Cathepsin E promotes pulmonary emphysema via mitochondrial fission

Abstract

Emphysema is characterized by loss of lung elasticity and irreversible air space enlargement, usually in the later decades of life. The molecular mechanisms of emphysema remain poorly defined. We identified a role for a novel cathepsin, cathepsin E, in promoting emphysema by inducing mitochondrial fission. Unlike previously reported cysteine cathepsins, which have been implicated in cigarette smoke-induced lung disease, cathepsin E is a nonlysosomal intracellular aspartic protease whose function has been described only in antigen processing. We examined lung tissue sections of persons with chronic obstructive pulmonary disease, a clinical entity that includes emphysematous change. Human chronic obstructive pulmonary disease lungs had markedly increased cathepsin E protein in the lung epithelium. We generated lung epithelial-targeted transgenic cathepsin E mice and found that they develop emphysema. Overexpression of cathepsin E resulted in increased E3 ubiquitin ligase parkin, mitochondrial fission protein dynamin-related protein 1, caspase activation/apoptosis, and ultimately loss of lung parenchyma resembling emphysema. Inhibiting dynamin-related protein 1, using a small molecule inhibitor in vitro or in vivo, inhibited cathepsin E-induced apoptosis and emphysema. To the best of our knowledge, our study is the first to identify links between cathepsin E, mitochondrial fission, and caspase activation/apoptosis in the pathogenesis of pulmonary emphysema. Our data expand the current understanding of molecular mechanisms of emphysema development and may provide new therapeutic targets.

Figure 1

Generation of lung epithelial-targeted constitutive Cat E Tg mice. A: Construct for generation of lung epithelial-targeted, constitutive CC10-Cat E Tg mice. B: Construct for generation of inducible CC10-Cat E Tg mouse. CMV, cytomegalovirus; hCat E, human Cat E; INS, insulator; rtTA, reverse tetracycline transactivator; Tet O, tetracycline operator.

Figure 2

Human COPD lung section and smoking mouse lungs showed increased Cat E expression. Representative immunohistochemical staining for Cat E in human lung tissue with (B) or without (A) COPD. Cat E protein is detected as red cytoplasmic staining (single arrow, airway epithelium; double arrow, lung alveolar epithelial cells). C: Cat E expression in human COPD lung alveolar epithelial cells [Cat E (red); SP-C (green); arrows, alveolar epithelial cells with Cat E expression]. D: Cat E expression is increased in mouse lungs after 6 months of CS compared with NS lungs. Data are expressed as means ± SEM. ^∗P < 0.05 versus mouse NS lungs. Original magnification: ×200 (A and B); ×400 (C). CS, cigarette smoke; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NS, without smoking; SP-C, surfactant protein C.

Figure 3

Lung epithelial-targeted constitutive Cat E Tg mice develop emphysema. A: Cat E protein expression in cCat E Tg⁺ mouse lungs and Tg⁻ WT littermates detected by immunohistochemistry (arrow, positive brown staining). B: Cat E protein expression in cCat E Tg⁺ mouse lungs compared with Tg⁻ mouse lungs detected by Western blot analysis. C: cCat E Tg⁺ mouse lungs showed increased Cat E enzymatic activity compared with Tg⁻ WT littermates. D: Representative lung histology of 3-month-old cCat E Tg⁺ mouse lungs with emphysema versus WT littermates. E: cCat E Tg⁺ and Tg⁻ mouse lung volumes at different ages. F: cCat E Tg⁺ and Tg⁻ mouse lung mean linear chord lengths at different ages. Data are expressed as means ± SEM. ^∗P < 0.05 versus cCat E Tg⁻ WT littermates. Original magnification: ×400 (A); ×100 (D). cCat E, constitutive CC10-Cat E.

Figure 4

Lung epithelial-targeted inducible Cat E Tg mice develop emphysema. A: Cat E protein expression in iCat E Tg⁺ mouse lungs (after 2 weeks of dox water) and Tg⁻ WT littermates detected by immunofluorescence (arrow, positive red staining). B: Cat E protein expression in iCat E Tg⁺ mouse lungs after 2 weeks of dox water compared with Tg⁻ mouse lungs detected by Western blot analysis. C: iCat E Tg⁺ mouse lungs showed increased Cat E enzymatic activity compared with Cat E Tg⁻ WT littermates. D: Representative lung histology of iCat E Tg⁺ mouse lungs (after 2 weeks of dox water) with emphysema versus WT littermates. E: iCat E Tg⁺ and Tg⁻ mouse lung volumes after a time course of dox water. F: iCat E Tg⁺ and Tg⁻ mouse lung mean linear chord lengths at different time points after a time course of dox water. Data are expressed as means ± SEM. ^∗P < 0.05 versus iCat E Tg⁻ WT littermates. Original magnification: ×200 (A); ×100 (D). iCat E, inducible CC10-Cat E.

Figure 5

Cat E causes caspase 3-mediated apoptosis, and caspase inhibition prevents the development of emphysema. A: Representative sections from iCat E Tg⁺ mouse lung stained for TUNEL after 2 weeks of dox water showed increased TUNEL positivity compared with Tg⁻ littermate WT mouse lung (TUNEL staining; arrow, nuclear blue staining). B: Quantitation of TUNEL-positive cells is expressed as percentage of total cells in lung sections. C: MLE12 cells were treated with 100 ng/mL human recombinant active Cat E for 48 hours and apoptosis was analyzed by flow cytometry. D: Representative caspase 3 activation detected by immunohistochemical staining in iCat E Tg⁺ mouse lung compared with Tg⁻ littermate WT mouse lung (arrow, positive red cytoplasmic staining for active caspase 3). E: iCat E Tg⁺ mouse lungs showed increased caspase 3 activity. F: MLE12 cells treated with recombinant active Cat E (100 ng/mL) for 48 hours showed increased caspase 3 activity. Z-VAD, a broad caspase inhibitor, was administered 1 day before dox water and repeated daily for 2 weeks via intraperitoneal injection (3 mg/kg body weight/day). Z-VAD treatment decreased lung caspase 3 activity in iCat E Tg⁺ mouse lungs (G), TUNEL-positive apoptotic cells in iCat E Tg⁺ mouse lungs (H), lung volume in iCat E Tg⁺ mouse lungs (I), and lung emphysema in iCat E Tg⁺ mice, as measured by mean linear chord length (J). Data are expressed as means ± SEM (B, C, and E–J). ^∗P < 0.05 versus MLE12 cells without active Cat E treatment CTRL (C and F), versus iCat E Tg⁻ WT littermates (B and E), versus iCat E Tg⁺ mice treated with Z-VAD or iCat E Tg⁻ WT littermates (G–J). Original magnification: ×400 (A); ×600 (D). CTRL, control; iCat E, inducible CC10-Cat E; RFU, relative fluorescence unit.

Figure 6

Cat E induces mitochondria fission and mitochondrial fission protein Drp1. A: MLE12 cells were treated with 100 ng/mL human recombinant active Cat E for 16 hours, and mitochondrial morphology was observed by transmission electron microscope. Cells treated with Cat E showed disorganized, smaller, and fragmented mitochondria compared with control cells (arrow, mitochondrion undergoing fission). B: MLE12 cells were treated with 100 ng/mL human recombinant active Cat E for 16 hours, and mitochondria morphology was observed by mitochondrial immunofluorescence. Cells treated with active Cat E showed disorganized, smaller, and fragmented mitochondria (single arrow, fragmented mitochondria) compared with control cells (double arrow, elongated mitochondria). C: iCat E Tg⁺ mouse lungs (after 2 weeks of dox water) showed increased Drp1 fission protein and proapoptotic Bax expression by Western blot analysis. D: iCat E Tg⁺ mouse lungs showed increased Drp1 mRNA expression detected by real-time reverse transcription PCR. Data are expressed as means ± SEM. ^∗P < 0.05 versus iCat E Tg⁻ WT littermates. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; iCat E, inducible CC10-Cat E.

Figure 7

Inhibiting Drp1 prevents Cat E-induced emphysema. A: MLE12 cells treated with 100 ng/mL human recombinant active Cat E showed increased activation of caspase 3 by demonstrating caspase 3 cleavage detected by Western blot analysis (lanes 5 and 6). Mdivi-1 was administered 1 day before dox water and repeated daily for 2 weeks via intraperitoneal injection (50 mg/kg body weight/day). B: Mdivi-1 treatment inhibits caspase 3 activity in iCat E Tg⁺ mouse lungs. C: MLE12 cells were transfected with pCC10-Cat E, empty vector, or Drp1K38A, a dominant-negative mutant of Drp1. After incubation, cells were cultured for an additional 48 hours, and apoptosis was analyzed by flow cytometry. D: Mdivi-1 treatment significantly decreases TUNEL-positive lung cells in iCat E Tg⁺ mouse lungs. E: Mdivi-1 treatment decreases lung volumes in iCat E Tg⁺ mice. F: Mdivi-1 treatment decreases lung emphysema in iCat E Tg⁺ mice, as measured by mean linear chord length. G: Representative lung histology of iCat E Tg⁺ mouse lungs with (right panel) or without (middle panel) Mdivi-1 treatment versus WT littermates (left panel). Data are expressed as means ± SEM (B–F). ^∗P < 0.05 versus Cat E Tg⁺ mice (B and D–F), ^∗∗P < 0.01 versus MLE12 cells transfected with pCC10-CatE (C). Original magnification, ×100. CTRL, MLE12 cells without Cat E treatment control; DMSO, dimethyl sulfoxide, solvent for Mdivi-1; iCat E, inducible CC10-Cat E; Mdivi-1, small molecule Drp1 inhibitor; Mdivi-1/Cat E, MLE12 cells treated with 50 μmol/L Mdivi-1 for 30 minutes followed by 100 ng/mL human recombinant active Cat E treatment for 48 hours; pCC10-CatE, Cat E overexpressing plasmid; RFU, relative fluorescence unit.

Figure 8

Parkin mediates Cat E-regulated mitochondrial fission and Drp1 induction. A: iCat E Tg⁺ mouse lungs (after 2 weeks of dox water) showed increased Parkin expression by Western blot analysis. B: MLE12 cells treated with 100 ng/mL human recombinant active Cat E for 16 hours showed increased Parkin expression by Western blot analysis. C: MLE12 cells transfected with 34 nmol/L NS siRNA or Cat E siRNA showed decreased Parkin expression as analyzed by real-time reverse transcription PCR. D: MLE12 cells transfected with 34 nmol/L NS siRNA or Parkin siRNA showed decreased Drp1 expression as analyzed by real-time reverse transcription PCR. MLE12 cells transfected with 34 nmol/L NS siRNA or Parkin siRNA showed decreased proteasome 20S activity. Data are expressed as means ± SEM. ^∗P < 0.05 versus NS siRNA (C) or Cat E (D and E) group. CTRL, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; iCat E, inducible CC10-Cat E; NS, nonspecific; RFU, relative fluorescence unit.

Figure 9

Summary of the effects of Cat E on emphysema. We postulate that Cat E overexpression leads to pulmonary emphysema by increasing mitochondrial fission via Parkin, UPS, and Drp1 induction, thereby disrupting the balance between mitochondrial fission and fusion, leading to increased caspase 3-mediated cell death and ultimately emphysema.