Rac1-mediated mitochondrial H2O2 generation regulates MMP-9 gene expression in macrophages via inhibition of SP-1 and AP-1

Our plain-English summary of Rac1-mediated mitochondrial H2O2 generation regulates MMP-9 gene expression in macrophages via inhibition of SP-1 and AP-1, by S. Murthy, A. Ryan, C. He, et al (The Journal of Biological Chemistry, 2010).

The study

• Authors: S. Murthy, A. Ryan, C. He, R.K. Mallampalli, A.B. Carter
• Journal: The Journal of Biological Chemistry
• Published: 2010;285(32):25062-73
• Cited by: 78 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Aberrant matrix deposition is a hallmark of pulmonary fibrosis and is characterized by an imbalance between matrix deposition and degradation. We have previously shown that mice harboring a conditional deletion of the GTP-binding protein, Rac1, in macrophages are protected from asbestos-induced pulmonary fibrosis. To investigate the contribution of aberrant matrix degradation, we addressed the role of Rac1 in regulating expression of macrophage-specific MMP-9 (matrix metalloproteinase-9). We found that MMP-9 gene transcription was significantly greater in Rac1 null macrophages. Deletion and mutational analysis of the MMP-9 promoter revealed that both SP-1 and AP-1 are essential for MMP-9 transcription. Overexpression of constitutive active Rac1 (V12) revealed that H(2)O(2) was derived from the mitochondria. Rac1-induced H(2)O(2) generation down-regulated MMP-9 gene transcription, whereas catalase overexpression in WT cells enhanced MMP-9 expression. SP-1 interacted directly with both c-Jun and c-Fos, and H(2)O(2) decreased this binding, suggesting that SP-1 and AP-1 function cooperatively to regulate MMP-9 transcription. Rac1-mediated H(2)O(2) inhibited the ERK MAPK, which was essential for activation of SP-1 and AP-1. ERK activation and MMP-9 expression were recovered by overexpressing catalase or transfecting siRNA for the mitochondrial iron-sulfur protein, Rieske. These observations were recapitulated in vivo. MMP-9 mRNA was higher in alveolar macrophages isolated from Rac1 null mice and wild type mice given catalase. Rac1 regulates MMP-9 transcription via mitochondrial H(2)O(2) generation, providing a potential mechanism by which Rac1 null mice fail to develop pulmonary fibrosis.

In plain English

This laboratory study used cultured mouse and human cells, and mice; it did not study people. The authors had earlier reported that mice whose macrophages (immune cells) lack the protein Rac1 are protected from asbestos-caused lung scarring (pulmonary fibrosis). They asked whether Rac1 controls MMP-9, an enzyme that breaks down tissue framework. In cultured macrophages, activity of the MMP-9 gene's control region was 7-fold greater in cells lacking Rac1 than in normal cells. In human THP-1 cells, active Rac1 raised mitochondrial hydrogen peroxide production more than 6.5-fold. In Rac1-lacking macrophages, added hydrogen peroxide lowered that control region's activity. Mice were given chrysotile asbestos into the windpipe. Lung macrophages from Rac1-lacking mice had significantly more MMP-9 messenger RNA (the gene's working copy) than those from normal mice. Asbestos-exposed normal mice given catalase, an antioxidant enzyme, had more than 7-fold more macrophage MMP-9 messenger RNA than those given water. Mice lacking MMP-9 developed fibrosis to a similar extent as normal mice. The authors concluded that Rac1-driven mitochondrial hydrogen peroxide suppresses MMP-9 and offers a mechanism for why Rac1-lacking mice are protected.

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 study: cell-culture experiments (macrophages, THP-1 cells, lung fibroblasts) plus animal experiments in mice
• Who was studied: The animals were normal (wild-type) C57BL/6 mice, C57BL/6 mice with the Rac1 gene disrupted in myeloid cells, and MMP-9 knockout mice from Jackson Laboratory. Macrophages were cultured from these mice. Human THP-1 cells and human lung fibroblasts (HFL-1) were also used. The work was done at the University of Iowa. Figure legends give 3 to 6 mice per group in the asbestos experiments.
• Exposure: Mice received chrysotile asbestos from the NAIMA Fiber Repository once into the trachea: 100 micrograms in 50 microlitres of saline. The article prints these as '100 g' and '50 l' because the micro sign was lost. Mice were examined 21 days later. Some normal mice also received catalase (2000 units per mouse) or water daily for 20 days. Cultured cells received chrysotile at 10 micrograms per square centimetre (printed '10 g/cm2') and, in some experiments, hydrogen peroxide.
• Main results:
  • In cultured macrophages, MMP-9 promoter activity was 7-fold greater in Rac1-lacking cells than in normal cells.
  • The effect was specific to MMP-9: MMP-12 mRNA did not differ between normal and Rac1-lacking macrophages.
  • In THP-1 cells expressing constitutively active Rac1, mitochondrial hydrogen peroxide generation increased more than 6.5-fold.
  • In normal macrophages overexpressing catalase, MMP-9 promoter activity was about 5-fold greater.
  • In Rac1-lacking macrophages carrying an MMP-9 promoter reporter, added hydrogen peroxide significantly lowered reporter (luciferase) activity.
  • In Rac1-lacking cells, added hydrogen peroxide reduced SP-1 binding and abolished AP-1 binding to the MMP-9 promoter.
  • In asbestos-exposed mice, MMP-9 mRNA in lung macrophages was significantly greater in Rac1-lacking mice than in normal mice.
  • In asbestos-exposed normal mice, macrophage MMP-9 mRNA was more than 7-fold greater in mice given catalase than in mice given vehicle (water).
  • Normal mice and MMP-9 knockout mice developed a similar extent of fibrosis after asbestos, measured by hydroxyproline in lung-wash fluid.
  • On lung staining 21 days after asbestos, normal and MMP-9 knockout mice had extensive collagen deposition, while Rac1-lacking mice had none.
  • Human lung fibroblasts cultured in medium from Rac1-lacking macrophages secreted significantly less procollagen and collagen I than fibroblasts cultured in medium from normal macrophages.
• The authors' conclusion: The authors concluded that Rac1-mediated mitochondrial hydrogen peroxide generation suppresses MMP-9 gene transcription. They said this provides a mechanism by which Rac1-lacking mice are protected from developing pulmonary fibrosis.

Topics

Medical terms in this study, explained in our Lexicon: Macrophage, Deposition, Pulmonary Fibrosis, Alveolar Macrophage.

Related studies

• Mitochondrial Rac1 GTPase import and electron transfer from cytochrome c are required for pulmonary fibrosis — H.L. Osborn-Heaford, A.J. Ryan, et al. (2012)
• Mitochondrial Cu,Zn-superoxide dismutase mediates pulmonary fibrosis by augmenting H2O2 generation — C. He, S. Murthy, et al. (2011)
• Cu,Zn-Superoxide Dismutase-Mediated Redox Regulation of Jumonji Domain Containing 3 Modulates Macrophage Polarization and Pulmonary Fibrosis — C. He, J.L. Larson-Casey, et al. (2016)
• NOX4 regulates macrophage apoptosis resistance to induce fibrotic progression — J.L. Larson-Casey, L. Gu, et al. (2021)
• Osteopontin is linked to p65 and MMP-9 expression in pulmonary adenocarcinoma but not in malignant pleural mesothelioma — A.B. Frey, A. Wali, et al. (2007)
• High levels of catalase and glutathione peroxidase activity dampen H2O2 signaling in human alveolar macrophages — A.B. Carter, L.A. Tephly, et al. (2004)

Browse more research in The Journal of Biological Chemistry, or return to medical research.