Mitochondrial calcium uniporter regulates PGC-1α expression to mediate metabolic reprogramming in pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with an increased mortality. Metabolic reprogramming has a critical role in multiple chronic diseases. Lung macrophages expressing the mitochondrial calcium uniporter (MCU) have a critical role in fibrotic repair, but the contribution of MCU in macrophage metabolism is not known. Here, we show that MCU regulates peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and metabolic reprogramming to fatty acid oxidation (FAO) in macrophages. MCU regulated PGC-1α expression by increasing the phosphorylation of ATF-2 by the p38 MAPK in a redox-dependent manner. The expression and activation of PGC-1α via the p38 MAPK was regulated by MCU-mediated mitochondrial calcium uptake, which is linked to increased mitochondrial ROS (mtROS) production. Mice harboring a conditional expression of dominant-negative MCU in macrophages had a marked reduction in mtROS and FAO and were protected from pulmonary fibrosis. Moreover, IPF lung macrophages had evidence of increased MCU and mitochondrial calcium, increased phosphorylation of ATF2 and p38, as well as increased expression of PGC-1α. These observations suggest that macrophage MCU-mediated metabolic reprogramming contributes to fibrotic repair after lung injury.

Keywords: Fatty acid oxidation (FAO); MCU; Metabolic reprogramming; Mitochondrial ROS; PGC-1α; Pulmonary fibrosis.

Graphical abstract

Fig. 1

MCU expression and mitochondrial Ca²⁺are increased in IPF lung macrophages. (A) Total RNA was isolated from lung macrophages of normal or IPF subjects. MCU mRNA was measured by real-time PCR, n = 10. (B) MCU was detected by immunoblot analysis in lung macrophage mitochondria from human subjects. (C) Statistical analysis, n = 6. (D) Ca²⁺ was measured in isolated mitochondria of lung macrophages from human subjects, n = 8. (E) The colocalization of MCU and F4/80 in lung tissue of normal and IPF subjects was detected by IHC-P. Representative micrograph of three individual subjects. Scale bars, 20 μm. (F) The colocalization of MCU and SPC in lung tissue from normal and IPF subjects was detected by IHC-P. Representative micrograph of three individual subjects. Scale bars, 20 μm. (G) WT mice were exposed to saline or bleomycin (1.75U/kg). Lung macrophages were harvested by BAL at indicated time points (day 5, 10, 15 and 21). Whole cell lysate was prepared for the detection of MCU by immuoblot analysis. (H) Reciprocal bone marrow (BM) chimera was performed with WT and MCU ^± mice. After 8 weeks mice were exposed to saline or bleomycin (1.75U/kg) for 21 days. Lungs were subjected to Sirius red staining. Represented micrographs from 6 mice per condition are shown. Scale bars, 200 μm at x5. (I) Lung collagen content was determined by hydroxyproline assay, n = 6. WT and DN-MCU-Lyz2-cre littermates were exposed to saline or bleomycin for 21 days. (J) Mitochondrial Ca²⁺ in lung macrophages from mice was measured in freshly isolated mitochondria, n = 4. (K) α-SMA expression was determined in lung tissue by IHC-P. Representative micrograph of four individual replicates. Scale bars, 200 μm at x5. (L) Quantification of α-SMA expression in mice lung tissue, n = 4 (saline) and n = 5 (bleomycin). (M) Representative (n = 5) microphaph of Sirius red staining of lung parenchyma, scale bars, 200 μm at x5 and (N) hydroxyproline assay, n = 5. All data are expressed as mean ± SEM. S, saline; B and Bleo, bleomycin; DN-MCU, DN-MCU-Lyz2-cre. One-way ANOVA with Tukey's post hoc comparison (I, J, L and N). Two-tailed student's t-test (A, C and D). ***p ≤ 0.001. See also Fig. S1.

Fig. 2

FAO enzymes in lung macrophages are regulated by MCU. (A) Immunoblot analysis for PGC-1α in lung macrophage nuclear extracts from human subjects. (B) Statistical analysis, n = 6. (C) Immunoblot analysis for CPT1A in lung macrophage mitochondria of human subjects. (D) Statistical analysis, n = 6. (E) WT mice were exposed to saline or bleomycin. Lung macrophages were harvested by BAL at indicated time points. Immunoblot analysis of PGC-1α was performed. (F) PGC-1α was determined by fluorescent microcopy in lung macrophages from saline- or bleomycin-exposed WT or DN-MCU-Lyz2-cre mice, scale bars, 20 μm. (G) Statistical analysis, n = 4 Inset: PGC-1α was detected by immunoblot analysis in isolated nuclear extracts from mice lung macrophages. Lung macrophage mitochondria were isolated from saline- or bleomycin-exposed WT or DN-MCU-Lyz2-cre mice, and subjected to (H) immunoblot analysis of Cpt1a. (I) Statistical analysis, n = 3. MH-S cells were transfected with empty, MCU_WT, or MCU_DN. (J) Cells were subjected to fluorescent microscopy for PGC-1α, scale bars, 10 μm. (K) Statistical analysis, n = 4. (L) Mitochondria were isolated for immunoblot analysis of Cpt1a. (M) Statistical analysis, n = 3. (N) MH-S cells were co-transfected with empty or MCU_WT in combination with scrambled or PGC-1α siRNA. Total RNA was isolated for the measurement of Cpt1a mRNA by real-time PCR, n = 4. MH-S cells were transfected with scrambled or PGC-1α siRNA, and exposed to vehicle or bleomycin (0.0126 U/ml, 3 h). (O) Immunoblot analysis of Cpt1a in isolated mitochondria. (P) Statistical analysis, n = 3. All data are expressed as mean ± SEM. Bleo, bleomycin; DN-MCU, DN-MCU-Lyz2-cre. One-way ANOVA with Tukey's post hoc comparison (G, I, K, M, N, and P). Two-tailed student's t-test (B and D). *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001. See also Fig. S2.

Fig. 3

PGC-1α is transcriptionally activated by MCU. (A) MH-S cells were co-transfected with renilla luciferease plasmid, pGL3-PGC-1α luciferase promoter combined with empty, MCU_WT, or MCU_DN. The PGC-1α promoter activity was determined by measuring firefly luciferase and renilla luciferase, and represented with the ratio of firefly to renilla, n = 6. MH-S cells were transfected with empty, MCU_WT, or MCU_DN. (B) Immunoblot analysis was performed for p-ATF2 and p-p38 in nuclear extracts and MCU in isolated mitochondria. (C) Statistical analysis for p-ATF2, n = 3. (D) Statistical analysis for p-p38, n = 3. (E) MH-S cells were transfected with empty, MCU_WT, or MKK6_CA, and treated with vehicle (V) or SB203580 (SB, 10 μm for 2 h). Immunoblot analysis for p-ATF2 and p-p38 was performed in nuclear extracts. (F) Statistical analysis for p-ATF2, n = 3. (G) Statistical analysis for p-p38, n = 3. (H) MH-S cells were transfected with empty, MKK6, or p38_DN in combination with empty or MCU_WT. Immunoblot analysis for p-ATF2 was performed in nuclear extracts. (I) Statistical analysis for p-ATF2, n = 3. (J) MH-S cells were transfected with empty, MKK6, or p38_DN in combination with empty or MCU_WT. Cells were also transfected with renilla luciferease plasmid and pGL3-PGC-1α luciferase promoter. The PGC-1α promoter activity is expressed as the ratio of firefly to renilla, n = 3. (K) WT and DN-MCU-Lyz2-cre littermates were exposed to saline or bleomycin (1.75U/kg) for 21 days. Lung macrophages were isolated by BAL. Immunoblot analysis for p-ATF2 and p-p38 was performed in nuclear extracts. (L) Statistical analysis for p-ATF2, n = 3. (M) Statistical analysis for p-p38, n = 3. (N) Statistical analysis of immunoblot analysis for p-ATF2 in lung macrophage nuclear extracts from normal and IPF subjects, n = 6. Inset, representative immunoblot analysis. (O) Statistical analysis of immunoblot analysis for p-p38 in lung macrophage nuclear extracts from normal and IPF subjects, n = 6. Inset, representative immunoblot analysis. (P) Schematic illustration showing site of p-ATF2 binding to the PGC-1α promoter in the cAMP response element (CRE) domain. WT and DN-MCU-Lyz2-cre littermates were exposed to saline or bleomycin (1.75U/kg) for 21 days. Lung macrophages were isolated by BAL and subjected to ChIP analysis of p-ATF2 bound to the PGC-1α promoter DNA by in the CRE domain with (Q) standard PCR or (R) quantitiative PCR, n = 6. All data are expressed as mean ± SEM. One-way ANOVA with Tukey's post hoc comparison. Two-tailed student's t-test (N and O). *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001. See also Fig. S3.

Fig. 4

MCU-mediated p38 MAPK activation and FAO enzymes are dependent on mitochondrial oxidative stress. THP-1 cells were co-transfected with empty or MCU_WT in combination with scrambled or Rieske siRNA. (A) Mitochondria were isolated for the quantitation of H₂O₂ by pHPA assay, n = 4; (B) Immunoblot analysis was performed for p-ATF2 and p-p38 in nuclear extracts. THP-1 cells were co-transfected with empty or MCU_WT with empty or catalase. Cells were subjected to quantitation of H₂O₂ by pHPA assay (C) in isolated cytosol, n = 4 and (D) in mitochondria, n = 4. (E) THP-1 cells were co-transfected with empty or MCU_WT with empty or catalase. Immunoblot analysis was performed for p-ATF2 and p-p38 in nuclear extracts. (F) THP-1 cells were transfected with empty, MCU_WT or MCU_DN. Cells were treated with CCCP (3 μM, 30 min) 24 h later. Cells were then labelled with JC-1 (10 μg/ml) for 20 min, and subjected to detection of ΔΨm by confocal imaging. Scale bars, 20 μm. (G) ΔΨm was quantitated by ImageJ and represented as RFU ratio of aggregate/monomer, n = 3. (H) WT and DN-MCU-Lyz2-cre littermates were exposed to saline or bleomycin for 21 days. Lung macropages were harvested by BAL for quantification of H₂O₂ by pHPA assay in isolated mitochondria, n = 5. MH-S cells were transfected with empty or MCU_WT in combination of scrambled or Rieske siRNA. Total RNA was extracted to measure (I) PGC-1α mRNA, n = 4 and (J) Cpt1a mRNA, n = 4. Inset, immunoblot analysis for Rieske. THP-1 cells were transfected with empty or MCU_WT, and treated with MitoTEMPO (50 μM, overnight). Cells were subjected to (K) immunoblot analysis for PGC-1α in nuclear extracts. (L) Statistical analysis, n = 4; THP-1 cells were transfected and treated as in (K). (M) Immunoblot analysis for Cpt1a in isolated mitochondria. (N) Statistical analysis, n = 3. All data are expressed as mean ± SEM. DN-MCU, DN-MCU-Lyz2-cre. One-way ANOVA with Tukey's post hoc comparison. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001. See also Fig. S4.

Fig. 5

MCU regulates metabolic reprogramming to FAO in macrophages. Reciprocal bone marrow chimeric mice were exposed to saline or bleomycin. Lung macrophages from were harvested by BAL. (A) OCR kinetics and (B) Max OCR are shown, n = 3. (C) WT and DN-MCU-Lyz2-cre littermates were exposed to saline or bleomycin for 21 days. Lung macrophages were harvested for the determination of OCR, n = 3 or 5. (D) Max OCR, n = 3 or 5. (E) WT and DN-MCU-Lyz2-cre littermates were exposed to saline or bleomycin for 21 days. Lung macropages were harvested by BAL. FAO was determined by measuring OCR with palmitate as substrate. Results are shown as max OCR, n = 4. MH-S cells were transfected with empty, MCU_WT, or MCU_DN. (F) Kinetics of OCR using palmitate as a substrate. (G) Max OCR, n = 3 or 4. (H) MH-S cells were transfected with empty, MCU_WT, or MCU_DN. Cells were subjected to seahorse assay to obtain ECAR, n = 4. (I) MH-S cells were transfected with empty or MCU_WT, in combination with empty or PGC-1α. FAO was determined by OCR using palmitate as a substrate. Max OCR, n = 4. (J) MH-S cells were transfected with empty or MCU_WT, and treated with etomoxir (40 μM) overnight. Cells were subjected to FAO by OCR measurement, n = 4–6. (K) MH-S cells were transfected with empty, MCU_WT, or MCU_DN and whole lysate was prepared for the determination of Cpt1a activity, n = 4. (L) MH-S cells were transfected with empty or MCU_WT, and treated with vehicle or malonyl CoA (100 μm, 3 h). Whole lysate was prepared for the determination of Cpt1a activity, n = 4. (M) MH-S cells were transfected with empty, MCU_WT, or MCU_DN. Immunoblot analysis for p-ACC2 in isolated mitochondria. (N) Statistical analysis, n = 3. (O) MH-S cells were transfected with empty or MCU_WT and treated with vehicle or malonyl CoA. FAO was determined by OCR using palmitate as a substrate, n = 4–5. All average data were represented as mean ± SEM. S, saline; B and Bleo, bleomycin; PMT, palmitate; DN-MCU, DN-MCU-Lyz2-cre. One-way ANOVA with Tukey's post hoc comparison. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001. See also Fig. S5.

Fig. 6

Metabolic reprogramming and macrophage phenotype are interdependent. (A) Lung macrophages from bone marrow chimeric mice were harvested to measure CD206 and MARCO by confocal analyses, scale bars, 20 μm. Statistical analyses of (B) CD206 and (C) MARCO were performed, n = 5. BAL fluid was harvested from WT and DN-MCU-Lyz2-cre mice for the determination of (D) active TGF-β1, n = 5, (E) PDGF-B, n = 5, and (F) TNF-α by ELISA, n = 5. (G) MH-S cells were transfected with scrambled or PGC-1α siRNA and exposed to bleomycin (0.0126 U/ml, 4 h). Total RNA was isolated for the quantitation of TGF-β1 mRNA by real-time quantitative PCR, n = 5. (H) MH-S cells were transfected with scrambled or Cpt1a siRNA and exposed to bleomycin (0.0126 U/ml, overnight). Conditioned media were collected for the detection of active TGF-β1 by ELISA, n = 6. (I) MH-S cells were transfected to with scrambled or PGC-1α siRNA. Cells were exposed to bleomycin (0.0126 U/ml, 4 h). Total RNA was isolated for the measurement of TNF-α mRNA by real-time PCR, n = 5. (J) MH-S cells were transfected with scrambled or Cpt1a siRNA. Cells were exposed to bleomycin (0.0126 U/ml, overnight). Conditioned media were collected for the measurement of TNF-α by ELISA, n = 6. (K) MH-S cells were co-transfected with empty or MCU_WT in combination of scrambled or Cpt1a siRNA. Cells were then subjected fluorescent microscopy for CD206 and MARCO; scale bars, 20 μm; Statistical analyses for (L) CD206, n = 3 and (M) MARCO, n = 3. (N) WT and Tgfb1^−/−Lyz2-cre mice were exposed to saline or bleomycin (1.75U/kg) for 21 days. Lung macrophages were harvested by BAL for the detection of PGC-1α by immunoblot analysis. (O) Statistical analysis, n = 3. (P) Total RNA was isolated from lung macrophages of Tgfb1^−/−Lyz2-cre mice and WT mice for the measurement of PGC-1α gene expression level by qRT-PCR, n = 5. All data are expressed as mean ± SEM. Sali, Saline; Bleo, Bleomycin; DN-MCU, DN-MCU-Lyz2-cre. One-way ANOVA with Tukey's post hoc comparison. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001.