B26-22 Cadmium Disrupts Circadian Clock and Epithelial Homeostasis in Alveolar Type 2 Cells via Rev-erbα-dependent Mechanisms

Rationale Cadmium (Cd) is a major environmental toxicant found in cigarette smoke and airborne particulates. Due to its slow elimination, Cd accumulates in lung tissue and contributes to chronic lung diseases, such as chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis. Cd exposure induces inflammation, profibrotic signaling, and extracellular matrix (ECM) remodeling. Alveolar type 2 (AT2) epithelial cells are progenitors essential for alveolar repair and the maintenance of epithelial integrity. Environmental toxicants impair AT2 cell proliferation, differentiation, and survival, thereby compromising lung regeneration. Disruption of the circadian rhythm has been implicated in chronic lung diseases. The circadian transcription factor REV-ERBα modulates inflammation, metabolism, and cellular homeostasis; however, its function in Cd-induced AT2 cell injury remains ambiguous. We hypothesized that Cd exposure perturbs circadian clock components, particularly REV-ERBα, resulting in dysregulated epithelial and mesenchymal marker expression, activation of profibrotic pathways, and increased apoptosis, and that pharmacologic activation of REV-ERBα mitigates these effects. Methods Mouse lung epithelial MLE-15 (AT2) cells were cultured in RPMI-1640 supplemented with insulin, transferrin, sodium selenite, hydrocortisone, β-estradiol, HEPES, L-glutamine, penicillin, streptomycin, and 5% fetal bovine serum (FBS). Cells were pretreated with the REV-ERBα agonists SR29065 (10 µM) or GSK4112 (20 µM) for 1 hour, followed by co-treatment with CdCl2 (10 µM) for 24 hours in 2% FBS medium. Whole-cell lysates were analyzed for circadian clock, epithelial, mesenchymal, profibrotic, and apoptotic markers by immunoblotting. Ongoing studies will assess these targets at the transcript level and in a preclinical mouse model of repeated Cd exposure-induced lung injury. Results Cd exposure significantly altered the expression of core clock proteins (BMAL1, REV-ERBα) and increased epithelial (E-CAD), mesenchymal (VIM), and profibrotic markers (FN1, αSMA), along with the apoptotic marker cleaved-PARP, in MLE-15 cells. Co-treatment with REV-ERBα agonists (SR29065 or GSK4112) effectively restored several of the Cd-induced alterations in these key markers, supporting a protective role of REV-ERBα signaling in maintaining epithelial homeostasis. Conclusions Cd exposure disrupts circadian clock signaling and promotes epithelial-mesenchymal and profibrotic responses in AT2 cells. Pharmacologic activation of REV-ERBα mitigates these effects, highlighting a critical role for the circadian clock in maintaining alveolar epithelial integrity during toxicant-induced injury. Ongoing studies using in vivo models aim to define the cell type-specific roles of clock molecules in Cd-induced lung injury and identify potential circadian-based therapeutic strategies for chronic lung disease.