Viranuj Sueblinvong, Lucian T. Marts, Stephen T. Mills, Tamara Murphy, David E. Green
We previously demonstrated that chronic alcohol ingestion augments TGFβ1 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 TGFβ1 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, TGFβ1 expression in the fibroblast are not known. MicroRNA (miR)-21 has been shown to inhibit Smad7, a TGFβ1 signaling inhibitor. In this study, we hypothesized that alcohol augments TGFβ1 expression through up-regulation of miR-21, which subsequently inhibits Smad7.
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 TGFβ1. NIH 3T3 fibroblasts were transfected with a miR-21 inhibitor and cultured ± alcohol. α-SMA, Smad7, and TGFβ1 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 TGFβ1 was assessed.
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 TGFβ1 and α-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 TGFβ1 gene expression in alcohol-treated NIH 3T3 fibroblasts.
Alcohol treatment increases TGFβ1 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.
In the murine lung fibroblast, alcohol exposure augments microRNA-21 (miR-21) expression, and miR-21 subsequently inhibits SMAD7, the canonical TGFβ1 signaling pathway inhibitor. This process leads to a relative increase in TGFβ1 signaling and expression. It appears the increase in miR-21 may be secondary to alcohol-mediated activation of STAT3. This pathway can be inhibited by activation of Nrf2 with sulforaphane, which ultimately mitigates alcohol-induced TGFβ1 expression.