TGFβ1 Mediates Alcohol‐Induced Nrf2 Suppression in Lung Fibroblasts

Abstract

Rationale Chronic alcohol ingestion induces the expression of TGFβ1, inhibits Nrf2-mediated activation of the anti-oxidant response element (ARE), depletes alveolar glutathione pools, and potentiates acute lung injury. In this study we examined the mechanistic relationship between TGFβ1 and Nrf2-ARE signaling in the experimental alcoholic lung. Methods Wild type mice were treated ± alcohol in drinking water for 8 weeks and their lungs were assessed for Nrf2 expression. In parallel, mouse lung fibroblasts were cultured ± alcohol and treated ± sulforaphane (an activator of Nrf2), ± TGFβ1, ± TGFβ1 neutralizing antibody, and/or ± ALK5 inhibitors (to block TGβ1 receptor signaling) and then analyzed for the expression of Nrf2, Keap1 and TGFβ1, Nrf2-ARE activity, and the expression of the Nrf2-ARE-dependent anti-oxidants glutathione s-transferase theta 2 (GSTT2) and glutamate-cysteine ligase catalytic subunit (GCLC). Finally, RNA silencing of Nrf2 was then performed prior to alcohol exposure and subsequent analysis of TGFβ1 expression. Results Alcohol treatment in vivo or in vitro decreased Nrf2 expression in murine whole lung and lung fibroblasts, respectively. In parallel, alcohol exposure in vitro decreased Keap1 gene and protein expression in lung fibroblasts. Further, alcohol exposure increased TGFβ1 expression but decreased Nrf2-ARE activity and expression of the ARE-dependent genes for GSTT2 and GCLC. These effects of alcohol were prevented by treatment with sulforaphane; in contrast, Nrf2 RNA silencing expression exacerbated alcohol-induced TGFβ1 expression. Finally, TGFβ1 treatment directly suppressed Nrf2-ARE activity whereas blocking TGFβ1 signaling attenuated alcohol-induced suppression of Nrf2-ARE activity. Conclusion Alcohol-induced oxidative stress is mediated by TGFβ1, which suppresses Nrf2-ARE-dependent expression of anti-oxidant defenses and creates a vicious cycle that feeds back to further increase TGFβ1 expression. These effects of alcohol can be mitigated by activation of Nrf2, suggesting a potential therapy in individuals at risk for lung injury due to alcohol abuse.

Alcohol‐Induced Interleukin‐17 Expression Causes Murine Lung Fibroblast‐to‐Myofibroblast Transdifferentiation via Thy‐1 Down‐Regulation

“…In contrast, the alcoholic lung expresses an excessive amount of TGF β 1 that renders it more susceptible to fibroproliferative disrepair (Sueblinvong et al., ). In that context, this study builds on and complements previous studies by our group and others that have identified that a combination of oxidative stress and activation of TGF β 1 promotes behavioral changes in lung fibroblasts (Neveu et al., ; Sueblinvong et al., ; Wynn and Ramalingam, ). TGF β 1 has been shown to regulate fibroblast‐to‐myofibroblast transdifferentiation, which is a critical step in normal wound healing responses and is dysregulated in fibrotic lung diseases (Wynn and Ramalingam, ).…”

“…Loss of Thy1 is an earlier step in the process of fibroblast‐to‐myofibroblast transdifferentiation (Hagood et al., ; Ramirez et al., ; Zhou et al., ). We previously showed that alcohol induces TGF β 1 expression and that TGF β 1 inhibits Thy‐1 expression in the lung fibroblasts (Neveu et al., ; Sueblinvong et al., ). We speculated that alcohol treatment can inhibit lung fibroblast Thy‐1 expression.…”

“…At 72 hours, cells were harvested for RNA, protein isolation, and flow cytometry. These time points were selected based on our preliminary experiments and published data in the literature (Neveu et al., ; Sueblinvong et al., ).…”

“…More recently, we showed that chronic alcohol ingestion primes the lung for fibroproliferative disrepair following bleomycin‐induced acute lung injury (ALI) in mice (Sueblinvong et al., ). We identified transforming growth factor β 1 (TGF β 1) as a proximal and critical mediator of the alcoholic lung phenotype (Bechara et al., ; Holguin et al., ; Sueblinvong et al., ). Specifically, the lung exhibits increased oxidative stress, TGF β 1 activation, disruption of the epithelial cell barrier, macrophage dysfunction, and fibroproliferative disrepair in response to injury (Bechara et al., ; Mehta and Guidot, ; Mehta et al., ; Sueblinvong et al., ).…”

Summary of the 2014 Alcohol and Immunology Research Interest Group (AIRIG) meeting

“…Many oxidative stress molecules in the lung are regulated by nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a transcription factor which specifically activates the anti-oxidant response element (ARE) in lung fibroblasts (Cho, Reddy, & Kleeberger, 2006). It has been shown that chronic alcohol intoxication leads to decreases in Nrf2 expression and nuclear binding in lung fibroblasts (Sueblinvong et al, 2014). Recently, thioredoxin-1 (Trx-1), a part of the Nrf2-ARE, was observed to be significantly decreased in the lung in vivo and in the nucleus and cytoplasm of lung fibroblasts in vitro following 60 mM alcohol exposure for 24–72 hours (unpublished data).…”