p62-Keap1-Nrf2 positive feedback loop protects against inorganic arsenic-induced senescence and EMT in alveolar type II cells.

Abstract

Inorganic arsenic (As) exposure is known to induce pulmonary fibrosis, yet the underlying mechanisms remain incompletely understood. This study investigated the crosstalk between sequestosome 1 (SQSTM1/p62) and nuclear factor erythroid 2-related factor 2 (Nrf2) in regulating cellular senescence and epithelial-mesenchymal transition (EMT) in As-exposed alveolar type II (AT2) cells. RNA sequencing analysis initially suggested that As exposure promotes EMT and senescence in MLE-12 cells. Subsequent experiments demonstrated that As exposure progressively decreased the epithelial marker E-cadherin while increasing the mesenchymal marker N-cadherin in MLE-12 cells, a mouse AT2 cell line, and murine lung tissues. In addition, elevated expression of transforming growth factor (TGF)-β and α-smooth muscle actin (SMA) was observed. Meanwhile, As exposure increased the number of SA-β-galactosidase (SA-β-gal)-positive MLE-12 cells and enhanced the transcriptional activity of senescence-associated secretory phenotype (SASP)-related genes, including Cxcl1, Cxcl11, Cxcl16, Ccl20, Mcp-1, Tgf-β, and Gm-csf. Moreover, the expression of senescence-associated markers p16 and p21 was upregulated in both As-exposed MLE-12 cells and murine lungs. Notably, Nrf2 knockdown exacerbated cellular senescence, as evidenced by increased SA-β-gal-positive cells, enhanced SASP gene expression, and elevated p16 and p21 levels, along with augmented EMT marker expression. To further verify the protective role of Nrf2, As-exposed MLE-12 cells were treated with the Nrf2 activator dimethyl fumarate (DMF). DMF activates Nrf2 by modifying Keap1, thereby disrupting Keap1-mediated Nrf2 degradation and promoting Nrf2 stabilization and nuclear translocation. DMF markedly reversed As-induced senescence, as shown by decreased SASP-related gene, p16, and p21 expression, while attenuating EMT, evidenced by restored E-cadherin and reduced N-cadherin and α-SMA. Moreover, pharmacological disruption of the p62-Keap1 interaction using K67 restored Keap1-mediated suppression of Nrf2 and further aggravated senescence and EMT phenotypes. Collectively, these findings demonstrate that As exposure induces cellular senescence and EMT in MLE-12 cells and murine lungs. The p62-Keap1-Nrf2 positive feedback loop functions as an essential cytoprotective axis that attenuates As-evoked senescence and EMT in AT2 cells, underscoring its potential as a therapeutic target for arsenic-associated pulmonary fibrosis.