calpain and Asthma

Allergic asthma is characterized by inflammation and airway remodelling. Airway remodelling with excessive deposition of extracellular matrix (ECM) and larger smooth muscle mass are correlated with increased airway responsiveness and asthma severity. Calpain is a family of calcium-dependent endopeptidases, which plays an important role in ECM remodelling. However, the role of calpain in airway smooth muscle remodelling remains unknown.. To investigate the role of calpain in asthmatic airway remodelling as well as the underlying mechanism.. The mouse asthma model was made by ovalbumin sensitization and challenge. Calpain conditional knockout mice were studied in the model. Airway smooth muscle cells (ASMCs) were isolated from smooth muscle bundles in airway of rats. Cytokines IL-4, IL-5, TNF-α, and TGF-β1, and serum from patients with asthma were selected to treated ASMCs. Collagen-I synthesis, cell proliferation, and phosphorylation of Akt in ASMCs were analysed.. Inhibition of calpain using calpain knockout mice attenuated airway smooth muscle remodelling in mouse asthma models. Cytokines IL-4, IL-5, TNF-α, and TGF-β1, and serum from patients with asthma increased collagen-I synthesis, cell proliferation, and phosphorylation of Akt in ASMCs, which were blocked by the calpain inhibitor MDL28170. Moreover, MDL28170 reduced cytokine-induced increases in Rictor protein, which is the most important component of mammalian target of rapamycin complex 2 (mTORC2). Blockage of the mTORC2 signal pathway prevented cytokine-induced phosphorylation of Akt, collagen-I synthesis, and cell proliferation of ASMCs and attenuated airway smooth muscle remodelling in mouse asthma models.. Our results indicate that calpain mediates cytokine-induced collagen-I synthesis and proliferation of ASMCs via the mTORC2/Akt signalling pathway, thereby regulating airway smooth muscle remodelling in asthma.

Topics: Airway Remodeling; Animals; Asthma; Calpain; Cell Proliferation; Collagen Type I; Cytokines; Dipeptides; Disease Models, Animal; Mechanistic Target of Rapamycin Complex 2; Mice; Mice, Knockout; Muscle, Smooth; Myocytes, Smooth Muscle; Phosphorylation; Proto-Oncogene Proteins c-akt; Rapamycin-Insensitive Companion of mTOR Protein; Signal Transduction

Obesity is known to increase asthma risk and severity. Increased levels of asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, are associated with mitochondrial toxicity, asthma, and metabolic syndrome. IL-4 upregulates the expression of protein arginine methyltransferases, which are essential for ADMA formation. Importantly, cross-talk between IL-4, ADMA, and mitochondrial dysfunction could explain how obesity and IL-4 can synergize to exacerbate allergic inflammation.. We sought to investigate how IL-4, a key asthma-associated cytokine, can influence ADMA-related effects on lungs.. BEAS2B (bronchial epithelial) cells were treated with IL-4 followed by ADMA and investigated for oxo-nitrative stress and resultant mitochondrial toxicity after 48 hours by using flow cytometry, confocal imaging, immunoblotting, and fluorimetric assays.. IL-4-induced mitotoxicity in BEAS2B cells was significantly higher in the presence of exogenous ADMA. IL-4 treatment led to proteolytic degradation of dimethylarginine dimethylaminohydrolase 2, which catabolizes ADMA. IL-4 pretreatment was associated with increased intracellular ADMA accumulation and increased ADMA-induced mitotoxicity. Airway epithelial cells treated with IL-4 followed by ADMA showed exaggerated oxo-nitrative stress and potent induction of the cellular hypoxic response, despite normoxic conditions. The hypoxic response was associated with reduced mitochondrial function but was reversible by overexpression of the mitochondrial biogenesis factor, mitochondrial transcription factor A.. We conclude that IL-4 promotes intracellular ADMA accumulation, leading to mitochondrial loss through oxo-nitrative stress and hypoxic response. This provides a novel understanding of how obesity, with high ADMA levels, and asthma, with high IL-4 levels, might potentiate each other and highlights the potential of mitochondrial-targeted therapeutics in obese subjects with asthma.

Topics: Amidohydrolases; Apoptosis; Arginine; Asthma; Calpain; Cell Line; Cells, Cultured; Cytokines; Epithelial Cells; Humans; Hypoxia; Inflammation Mediators; Interleukin-4; Mitochondria; Nitric Oxide; Oxidative Stress; Peroxynitrous Acid; Proteolysis; Reactive Oxygen Species; Respiratory Mucosa

Inositol polyphosphate phosphatases regulate the magnitude of phosphoinositide-3 kinase signalling output. Although inositol polyphosphate-4-phosphatase is known to regulate phosphoinositide-3 kinase signalling, little is known regarding its role in asthma pathogenesis. Here we show that modulation of inositol polyphosphate-4-phosphatase alters the severity of asthma. Allergic airway inflammation in mice led to calpain-mediated degradation of inositol polyphosphate-4-phosphatase. In allergic airway inflammation models, preventing inositol polyphosphate-4-phosphatase degradation by inhibiting calpain activity, or overexpression of inositol polyphosphate-4-phosphatase in mouse lungs, led to attenuation of the asthma phenotype. Conversely, knockdown of inositol polyphosphate-4-phosphatase severely aggravated the allergic airway inflammation and the asthma phenotype. Interestingly, inositol polyphosphate-4-phosphatase knockdown in lungs of naive mice led to spontaneous airway hyper-responsiveness, suggesting that inositol polyphosphate-4-phosphatase could be vital in maintaining the lung homeostasis. We suggest that inositol polyphosphate-4-phosphatase has an important role in modulating inflammatory response in asthma, and thus, uncover a new understanding of the complex interplay between inositol signalling and asthma, which could provide alternative strategies in asthma management.

Topics: Animals; Asthma; Calpain; Cell Line; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Humans; Hypersensitivity; Inflammation; Lung; Male; Mice; Mice, Inbred BALB C; Phosphoric Monoester Hydrolases; Real-Time Polymerase Chain Reaction; RNA, Small Interfering

Asthma is a chronic airway inflammatory disorder which is characterized by reversible airway obstruction, airway hyperresponsiveness and airway inflammation. Oxidative stress has been shown to be strongly associated with most of the features of asthma and leads to accumulation of phosphatidyl inositol (3,4) bis-phosphate {PtdIns(3,4)P2} which is the major substrate for inositol polyphosphate 4 phosphatase (INPP4A). PtdIns(3,4)P2 in turn activates PI3K pathway and contributes to oxidative stress. Thus, there exists a vicious loop between oxidative stress and lipid phosphatase signaling. In this context, we have recently shown that INPP4A, a crucial molecular checkpoint in controlling PI3K-Akt signaling pathway, is downregulated in allergic airway inflammation. Resveratrol, a potent antioxidant found in red wines, has been shown to attenuate asthma features in murine model of allergic airway inflammation (AAI), however the underlying mode of its action was not completely understood. In this study, the effect of resveratrol on mitochondrial dysfunction, PI3K-Akt signaling and inositol polyphosphate 4 phosphatase was studied in murine model of allergic airway inflammation. We observed that resveratrol treatment of allergic mice was found to significantly downregulate oxidative stress and restore mitochondrial function. It also decreased calpain activity and restored the expression of INPP4A in lungs which in turn reduced Akt kinase activity and Akt phosphorylation. These results suggest a novel mechanism of action of resveratrol in attenuating asthma phenotype by downregulating PI3K-Akt pathway via upregulating INPP4A.

Topics: Animals; Anti-Asthmatic Agents; Asthma; Calpain; Gene Expression Regulation; Male; Mice; Mice, Inbred BALB C; Ovalbumin; Oxidative Stress; Phosphatidylinositol 3-Kinases; Phosphoric Monoester Hydrolases; Phosphorylation; Proto-Oncogene Proteins c-akt; Resveratrol; Stilbenes

We recently showed that poly(ADP-ribose)polymerase-1 (PARP-1) may play a role in allergen (ovalbumin)-induced airway eosinophilia, potentially through a specific effect on IL-5 production. We also reported that while IL-5 replenishment promotes reversal of eosinophilia in lungs of PARP-1(-/-) mice, IL-4 or Immunoglobulin E replenishment do not, suggesting a potentially significant regulatory relationship between PARP-1 and IL-5.. To explore the mechanism by which PARP-1 regulates IL-5 production and to determine how PARP-1 inhibition blocks allergen-induced eosinophilia.. This study was conducted using a murine model of allergic airway inflammation and primary splenocytes.. PARP-1 knockout-associated reduction in IL-5 upon allergen exposure occurs at the mRNA level. Such an effect appears to take place after IL-4 receptor activation as PARP-1 inhibition exerted no effect on JAK1/JAK3 activation. Signal transducer and activator of transcription-6 (STAT-6) protein was severely downregulated in spleens of PARP-1(-/-) mice without any effect on mRNA levels, suggesting an effect on protein integrity rather than gene transcription. Interestingly, the degradation of STAT-6 in PARP-1(-/-) mice required allergen stimulation. Additionally, PARP-1 enzymatic activity appears to be required for STAT-6 integrity. The downregulation of STAT-6 coincided with mRNA and protein reduction of GATA-binding protein-3 and occupancy of its binding site on the IL-5 gene promoter. IL-4 was sufficient to induce STAT-6 downregulation in both PARP-1(-/-) mice and isolated splenocytes. Such degradation may be mediated by calpain, but not by proteasomes.. These results demonstrate a novel function of PARP-1 in regulating IL-5 expression during allergen-induced inflammation and explain the underlying mechanism by which PARP-1 inhibition results in IL-5 reduction.

Topics: Allergens; Animals; Asthma; Calpain; Disease Models, Animal; Eosinophilia; Female; Humans; Inflammation; Interleukin-5; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Respiratory System; STAT6 Transcription Factor