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Published on: August 16, 2013
p47phox and reactive oxygen species production modulate expression of microRNA-451 in macrophages
R Ranjan1, Y G Lee, M Karpurapu
1Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Illinois , Chicago, IL , USA.
Abstract:
The production of microRNAs (miRNA) is influenced by various stimuli, including environmental stresses. We hypothesized that reactive oxygen species (ROS)-associated stress could regulate macrophage miRNA synthesis. miRNAs undergo unique steps of maturation processing through either one of two pathways of cytoplasmic processing. Unlike the canonical pathway, the regulation of alternative cytoplasmic processing of miRNA has not been fully elucidated yet. We cultured bone marrow derived macrophages (BMDM) from wild type (WT) and p47(phox-/-) mice and profiled miRNA expression using microarrays. We analyzed 375 miRNAs including four endogenous controls to normalize the data. At resting state, p47(phox-/-) BMDM has the markedly reduced expression of miR-451 compared to WT BMDM, without other significant differences. Unlike majority of miRNAs, miR-451 goes through the unique alternative processing pathway, in which Ago2 plays a key role. In spite of significant reduction of mature miR-451, however, its precursor form, pre-mir-451, was similar in both BMDMs, suggesting that the processing of pre-mir-451 is impaired in p47(phox-/-) BMDM. Moreover, p47(phox-/-) BMDM expressed significantly reduced level of Ago2. In contrast, Ago2 mRNA levels were similar in WT and p47(phox-/-) BMDM, suggesting a post-transcriptional defect of Ago2 production in p47(phox-/-) macrophages, which resulted in impaired processing of pre-miR-451. In order to examine the functional significance of miR-451 in macrophages, we cultured BMDMs from miR-451 knock-out mice. Of interest, miR-451-deficient BMDM exhibited reduced ROS generation upon zymosan stimulation, compared to WT BMDM. Our studies suggest functional crosstalk between ROS and miR-451 in the regulation of macrophage oxidant stress.
Insights
Reactive oxygen species (ROS) stress impairs microRNA-451 (miR-451) processing in macrophages by reducing Ago2 levels. miR-451 deficiency lowers ROS generation, indicating a functional link between ROS and macrophage oxidative stress regulation.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- MicroRNA (miRNA) production is modulated by environmental factors, including oxidative stress.
- Macrophage miRNA synthesis and maturation, particularly via alternative pathways, remain incompletely understood.
- Reactive oxygen species (ROS) are implicated in cellular signaling and stress responses.
Purpose of the Study:
- To investigate the hypothesis that ROS-associated stress regulates macrophage miRNA synthesis.
- To elucidate the role of the alternative miRNA processing pathway in ROS-mediated stress.
- To explore the functional significance of miR-451 in macrophage oxidative stress.
Main Methods:
- Bone marrow-derived macrophages (BMDM) from wild-type and p47(phox-/-) mice were cultured and subjected to miRNA expression profiling via microarrays.
- Analysis included 375 miRNAs and assessment of precursor and mature miR-451 levels, as well as Ago2 expression.
- miR-451 deficient BMDM were generated and stimulated to assess ROS generation.
Main Results:
- p47(phox-/-) BMDM exhibited significantly reduced mature miR-451 expression compared to wild-type, with similar precursor levels, indicating impaired processing.
- A marked decrease in Ago2 protein levels was observed in p47(phox-/-) BMDM, without changes in Ago2 mRNA, suggesting post-transcriptional regulation.
- miR-451 deficient BMDM showed reduced ROS generation upon stimulation, highlighting a functional role for miR-451 in oxidative stress response.
Conclusions:
- ROS-associated stress, specifically via the p47(phox) pathway, impairs the alternative processing of miR-451 in macrophages.
- Post-transcriptional downregulation of Ago2 is a key mechanism underlying impaired miR-451 maturation in ROS-stressed macrophages.
- A functional crosstalk exists between ROS and miR-451, suggesting miR-451 plays a role in regulating macrophage oxidative stress.
