C71-32 Zinc Deficiency Mediates Type 2 Alveolar Epithelial Cell Regeneration via Activator Protein 1 Signaling

Rationale

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, age-related lung disease characterized by impaired alveolar regeneration and irreversible extracellular matrix remodeling. Type 2 alveolar epithelial cells (AEC2s) serve as progenitor cells that maintain epithelial homeostasis and mediate repair following lung injury. However, persistent aging and fibrotic stress disrupt AEC2 proliferation and differentiation into alveolar type 1 cells (AEC1s), leading to accumulation of damage-associated transient progenitors accumulation, thereby contributing to progressive inflammation and fibrosis. The zinc transporter SLC39A8 (ZIP8) is highly expressed in the alveolar epithelium and plays a critical role in maintaining intracellular zinc homeostasis. The expression level of ZIP8 in AEC2s declines with aging. Zinc signaling regulates stress-responsive transcription factors, including activator protein-1 (AP-1), which integrates inflammatory, oxidative, and profibrotic signals to regulate epithelial regeneration. Previous transcriptomic analyses have demonstrated increased expression of AP-1 component genes, such as JUN and FOS, in AEC2s from aged lungs. However, how AP-1 activity involving in ZIP8-dependent zinc signaling modulating AEC2 regeneration remains unclear. In this study, we aim to investigate the role of AP-1 signaling in AEC2s regeneration in ZIP8 knockout old mice.

Methods

Single-cell RNA sequencing was performed to assess AP-1-related gene expression in AEC2s from human and ZIP8 knockout aged and control aged mice. Primary AEC2s were isolated from ZIP8 knockout and control aged mice and cultured ex vivo with or without AP-1 inhibitor (10 or 15 μM) for 14 days. Colony-forming efficiency (CFE) and organoid size were quantified.

Results

Transcriptomic analysis revealed elevated expression of AP-1 component genes and inflammatory genes in AEC2s from IPF patients. In human samples, ZIP8low AEC2s exhibited a higher proportion of transitional cells compared with ZIP8high AEC2s, suggesting that ZIP8 deficiency promotes transitional cell accumulation. Consistently, ZIP8 knockout AEC2s from mouse lung showed increased AP-1 and inflammatory gene expression, along with an expansion of Krt8⁺ transitional cells. Ex vivo three-dimensional organoid cultures showed significantly decreased CFE in ZIP8 knockout aged mice compared with controls; notably, this reduction was not observed following AP-1 inhibitor treatment. Moreover, treatment with 10 μM AP-1 inhibitor significantly increased organoid size in ZIP8 knockout aged mice.

Conclusions

These findings indicate that ZIP8 deficiency leads to the accumulation of transitional cells and activation of AP-1 signaling. AP-1 inhibition partially restores AEC2 proliferative capacity, implicating AP-1 as a key downstream mediator of ZIP8 regulation of alveolar epithelial regeneration in IPF.

This abstract is funded by: National Institutes of Health