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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 23, 2005 as doi:10.1096/fj.04-3309fje. |
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,1
* Department of Internal Medicine,
Research Center for Allergic Immune Diseases,
Department of Pediatrics,
Department of Anatomy, and
|| Department of Biochemistry, Chonbuk National University Medical School, Jeonju, South Korea; and
¶ Division of Endocrinology and Metabolism, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea
1 Correspondence: Department of Internal Medicine, Chonbuk National University Medical School, 634-18, Keumamdong, Jeonju 561-712, South Korea. E-mail: leeyc{at}chonbuk.ac.kr
SPECIFIC AIMS
The peroxisome proliferator-activated receptor 
(PPAR) has been shown to regulate phosphatidylinositol 3-kinase (PI3K) signaling by modulating phosphatase and tensin homologue deleted on chromosome ten (PTEN) expression in inflammatory cells. In the present study, we used a mouse model for asthma to determine the effect of PPAR agonists (rosiglitazone or pioglitazone) and adenovirus (Ad) gene transfer vector expressing a PPAR cDNA (AdPPAR) on allergen-induced bronchial inflammation and airway hyperresponsiveness.
PRINCIPAL FINDINGS
1. Administration of PPAR agonists or AdPPAR reduces airway inflammation
Numbers of total cells and eosinophils in bronchoalveolar lavage (BAL) fluids were increased significantly 72 h after ovalbumin (OVA) inhalation compared with levels after saline inhalation. Increased numbers of eosinophils 3 days after OVA inhalation were significantly reduced by administration of rosiglitazone, pioglitazone, or AdPPAR. Histologic analyses revealed typical pathologic features of asthma in OVA-exposed mice. Numerous inflammatory cells, including eosinophils, infiltrated around bronchioles; the airway epithelium was thickened and mucus and debris accumulated in the lumen of bronchioles compared with the control. Mice treated with rosiglitazone, pioglitazone, or AdPPAR showed marked reductions in the thickening of airway epithelium, infiltration of inflammatory cells in the peribronchiolar region, number of inflammatory cells, and amount of debris in airway lumen. No significant changes were observed in AdLacZ-treated mice.
2. Increased IL-4, IL-5, IL-13, and eosinophil cationic protein (ECP) levels after OVA inhalation are significantly reduced by PPAR agonists or AdPPAR
Western blot analysis revealed that IL-4, IL-5, and IL-13 protein levels in lung tissues were increased significantly 72 h after OVA inhalation compared with levels following saline inhalation. Increased IL-4, IL-5, and IL-13 levels 72 h after OVA inhalation were significantly reduced by administration of rosiglitazone, pioglitazone, or AdPPAR. Consistent with results from Western blot analysis, enzyme immunoassays revealed that increased IL-4, IL-5, and IL-13 levels 72 h after OVA inhalation were significantly reduced by administration of rosiglitazone, pioglitazone, or AdPPAR. RT-PCR analysis revealed that increased IL-4, IL-5, and IL-13 mRNA expression in lung tissues was increased significantly 72 h after OVA inhalation compared with levels after saline inhalation. Increased IL-4, IL-5, and IL-13 mRNA expression in lung tissues 72 h after OVA inhalation was decreased by administration of rosiglitazone, pioglitazone, or AdPPAR.
Increased ECP levels in BAL fluids 72 h after OVA inhalation were decreased significantly by administration of rosiglitazone, pioglitazone, or AdPPAR, indicating these agents attenuate the antigen-induced release of soluble mediators of inflammation into the lungs.
3. Administration of PPAR agonists or AdPPAR reduces airway hyperresponsiveness
Airway responsiveness was assessed as a percent increase of enhanced pause (Penh) in response to increasing doses of methacholine. In OVA-sensitized and -challenged mice, the dose-response curve of percent Penh shifted to the left vs. that of control mice. The percent Penh produced by methacholine administration (doses of 2.5–50 mg/mL) increased significantly in OVA-sensitized and -challenged mice compared with controls. OVA-sensitized and -challenged mice treated with rosiglitazone, pioglitazone, and AdPPAR showed a dose-response curve of percent Penh that shifted to the right compared with that of untreated mice or OVA-sensitized and -challenged mice treated with AdLacZ, indicating that rosiglitazone, pioglitazone, or AdPPAR treatment reduces OVA-induced airway hyperresponsiveness.
4. Expression of PPAR is increased by OVA inhalation, and the increase is further enhanced by administration of PPAR agonists or AdPPAR
Western blot analysis revealed that PPAR levels in lung tissues were increased significantly 72 h after OVA inhalation compared with levels after saline inhalation. Increased PPAR levels were further increased by administration of rosiglitazone, pioglitazone, or AdPPAR.
5. Activation of PPAR up-regulates PTEN expression in allergen-induced asthmatic lungs. This up-regulation correlates with decreased PI3K activity as measured by reduced phosphorylation of Akt
Western blot analysis revealed that PTEN protein levels were decreased significantly 72 h after OVA inhalation compared with levels after saline inhalation (Fig. 1
A, B). Administration of rosiglitazone, pioglitazone, or AdPPAR resulted in elevation of PTEN protein levels similar to control levels. PTEN enzyme assays revealed that PTEN activity was decreased significantly 72 h after OVA inhalation compared with levels after saline inhalation (Fig. 2C
). Decreased PTEN activity 72 h after OVA inhalation was increased by administration of rosiglitazone, pioglitazone, or AdPPAR.
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Immunoreactive PTEN localized in epithelial layers around the bronchioles of control mice. This immunoreactive PTEN disappeared in allergen-induced asthmatic lungs. Intratracheal administration of AdPPAR restored PTEN expression in allergen-induced asthmatic mice lungs whereas AdLacZ did not.
Expression of PTEN in lung epithelial cells after isolation and primary culture of murine tracheal epithelial cells was observed. Immunocytologic analysis showed localization of immunoreactive PTEN in tracheal epithelial cells from control mice. However, immunoreactive PTEN was markedly reduced in tracheal epithelial cells from OVA-exposed mice. Intratracheal administration of AdPPAR restored PTEN expression in tracheal epithelial cells from allergen-induced asthmatic mice, but AdLacZ did not.
Immunocytologic analysis of BAL fluids showed localization of PTEN in BAL cells from control mice. However, immunoreactive PTEN was markedly reduced in precipitated cells from OVA-exposed mice. Intratracheal administration of AdPPAR restored PTEN expression in BAL cells from allergen-induced asthmatic mice whereas AdLacZ did not.
Expression of PTEN in BAL fluid eosinophils isolated by Percoll gradients was examined by immunocytology. Immunoreactive PTEN was markedly reduced in eosinophils from OVA-exposed mice. Intratracheal administration of AdPPAR restored immunoreactive PTEN expression in BAL fluid eosinophils from allergen-induced asthmatic mice; additional AdLacZ treatment did not affect immunoreactivity.
To support the contention that the effects of rosiglitazone, pioglitazone, or AdPPAR on allergen-induced bronchial inflammation and airway hyper-responsiveness were specifically directed through the PI3K pathway, we used Western blot analysis to determine Akt phosphorylation. Levels of p-Akt protein in lung tissues were increased 72 h after OVA inhalation compared with levels in control mice; no significant changes in Akt protein levels were observed in any group tested. Increased p-Akt but not Akt protein levels in lung tissues 72 h after OVA inhalation were significantly reduced by rosiglitazone, pioglitazone, or AdPPAR.
CONCLUSIONS AND SIGNIFICANCE
Recent studies have indicated that PPAR plays an important role in anti-inflammatory responses and that PPAR signaling is associated with regulation of PTEN expression. It is known that up-regulation of PTEN expression reduces asthmatic pathogenesis. However, interrelationship between these proteins in regulation of anti-inflammatory function has not been examined with an in vivo asthma model. Our present study with OVA-induced murine model of asthma has revealed that activation of PPAR with the agonists enhances expression of PPAR and PTEN, resulting in reduction of eosinophilic inflammation and airway hyperresponsiveness. We found that overexpression of PPAR by administration of AdPPAR increases PTEN expression while decreasing levels of ECP and various cytokines (IL-4, IL-5, and IL-13) induced by OVA inhalation. These findings suggest that PPAR uses PTEN to modulate asthmatic responses.
Studies have demonstrated that activation of PPAR reduces expression of various cytokines, airway hyperresponsiveness, and activation of eosinophils which are increased by induction of asthma. Consistent with these observations, our results showed that administration of PPAR agonists or AdPPAR substantially inhibited expression of cytokines (IL-4, IL-5, and IL-13), airway hyperresponsiveness, and eosinophilic inflammation. Induction of asthma through OVA challenge increased expression of PPAR itself and administration of the agonists further increased receptor expression. Up-regulation of PPAR expression is observed in human asthmatic airways. Overexpression of PPAR by administration of AdPPAR resulted in reduction of all asthmatic features. These findings indicate that PPAR is associated with anti-inflammatory responses in asthma.
Studies have demonstrated that inflammatory mediators attract and activate eosinophils via signal transduction pathways involving PI3K. PTEN functions primarily as a lipid phosphatase that regulates crucial signal transduction pathways mediated by phosphatidylinositol 3,4,5-triphosphate (PIP3). PTEN has been implicated in regulating cell survival signaling through the PI3K/Akt pathway. PTEN opposes the action of PI3K by dephosphorylating the signal lipid PIP3. PIP3, produced by PI3K after activation by receptor tyrosine kinases, activated Ras, or G-proteins leads to stimulation of several downstream targets, including the serine/threonine protein kinase Akt (Fig. 2
). We previously demonstrated that PI3K activity is up-regulated and PTEN expression is reduced in allergen-induced model of asthma. Consistent with these previous findings, in the present OVA-induced model of asthma, PTEN protein level is substantially reduced whereas Akt phosphorylation is increased. Administration of PPAR agonists or AdPPAR results in increase of PTEN expression up to the level of the control. In support, Akt phosphorylation increased by induction of asthma with OVA is significantly reduced by administration of PPAR agonists or AdPPAR. The signaling mechanism by which stimulation of PPAR with the agonists regulates PTEN expression as well as Akt phosphorylation remains to be elucidated. However, our results agree with the observation that the anti-inflammatory action of PPAR agonists is mediated via up-regulation of PTEN. Mounting evidence indicates that many inflammatory mediators attract and activate eosinophils via signal transduction pathways involving PI3K. Studies have shown that administration of PI3K inhibitors such as wortmannin suppresses generation of various inflammatory mediators in eosinophils. We recently demonstrated that administration of AdPTEN cDNA or PI3K inhibitor significantly attenuates bronchial inflammation and airway hyperresponsiveness. Collectively, these findings suggest a protective role for PPAR in the pathogenesis of the asthma phenotype through regulation of PTEN expression.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3309fje; doi: 10.1096/fj.04-3309fje
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