Abstract
GM-CSF is an endogenous pulmonary cytokine produced by normal alveolar epithelial cells (AEC) that is a key defender of the alveolar space. AEC GM-CSF expression is suppressed by oxidative stress through alternations in mRNA turnover, an effect that is reversed by treatment with recombinant GM-CSF. We hypothesized that specific microRNA (miRNA) would play a key role in AEC GM-CSF regulation. A genome-wide miRNA microarray identified 19 candidate miRNA altered in primary AEC during oxidative stress with reversal by treatment with GM-CSF. Three of these miRNA (miR 133a, miR 133a*, and miR 133b) are also predicted to bind the GM-CSF 3'-untranslated region (UTR). PCR for the mature miRNA confirmed induction during oxidative stress that was reversed by treatment with GM-CSF. Experiments using a GM-CSF 3'-UTR reporter construct demonstrated that miR133a and miR133b effects on GM-CSF expression are through interactions with the GM-CSF 3'-UTR. Using lentiviral transduction of specific mimics and inhibitors in primary murine AEC, we determined that miR133a and miR133b suppress GM-CSF expression and that their inhibition both reverses oxidant-induced suppression of GM-CSF expression and increases basal expression of GM-CSF in cells in normoxia. In contrast, these miRNAs are not active in regulation of GM-CSF expression in murine EL4 T cells. Thus, members of the miR133 family play key roles in regulation of GM-CSF expression through effects on mRNA turnover in AEC during oxidative stress. Increased understanding of GM-CSF gene regulation may provide novel miRNA-based interventions to augment pulmonary innate immune defense in lung injury.
Highlights
Accelerated mRNA turnover results in suppression of lung epithelial cell GM-CSF expression during oxidative stress
Expression of GM-CSF mRNA in whole lung and in the alveolar wall is suppressed by exposure of mice to hyperoxia [8]
This strongly suggests that the GM-CSF identified with staining was produced by alveolar epithelial cells (AEC) and that AEC are the major source of GM-CSF in the lung
Summary
Accelerated mRNA turnover results in suppression of lung epithelial cell GM-CSF expression during oxidative stress. Members of the miR133 family play key roles in regulation of GM-CSF expression through effects on mRNA turnover in AEC during oxidative stress. We report that a family of specific miRNA expressed in AEC plays a key role both in regulating constitutive GM-CSF expression at baseline and in suppressing GM-CSF expression during oxidative stress through interactions with the 3Ј-untranslated region of the GM-CSF mRNA. Detailed understanding of these mechanisms may afford a therapeutic opportunity for targeted manipulation of endogenous expression of GM-CSF in the lung
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have