Pulmonary fibrosis (PF) is characterized by failed alveolar re-epithelialization and fibroblast activation.1 A continuing struggle in the field has been how to diagnose and treat the disease early and effectively on the basis of shared pathogenetic mechanisms. Transforming growth factor-β (TGF-β) signaling and mitochondria are involved in PF pathogenesis.2 Mitochondrial respiratory chain complexes (RCCs) I, III, IV, and V comprise both nuclear DNA (nDNA)- and mitochondrial DNA (mtDNA)-encoded subunits, and their biogenesis depends on the cooperation of cytoplasmic (cyto) and mitochondrial (mito) translation (Supplementary Fig. 1).3 To date, a correlation between PF and RCC biogenesis has not been reported.
We first examined the TGF-β levels and the enzymatic activity of five RCCs in blood samples of healthy controls (HCs) and patients with the two most common forms of PF,4 idiopathic PF (IPF) and connective tissue disease PF (CTD-PF). Decreased RCCs activities, especially complex V, were observed in PF patients. Decreased complex V activity was significantly correlated with increased expression of TGF-β. Further receiver-operating characteristic (ROC) analysis revealed that the PF group and the HC group were well distinguished based on complex V activity levels and especially based on the combination of complex V activity and serum TGF-β levels. In addition, the extent of the fibrotic pattern on high-resolution computed tomography (HRCT) scans was correlated with complex V activity. These data suggest a potential role for the TGF-β +/complex V− combination as a PF blood biomarker (Fig. 1a–f, Supplementary Fig. 2, and Supplementary Table 1). Data from a bleomycin (BLM)-induced PF mouse model supported the above results, quantitatively and qualitatively indicating increased expression of TGF-β as well as decreased activity of complex V in both pulmonary tissue and blood samples. Besides, correlation analyses also reflected that there was the correlation between TGF-β, complex V activity and fibrotic lesions (Fig. 1g, Supplementary Figs. 3, S4, and Supplementary Tables 2 and 3).
The human alveolar epithelial cell line A549, the epithelial-like cell line H1299, the bronchial epithelial cell line 16HBE, human lung fibroblasts (HLFs), and mouse primary type II alveolar epithelial cells (AECII) were used to explore the cellular details and molecular mechanism. As the concentration of TGF-β increased, more cells exhibited a spindle-shaped mesenchymal cell morphology similar to that of fibroblasts. Under transmission electron microscopy (TEM), the mitochondria displayed a swollen morphology and disordered cristae structure. Reduced numbers of cristae, vacuolization and slight staining in the bottom edge regions (where complex V is located) suggested a critical complex V defect (Fig. 1h, Supplementary Fig. 5, and Supplementary Video 1). To examine each complex quantitatively and qualitatively, blue native gel (BNG), in-gel activity (IGA) assays and western blotting (WB) were performed. In general, all complexes displayed decreases in both amount and activity as the TGF-β concentration increased. However, complex V decreased first and most significantly. Adenosine triphosphate (ATP) production also decreased, but a reactive oxygen species (ROS) burst occurred (Fig. 1i–k and Supplementary Figs. 6 and 7). Analysis of transcriptomic sequencing data with the Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that the phosphatidylinositol 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathway was the most highly enriched pathway, and translation enhancement was observed in the increased portion of the polysomes in ribosomal profiling. However, the total amount of ribosomes decreased because the amounts of monomers plummeted (Fig. 1l, m and Supplementary Fig. 8).
Considering that the cytoribosome (80 S) has an RNA outer layer but that the mito-ribosome (55 S) is covered by a tight protein enclosure that protects it from the frequently changing redox state in the mitochondrial matrix, we hypothesized that there were differences in the tolerance of the two ribosomes to a TGF-β + /complex V− state and subsequent intracellular stress. Cyto-80S protein S6 and mito-55S protein L48 were stained, and confocal microscopy and superresolution-stimulated emission depletion (STED) microscopy with SVl Htygens deconvolution to reach a 2-dimensional resolution of approximately 30 nm showed decreased 80 S levels but stable 55 S levels (Fig. 1n–u and Supplementary Fig. 9).
The de novo protein biosynthesis and product stability of both the cyto- and mito-translation systems were examined by click-iT l-azidohomoalanine (AHA) and chase assays. Cyto-translation exhibited enhanced productivity, in agreement with the PI3K-mTOR upregulation data above, but a very fast turnover rate and low stability of the protein products were observed. In contrast, mito-translation in both untreated and TGF-β-treated samples exhibited constant values for all 13 mtDNA-encoded polypeptides. More interestingly, the stability of ATP6, a complex V subunit, differed from that of the other complexes. Compared to the ND5 band, which had a similar expression level as ATP6 under TGF-β stimulation after 1 hour of chase, the ATP6 band was nearly undetectable after 2 h of chase (Fig. 1v, w and Supplementary Fig. 10). These results were consistent with our findings that TGF-β signaling enhanced cyto-translation but ultimately damaged cyto-ribosomes. The consequence of such cyto-translation was a quantitative increase in protein products but a decrease in the quality of the newly synthesized polypeptides, which might have been of normal length but lacked complete folding or maturation and prompt degradation. In contrast, the mito-ribosomes were much more stable, and the mtDNA-encoded respiratory chain subunits were biosynthesized in a relatively constant state (Fig. 1x). One of the two IPF drugs, pirfenidone (PFD), was able to prevent the progression of PF by efficiently rescuing complex V activity (Supplementary Fig. 11).
Here, we found that elevated TGF-β expression was associated with impairment of complex V activity in IPF and CTD-PF patients’ blood as well as in a BLM-induced PF mouse model. Further mechanistic study in human pulmonary epithelial cells revealed a cyto-/mito-translation imbalance. Specifically, we observed mTOR activation but cyto-translation damage and protein product instability, in contrast to mito-translation stability, that impaired the balanced biogenesis of multisubunit RCCs, mainly complex V, in the mitochondria of fibrotic cells. Decreases in complex V and ATP and a burst of ROS induced alveolar epithelial cell dedifferentiation.5 On the basis of these findings, TGF-β + /complex V− may be a potential biomarker of PF and a drug target for pulmonary regeneration in the post-COVID era.
The raw data from the RNA-Seq analysis have been deposited in the Sequence Read Archive (SRA) database (Accession number: PRJNA932932). The data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.
All codes in this study are available upon reasonable request.
Richeldi, L., Collard, H. R. & Jones, M. G. Idiopathic pulmonary fibrosis. Lancet 389, 1941–1952 (2017).
Distler, J. H. W. et al. Shared and distinct mechanisms of fibrosis. Nat. Rev. Rheumatol. 15, 705–730 (2019).
Scarpulla, R. C. Transcriptional paradigms in mammalian mitochondrial biogenesis and function. Physiol. Rev. 88, 611–638 (2008).
Noble, P. W., Barkauskas, C. E. & Jiang, D. Pulmonary fibrosis: patterns and perpetrators. J. Clin. Investig. 122, 2756–2762 (2012).
Wang, Q., Wang, Y., Li, S., Zhou, A. & Qin, Y. Organelle biogenesis: ribosomes as organizer and performer. Sci. Bull. 67, 1614–1617 (2022).
This work was supported by the National Key R&D Program of China (2018YFA0106900, 2018YFA0106901) and the National Natural Science Foundation of China (NSFC) (31830043). The authors acknowledge Prof. Dr. Guangbiao Zhou (Chinese Academy of Medical Sciences), Dr. Xue Li (Tianjin Key Laboratory of Lung Regenerative Medicine, Tianjin University Haihe Hospital) for providing materials and help; Prof. Dr. Taotao Wei (IBP, CAS) for discussion and draft polishing; Dr. Xixia Li and Ms. Yan Teng from the Center for Biological Imaging (CBI), IBP, CAS for their help with collecting and analyzing the TEM and SIM images; Ms. Junfeng Hao, Xiaoxiao Zhu, Dongdong Fan and Jiajia Hou from Laboratory Animal Center, IBP, CAS for their help with mouse experiments; Prof. Dr. Dong Li and Dr. Yanan Gao from Department of Radiology, Tianjin Medical University General Hospital for chest HRCT assessment.
The authors declare no competing interests.
All studies were performed according to the guidelines of the Animal Care and Use Committee, IACUC from the Institute of Biophysics, Chinese Academy of Sciences, China (approval number SYXK2021108). The study was approved by the local ethics committee of Tianjin Medical University General Hospital (approval number IRB2021-YX-003-01). All participants provided written informed consent prior to study inclusion.
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Zhang, N., Zhao, Z., Zhao, Y. et al. TGF-β changes cyto/mito-ribosome balance to target respiratory chain complex V biogenesis in pulmonary fibrosis therapy. Sig Transduct Target Ther 8, 136 (2023). https://doi.org/10.1038/s41392-023-01370-2