Airway hydration and ciliary function are critical to airway homeostasis and dysregulated in chronic obstructive lung disease. COPD is impacted by cigarette smoking with no therapeutic options. We utilized a high copy cDNA library genetic selection approach in the amoeba Dictyostelium discoideum to identify genetic protectors from cigarette smoke (CS). Adenine nucleotide translocase (Salvioli et al.), a mitochondrial ADP/ATP transporter, was protective against CS in Dictyostelium and human bronchial epithelial cells. ANT2 gene expression is reduced in lung tissue from COPD patients and in a mouse smoking model. ANT1 and ANT2 overexpression resulted in enhanced oxidative respiration and ATP flux. In addition to ANT's presence in the mitochondria, ANT resides at the plasma membrane in airway epithelial cells and regulates airway homeostasis. ANT2 overexpression stimulates airway surface hydration by ATP and maintains ciliary beating after CS exposure, which are key functions of the airway. Our study highlights the potential of ANT upregulation and/or agonists in protecting from dysfunctional mitochondrial metabolism, airway hydration, and ciliary motility in COPD.
Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease associated with aberrant activation and differentiation of fibroblasts, leading to abnormal extracellular matrix production. Currently, it is still an untreatable disease (except for lung transplantation). Here, we demonstrate that the Raf1 inhibitor GW5074 ameliorates lung fibrosis in bleomycin-induced pulmonary fibrosis. Posttreatment with GW5074 reduced fibronectin (FN) expression, collagen deposition, and inflammatory cell infiltration in bleomycin-challenged mice, suggesting an antifibrotic property of GW5074. To determine the molecular mechanisms by which inhibition of Raf1 ameliorates lung fibrosis, we investigated the role of Raf1 in TGF-β1 signaling in human lung fibroblasts. GW5074 or downregulation of Raf1 by siRNAs significantly attenuated TGF-β1-induced smooth muscle actin, FN, and collagen I expression, whereas overexpression of Raf1 promoted the effects of TGF-β1 in lung fibroblasts. Furthermore, we found that Raf1-promoted TGF-β1 signaling was through the Raf1/ERK/Smad pathway and contributed to the cell proliferation and migration in human lung fibroblasts. This study provides preclinical and mechanistic evidence for development of Raf1 inhibitors as potential antifibrotic drugs for the treatment of IPF.
The authors have declared no conflicts of interest exist. AbstractAirway hydration and ciliary function are critical to airway homeostasis and dysregulated in chronic obstructive lung disease (COPD). COPD is the 4 th leading cause of death in the US and is impacted by cigarette smoking with no therapeutic options. We utilized a genetic selection approach in the amoeba Dictyostelium discoideum as a comparative discovery tool in lung biology to identify genetic protectors from cigarette smoke (CS). Adenine nucleotide translocase (ANT), a mitochondrial ADP/ATP transporter, was protective against CS in Dictyostelium and human bronchial epithelial cells. ANT2 gene expression is reduced in lung tissue from COPD patients and in a mouse smoking model. ANT1and ANT2 overexpression resulted in enhanced oxidative respiration and ATP flux. In addition to ANT's presence in the mitochondria, ANT1 and ANT2 reside at the plasma membrane in airway epithelial cells and this localization plays a role in how ANTs regulate airway homeostasis. ANT2 overexpression stimulates airway surface liquid hydration by ATP and maintains ciliary beating after CS exposure, which are key functions of the airway. Our study highlights the potential of ANT modulation in protecting from dysfunctional mitochondrial metabolism, airway hydration, and ciliary motility in COPD.
Chronic obstructive pulmonary disease (COPD) is a destructive lung disease resulting in alveolar tissue loss, airway remodeling and inflammation with cigarette smoke (CS) being a major causative factor. Mitochondrial dysfunction can lead to changes in cell fate implicated in COPD. Cellular senescence, a state of cell cycle arrest, results in abnormal responses in lung epithelial cells. An important protein in mitochondrial metabolism and cell fate is adenine nucleotide translocase (ANT), a mitochondrial ATP transporter. Our studies suggest that levels of ANT are altered in COPD. We hypothesize that loss of ANTs results in increased cellular senescence thus altering COPD pathogenesis and airway dysfunction. Methods Utilizing siRNA knockdown of SLC25A4 (ANT1) and SLC25A5 (ANT2) in human bronchial epithelial cells, Beas‐2bs, we assessed cellular senescence (p53, p21, p16) via western blot, qt‐PCR and B‐galactocidase analysis. Cells were also exposed to CS extract. Wildtype (WT) and ant1‐null mice were exposed to CS for 2 and 6mo or air control with lung histology and morphometry analysis. We performed single cell RNA sequencing on lungs from 2mo air and CS‐exposed mice. Single‐cell library preparation was performed using 10X Genomics 5prime v1 reagents to analyze 5000 cells/mouse (n=3 mice/group). Alignment was performed using Cell Ranger and standard workflow analysis using R package Seurat v3.2. Cell clusters were generated using unsupervised UMAP. Pathway analysis on differential gene expression lists by cell type was performed using Ingenuity Pathway Analysis. Results Knockdown of ANT1 or ANT2 in Beas‐2b cells results in an increase in cell senescence proteins p16, p21, p53 and beta‐galactosidase staining. Surprisingly, ant1‐null mice exposed to 6months of CS exposure are protected from alveolar destruction (fig.1), airway remodeling and chronic inflammation with fewer macrophages. Single cell RNA sequencing of WT lung cells demonstrates that CS (2 mo) causes a significant increase in genes associated with senescence in alveolar type 1 epithelial cells (AT1, p‐value 1.0x10‐6) with dysregulation of mitochondrial‐related gene pathways when compared to air controls: mitochondrial dysfunction, upregulation of Eif2 signaling and NRF2‐mediated oxidative stress, and downregulation in sirtuin signaling. Alveolar type 2 cells (AT2) from WT‐smoked mice show downregulation of Eif2 signaling, unfolded protein response, NRF2‐mediated oxidative stress, and sirtuin signaling. Expression of slc25a4 (ant1) is upregulated in AT2 cells in WT‐smoked mice (p‐value 1.7x10‐17). Mitochondrial and cell fate pathways were significantly altered in AT1 and AT2 cells from WT‐smoked mice but not from ant1‐null mice, suggesting a key role in lung epithelial responses in COPD. Conclusions ANTs alter cell senescence in alveolar epithelial cells. An In vivo mouse model shows that ant1 is upregulated in AT2 cells after CS and loss of ant1 is protective against CS lung injury. Ant1 regulates epithelial cell fate, mitochondrial function and CS‐rela...
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