Post-translational regulation of PGC-1α modulates fibrotic repair

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease associated with mitochondrial oxidative stress. Mitochondrial reactive oxygen species (mtROS) are important for cell homeostasis by regulating mitochondrial dynamics. Here, we show that IPF BAL cells exhibited increased mitochondrial biogenesis that is, in part, due to increased nuclear expression of peroxisome proliferator-activated receptor-ɣ (PPARɣ) coactivator (PGC)-1α. Increased PPARGC1A mRNA expression directly correlated with reduced pulmonary function in IPF subjects. Oxidant-mediated activation of the p38 MAPK via Akt1 regulated PGC-1α activation to increase mitochondrial biogenesis in monocyte-derived macrophages. Demonstrating the importance of PGC-1α in fibrotic repair, mice harboring a conditional deletion of Ppargc1a in monocyte-derived macrophages or mice administered a chemical inhibitor of mitochondrial division had reduced biogenesis and increased apoptosis, and the mice were protected from pulmonary fibrosis. These observations suggest that Akt1-mediated regulation of PGC-1α maintains mitochondrial homeostasis in monocyte-derived macrophages to induce apoptosis resistance, which contributes to the pathogenesis of pulmonary fibrosis.

Keywords: PGC-1α; mitochondrial biogenesis; monocyte-derived macrophages; pulmonary fibrosis.

Conflict of interest statement

The authors declare no conflicts of interest exists.

© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.

Figures

FIGURE 1

IPF BAL cells have increased mitochondrial biogenesis. A, Nuclear immunoblot analysis and B, quantification of PGC‐1α in BAL cells from normal and IPF subjects (n = 7). mRNA expression in BAL cells from normal (n = 5‐7) or IPF subjects (n = 5‐8) for C, PPARGC1A, TFAM, CS, and 12s rRNA. D, Ratio of mtDNA to nuclear DNA in normal and IPF BAL cells (n = 5). E, mRNA analysis of PPARGC1A in IPF BALC with silencing of PGC‐1α (n = 8). Inset, PGC‐1α immunoblot analysis. F, TFAM, CS, and COX4I1 expression (n = 8) in IPF BALC with silencing of PGC‐1α. Pearson’s correlation of PPARGC1A mRNA in BAL cells and G, FVC or H, FVC % predicted in IPF subjects (n = 5). I, Nuclear immunoblot of PGC‐1α in BAL cells from saline or bleomycin‐exposed mice at 21 days. J, mRNA analysis Ppargc1a, Tfam, Cs, and Cox4i1 mRNA in BAL cells from saline or bleomycin‐exposed mice at 21 days (n = 5). *P < .05; **P < .001; ***P < .0001. Values shown as mean ± S.E.M. Two‐tailed t‐test statistical analysis was utilized for B‐F, J. Pearson’s coefficient was used for G, H

FIGURE 2

Akt1 promotes mitochondrial biogenesis. A, MitoTracker green staining of MH‐S cells transfected with empty (Emp) (n = 4) or Akt1CA (n = 4). Scale bars represent 10 μm. B, Flow cytometry of transfected THP‐1 cells (n = 3). BAL cells were isolated at indicated days after saline (S) (n = 5/time point) or bleomycin (B, bleo) exposure (n = 5/time point) from Akt1fl/fl or Akt1−/−Lyz2‐cre mice (Akt1−/−). C, Immunoblot analysis and D, quantification of p‐Akt1 (n = 5). E, Representative histology of lung sections with Masson’s trichome staining (n = 5). Scale bar = 500 μm. F, Hydroxyproline analysis of lung homogenates (n = 5). mRNA analysis of G, Ppargc1a (n = 5), H, Tfam (n = 5), and I, Cs (n = 5). J, TEM analysis of BAL cells (n = 8) from Akt1fl/fl and Akt1−/−Lyz2‐cre mice. Scale bar represents 1 μm. K, Total number of mitochondria per BAL cell (n = 8). *P < .05; **P < .001; ***P < .0001. Values shown as mean ± S.E.M. Two‐tailed t test statistical analysis was utilized for B. One‐way ANOVA followed by Tukey’s multiple comparison test was utilized for D, F, K. Two‐way ANOVA followed by Bonferroni post‐test was utilized for G‐I. ***with open line in G‐I refer to Akt1fl/fl bleomycin vs Akt1fl/fl saline at 5, 10, 14, and 21 days and Akt1fl/fl bleomycin vs Akt1−/−Lyz2‐cre bleomycin at 5, 10, 14, and 21 days. Bracketed lines in G‐I is comparing Akt1fl/fl bleomycin at indicated timepoints

FIGURE 3

Monocyte‐derived macrophages are resistant to apoptosis. A, Caspase‐3 activity (n = 4‐6) in IPF BALC with silencing of PGC‐1α. BAL cells were isolated at indicated days after saline (n = 5/time point) or bleomycin exposure (n = 5/time point) from Akt1fl/fl or Akt1−/−Lyz2‐cre mice. B, TUNEL staining (n = 5) and C, quantification (n = 5). Scale bar represents 40 μm. BAL cells were isolated 21 days after exposure. D, Representative flow cytometry plots of monocyte‐derived macrophages (MDM, CD45+CD11b+/−Ly6G−CD64+Ly6c−Siglec Flow) and resident alveolar macrophages (RAM, CD45+CD11b+/−Ly6G−CD64+Ly6c−Siglec Fhi) from saline or bleomycin‐exposed Akt1fl/fl and Akt1−/−Lyz2‐cre mice. Total cell number of (E) MDM (n = 5), F, annexin V positive MDM (n = 5), (G) RAM (n = 5), and H, annexin V positive RAM from Akt1fl/fl and Akt1−/−Lyz2‐cre BAL (n = 5). **P < .001; ***P < .0001. Values shown as mean ± S.E.M. Two‐tailed t‐test statistical analysis was utilized for A. One‐way ANOVA followed by Tukey’s multiple comparison test was utilized for C, E‐H

FIGURE 4

Pulmonary fibrosis does not require Akt2. Akt2fl/fl or Akt2−/−Lyz2‐cre mice were exposed to saline or bleomycin (Bleo). BAL cells were isolated 21 days later. A, TUNEL staining (n = 5), B, TUNEL quantification (n = 4‐5), and C, caspase‐3 activity (n = 5). Scale bar represent 50 μm. mRNA analysis of D, Ppargc1a (n = 5‐6), E, Tfam (n = 5‐6), F, Cs (n = 5‐6), and G, Cox4i1 expression (n = 5‐6) in BAL cells. H, Histology of lung sections with Masson’s trichrome staining (n = 5) and I, hydroxyproline analysis of homogenized lung (n = 5‐6). ***P < .0001. Values shown as mean ± S.E.M. One‐way ANOVA followed by Tukey’s multiple comparison test was utilized

FIGURE 5

Akt1‐mediated mtROS activates PGC‐1α via phosphorylation of p38 MAPK. A, Nuclear immunoblot analysis for p‐PGC‐1α (S570) and PGC‐1α in transfected macrophages. B, PPARGC1A promoter activity in transfected macrophages (n = 8). Inset, p‐Akt1 and p‐Akt2 immunoblot. C, Nuclear immunoblot analysis in transfected macrophages. D, Schematic of Akt2 phosphorylation and mutation sites on PGC‐1α. E, NFE2L2 mRNA analysis in macrophages expressing empty (emp), Akt1C(A) or Akt2CA together with PGC‐1αWT (WT) or PGC‐1αS570A (S570A) (n = 3). Macrophages were transfected with empty or Akt1CA together with MKK6(Glu) or p38DN. F, PPARGC1A mRNA expression (n = 3), G, nuclear immunoblot analysis, H, quantification of PGC‐1α expression (n = 3); inset, Akt1 immunoblot analysis, I, NRF1 mRNA expression (n = 3). J, Schematic of p38MAPK phosphorylation and mutation sites on PGC‐1α. K, NFE2L2 mRNA analysis of macrophages expressing empty or Akt1CA together with PGC‐1αWT (WT), PGC‐1αT262A (T262A), PGC‐1αS265A (S265A), PGC‐1αT298A (T298A) (n = 3). L, Immunoblot analysis of BAL cells from saline or bleomycin‐exposed Akt1fl/fl and Akt1−/−Lyz2‐cre mice. Macrophages were transfected with scramble or PGC‐1α siRNA and empty or Akt1CA. M, Mitochondrial DNA content (n = 3) and N, Tfam mRNA expression (n = 3). Inset, PGC‐1α mRNA expression. O, Nuclear immunoblot analysis in macrophages expressing empty or Akt1CA and treated with vehicle or mitoTEMPO (10 µM, overnight). **P < .001; ***, P < .0001. Values shown as mean ± S.E.M. One‐way ANOVA followed by Tukey’s multiple comparison test was utilized. CPD = Cdc4 phosphodegron motifs

FIGURE 6

Mitochondrial division inhibitor prevents bleomycin‐induced pulmonary fibrosis. A, Immunoblot analysis and B, quantification of p‐Drp1 (S616) expression in BAL cells from normal (n = 5) or IPF subjects (n = 4). C, TUNEL staining (n = 5) and D, TUNEL quantification (n = 5) of IPF BAL cells treated with vehicle or mdivi‐1 (20 µM, overnight). Scale bar represents 50 μm. C57BL/6J WT mice were exposed to saline or bleomycin (bleo), 10 days after exposure mice were administered daily ip injections of vehicle or mdivi‐1. BAL cells were isolated 21 days later. E, Immunoblot analysis in BAL cells. F, TUNEL staining (n = 5), G, TUNEL quantification (n = 5), and H, Caspase‐3 activity (n = 6), Scale bar represent 50 μm. mRNA analysis of I, Ppargc1a (n = 5), J, Tfam (n = 5), K, Cs (n = 5), and L, Cox4i1 expression (n = 5). M, Masson’s trichrome staining of lung sections (n = 5) and N, hydroxyproline analysis (n = 5). **P < .001; ***P < .0001. Values shown as mean ± S.E.M. Two‐tailed t‐test statistical analysis was utilized for B and D. One‐way ANOVA followed by Tukey’s multiple comparison test was utilized for G‐L, N

FIGURE 7

Deletion of PGC‐1α in monocyte‐derived macrophages protects against pulmonary fibrosis. Ppargc1afl/fl or Ppargc1a−/−Csf1rMeriCreMer mice were administered tamoxifen (TAM) or regular (Reg) chow and exposed to saline or bleomycin (bleo). BAL cells were isolated 21 days later. A, mRNA analysis of PGC‐1α in BAL cells (n = 5‐6). B, TUNEL staining (n = 5‐6), and C, TUNEL quantification (n = 5‐6). Scale bar represents 50 μm. D, Tfam (n = 5), E, Cs (n = 5‐6), and F, Cox4i1 mRNA expression (n = 5‐6). G, mtDNA/nDNA in BAL cells (n = 4‐5). H, Masson’s trichrome staining of lung sections (n = 5‐6) and I, hydroxyproline analysis (n = 5). BAL cells were isolated from IPF patients by BAL. **P < .001; ***P < .0001. Values shown as mean ± S.E.M. One‐way ANOVA followed by Tukey’s multiple comparison test was utilized

FIGURE 8

PGC‐1α regulates fibrotic progression in monocyte‐derived macrophages. Lung injury promotes Akt1 activation and the generation of the mitochondrial ROS (mtROS), which induces p38 MAPK. The p38‐mediated increase of PGC‐1α activation occurs via Akt1 and augments the transcription of mitochondrial DNA genes, including TFAM and CS (citrate synthase) to induce apoptosis resistance in monocyte‐derived macrophages and fibrotic progression