American Journal of Respiratory Cell and Molecular Biology最新文献

The efficacy of β-agonists in asthma is severely limited by β-adrenoceptor desensitization which results in poorly managed symptoms and refractory bronchoconstriction. Thus, there is a need to identify novel therapeutic pathways and to clarify the relationship between novel therapeutics and functional β-adrenoceptor responsiveness. We have previously demonstrated that acute antagonism of the calcium activated chloride channel, transmembrane member 16A (TMEM16A), relaxes airway smooth muscle (ASM). We sought to determine the efficacy and role of TMEM16A antagonism in the context of desensitization β - adrenoceptor responsiveness. For these studies, we exposed murine tracheal rings on wire myography and precision cut lung slices to contractile mediators in the presence or absence of TMEM16A antagonists and β-agonists with or without prior β-adrenoceptor desensitization. Contractile studies were also performed with human tracheal and bronchial ASM. Finally, the ability of TMEM16A antagonism to prevent desensitization of β₂-adrenoceptor-induced cyclic AMP synthesis was measured in human ASM cells. From these studies we demonstrate that acute TMEM16A antagonism is effective in relaxing β-agonist desensitized ASM in central and peripheral murine ASM and human ASM. Furthermore, we demonstrate that chronic pretreatment with TMEM16A antagonists prevents functional desensitization of β-agonist responsiveness in mouse and human upper airways and prevents desensitization of β-agonist-mediated cyclic AMP production in human ASM cells. Taken together, the present study demonstrates a favorable therapeutic profile of TMEM16A antagonism for airway smooth muscle relaxation despite functional desensitization of β-agonist responsiveness which may be a novel therapeutic approach in the face of β-adrenoceptor tachyphylaxis.

ADP-ribosylation factor 6 (ARF6), a GTPase associated with cancer metastasis, is activated in the lung endothelium in pulmonary arterial hypertension (PAH). To identify ARF6-regulated pathways relevant to PAH, we performed a state-of-the-art proteomic analysis of human pulmonary artery endothelial cells (HPAECs) overexpressing the wildtype, constitutively active, fast-cycling and dominant negative mutants of ARF6. The analysis revealed a novel link of ARF6 with hypoxia-inducible factor (HIF), in addition to endocytotic vesicle trafficking, cell proliferation, angiogenesis, oxidative stress and lipid metabolism. Active ARF6 markedly increased expression and activity of HIF-2, critical in PAH, with HIF-1 relatively unaffected. Hypoxic ARF6 activation was a prerequisite for HIF-2 activation and HIF-dependent gene expression in HPAECs, PAH blood-derived late outgrowth endothelial colony forming cells (ECFCs) and hypoxic mouse lungs in vivo. A novel ARF6 inhibitor, chlortetracycline (CTC), reduced hypoxia-induced HIF-2 activation, proliferation and angiogenesis in HPAECs and reduced HIF-2 expression in lung and heart tissues of hypoxic mice. PAH ECFCs showed elevated expression and activity of ARF6 and HIF2, which was attenuated by CTC, and oral CTC attenuated development of PH in chronically hypoxic mice. We identify epidermal growth factor receptor (EGFR) as a direct interactor of ARF6, and EGFR signalling as a crucial mechanism linking ARF6 and HIF activation. In conclusion, we are first to demonstrate a key role of ARF6 in the regulation of HIF-2α activation in vitro and in vivo and show that HIF-2α, a master-regulator of vascular remodelling in PAH, can be targeted by a clinically approved antibiotic chlortetracycline.

Tubulogenesis depends on precise cell shape changes driven by asymmetric tension from the actin cytoskeleton. How actin asymmetry is dynamically controlled to coordinate epithelial cell shape changes required for respiratory tubulogenesis remains unknown. Herein, we unveiled a critical role for the transcription factor KLF5, regulating actin asymmetry, inducing epithelial cell shape changes by balancing RHOA and CDC42 GTPase activity via RICH2. Conditional Klf5 expression or deletion in pulmonary epithelial cells affected apical actin organization and the positioning of apical polarity proteins in cell membranes, disrupting branching and sacculation of respiratory tubules during mouse lung morphogenesis. Increased KLF5 levels were observed in epithelial cells lining dilated tubules in lungs from patients with congenital pulmonary airway malformation (CPAM). Together, our study demonstrates that dynamic regulation of apical actin organization by KLF5 is essential for respiratory tubulogenesis, providing a mechanistic framework for comprehending the morphogenesis of respiratory tubules.

Spatially coordinated ERK signaling events ("SPREADs") transmit radially from a central point to adjacent cells via secreted ligands for EGFR and other receptors. SPREADs maintain homeostasis in non-pulmonary epithelia, but it is unknown whether they play a role in the airway epithelium or are dysregulated in inflammatory disease. To address these questions, we measured SPREAD activity with live-cell ERK biosensors in human bronchial epithelial cell lines (HBE1 and 16HBE) and primary human bronchial epithelial (pHBE) cells, in both submerged and biphasic Air-Liquid Interface (ALI) culture conditions (i.e., differentiated cells). Airway epithelial cells were exposed to pro-inflammatory cytokines relevant to asthma and chronic obstructive pulmonary disease (COPD). Type 1 pro-inflammatory cytokines significantly increased the frequency of SPREADs, which coincided with epithelial barrier breakdown in differentiated pHBE cells. Furthermore, SPREADs correlated with IL-6 peptide secretion and the appearance of localized clusters of phospho-STAT3 immunofluorescence. To probe the mechanism of SPREADs, cells were co-treated with pharmacological treatments (gefitinib, tocilizumab, hydrocortisone) or metabolic modulators (insulin, 2-deoxyglucose). Hydrocortisone, inhibitors of receptor signaling, and suppression of metabolic function decreased SPREAD occurrence, implying that pro-inflammatory cytokines and glucose metabolism modulate SPREADs in human airway epithelial cells via secreted EGFR and IL6R ligands. We conclude that spatiotemporal ERK signaling plays a role in barrier homeostasis and dysfunction during inflammation of the airway epithelium. This novel signaling mechanism could be exploited clinically to supplement corticosteroid treatment for asthma and COPD.

Acute respiratory distress syndrome (ARDS) is a serious illness accounting for 10% of ICU admissions and high mortality of 31-45% with a paucity of pharmacologic treatment options. Dysregulated inflammation and oxidative stress are hallmark features of ARDS. We previously showed that transgenic mice expressing a naturally occurring polymorphism of the antioxidant enzyme extracellular superoxide dismutase (EC-SOD), are protected against Staphylococcus aureus (S. aureus) pneumonia, acute lung injury, and pulmonary neutrophilia. In this mouse strain, an R213G amino acid substitution leads to lower tissue binding affinity and elevated alveolar and plasma EC-SOD levels, though the redox-regulated mechanisms responsible for protection against S. aureus are not yet elucidated. Neutrophils are recruited to the areas of injury and inflammation, in part by activated platelets, which contain multiple redox-sensitive targets. Thus, we hypothesize that increased circulating EC-SOD due to the EC-SOD R213G variant protects against S. aureus pneumonia by reducing platelet activation and subsequent neutrophil recruitment to the lung. We demonstrate that, compared to WT mice with S. aureus pneumonia, platelet activation, formation of platelet-neutrophil aggregates (PNAs), and influx of neutrophils and PNAs into the lung are decreased in the infected R213G mice. Furthermore, pre-treatment with a MnTE-2-PyP SOD mimetic protects against S. aureus-induced platelet activation, pulmonary neutrophilia, and acute lung injury. Our data highlight the redox regulation of platelet activation as a driver of S. aureus-induced acute lung injury.

Mutations in the Tuberous Sclerosis Complex (TSC) genes result in the hyperactivation of the mechanistic/mammalian target of rapamycin 1 (mTORC1) growth pathway in mesenchymal pulmonary cells. Rapamycin (Sirolimus^TM), a naturally occurring macrolide, is the only therapeutic approved for women with lymphangioleiomyomatosis (LAM), a progressive, destructive lung disease caused by TSC gene mutations and mTORC1 hyperactivation. However, on cessation of the drug, lung function decline continues. We demonstrated here that pulmonary LAM cancer stem-like cells (SLS) most highly expressed the eukaryotic translation initiation factor 4E (eIF4E)-dependent translation initiation genes. We also showed that the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) gene has the lowest expression in these cells, indicating that the 4E-BP1/eIF4E ratio in LAM SLS cells favors unrestrained eIF4E oncogenic mRNA translation. The bi-steric mTORC1-selective compound RMC-5552 prevented growth of LAM-associated fibroblasts (LAFs) and phosphorylation of proteins in the ribosomal protein S6K1/ribosomal protein S6 (S6K1/S6) and 4E-BP1/eIF4E translation mTORC1-driven pathways, whereas rapamycin only blocked the S6K/S6 axis. Rapamycin inhibition of LAF growth was rapidly reversed, but RMC-5552 inhibition was more durable. RMC-5552, through its potential to eradicate LAM cancer SLS cells, may have therapeutic benefit in LAM and other diseases with mTORC1 hyperactivity.