ABSTRACT: BACKGROUND:We previously demonstrated that chronic alcohol ingestion augments TGF?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?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?1 expression in the fibroblast are not known. MicroRNA (miR)-21 has been shown to inhibit Smad7, a TGF?1 signaling inhibitor. In this study, we hypothesized that alcohol augments TGF?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?1. NIH 3T3 fibroblasts were transfected with a miR-21 inhibitor and cultured ± alcohol. ?-SMA, Smad7, and TGF?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?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 ~30% in alcohol-treated fibroblasts. In parallel, inhibition of miR-21 attenuated alcohol-mediated decrease in Smad7 and increase in TGF?1 and ?-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?1 gene expression in alcohol-treated NIH 3T3 fibroblasts. CONCLUSIONS:Alcohol treatment increases TGF?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.
