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Am J Physiol Gastrointest Liver Physiol 293: G319-G326, 2007. First published April 26, 2007; doi:10.1152/ajpgi.00056.2007
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INFLAMMATION/IMMUNITY/MEDIATORS

PPAR-{gamma} knockout in pancreatic epithelial cells abolishes the inhibitory effect of rosiglitazone on caerulein-induced acute pancreatitis

C. Y. Ivashchenko,1 S. Z. Duan,1 M. G. Usher,1 and R. M. Mortensen1,2,3

Departments of 1Molecular and Integrative Physiology, 2Pharmacology, and 3Internal Medicine, Metabolism, Endocrinology, and Diabetes Division, University of Michigan Medical School, Ann Arbor, Michigan

Submitted 31 January 2007 ; accepted in final form 16 April 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) agonists, such as the thiazolidinediones (TZDs), decrease acute inflammation in both pancreatic cell lines and mouse models of acute pancreatitis. Since PPAR-{gamma} agonists have been shown to exert some of their actions independent of PPAR-{gamma}, the role of PPAR-{gamma} in pancreatic inflammation has not been directly tested. Furthermore, the differential role of PPAR-{gamma} in endodermal derivatives (acini, ductal cells, and islets) as opposed to the endothelial or inflammatory cells is unknown. To determine whether the effects of a TZD, rosiglitazone, on caerulein-induced acute pancreatitis are dependent on PPAR-{gamma} in the endodermal derivatives, we created a cell-type specific knock out of PPAR-{gamma} in pancreatic acini, ducts, and islets. PPAR-{gamma} knockout animals show a greater response in some inflammatory genes after caerulein challenge. The anti-inflammatory effect of rosiglitazone on edema, macrophage infiltration, and expression of the proinflammatory cytokines is significantly decreased in pancreata of the knockout animals compared with control animals. However, rosiglitazone retains its effect in the lungs of the pancreatic-specific PPAR-{gamma} knockout animals, likely due to direct anti-inflammatory effect on lung parenchyma. These data show that the PPAR-{gamma} in the pancreatic epithelia and islets is important in suppressing inflammation and is required for the anti-inflammatory effects of TZDs in acute pancreatitis.

peroxisome proliferator-activated receptor-{gamma}; caerulein-induced acute pancreatitis; pancreatic epithelial cell-type specific PPAR-{gamma} knockout; proinflammatory cytokines; rosiglitazone

ACUTE PANCREATITIS (AP) is a common condition that carries a significant risk of morbidity and mortality. It is characterized by intra-acinar cell activation of digestive enzymes, pancreatic inflammation, cell damage, and a systemic inflammatory response governed by the release of proinflammatory cytokines (37). The severity of acute pancreatitis can vary over a wide range, and severe pancreatitis has a mortality rate between 15 and 20% (51).

The factors that lead to the autodigestive, inflammatory condition of AP include occlusion of the pancreatic duct with subsequent block in the drainage of the digestive enzymes, mechanical tissue injury, hypercalcemia, autoimmune diseases, and certain viral infections. Certain insults, such as long-term ethanol consumption, may sensitize the pancreas to development of AP (28, 45). Initial acinar cell injury may be due to the inappropriate activation of proteases, in particular trypsin, within the pancreas and this is one of the earliest events in the pathophysiology of experimental AP (24, 55).

One of the most widely used models of AP in mice is injection of caerulein, a cholecystokinin (CCK) analog and pancreatic secretagogue. Whereas CCK stimulates acinar cells to secrete digestive enzymes and pancreatic fluid, hyperstimulation with caerulein leads to edematous pancreatitis characterized by a higher serum amylase level, edema, leukocyte infiltration, and vacuolation of acinar cells (52). Unchecked release of the activated digestive enzymes also leads to increased expression and release of the inflammatory cytokines and chemokines such as IL-1beta, IL-6, MCP-1, and IL-8 that are not normally detected in pancreas at high levels (6, 49). These molecules are not exclusively synthesized by infiltrating lymphocytes. Both exocrine and endocrine cells of the pancreas, including acinar, ductal cells, and pancreatic stellate cells, as well as fibroblasts, are thought to be important in the production of inflammatory cytokines (14). The combined release of the inflammatory mediators by pancreatic and immune system cells during AP leads to amplification of the inflammatory reaction and supports further leukocyte infiltration and activation of pancreas. At the same time, increased vascular permeability leads to increased edema and ischemia of the pancreas. The sharp increase in the serum levels of inflammatory mediators has the potential to transform AP into systemic inflammatory response which affects lungs, liver, circulatory system, and other organs. In fact, pancreatitis-associated pulmonary injury results in significant morbidity by itself (30, 39).

Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}), a nuclear transcription factor that regulates adipocyte differentiation and fatty acid metabolism and is a target of the anti-diabetic thiazolidinediones (TZDs), is expressed in both endocrine and exocrine pancreatic cell types (43, 56). The role of PPAR-{gamma} in pancreas is not restricted to insulin signaling pathways. As a transcriptional activator and sometimes a transcriptional repressor, it has been shown to be important in cell growth, metabolism (in particular responses to altered energy homeostasis), apoptosis, and inflammation (for review, see Refs. 46 and 50). Both specific PPAR-{gamma} agonists, such as TZDs, and less specific ones, such as 15d-PGJ2, have been shown to attenuate the severity of the inflammation both in mouse models of AP (9, 27, 43) and chronic pancreatitis (48). In in vitro experiments, PPAR-{gamma} agonists inhibit expression of proinflammatory cytokines and adhesion molecules in various cell types, such as endothelial cells (33, 54), intestinal epithelial cells (26), macrophages (1, 40), and monocytes (25).

It has not been established which of the pancreatic cell types are responsible for the anti-inflammatory effects of PPAR-{gamma} activation in pancreatitis. Furthermore, since TZDs have been shown to have PPAR-{gamma}-independent effects depending on the cell type and the response tested (2, 7, 31), their anti-inflammatory action in the pancreas cannot be proved to be PPAR-{gamma} dependent by use of agonists alone. To determine whether PPAR-{gamma} expression in the pancreatic acini, ductal cells, and islets is necessary for the anti-inflammatory effects of TZDs, we utilized a cell-type-specific knockout model of PPAR-{gamma} in pancreatic epithelia to study the severity of AP induced by caerulein.


   METHODS
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 METHODS
RESULTS
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Animal work. Mice deleted for PPAR-{gamma} selectively in beta-cells, pancreatic ducts, and acinar cells were generated by crossing a Pdx-1 Cre mouse line (kind gift of D. A. Melton, described in Ref. 21) to mouse line with two floxed PPAR-{gamma} alleles PPAR-{gamma} fl/fl (kind gift of F. Gonzalez) to create pancreatic epithelial-specific PPAR-{gamma} knockout (PANC-PGKO) mice. Age-matched sibling floxed control (FC) or PANC-PGKO mice received either rosiglitazone (Cayman Chemical, Ann Arbor, MI) (50 mg·kg–1·day–1) in chow or vehicle for 10 days before induction of AP. AP was induced by intraperitoneally injected caerulein (Sigma, no. C-9026) at 50 µg/kg hourly for 8 h. Control animals received saline injections. Each of eight experimental groups consisted of three to six animals and the experiments were repeated three times. Two hours after the last caerulein injection, mice were killed and dissected. Pancreas was divided into several parts. One part was used to quantitate the extent of edema by measuring tissue water content. Briefly, pancreatic and lung tissue was weighed before and after desiccation at 95°C for 24 h. The difference between wet and dry weight was expressed as percent of the wet weight. The other parts of the pancreas and lung were frozen in liquid nitrogen for later RNA and protein extraction, fixed in formalin, or frozen in optimum cutting temperature compound for histological analysis.

Histological analysis. Formalin-fixed paraffin embedded sections were stained with hematoxylin and eosin (H&E) using an H&E kit (Lab Visions) according to manufacturer's instructions. Slides were photographed using an Olympus BX microscope. Numbers of infiltrating neutrophils and the severity of inflammation were evaluated by two skilled blinded evaluators. Infiltrating neutrophils were counted in three to four random fields per slide, three slides per condition, as described previously (5). Similarly, to evaluate the severity of inflammation, four H&E slides per condition were inspected by the evaluators.

Immunohistochemistry. F4/80, differentiated macrophage marker was used to quantify the level of macrophage infiltration of frozen pancreatic tissue sections. Briefly, sections were digested for 10 min in proteinase K (Qiagen) in PBS, washed, blocked with SuperBlock reagent in Tris-buffered saline (Pierce) supplemented with 0.1% Tween 20 and incubated with 1:200 dilution of F4/80 antibody (BMA) in blocking buffer. Fluorescent secondary antibody was used at 1:1,000 dilution and sections were photographed with use of a Leica inverted microscope. Numbers of infiltrating macrophages were counted per picture; three pictures/slide were randomly chosen.

QRT-PCR. RNA was isolated from frozen tissues with an RNeasy kit from Qiagen (with Tissue Rotor to homogenize samples) in buffer RLT supplemented with 2-mercaptoethanol. Samples were treated (on column) with DNase (Qiagen), eluted with water, and quantified by use of a UV spectrophotometer. Reverse transcription reactions using a Applied Biosciences kit with random hexamers as primers were done with 1 µg of RNA for each sample. Quantitative real-time PCR (QRT-PCR) was performed by using Bio-Rad iCycler, with the use of SYBRgreen. Samples were normalized to levels of beta-actin, 18S RNA, and GAPDH. Primer sequences of the following primers are available upon request: beta-actin, 18S RNA, GAPDH, ICAM-1, CXCL1, MCP-1, IL-6, IL-10, neutrophil elastase (ELA2).

Western blot analysis. Tissues were prepared by homogenization in standard lysis buffer supplemented with protease and phosphatase inhibitors. For nuclear and cytoplasmic isolation of protein, an Active Motif Nuclear Extract kit was used as per manufacturer's protocol. Five to 15 µg of each protein sample were subjected to (SDS-PAGE) gel electrophoresis followed by transfer to nitrocellulose membrane (Bio-Rad). Membranes were probed with antibodies against PPAR-{gamma} (E-8), p65/RelA (C-20), p50/p105, c-Jun (Santa Cruz Biotechnology) at 1:500, ICAM-1 (Santa Cruz Biotechnology) 1:2,000, or beta-actin (Sigma) (1:2,000); followed by appropriate HRP-conjugated secondary antibodies (Pierce) at the dilution 1:2,000, all in Tris-buffered saline with 5% milk and 0.2% Tween 20. After incubation with SuperSignal reagent (Pierce), bands were detected on a Bio-Rad FluorSMax imager and quantified with Bio-Rad Quantity One software. Normalization to either actin bands or {alpha}-tubulin was performed unless quantification of the Coomassie showed no difference between samples.

Statistics. For evaluations of histological sections, two skilled blinded observers looked at three to four slides of pancreata from animals of each experimental group and scored inflammation. Western blot, QRT-PCR, and immunohistochemistry experiments were repeated a minimum of three times. Statistical analyses were performed by ANOVA and unpaired two-tailed Student's t-test. Data are presented as means ± SE. The value for P ≤ 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
To examine the role of PPAR-{gamma} in pancreatic inflammation, viable mice deleted for PPAR-{gamma} in acinar cells, pancreatic ducts and islets of Langerhans were generated using a Pdx-1 Cre mouse line crossed to mice with second exon of the PPAR-{gamma} gene flanked by the LoxP sites, to create PANC-PGKO mice. Pdx-1 is required for pancreas formation and is expressed in the endodermal precursor but not in endothelial or mesodermal derivatives such as fibroblasts. During early stages of mouse embryogenesis, Pdx-1 is highly expressed throughout the entire pancreatic epithelium, including both endocrine and exocrine cells, as well as subpopulations of duodenal and gastric enteroendocrine cells (16, 21). It has been shown that Pdx-1 Cre mice crossed to reporter line Z/AP mice demonstrate a uniform HPAP staining in pancreatic acinar, ductal, and islet cells (21). Therefore, our PANC-PGKO mice lack functional PPAR-{gamma} in pancreatic acini, ducts, and islets. Acinar cells are thought to represent ~80% of the tissue volume, ductal cells contribute ~10%, and islets only 1–2% of the volume of the pancreas. The expression of PPAR-{gamma} in pancreas varies by cell type. For instance, the amount of PPAR-{gamma} protein in the exocrine pancreas has been shown to be lower than in the endocrine pancreas (13). Western blotting for PPAR-{gamma} protein (Fig. 1A) shows 80–90% decrease in the amount of PPAR-{gamma} in the knockout animals' pancreata, which is consistent with the proposed amounts of epithelial cells in the pancreas and with the activity of the Cre recombinase response.


Figure 1
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  		Fig. 1. Pancreatic epithelial-specific PPAR-{gamma} knockout (PANC-PGKO) animals show markedly reduced PPAR-{gamma} levels compared with controls. A: in Pdx-1 Cre mice crossed to PPAR-{gamma} fl/fl mice, 80–90% of the PPAR-{gamma} protein is knocked out of the pancreata of the transgenic mice. Representative Western blot of PPAR-{gamma} in floxed control (FC) and knockout (KO) animals' pancreas and quantification of band intensities of PPAR-{gamma} to a-tubulin signal (n = 4). B: PPAR-{gamma} KO animals' pancreas is histologically normal [hematoxylin and eosin stain (H&E)]. C: the weight of the pancreas is increased. PW, pancreas weight; BW, body weight.

 
Characterization of the PANC-PGKO mice shows that inactivation of PPAR-{gamma} in pancreatic epithelial cells leads to normal pancreatic histology (Fig. 1B) but significantly increased weight of pancreas (Fig. 1C) and enlarged islets (data not shown), consistent with the previously published reports that targeted PPAR-{gamma} knockout in beta-cells leads to islet hyperplasia (11, 44) and that heart PPAR-{gamma} knockout leads to increased heart size (11, 12).

Administration of rosiglitazone has been previously shown to ameliorate the severity of acute pancreatitis induced by caerulein administration (9). FC and PANC-PGKO mice were untreated or pretreated with rosiglitazone for 10 days at dose of 10 mg/kg. On the day of the caerulein challenge, AP was induced by eight consecutive injections of caerulein. Two hours after the last caerulein injection, mice were killed and their tissues collected. Representative tissue sections from one of the three experiments are shown (Fig. 2A). Histologically, there is no observable difference between untreated or rosiglitazone-treated PANC-PGKO and FC mice. Stimulation with caerulein induced pancreatic and lung inflammation. Ten-day pretreatment with rosiglitazone significantly decreased AP induced by caerulein in FC mice but not in the PANC-PGKO mice. Scoring of inflammation, defined as accumulations of neutrophils and mononuclear cells, perivascular infiltrate, and hemorrhage as well as edema and tissue necrosis was performed by two skilled observers on slides from at least three animals per condition from a representative experiment (Fig. 2B).


Figure 2
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  		Fig. 2. PPAR-{gamma} is required for rosiglitazone suppression of caerulein-induced AP as measured by histology and quantitative real-time PCR (QRT-PCR). Control (Cont), sham injected; Rosi, 10 days rosiglitazone in chow; Cer, 8 hourly injections of caerulein; Rosi + Cer, 10 days rosiglitazone in chow followed by 8 hourly caerulein injections. A: H&E staining of the pancreas tissue slides. NT, not treated. B: histological score of inflammation, 0 being no inflammation, 5 the highest degree of inflammation observed; 2 skilled blinded evaluators, 4 H&E slides per condition. C: quantification of F4/80 macrophage marker staining: counts of macrophages/field, 3–4 slides per condition, 3–4 fields/slide. D: QRT-PCR for neutrophil marker, neutrophil elastase (ELA2), n = 6 per group. E: neutrophil count, 3–4 H&E slides/condition.

 
Immunohistochemistry analysis of cells positive for F4/80, an activated macrophage marker, shows that rosiglitazone decreases numbers of macrophages infiltrating pancreata in caerulein-stimulated FC but not KO mice (Fig. 2C). In addition, significant presence of vacuolated cells was noted in slides from caerulein-treated animals but not scored. Caerulein-induced pancreatic neutrophil infiltration was quantified both by quantitative real time PCR for ELA2 marker (10) and by neutrophil count in the H&E sections. ELA2 expression is significantly increased by caerulein in pancreas of both FC and PANC-PGKO animals. However, whereas its high levels are suppressed by rosiglitazone in the FC pancreas, they remain unaffected by rosiglitazone treatment in the PANC-PGKO (Fig. 2D). Neutrophil count in H&E sections supported the pattern seen in QRT-PCR of ELA2 (Fig. 2E).

Pancreatic and lung edema are well known hallmarks of acute pancreatitis and have been shown to be induced by caerulein. Pancreatic edema induced by caerulein is suppressed by rosiglitazone in FC but not PANC-PGKO animals (Fig. 3A). However, lung edema was suppressed by rosiglitazone in both FC and KO mice (Fig. 3B). Since PPAR-{gamma} is still present in the lung parenchyma, TZDs are able to suppress lung inflammation directly.


Figure 3
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  		Fig. 3. PPAR-{gamma} is required for rosiglitazone-mediated decrease of caerulein-induced edema. Effects of rosiglitazone are maintained in the lungs of PANC-PGKO mice. Each experimental group n = 6–8. A: pancreatic edema. B: lung edema.

 
The initial inflammatory response in pancreas is augmented by the recruitment of the inflammatory cells from the circulation to the organ. This recruitment is partially mediated by the expression of adhesion molecules on the surface of vascular endothelial cells in pancreas. In turn, the leukocyte infiltrate contributes to the maintenance and amplification of the inflammatory response. In addition to measures of macrophage and neutrophil infiltration, we measured the expression of a number of proinflammatory cytokines and adhesion molecules, which can be expressed both by the cells of the pancreas and by the leukocytes and which have previously been shown to be both induced by the caerulein and inhibited by the administration of the PPAR-{gamma} agonists. In particular, we used QRT-PCR to examine the expression of a canonical NF-{kappa}B target genes ICAM-1 and CXCL1, shown to be induced in AP (15, 32), and of two other genes, MCP-1, a putative AP-1 target, and IL-6, whose transcriptional control is more complex including regulation by NF-{kappa}B, AP-1, and C/EBP-{alpha} (4).

In lungs of both FC and PANC-PGKO animals, caerulein induced significant increases in ICAM-1, MCP-1, and ELA2. Approximately threefold induction of ICAM-1 was observed in both FC and PANC-PGKO animals. Rosiglitazone pretreatment abrogated this induction in both FC and PANC-PGKO animals (Fig. 4A). MCP-1 was induced to a significantly different degree in the lungs of the FC (3-fold) and PANC-PGKO (5-fold) animals. This suggests that PPAR-{gamma} absence may have a potentiating effect on the induction or progression of the inflammatory response. Nevertheless, rosiglitazone pretreatment still suppressed, likely through direct effects as described above, the proinflammatory gene expression in lungs of both FC and PANC-PGKO animals (Fig. 4, B and C).


Figure 4
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  		Fig. 4. Rosiglitazone suppresses caerulein-induced inflammatory molecules in lung. QRT-PCR in lung samples of FC and KO mice. Each experimental group n = 9–16. A: ICAM-1. B: MCP-1. C: ELA2.

 
In pancreas, some genes consistently show significantly higher baseline levels of expression in the PANC-PGKO animals compared with FC (e.g., MCP-1 see Fig. 5B) whereas others showed a trend toward higher levels (e.g., ICAM-1, CXCL1; Fig. 5, A and C). This correlates with the recent data indicating that the expression of PPAR-{gamma} inversely correlates with pancreatitis severity and expression of the proinflammatory cytokines in the pancreas (43). Significantly, caerulein-induced increase in expression of proinflammatory cytokines in pancreas was statistically different between the FC and the PANC-PGKO groups for MCP-1 and CXCL1 (Fig. 5, B and C) and a trend toward increased caerulein-induced inflammation was observed for ICAM-1 in some experiments. The inhibitory effect of PPAR-{gamma} activation on caerulein-induced inflammation is present in the FC mice, where rosiglitazone reduces the expression of proinflammatory cytokines back to basal levels. However, in the PANC-PGKO animals rosiglitazone did not significantly decrease the levels of these cytokines. Therefore, PPAR-{gamma} in the epithelial and/or islet cells of the pancreas is necessary for the antagonistic effects of rosiglitazone on the expression of proinflammatory cytokines in experimental model of acute pancreatitis.


Figure 5
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  		Fig. 5. Rosiglitazone suppresses caerulein-induced inflammatory molecules in pancreas of FC but not PANC-PGKO animals. QRT-PCR in pancreas of FC and KO animals. Each experimental group n = 9–16. NS, not significant. A: ICAM-1. B: MCP-1. C: CXCL1. D: IL-6.

 
Mechanisms of the anti-inflammatory effect of PPAR-{gamma} activation are not well understood. After determining that its activation in the epithelial, endothelial, and/or islet cells of the pancreas leads to suppression of AP, we looked for the possible mechanism of this effect. Since PPAR-{gamma} induction of a potent anti-inflammatory gene has not been demonstrated, we considered its possible involvement in several previously described mechanisms in pancreatitis. Changes in the NF-{kappa}B expression levels in a model of chronic pancreatitis (22) have been described and adenoviral overexpression of RelA/p65 subunit of the NF-{kappa}B is sufficient to induce pancreatic and systemic inflammatory response (8). Therefore we examined total levels of p65, p50, I{kappa}B-{alpha}, and phosphorylated I{kappa}B-{alpha} by Western blotting. Protein levels of the NF-{kappa}B pathway members were not significantly different between the FC and the PANC-PGKO animals treated or untreated with rosiglitazone or caerulein at the time point tested (data not shown). However, increased transcription of the NF-{kappa}B target genes (ICAM-1, CXCL1, MCP-1) after caerulein treatment is inhibited by PPAR-{gamma} activation in FC but not PANC-PGKO animals. This suggests that PPAR-{gamma} acts either by direct binding to NF-{kappa}B or by indirect effects at the promoters of NF-{kappa}B target genes.

Another proinflammatory transcription factor, AP-1, does appear to be affected by PPAR-{gamma} activation in the context of pancreatic inflammation. c-Jun, a component of AP-1 is induced by the caerulein treatment in both the FC and the PANC-PGKO animals. Since rosiglitazone retains its suppressive effect on the c-Jun levels in the PANC-PGKO animals (Fig. 6), we conclude that inhibition of c-Jun is not required for TZD anti-inflammatory activity. Furthermore, TZD's modulation of c-Jun may be a PPAR-{gamma}-independent event. Alternatively, it may occur in pancreatic endothelial cells although it seems unlikely because of the small number of endothelial cells. This result offers a glimpse of the complexity of the PPAR-{gamma} activity in the pancreatic inflammation: multiple inflammatory pathways are involved and are being affected by rosiglitazone treatment.


Figure 6
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  		Fig. 6. Rosiglitazone inhibits caerulein-induced increase in c-jun in both FC and PANC-PGKO mice. A: representative Western blot of c-Jun. B: quantification of band intensities of c-Jun to a-tubulin signal (n = 6).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this report, we demonstrate that rosiglitazone acts directly through PPAR-{gamma} in acini, ducts, and islets to suppress markers of the inflammatory response developed during acute pancreatitis. Increased chemokine production by the acinar cells is an important early stage event in the AP (19). In islets, CCK-producing cells within the pancreatic islets could also be involved in the amplification of the inflammatory response (47). However, the relative importance of the contribution of these cell types as opposed to macrophages and endothelial cells to inflammation in pancreatitis has not been evaluated.

The positive feedback loop of proinflammatory cytokine production in which endothelial cells, macrophages, and neutrophils participate is an important determinant of the extent of inflammation in many organs. In particular, vascular endothelial cell activation and injury caused by sustained exposure to inflammatory cytokines plays a significant role in the pathogenesis of atherosclerosis (for review, see Ref. 18). Recent studies suggest that TZDs have antiatherogenic effects at least partially due to their anti-inflammatory actions in endothelial cells (34, 41). Neutrophils express almost no PPAR-{gamma} and are therefore unlikely targets for TZDs (3, 20). Therefore, we considered the possibility that TZDs would still modify inflammation in pancreas when the PPAR-{gamma} is removed from all the pancreatic cell populations derived from endoderm but remains in the endothelium. However, we show that cell-type-specific knockout of PPAR-{gamma} in pancreatic epithelia and islets removes anti-inflammatory effects of rosiglitazone. Although endothelial or macrophage PPAR-{gamma} may still have an important role, its activation by TZDs is not sufficient to suppress inflammation. Therefore, PPAR-{gamma} in endodermal-derived cells plays a major role in the development and modification of the pancreatic inflammatory response.

Locally increased production of the proinflammatory cytokines and leukocyte activation by pancreatitis can promote inflammation in distant organs, including the lung. In the PANC-PGKO animals, the anti-inflammatory effect of rosiglitazone in the lung is maintained despite full pancreatitis. Elevated cytokines and activated leukocytes still provide a stimulus for lung inflammation. However, lung expresses PPAR-{gamma} at high levels and TZDs can suppress this inflammatory response (3, 53). Therefore the anti-inflammatory effects of rosiglitazone are likely due to direct effects on the PPAR-{gamma} in the lung parenchyma.

Although the mechanisms of the TZDs' anti-inflammatory effects are not completely deciphered, it is known that PPAR-{gamma} activation in the context of inflammation has pleiotropic effects. These PPAR-{gamma} effects may involve direct change in transcription through binding of the PPAR-{gamma}/RXR-{alpha} heterodimer to the PPAR-{gamma} response elements in the promoters of the target genes (17) or alteration of activity of other transcription factors or regulators (36, 42). The anti-inflammatory activity of PPAR-{gamma} may, therefore, be due to the induction of a potent anti-inflammatory mediator or to its antagonism of the proinflammatory transcription factors. To date, no direct anti-inflammatory genes have been found to be activated by PPAR-{gamma} in pancreas. However, it has been shown that in intestinal epithelial cells, direct binding of PPAR-{gamma} to the NF-{kappa}B subunit p65/RelA and the shuttling of the activated transcription factors from the nucleus is partially responsible for the reduction in expression of proinflammatory genes after PPAR-{gamma} activation (26). In macrophages, PPAR-{gamma} becomes SUMOylated upon its activation, leading to the transrepression of the inflammatory genes through targeting nuclear receptor corepressor–histone deacetylase-3–PPAR-{gamma} complexes to the response elements occupied by NF-{kappa}B (38). Which of these, or other mechanisms play a role in the pancreatic epithelial cells remains to be shown.

We were not able to find changes in the basal levels of proinflammatory transcription factors such as p65/RelA and AP-1 in the pancreas. However, higher baseline expression of some proinflammatory molecules such as MCP-1 suggests that PPAR-{gamma} knockout does have a permissive effect on inflammation. The role of PPAR-{gamma} is complex as we do show that inhibition of c-Jun, a component of the AP-1, by rosiglitazone is maintained in the PANC-PGKO animals. Involvement of AP-1 in the caerulein-induced inflammation has been suggested. Caerulein induces c-Jun expression through the activation of stress pathways (activation of MAPKs, through increased JNK activity and phosphorylation and activation of AP-1, that in turn leads to increased levels of c-Jun). JNK inhibitors have a protective effect in caerulein-induced AP (35). At the same time rosiglitazone can directly inhibit stress-activated protein kinases (23, 29). Therefore, it is possible that rosiglitazone has a weak anti-inflammatory effect through AP-1 inhibition that is most probably PPAR-{gamma} independent, but our data indicate that this mechanism does not contribute significantly to inhibition of inflammation in AP. This warrants further investigation of the anti-inflammatory PPAR-{gamma} mechanism.

In summary, we show that cell-type-specific knockout of PPAR-{gamma} in epithelial cells of the pancreas removes the anti-inflammatory effects of rosiglitazone on proinflammatory cytokine production, pancreatic edema, and infiltration of the tissues with the leukocytes. Therefore, PPAR-{gamma} in pancreatic epithelial cells plays a major role in the development and modification of the pancreatic inflammatory response. Our study contributes to the growing body of data that suggests that PPAR-{gamma} activation might provide a new approach to modifying pancreatic inflammation and be useful in the management of AP.


   GRANTS
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 ABSTRACT
 METHODS
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 DISCUSSION
 GRANTS
REFERENCES
 
This work was funded by a pilot project and cores of the Michigan Diabetes Research and Training Center (NIH5P60 DK-20572 from the National Institute of Diabetes and Digestive and Kidney Diseases) and R01HL-083201 (R. M. Mortensen) from the National Heart, Lung, and Blood Institute.


   ACKNOWLEDGMENTS
 
The authors thank Dr. J. A. Williams and Dr. Y. D. Ivashchenko for critical reading of the manuscript and Debora VanHeyningen for expert technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Mortensen, 1150 W. Medical Ctr. Dr., Med. Sci. II, Rm. 7744, Ann Arbor, MI 48109 (e-mail: rmort{at}umich.edu)

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


   REFERENCES
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