Abstract
BackgroundThe airway epithelial barrier function is disrupted in the airways of asthmatic patients. Abnormal mitochondrial biogenesis is reportedly involved in the pathogenesis of asthma. However, the role of mitochondrial biogenesis in the airway barrier dysfunction has not been elucidated yet. This study aimed to clarify whether the peroxisome proliferator-activated receptor γ coactivator-1alpha (PGC-1α), a central regulator of mitochondrial biogenesis, is involved in the disruption of the airway barrier function induced by aeroallergens.MethodsBEAS-2B cells were exposed to house dust mite (HDM) and the expressions of PGC-1α and E-cadherin, a junctional protein, were examined by immunoblotting. The effect of SRT1720, a PGC-1α activator, was investigated by immunoblotting, immunocytochemistry, and measuring the transepithelial electrical resistance (TEER) on the HDM-induced reduction in mitochondrial biogenesis markers and junctional proteins in airway bronchial epithelial cells. Furthermore,the effects of protease activated receptor 2 (PAR2) inhibitor, GB83, Toll-like receptor 4 (TLR4) inhibitor, lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS), protease inhibitors including E64 and 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) on the HDM-induced barrier dysfunction were investigated.ResultsThe amounts of PGC-1α and E-cadherin in the HDM-treated cells were significantly decreased compared to the vehicle-treated cells. SRT1720 restored the expressions of PGC-1α and E-cadherin reduced by HDM in BEAS-2B cells. Treatment with SRT1720 also significantly ameliorated the HDM-induced reduction in TEER. In addition, GB83, LPS-RS, E64 and AEBSF prevented the HDM-induced reduction in the expression of PGC1α and E-cadherin.ConclusionsThe current study demonstrated that HDM disrupted the airway barrier function through the PAR2/TLR4/PGC-1α-dependent pathway. The modulation of this pathway could be a new approach for the treatment of asthma.
Highlights
The airway epithelial barrier function is disrupted in the airways of asthmatic patients
Materials The following reagents were used in this study: purified house dust mite (HDM) extract from Dermatophagoides pteronyssinus was purchased from LSL (Tokyo, Japan); SRT1720 was from Selleck Chemicals (Houston, TX); GB83 was from Axon Medchem (Groningen, Netherlands); Lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS) was from Invivogen (San Diego, CA); Dexamethasone, E64, 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), and mouse monoclonal anti-β-actin antibody were from Sigma (St Louis, MO)
A blocking reagent, was from Dako (Kyoto, Japan); Rabbit polyclonal antiPGC-1α antibody, rabbit monoclonal anti-TFAM antibody, rabbit monoclonal anti-PINK1 antibody, rabbit monoclonal anti-E-cadherin antibody, rabbit polyclonal anti-zonula occludens (ZO)-1 antibody, FITC-conjugated goat anti-rabbit secondary antibody, and Alexa Fluor 647-conjugated goat anti-rabbit secondary antibody were from Abcam (Cambridge, MA); horseradish peroxidase–conjugated secondary antibodies and mouse monoclonal anti-inducible nitric oxide synthases antibody were from Santa Cruz Biotechnology (Dallas, TX); MitoTracker Red probe and Hoechst 33,342 were from Invitrogen Life Technologies (Eugene, OR); Keratinocyte-SFM and Human Keratinocyte Growth Supplements were from Gibco (Grand Island, NY)
Summary
The airway epithelial barrier function is disrupted in the airways of asthmatic patients. Abnormal mitochondrial biogenesis is reportedly involved in the pathogenesis of asthma. This study aimed to clarify whether the peroxisome proliferator-activated receptor γ coactivator-1alpha (PGC-1α), a central regulator of mitochondrial biogenesis, is involved in the disruption of the airway barrier function induced by aeroallergens. A reduction in the expression of E-cadherin and ZO-1 is reportedly observed in the airways of asthmatic patients [7, 8]. Allergens with proteolytic activity such as HDM can directly cleave epithelial tight junctions and disrupt barrier structures [9, 10]. The major HDM, Dermatophagoides pteronyssinus allergen Der p 1 is known to cleave tight junctions directly and indirectly through protease-activated receptor-2 activation [11]. The precise mechanisms by which epithelial junctions are disrupted are not fully understood
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