Rac1-mediated Mitochondrial H2O2 Generation Regulates MMP-9 Gene Expression in Macrophages via Inhibition of SP-1 and AP-1

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.

Mitochondrial Rac1 GTPase Import and Electron Transfer from Cytochrome c Are Required for Pulmonary Fibrosis

“…Rac1 is ubiquitously expressed, and its activation increases the generation of H 2 O 2 (3,6,16,17). Pulmonary fibrosis is characterized by aberrant tissue remodeling in response to injury, and this process can be influenced by H 2 O 2 .…”

“…Determination of H 2 O 2 Generation-H 2 O 2 concentration was determined as described previously (3,4).…”

“…We have previously shown that overexpression of constitutively active Rac1 in macrophages increased mitochondrial H 2 O 2 (3,6); however, the mechanism by which Rac1 mediates mitochondrial H 2 O 2 generation in macrophages has not been described.…”

“…The generation of reactive oxygen species (ROS), 3 including H 2 O 2 , plays a critical role in tissue injury and consequent fibrosis by modulating extracellular matrix deposition (3,4). The production of ROS is accentuated by the inefficient phagocytosis of asbestos fibers by alveolar macrophages (5).…”

“…Rac1 activation increases the generation of H 2 O 2 in multiple cell types (3,16,17), and Rac1 has been shown to indirectly increase mitochondrial ROS generation via engagement of integrins or by increasing ceramide levels (18 -20). We have previously shown that overexpression of constitutively active Rac1 in macrophages increased mitochondrial H 2 O 2 (3,6); however, the mechanism by which Rac1 mediates mitochondrial H 2 O 2 generation in macrophages has not been described.…”