Mitochondrial Rac1 GTPase import and electron transfer from cytochrome c are required for pulmonary fibrosis

Our plain-English summary of Mitochondrial Rac1 GTPase import and electron transfer from cytochrome c are required for pulmonary fibrosis, by H.L. Osborn-Heaford, A.J. Ryan, S. Murthy, et al (The Journal of Biological Chemistry, 2012).

The study

• Authors: H.L. Osborn-Heaford, A.J. Ryan, S. Murthy, A.M. Racila, C. He, J.C. Sieren, D.R. Spitz, A.B. Carter
• Journal: The Journal of Biological Chemistry
• Published: 2012;287(5):3301-12
• Cited by: 77 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

The generation of reactive oxygen species, particularly H(2)O(2), from alveolar macrophages is causally related to the development of pulmonary fibrosis. Rac1, a small GTPase, is known to increase mitochondrial H(2)O(2) generation in macrophages; however, the mechanism by which this occurs is not known. This study shows that Rac1 is localized in the mitochondria of alveolar macrophages from asbestosis patients, and mitochondrial import requires the C-terminal cysteine of Rac1 (Cys-189), which is post-translationally modified by geranylgeranylation. Furthermore, H(2)O(2) generation mediated by mitochondrial Rac1 requires electron transfer from cytochrome c to a cysteine residue on Rac1 (Cys-178). Asbestos-exposed mice harboring a conditional deletion of Rac1 in macrophages demonstrated decreased oxidative stress and were significantly protected from developing pulmonary fibrosis. These observations demonstrate that mitochondrial import and direct electron transfer from cytochrome c to Rac1 modulates mitochondrial H(2)O(2) production in alveolar macrophages pulmonary fibrosis.

In plain English

This laboratory study examined how Rac1, a small GTPase, drives mitochondrial hydrogen peroxide (H2O2) production in alveolar macrophages and its relation to asbestos-induced lung fibrosis. Alveolar macrophages were obtained by bronchoalveolar lavage from asbestosis patients and healthy non-smoking volunteers; those from asbestosis patients showed about 2.5-fold more mitochondrial Rac1 relative to whole-cell Rac1 (normals n=5, patients n=4, p less than 0.024) and nearly 4-fold higher mitochondrial Rac1 activity (p less than 0.0016), with no significant difference in whole-cell Rac1. Wild type mice and mice with Rac1 disrupted in cells of the myeloid lineage received 100 micrograms of chrysotile asbestos intratracheally; 21 days later wild type mice had higher macrophage and mitochondrial H2O2 generation, a higher percentage of total lung glutathione in the oxidised disulfide form (p less than 0.0110) and more lung hydroxyproline (p less than 0.0208), with micro-CT and trichrome changes of fibrosis. Cell experiments indicated Rac1 mitochondrial import required cysteine 189 and its geranylgeranylation, and that H2O2 generation mediated by mitochondrial Rac1 required electron transfer from cytochrome c to cysteine 178. The authors concluded that mitochondrial Rac1 activity in alveolar macrophages is associated with oxidative stress, which modulates fibrosis development.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory mechanistic study combining human alveolar macrophage samples, a mouse asbestos-exposure experiment with conditional Rac1 deletion in myeloid cells, and cell-culture/biochemical experiments
• Who was studied: Alveolar macrophages from normal volunteers (75% male, mostly 30-55 years) and from asbestosis patients (all male, 50-65 years), plus wild type and Rac1 null C57BL/6 mice (Rac1 disrupted in cells of the myeloid lineage), and THP-1 and MH-S cell lines; University of Iowa
• Exposure: Chrysotile asbestos: 100 micrograms intratracheally in mice, and 10 micrograms per cm2 for 3 h (or 0-60 min) to macrophages in vitro
• Main results:
  • Mitochondrial Rac1 expression (as a ratio of whole cell Rac1) was nearly 2.5-fold greater in alveolar macrophages from asbestosis patients than from normal subjects
  • Normal subjects (n=5) versus asbestosis patients (n=4) for the mitochondrial to whole cell Rac1 ratio, p less than 0.024
  • No significant difference in Rac1 expression was found in whole cell lysates between normal subjects and asbestosis patients
  • Mitochondrial Rac1 activity was nearly 4-fold higher in asbestosis patients' alveolar macrophages than in normal subjects (n=4 each, p less than 0.0016)
  • Mitochondria from normal subjects' alveolar macrophages showed significantly greater Rac2 than those from asbestosis patients (opposite pattern to Rac1)
  • In asbestos-exposed mice, alveolar macrophage H2O2 generation was higher in wild type (n=8) than Rac1 null (n=8) mice, p less than 0.0055; mitochondrial H2O2 higher in wild type (n=8) than Rac1 null (n=12), p less than 0.0023
  • Lungs of wild type mice (n=6) had a higher percentage of total glutathione in the disulfide form than Rac1 null mice (n=7), p less than 0.0110, which the authors read as increased oxidative stress
  • Lung hydroxyproline was significantly higher in wild type (n=4) than Rac1 null (n=4) asbestos-exposed mice, p less than 0.0208; micro-CT (3 mice each) and Masson's trichrome (10 mice each) showed fibrotic change in wild type and essentially normal lungs in Rac1 null mice
  • All Flag-Rac1 cysteine-to-serine mutants were imported into mitochondria except C189S, and simvastatin abrogated import of wild type Rac1, indicating geranylgeranylation of Cys-189 is needed for import
  • Mitochondria from cells expressing the C178S mutant had H2O2 reduced to levels similar to the non-imported C189S mutant (C178S p less than 0.0023 and C189S p less than 0.0026 compared with wild type)
  • Asbestos exposure increased cytochrome c co-immunoprecipitating with Rac1 in mitochondria, and cytochrome c was retained in mitochondria rather than released to the cytoplasm
  • Asbestos exposure and constitutively active Rac1 (Ad5.V12Rac1) each increased active caspase 3 in macrophages
  • In vitro, diamide inhibited H2O2 from Rac1-V5-His wild type and C6S but had no effect on C178S; beta-mercaptoethanol increased H2O2 more than 3-fold with wild type
• The authors' conclusion: The authors concluded that Rac1 is imported into macrophage mitochondria, is active in the intermembrane space where it interacts with and accepts electrons from cytochrome c via Cys-178 to generate H2O2, and that mitochondrial Rac1 activity in alveolar macrophages is associated with increased oxidative stress that modulates the development of pulmonary fibrosis
• Limitations the authors state:
  • The authors state that Rac1 is not the only control on H2O2, since Rac1 null macrophages still produced some H2O2 and other sources exist
  • The authors state that whether Rac1 binds cytochrome c directly, and which Rac1 domain does so, is still under study

Topics

Medical terms in this study, explained in our Lexicon: Pulmonary Fibrosis, Reactive Oxygen Species (ROS), Alveolar Macrophage, Macrophage, Oxidative Stress.

Related studies

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• Changes in autacoid and neuropeptide contents of lung cells in asbestos-induced pulmonary fibrosis — R. Day, S. Lemaire, et al. (1987)
• Alternative activation of macrophages and pulmonary fibrosis are modulated by scavenger receptor, macrophage receptor with collagenous structure — S. Murthy, J.L. Larson-Casey, et al. (2015)

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