Mitochondrial dysfunction and impaired DNA damage repair through PICT1 dysregulation in alveolar type II cells in emphysema

Abstract

Background: Alveolar type II (ATII) cells have a stem cell potential in the adult lung and repair the epithelium after injury induced by harmful factors. Their damage contributes to emphysema development, characterized by alveolar wall destruction. Cigarette smoke is the main risk factor for this disease development.

Methods: ATII cells were obtained from control non-smoker and smoker organ donors and emphysema patients. Isolated cells were used to study the role of PICT1 in this disease. Also, a cigarette smoke-induced murine model of emphysema was applied to define its function in disease progression further.

Results: Decreased PICT1 expression was observed in human and murine ATII cells in emphysema. PICT1 was immunoprecipitated, followed by mass spectrometry analysis. We identified MRE11, which is involved in DNA damage repair, as its novel interactor. PICT1 and MRE11 protein levels were decreased in ATII cells in this disease. Moreover, cells with PICT1 deletion were exposed to cigarette smoke extract. This treatment induced cellular and mitochondrial ROS, cell cycle arrest, nuclear and mitochondrial DNA damage, decreased mitochondrial respiration, and impaired DNA damage repair.

Conclusions: This study indicates that PICT1 dysfunction can negatively affect genome stability and mitochondrial activity in ATII cells, contributing to emphysema development. Targeting PICT1 can lead to novel therapeutic approaches for this disease.

Keywords: ATII cells; Apoptosis; COPD; Cell cycle; Double strand breaks; Mitochondrial respiration; OXPHOS; Oxidative stress.

Fig. 1

Decreased PICT1 protein expression in ATII cells in emphysema patients. Lung tissue and ATII cells were obtained from control non-smoker (N) and smoker (S) organ donors and emphysema patients (E). Panel I: A—PICT1 mRNA levels in lung tissue by RT-PCR. B—Representative Western blot images of PICT1 expression in lung tissue. C—Quantification of protein expression normalized to β-actin is shown. Panel II: A—PICT1 mRNA levels in ATII cells by RT-PCR. B—Representative Western blot images of PICT1 expression in ATII cells. C—Quantification of protein expression. Panel III: A – PICT1 was immunoprecipitated in lung tissue, followed by mass spectrometry analysis. Representative PICT1 (A) and TRIM22 spectrum (B) are shown. C TRIM22 mRNA expression in ATII cells by RT-PCR. D ATII cells were stained in lung tissue sections using SP-C (magenta), PICT1 (red), and TRIM22 (green) antibodies and DAPI (blue) followed by analysis by immunofluorescence (scale bar—5 μm). PICT1 fluorescence intensity in the nucleus (E) and cytoplasm (F) was quantified. G The ratio of nuclear to cytoplasmic PICT1 fluorescence intensity. H Pearson’s correlation coefficient for PICT1 and MRE11 fluorescence co-localization in ATII cells. Data are shown as means ± SEM (N = 3—14 lungs per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 2

Reduced MRE11 expression in ATII cells in emphysema. Lung tissue and ATII cells were obtained from non-smokers (NS), smokers (S), and emphysema patients (E). Panel I: PICT1 was immunoprecipitated in lung tissue, followed by mass spectrometry analysis. A Representative MRE11 spectrum is shown, which was identified as a PICT1 interactor. B MRE11 mRNA levels in ATII cells by RT-PCR. C Representative Western blot images of MRE11 expression in ATII cells. D Quantification of protein expression normalized to β-actin is shown. E MRE11 (green) and PICT1 (red) antibodies and DAPI (blue) were applied using immunofluorescence. ATII cells were identified in lung tissue sections using SP-C (magenta, scale bar 5 μm). F Pearson’s correlation coefficient for PICT1 and MRE11 fluorescence co-localization in ATII cells is shown. Panel II: A MRE11 mRNA levels in lung tissue by RT-PCR. B Representative Western blot image of MRE11 expression in lung tissue. C—Quantification of protein expression is shown. Data are shown as means ± SEM (N = 3 – 19 lungs per group). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

Decreased MRE11 protein levels in ATII cells in a murine model of emphysema. Wild-type mice were exposed to cigarette smoke for 8 months, as described in the Methods section, to induce emphysema. A Hematoxylin and eosin staining in murine lung tissue (scale bar 50 μm). Minimum (B), maximum (C), and mean (D) alveolar diameters were measured in lung tissue sections. E Representative micro-CT of the murine lung (scale bar-100 μm). F The intersection surface was quantified using micro-CT images. G Pict1 and H Mre11 mRNA levels were evaluated in lung tissue by RT-PCR. I Representative Western blotting images of PICT1 and MRE11 expression in lung tissue. PICT1 (J) and MRE11 expression (K) are quantified. L ATII cells in lung tissue sections were identified using SP-C (green). PICT1 (magenta), and MRE11 (red) antibodies, and DAPI (blue) by immunofluorescence (scale bar—5 μm). Quantification of PICT1 (M) and MRE11 (N) fluorescence intensity in ATII cells is shown. O Pearson’s correlation coefficient for PICT1 and MRE11 fluorescence co-localization. Data are shown as means ± SEM (N = 3 – 8 mice per group). p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 4

PICT1 deletion impairs DNA damage repair in A549 cells. Panel I: CRISPR-Cas9 strategy was used to generate cells with PICT1 deletion as described in the Methods section. A Representative Western blotting image of wild-type A549 cells and cells with PICT1 deletion. B Cells were treated with 20% cigarette smoke extract (CSE) for 24 h. Histograms were obtained by flow cytometry analysis using a DCF-DA probe. C DCF fluorescence intensity was quantified. D Representative images of comets (scale bar—5 µm). E Olive tail moments were quantified. Panel II: Wild-type A549 cells and cells with PICT1 deletion were treated with 20% CSE for 24 h. A Representative Western blotting images of MRE11, 53BP1, LIGASE IV, and KU80 expression. B-E Quantification of protein levels is shown. Panel III: mRNA expression of MRE11 (A), 53BP1 (B), KU80 (C), and LIGASE IV (D) by RT-PCR. Data are shown as means ± SEM (C – control; N = 3 – 9 experimental replicates). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 5

PICT1 deletion sensitizes A549 cells to replicative stress and cell death. A Representative cell cycle histograms showing sub-G1 (blue), G0/G1 (red), S (green), and G2/M (brown) phases in wild-type A549 cells and cells with PICT1 deletion using propidium iodide (PI) staining and flow cytometry analysis. B Quantification is also shown. C Representative flow cytometry plots of wild-type A549 cells and cells with PICT1 deletion treated with hydroxyurea for 24 h and stained using PI and Annexin V. D Cell death quantification. E Representative flow cytometry plots of wild-type A549 cells and cells with PICT1 deletion treated with 20% cigarette smoke extract (CSE) for 24 h. F Cell death quantification is shown. Data represent means ± SEM (N = 3 – 9 experimental replicates). *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.001 compared to all tested conditions

Fig. 6

Mitochondrial dysfunction in human primary ATII cells, A549 cells, and MLE15 cells. A PICT1 (red), TOM20 (green), and DAPI (blue) staining in lung tissue sections obtained from non-smokers (N), smokers (S), and emphysema patients (E) by immunofluorescence (scale bar—5 μm). ATII cells were identified using SP-C (magenta). B Pearson’s correlation coefficient for PICT1 and TOM20 co-localization in ATII cells is shown (N = 3 lungs per group). C Quantification of mitochondrial networks in ATII cells. D Mitochondrial respiration analysis in wild-type A549 cells and cells with PICT1 deletion treated with 20% cigarette smoke extract (CSE) for 24 h. Quantification of basal respiration (E) and maximum respiration (F) in A549 cells. G ATP-linked respiration after exposure to CSE relative to controls in A549 cells. QPCR was used to determine mtDNA amount (H), mtDNA damage (I), and common deletions (CD, J) in A549 cells. Representative histograms using MitoSOX staining and flow cytometry analysis (K) and the quantification of fluorescence intensity (L) in A549 cells. M Representative Western blotting images of MLE15 cells treated with NT (non-target) or PICT1 siRNA. N Histograms of MitoSOX staining by flow cytometry analysis (N) and quantification (O) in MLE15 cells. Data are shown as means ± SEM (KD – knockdown, N = 3 – 10 experimental replicates). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 7

The role of PICT1 in nuclear DNA damage and mitochondrial (mt) function. Increased PICT1/TRIM22 interaction induced by smoking leads to decreased PICT1 levels and high ROS production. This caused nuclear and mtDNA damage, common deletions, mitochondrial superoxide generation, and reduced mtDNA amount and respiration, contributing to ATII cell death and emphysema development