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.

Keywords: UCP2; cellular senescence; fibroblast; fibrosis; myofibroblast; oxidative stress; uncoupling protein-2.

FIGURE 1

UCP2 is highly expressed in IPF lungs and (myo)fibroblasts. (a) Mesenchymal stromal cells (MSCs) were isolated and cultured from broncho‐alveolar lavage (BAL) fluid of patients with stable (FVC loss ≤5% in the preceding 6 months) or progressive (FVC loss >10% in the preceding 6 months) IPF. qPCR was performed to assess the expression of UCP2. Graph represents mean ± SEM (n = 7–8); **p < 0.01. (b) Whole lung tissue obtained from explants of rejected donor lungs (non‐IPF) and IPF lungs were assessed for UCP2 expression by qPCR. Graph represents mean ± SEM (n = 4); *p < 0.05. (c) Fibroblasts were isolated from explants of rejected donor lungs (non‐IPF) and IPF lungs and cultured ex vivo. qPCR was performed to assess the expression of UCP2. Graph represents mean ± SEM (n = 6–8); *p < 0.05. (d–h) Immunohistochemical staining for UCP2 was performed on non‐IPF and IPF lung sections. Representative images (20× magnification) are shown in (d,e), respectively. A fibroblastic focus was identified in the IPF lung in (e) (dotted rectangle); (f–h) high magnification (40×) view of the fibroblastic focus stained with UCP2, α smooth muscle actin (α‐SMA) and secondary IgG antibody control, respectively. Black arrowhead in (f) shows fibroblasts in the fibroblastic focus. (i) Serum‐starved human diploid lung (IMR‐90) fibroblasts were treated with transforming growth factor‐β1 (TGF‐β1) 2 ng/ml for 24 h. UCP2 expression was assessed by real‐time PCR. Graph represents mean ± SEM (n = 3); **p < 0.01. (j) IMR‐90 fibroblasts at low population doubling length (low PDL; PDL < 20), and those undergoing replicative senescence at high PDL (PDL > 40) were assessed for UCP2 expression by real‐time PCR. Graph represents mean ± SEM (n = 3); **p < 0.01. (k) Non‐IPF human lung fibroblasts were treated with bleomycin 25 μg/ml for 72 h. UCP2 expression was assessed by real‐time PCR. Graph represents mean ± SEM (n = 3); **p < 0.01

FIGURE 2

UCP2 uncouples oxidative phosphorylation and decreases ATP synthesis. (a) Whole lung homogenates from non‐IPF and IPF lungs were assessed for ATP content. Graph represents mean ± SEM (n = 4); **p < 0.01. (b) Graphical representation of the correlation between ATP content and UCP2 mRNA expression in lung homogenates. (c) Non‐IPF and IPF lung fibroblasts grown ex vivo were assessed for ATP content. Graph represents mean ± SEM (n = 3); **p < 0.01. (d) IMR‐90 fibroblasts were treated with non‐targeting (NT) or UCP2‐targeting siRNA for 72 h and incubated with JC‐1 dye (2.5 μg/ml) for 30 min, with/without prior treatment with carbonyl cyanide‐4‐(trifluoromethoxy) phenylhydrazone (FCCP) (5 μM, for 30 min prior to JC‐1) for negative control. Orange/green fluorescence (indicative of JC‐1 aggregates/monomers) was captured by fluorescence microscopy. Scale bars = 50 μm. (e) IPF fibroblasts subjected to siRNA‐mediated knockdown of UCP2 for 72 h were incubated with JC‐1 dye (10 μg/ml) for 10 min, and orange/green fluorescence was analyzed by flow cytometry. The representative graphs depict the percentage of fibroblasts over threshold intensity of orange fluorescence on Y axis, with NT siRNA, 85.44 ± 0.8% vs. UCP2 siRNA, 92.12 ± 0.28%; mean ± SEM; p < 0.01; n = 5 replicates per group; 20,000 events recorded for each replicate. (f–h) Lung fibroblasts isolated and cultured from explants of 3 non‐IPF and 3 IPF subjects were subjected to siRNA‐mediated knockdown of UCP2 for 72 h. Real‐time PCR was performed to assess UCP2 mRNA expression (f); graph represents mRNA expression relative to non‐IPF cells treated with NT siRNA; mean ± SEM (n = 3); **p < 0.01, *p < 0.05. In parallel, ATP content was assessed in these fibroblasts (g); graph represents mean ± SEM (n = 3); **p < 0.01, *p < 0.05. (h) Graphical representation of correlation between ATP content and UCP2 mRNA expression in these fibroblasts

FIGURE 3

Constitutive high‐level expression of UCP2 impairs fatty acid oxidation and alters cellular redox state. (a–c) Non‐IPF and IPF lung fibroblasts (derived from three explants each) were cultured ex vivo and lysates were subjected to metabolomics analyses. (a) Partial least squares discriminant analysis (PLS‐DA) shows significant separation between the two groups of fibroblasts, (b) variable importance in projection (VIP) scores showing the top 25 metabolites that contribute to the PLS‐DA model, and (c) heatmap of the top 50 metabolites that are significantly different between the two groups; metabolites with lower concentrations are in blue and those with higher concentrations are in red. (d,e) IPF fibroblasts were subjected to siRNA‐mediated knockdown of UCP2 for 24 h. Lysates were subjected to metabolomics analyses. (d) PLS‐DA shows significant separation between the fibroblasts treated with non‐targeting siRNA (siNT) and UCP2‐targeting siRNA (siUCP2); (e) VIP scores showing the top 25 metabolites that contribute to the PLS‐DA model. (f) IPF fibroblasts were subjected to siRNA‐mediated knockdown of UCP2 for 72 h, stained with LipidTOX™ Red Neutral Lipid Stain and immunofluorescence imaging performed (representative images shown). Scale bars = 50 μm. (g) Quantification of LipidTOX™ fluorescence [of the fibroblasts in (f)] was performed by randomly selecting 25 individual fibroblasts each in NT siRNA and UCP2 siRNA groups and assessing their corrected total cellular fluorescence, CTCF [CTCF = Integrated density−(Area of selected cell × Mean fluorescence of background readings)], depicted graphically; boxes represent median and extend from 25th to 75th percentiles, and whiskers represent minimum to maximum values in arbitrary units (AU), n = 25, ****p < 0.0001. NT, non‐targeting

FIGURE 4

Chronic elevation of UCP2 induces increased production of reactive oxygen species in IPF lung fibroblasts. (a–c) IPF fibroblasts were subjected to siRNA‐mediated silencing of UCP2 for a total of 72 h. The fibroblasts were seeded 15,000 cells per well prior to measurement of oxygen consumption rate (OCR) in an XFe96 analyzer (Seahorse Bioscience); computed results for ATP‐linked OCR (a), Proton Leak OCR (b), and their ratio depicting the coupling efficiency (c) depicted graphically. Error bars represent mean ± SEM (n = 8); *p < 0.05, **p < 0.01, ****p < 0.0001. (d) IPF fibroblasts were subjected to siRNA‐mediated silencing of UCP2 for a total of 72 h, seeded at 15,000 cells per well, incubated with a substrate‐restricted medium and treated with etomoxir 4 µM or vehicle. Basal OCR due to intrinsic fatty acid oxidation (FAO) was calculated based on the difference between the OCR just prior to treatment with etomoxir and the OCR 30 min after treatment with etomoxir (depicted graphically). Error bars represent mean ± SEM (n = 10); ****p < 0.0001. (e) IPF fibroblasts were subjected to siRNA‐mediated silencing of UCP2 for a total of 72 h, seeded at 15,000 cells per well, incubated with a substrate‐restricted medium and treated with bovine serum albumin (BSA) alone or with BSA‐Palmitate conjugate as per the kit manufacturer's instructions. Basal OCR due to extrinsic FAO was calculated by the difference between the basal OCR of Palmitate‐treated cells and the cells treated with BSA alone, and subsequently subtracting the OCR due to excess proton leak in the Palmitate‐treated cells; thus, calculated basal OCR due to extrinsic FAO is depicted graphically. Error bars represent mean ± SEM (n = 4); ***p < 0.001. (f) IPF fibroblasts were subjected to siRNA‐mediated silencing of UCP2, or CPT1a, or both, for a total of 72 h. The fibroblasts were seeded 15,000 cells per well and incubated in a substrate‐restricted medium prior to measurement of OCR; computed results for reserve capacity depicted graphically. Error bars represent mean ± SEM (n = 10); ****p < 0.0001. (g) IPF fibroblasts with similar experimental conditions as in (a–c) were assessed for extracellular acidification rate (ECAR) in an XFe96 analyzer; ECAR denoting basal glycolysis depicted graphically. Error bars represent mean ± SEM (n = 8); ***p < 0.001. (h) IPF fibroblasts subjected to siRNA‐mediated knockdown of UCP2 for 72 h were incubated with MitoSOX™ Red (5 µM) and MitoTracker™ Green (100 nM) dyes for 15 min, and red–green fluorescence was analyzed by flow cytometry. The representative graphs depict the percentage of fibroblasts over threshold intensity of red fluorescence on Y axis, with NT siRNA, 19.03 ± 1.45% vs. UCP2 siRNA, 8.90 ± 0.38%; mean ± SEM; p < 0.0001; n = 6 replicates per group; 20,000 events recorded for each replicate. No significant difference noted in green fluorescence between groups. (i) IPF fibroblasts were subjected to siRNA‐mediated knockdown of UCP2 for 72 h. The fibroblasts were incubated with MitoSOX™ Red (5 µM) alone for 15 min, and red fluorescence was analyzed by flow cytometry, with change in intensity depicted graphically and graph representative of 5 replicates. (j) IPF fibroblasts were subjected to siRNA‐mediated knockdown of UCP2 for 24 h. Electron paramagnetic resonance (EPR) spectroscopy was performed with cyclic hydroxylamine spin probes to assess levels of nitroxide formation, reflecting levels of free radicals, chiefly superoxide in the cells (depicted graphically). Graph represents mean ± SEM (n = 6); *p < 0.05. (k) IPF fibroblasts were subjected to siRNA‐mediated knockdown of UCP2 for 72 h. Mitochondria were isolated and the rate of hydrogen peroxide production assessed using the p‐hydroxyphenylacetic acid (pHPA) assay; graph represents mean ± SEM (n = 3); ***p < 0.001. NT, non‐targeting; CPT1a, carnitine palmitoyl transferase‐1a

FIGURE 5

UCP2 regulates fibroblast senescence and myofibroblast differentiation. (a–l) In these experiments, IPF fibroblasts were subjected to siRNA‐mediated silencing of UCP2 for 72 h. (a) Fibroblast cell counting was performed at 72 h (10⁵ cells/well were seeded in both conditions at the start of each experiment); graph represents mean ± SEM (n = 5); ****p < 0.0001. (b) Western blotting was performed to assess the steady‐state expression of markers of cell‐cycling, cyclin‐D1 and phosphorylated Rb; myofibroblast markers, α‐smooth muscle actin (α‐SMA) and collagen 1a1 (COL1a1); representative blots are shown; densitometric analyses are shown in (c–f), respectively; graphs represent mean ± SEM (n = 4); *p < 0.05, **p < 0.01, ****p < 0.0001. (g) Cells were fixed and stained for DAPI and Ki‐67, a nuclear marker for cell proliferation; immunofluorescence imaging was performed; representative images (10×) are shown. (h) Senescence‐associated β‐galactosidase (SA‐β‐gal) staining with representative light microscopy images (10×) shown. (i,j) Senescence‐associated secretory phenotype (SASP) markers, interleukin 6 (IL‐6) (i) and interleukin 1β (IL‐1β). (j) Gene expression was assessed by real‐time PCR; graphs represent mean ± SEM (n = 4); ***p < 0.001, ****p < 0.0001. (k) Antimycin‐A 100 μM was added to the cells for 6 h prior to harvest. Western blotting was performed to assess the steady‐state levels of the apoptosis marker, cleaved poly (ADP‐ribose) polymerase (PARP); densitometric analysis shown in (l); graph represents mean ± SEM (n = 3); ***p < 0.001. The effects of UCP2 silencing on these fibroblast phenotypes were confirmed to be similar in fibroblasts derived from lung explants of at least 3 different IPF patients

FIGURE 6

Therapeutic targeting of UCP2 promotes resolution of experimental lung fibrosis. (a) 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 at 3 weeks after bleomycin injury, and fibroblasts were isolated and assessed for gene expression of UCP2 (depicted graphically). Graph represents mean ± SEM (n = 3 in each group); *p < 0.05. (b) Schematic depicting experimental design. 18‐month‐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). They were treated with UCP2‐targeting or non‐targeting (NT) siRNA, administered oropharyngeally every other day for 3 weeks, starting on day 22 after injury. Lungs were harvested at 6 weeks after injury, and the following analyses were performed. (c–h) Masson's trichrome histochemical staining for collagen was performed. Top panels (c,e,g) show whole lung sections, and bottom panels (d,f,h) show 20× magnification of selected areas. Images are representative of n = 3 in each group. (i) Hydroxyproline content of the lungs was assessed (depicted graphically); graphs represent mean ± SEM (n = 6–13); **p < 0.01, ***p < 0.001. (j,k) Representative images showing fluorescence patterns of α‐SMA positive fibroblasts (green), senescence marker p16 (red) and nuclei (DAPI‐blue). Scale bars = 50 μm. (l,m) Box plots show fluorescence intensity ratios of p16/α‐SMA and p16/nuclei from regions of enhanced fibrotic remodeling, n = 7 per group, 2 mice for each condition. **p < 0.01. (n–q) Representative images show fluorescence patterns of α‐SMA (green), apoptosis marker TUNEL (red) and nuclei (DAPI‐blue). Scale bars = 50 μm. Box plots show relative fluorescence intensity ratios, n = 7 per each group, **p < 0.01. NT, non‐targeting; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling