MiR-21-Mediated Suppression of Smad7 Induces TGFβ1 and Can Be Inhibited by Activation of Nrf2 in Alcohol-Treated Lung Fibroblasts

Background: We previously demonstrated that chronic alcohol ingestion augments TGF beta 1 expression in the lung fibroblast and increases the risk of fibroproliferative disrepair in a mouse model of acute lung injury. The effect of alcohol on TGF beta 1 is mitigated by treatment with sulforaphane (SFP), which can activate nuclear factor (erythroid-derived 2)-like 2 (Nrf2). However, the mechanisms by which alcohol amplifies, or SFP attenuates, TGF beta 1 expression in the fibroblast are not known. MicroRNA (miR)-21 has been shown to inhibit Smad7, a TGF beta 1 signaling inhibitor. In this study, we hypothesized that alcohol augments TGF beta 1 expression through up-regulation of miR-21, which subsequently inhibits Smad7. Methods: Primary mouse lung fibroblasts were cultured +/- alcohol +/- SFP and assessed for gene expression of miR-21, and gene and/or protein expression of Nrf2, Nrf2-regulated antioxidant enzymes, Smad7, STAT3, and TGF beta 1. NIH 3T3 fibroblasts were transfected with a miR-21 inhibitor and cultured +/- alcohol. alpha-SMA, Smad7, and TGF beta 1 protein expression were then assessed. In parallel, NIH 3T3 lung fibroblasts were transfected with Nrf2 silencing RNA (siRNA) and cultured +/- alcohol +/- SFP. Gene expression of miR-21, Nrf2, Smad7, and TGF beta 1 was assessed. Results: MiR-21 gene expression was increased by 12-fold at 48 hours, and Smad7 gene expression and protein expression were reduced by similar to 30% in alcohol-treated fibroblasts. In parallel, inhibition of miR-21 attenuated alcohol-mediated decrease in Smad7 and increase in TGF beta 1 and alpha-SMA protein expression. Treatment with SFP mitigated the effect of alcohol on miR-21, Smad7 and total and phosphorylated STAT3, and restored Nrf2-regulated antioxidant gene expression. Silencing of Nrf2 prevented the effect of SFP on miR-21, Smad7, and TGF beta 1 gene expression in alcoholtreated NIH 3T3 fibroblasts. Conclusions: Alcohol treatment increases TGF beta 1 in fibroblasts, at least in part, through augmentation of miR-21, which then inhibits Smad7 expression. These effects can be attenuated by activation of Nrf2 with SFP.