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.
Highlights
We demonstrate for the first time that Rac1-mediated mitochondrial H2O2 generation inhibits Matrix metalloproteinases (MMPs)-9 transcriptional activity via inhibiting SP-1 and AP-1 DNA binding, transcriptional activity, and direct interaction in macrophages
That confers Rac1 inhibitory response elements; (iii) Rac1-induced H2O2 generation was derived from the mitochondria; (iv) MMP-9 transcriptional activity was suppressed by Rac1
Induced H2O2; (v) Rac1 and H2O2 inhibited ERK MAPK, which is essential for SP-1 and AP-1 transcriptional activation; (vi) collagen deposition by human lung fibroblasts is abolished when cultured in conditioned medium from Rac1 null cells; and (vii) targeted deletion of Rac1, knockdown of the mitochondrial iron-sulfur complex III subunit, Rieske, and modulation of H2O2 in mice differentially regulates expression of MMP-9
Summary
MMP-9 protein and activity were dramatically increased in conditioned medium collected from Rac null macrophages compared with WT cells (Fig. 1A). This difference was observed at the mRNA level Address the specificity of the effects of Rac on MMP-9, we Immunoblot Analysis—Extraction of nuclear proteins and measured relative mRNA expression of MMP-2 and MMP-12, whole cell lysates were performed as described previously which are other macrophage-specific MMPs. MMP-2 was not [31]. Two AP-1 binding sites are present in the significant
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