (Fig. 3H).

These results indicate that PPAR{gamma} aggregates in CFTR-defective cells are induced by TG2.

TG2 inhibition controls inflammation in CF epithelial cells

TG2 activity does not only control the levels and function of PPAR{gamma}, but also influences other markers of inflammation. Inhibition of TG2 with either R283 or KCC009 (another TG2-specific inhibitor, gift from C. Khosla, Stanford University, Palo Alto, CA; data not shown) induced a significant reduction of the phosphorylated p42/p44 MAPKs (Fig. 4A), ICAM-1, and NF-{kappa}B activation (data not shown) both in CF tissues and IB3-1 cells. Inhibition of TG2 by siRNA also induced a significant decrease of the phosphorylated p42/p44 (Fig. 4B) and TNF-{alpha} release (Fig. 4C) in IB3-1 cells.


Figure 4
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  		FIGURE 4. Inhibition of TG2 activity controls inflammation in human airway CF epithelia and CFTR-defective cells. A, Confocal images of phospho-p42/p44 MAPK expression (green) by CF nasal mucosa and IB3-1 cells cultured with or without R283; CyTRAK Orange (red) nuclear counterstaining. B, Immunoblot of phospho-p42/p44 MAPKs in IB3-1 cells after TG2 gene silencing or incubation with R283. Scramble siRNA was used as a negative control. C, TNF-{alpha} ELISA in IB3-1 cells after TG2 silencing or incubation with R283. The experiment was repeated three times (*, p < 0.05 vs IB3-1 cells cultured in medium alone). Error bars represent SEMs. Confocal images of D, phospho-tyr expression (Py99 Ab) by CF nasal mucosa after incubation with R283 or R283, followed by GW9662, and E, TG2 activity (blue) in IB3-1 cells with Ca2+-chelator BAPTA-AM and in C38 cells with Ca2+-ionophore ionomycin. Phospho-p42/p44 MAPK expression (green) by IB3-1 cells cultured with or without BAPTA-AM and by C38 cells cultured with or without ionomycin. CyTRAK Orange (red) nuclear counterstaining. F and G, NAC decreases expression of TG2 activity (blue), phospho-tyr, phospho-p42/p44 (green) in CF nasal polyp mucosa (F), and in IB3-1 cells (G). Expression of TG2 protein (green) in both CF nasal polyp mucosa and IB3-1 is not affected by NAC treatment (F and G). H, TNF-{alpha} secretion in IB3-1 cell line cultured for 48 h with or without NAC. The experiment was repeated three times (*, p < 0.05 vs IB3-1 cells cultured in medium alone). Error bars represent SEMs. A and D–G, Scale bar, 10 µm.

 
The inhibitory activity of R283 on tyrosine phosphorylation, a well-established marker of epithelial inflammation in human CF airway mucosa (9), was abrogated by 4-h incubation with the PPAR{gamma} antagonist GW9662 (Fig. 4D), further indicating that TG2 controls inflammation through PPAR{gamma}.

Intracellular Ca2+ and ROS levels modulate TG2 activity and inflammation in CFTR-defective cells

We then investigated the mechanisms underlying TG2 up-regulation in CFTR-defective cells. Because TG2 activity is strictly Ca2+ dependent (22), and high Ca2+ mobilization has been shown in CF nasal or bronchial epithelia (26), we looked at the role of Ca2+ in CFTR-defective cells.

Ca2+ decrease, induced by the Ca2+ chelator BAPTA-AM (27), significantly reduced TG2 activity (Fig. 4E) as well as p42/44 phosphorylation (Fig. 4E) in IB3-1 cells. The Ca2+ ionophore ionomycin (27), on the contrary, increased TG2 activity (Fig. 4E) and phospho-p42/p44 (Fig. 4E) in C38 cells. These results demonstrated that Ca2+ ions drive TG2 activation and thus inflammation in CF epithelium.

Because TG2 activity is also regulated by ROS (27), we tested whether ROS influenced the TG2-induced inflammation in CF.

In agreement with previous reports, high levels of ROS were only detected in CF tissues and CFTR-defective cells (data not shown) (28). The ROS scavenger NAC significantly reduced TG2 activity (Fig. 4, F and G), phosphotyrosine expression (Fig. 4F), p42/44 phosphorylation (Fig. 4G), and TNF-{alpha} release (Fig. 4H) in both CF tissues and IB3-1 cells. Similar results were obtained after inhibition of ROS by another ROS scavenger, EUK 134 (data not shown).

CFTR inhibition induces TG2 up-regulation and inflammation in normal bronchial 16HBE cells

To assess that the pathway described to date was a direct consequence of CFTR dysfunction, we blocked the functional CFTR in the normal bronchial 16HBE cell line with the selective inhibitor CFTRinh-172 (8). We observed all the modifications we have reported above, such as increase of intracellular ROS (Fig. 5A), increase of TG2 protein and its activity (Fig. 5B), decrease of PPAR{gamma} (Fig. 5C), and increase of p42/44 phosphorylation (Fig. 5C).


Figure 5
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  		FIGURE 5. CTR inhibition induces airways inflammation. The 16HBE cells were cultured with or without CFTRinh-172 inhibitor. A, Confocal microscopy of intracellular ROS (CM-H2DCFDA detection, green) or a Wallac 1420 multilabel counter (each bar represents the mean plus SEMs of three separate experiments, each with n = 8; *, p < 0.01 compared with C38 cells). B, Confocal images of TG2 (green) and its activity (blue). Merge indicates overlay of individual channels (cyan). C, Confocal images of PPAR{gamma} and phospho-p42/44 MAPKs (green); CyTRAK Orange (red) nuclear counterstaining. Immunoblot analysis of PPAR{gamma} (55 kDa) and phospho-p42/44 MAPKs (42 and 44 kDa). A–C, Scale bar, 10 µm.

 
Altogether, these results clearly demonstrated how CFTR disruption directly leads to the development of airway inflammation in CF.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The novel finding of the present study is the demonstration that CFTR mutations lead to an increase of TG2 levels and activity that, in turn, down-regulate the anti-inflammatory effects of PPAR{gamma}. In cftr–/– mice, PPAR{gamma} expression is down-regulated both at the mRNA and protein levels, and its function is reduced compared with wild-type littermates (29). Therefore, PPAR{gamma} agonists have been exploited in therapeutic approaches to control inflammation in airway diseases in animal models (30, 31).

We have shown that PPAR{gamma} is not only reduced in CF epithelium and CFTR-defective cell lines, but also confined to perinuclear aggresomes, prominent pathological features common to many neurodegenerative conditions such as Huntington and Parkinson’s diseases (32). Aggresomes are generated in response to alterations of the ubiquitin proteasome system, the principal cellular mechanism to eliminate misfolded proteins before they aggregate (33). Aggresomes often develop when a threshold of misfolded protein is exceeded, and are usually enriched of molecular chaperone, proteasome subunits, ubiquitin conjugates, and the histone deacetylase HDAC6. In CF, the defective CFTR is often not transported beyond the endoplasmic reticulum and accumulates in aggresomes (16, 33). The data shown in Fig. 1C clearly indicate that also PPAR{gamma} colocalizes in aggresomes with ubiquitin and HDAC6. In CFTR-defective cells, the specific inhibition of the ubiquitin-proteasome system with MG-132 led to a significant increase of PPAR{gamma}, HDAC6-, and ubiquitin-PPAR{gamma} colocalization in aggresomes (Fig. 1, D–F). This suggests that the formation of these aggresomes in CF is caused by the accumulation of misfolded proteins fated to final degradation by the proteasome-ubiquitination system.

The report that in Parkinson’s disease TG2 cross-links {alpha}-synuclein and induces its aggregation in high molecular mass aggresomes (34) prompted us to investigate whether TG2 also had a role in the aggresome formation in CF. TG2 is a pleiotropic enzyme with a calcium-dependent transamidating activity that results in cross-linking of proteins via {epsilon}({gamma}-glutamyl) lysine bonds (22, 23). Our results clearly demonstrated a significant increase of TG2 protein and enzymatic activity in CF epithelium and CFTR-defective cell lines. TG2 up-regulation was also responsible for the aggresome formation in CF. Importantly, TG2 induced cross-linking of PPAR{gamma} and its sequestration into aggresomes (Fig. 3, A–D), because the TG2-inhibitor R283 produced a significant reduction of PPAR{gamma} protein aggregates and restored a wider intracellular protein distribution (Fig. 3, E–G). We have proven the unequivocal role of TG2 in controlling PPAR{gamma} and inflammation by blocking TG2 enzymatic activity (R283) as well as its translation (siRNA). Furthermore, the inhibition of the normal CFTR in a normal 16HBE bronchial cell line led to a dramatic up-regulation of TG2 and inflammation, clearly showing the direct link between CFTR defectiveness and TG2 up-regulation and inflammation.

Collectively, our study provides a molecular explanation of the reported association between CFTR malfunction and sterile inflammation. Highlighting the role of TG2 in CF, we have also indicated novel ways to modulate inflammation, a key component in CF pathogenesis.

Furthermore, increased amounts of TG2 have been recently described in other diseases, including cancer, in which up-regulation of TG2 is associated with an increased metastatic activity (35) or drug resistance, as in breast cancer, through the activation of NF-{kappa}B (36, 37). Because of its capacity to activate NF-{kappa}B, a crucial mediator of inflammation-induced tumor growth and metastatic progression, through depletion of free I{kappa}B{alpha} (38), TG2 provides a mechanistic link between inflammation and cancer.

In this view, our study may have a wider impact in the whole area of inflammation and cancer.


   Acknowledgments
 
We thank Martin Griffin (Aston University, Birmingham, U.K.) and Chaitan Khosla (Stanford University, Palo Alto, CA) for the gift of the TG2-specific inhibitors R283 and KCC009, respectively.


   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 the European Institute for Cystic Fibrosis Research, Cancer Research U.K., and Rothschild Trust. Back

2 Address correspondence and reprint requests to Dr. Sonia Quaratino, Cancer Research UK Oncology Unit, University of Southampton, SO16 6YD Southampton, UK. E-mail address: sq{at}soton.ac.uk or Luigi Maiuri, European Institute for Cystic Fibrosis Research, San Raffaele Scientific Institute, via Olgettina 58, 20132, Milan, Italy. E-mail address: maiuri{at}unina.it Back

3 Abbreviations used in this paper: CF, cystic fibrosis; bio-MDC, biotinylated monodansylcadaverine; CFTR, CF transmembrane conductance regulator; CM-H2DCFDA, 5(and 6)-chloromethyl-2'7'-dichlorodihydrofluorescein diacetate acetyl ester; HDAC6, histone deacetylase 6; NAC, N-acetyl-cysteine; PPAR{gamma}, peroxisome proliferator-activated receptor-{gamma}; ROS, reactive oxygen species; siRNA, small interfering RNA; TG2, tissue transglutaminase. Back

Received for publication January 8, 2008. Accepted for publication March 28, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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