Mitochondrial uncoupling protein‐2 reprograms metabolism to induce oxidative stress and myofibroblast senescence in age‐associated lung fibrosis

Abstract Mitochondrial dysfunction has been associated with age‐related diseases, including idiopathic pulmonary fibrosis (IPF). We provide evidence that implicates chronic elevation of the mitochondrial anion carrier protein, uncoupling protein‐2 (UCP2), in increased generation of reactive oxygen species, altered redox state and cellular bioenergetics, impaired fatty acid oxidation, and induction of myofibroblast senescence. This pro‐oxidant senescence reprogramming occurs in concert with conventional actions of UCP2 as an uncoupler of oxidative phosphorylation with dissipation of the mitochondrial membrane potential. UCP2 is highly expressed in human IPF lung myofibroblasts and in aged fibroblasts. In an aging murine model of lung fibrosis, the in vivo silencing of UCP2 induces fibrosis regression. These studies indicate a pro‐fibrotic function of UCP2 in chronic lung disease and support its therapeutic targeting in age‐related diseases associated with impaired tissue regeneration and organ fibrosis.

. UCP2 is highly expressed in IPF lungs and senescent fibroblasts.
(a) A publicly available GEO dataset, GSE24206 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE24206) was queried for UCP2 gene expression in whole lung tissue derived from non-IPF patients and those with early and advanced IPF using the GEO2R tool, graphical output shown. (b) A publicly available GEO dataset, GSE13330 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE13330) was queried for UCP2 gene expression in human foreskin fibroblasts that were either non-senescent or subjected to replicative senescence or stress-induced senescence (via exposure to bleomycin) using the GEO2R tool, graphical output shown. (c) UCP2 gene expression in various normal tissues in humans was obtained from the Genotype-Tissue Expression (GTEx) project online portal (https://www.gtexportal.org/home/gene/UCP2). The graphical output is in log scale sorted in ascending order of median expression in tissues. (d-h) The Fibroexplorer dataset (an online resource tool derived from fibroblast atlases by integration of single-cell transcriptomic data from approximately 230,000 fibroblasts across 17 tissues, 50 datasets, 11 disease states and 2 species) for perturbed state (human), available at https://www.fibroxplorer.com/perturbed-statehuman, was queried for UCP2 gene expression; output shown. (d) shows the clusters of fibroblasts characterized in the dataset (X axis) in various conditions (Y axis) (ulcerative colitis; PDAC = pancreatic ductal adenocarcinoma; NSCLC = non-small cell lung cancer; IPF = idiopathic pulmonary fibrosis; COVID = covid-19 pneumonia; the latter three fibroblast sets isolated from lungs). The sizes of the black boxes show relative expression of UCP2 in the cluster/condition.   An siRNA sequence targeting human UCP2 was designed, and its efficacy tested on non-IPF and IPF fibroblasts (derived from one lung explant each) (a). The fibroblasts were treated with the UCP2 siRNA or non-targeting (NT) siRNA (100 nM each) for 72 hours; steady-state mRNA levels assessed by qPCR. Graph represents mean ± SEM (n=3 each group); ***p<0.001. (b) IPF fibroblasts were treated with UCP2 or NT siRNA for 72 hours; western blotting was performed to assess steady-state expression of UCP2; representative blots shown, with their densitometric analysis in (c); error bars represent mean ± SEM (n=3); **p<0.01. Please note that the UCP2 band appears at ~95 kDa which represents 3x molecular weight of the UCP2 monomer. (d, e) A mouse/rat siRNA sequence that is orthologous to the human UCP2 siRNA sequence was generated. This sequence works for both mouse (Mus musculus) and rat (Rattus norvegicus) and differs from the human sequence in only 2 (out of 19) bases (see Key Resources Table). Mouse lung fibroblasts (d) and rat lung epithelial (RLE) cells (e) were treated with this siRNA (or non-targeting siRNA) for 72 hours; steady-state mRNA levels assessed by qPCR. Graphs represent mean ± SEM (n=3 each group); **p<0.01, ***p<0.001. NT = non-targeting.

Figure S3
Rep NT siRNA UCP2 siRNA Figure S3. Heatmap of metabolites in IPF fibroblasts treated with UCP2 siRNA.
IPF fibroblasts were subjected to siRNA-mediated knockdown of UCP2 for 72 hours. Lysates (experimental triplicate) were subjected to metabolomics analyses (same experiment described in Figure 3d OXPHOS Short exposure Long exposure Figure S4 continued…      (a) A publicly available GEO dataset, GSE6591 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE6591), was queried for UCP2 gene expression in the lungs among various age groups in C57BL/6J mice using the GEO2R tool; graphical output shown. (b-g) In the experiment described in Figure 6b, whole lung lysates (from the aged mice subjected to bleomycin injury and then treated with UCP2-or non-targeting siRNA) were assessed for gene expression of UCP2 (b) and collagen 1a1 (c) by qPCR. Graphs represent mean ± SEM (n=3 each group); *p<0.05. (d) Lung function was assessed using the flexiVent TM apparatus; static compliance measured; graph represents mean ± SEM (n=4-5 each group); ***p<0.001. (e, f) Representative images show the areas of fibrotic remodeling and fluorescence patterns of myeloperoxidase (MPO, red), type 1 epithelial cells (T1-, green) and nuclei (DAPI-blue) in indicated groups of mice.
Scale bars = 50 m. (g) Fibroblasts were isolated from the lungs and western blotting was performed to assess the steady state levels of myofibroblast markers -smooth muscle actin (-SMA) and collagen 1a1 (COL1a1); densitometric analyses shown in (h) and (i) respectively; graphs represent mean ± SEM (n=3); *p<0.05, **p<0.01. (j) In a separate experiment, young (2 months) and aged (18 months old) C57BL/6 mice were subjected to lung injury by instillation of oropharyngeal bleomycin (1.5 U/kg) (or no injury by instillation of PBS control). Lungs were harvested starting at 3 weeks up to 2 months after bleomycin injury; fibroblasts were isolated from the lungs and assessed for gene expression of UCP2 and collagen 1a1 (COL1a1). The graph represents correlation between mRNA expression of UCP2 and COL1a1.