# Impact of Cellular Senescence on LCN2 Expression in Salivary Gland Epithelial Cells and Oral Keratinocytes

## TL;DR

This study explores how aging affects LCN2 levels in oral cells, finding that increased LCN2 in older mice is likely due to IL-1β from macrophages, not cell senescence.

## Contribution

The study reveals that IL-1β from M1 macrophages, not senescence or SASP factors, drives age-related LCN2 upregulation in oral epithelial cells.

## Key findings

- LCN2 protein levels in salivary glands and oral epithelial cells increase with age in mice.
- Replicative and DNA damage-induced senescence do not increase LCN2 expression in oral epithelial cells.
- IL-1β from M1 macrophages, not senescent cells, induces LCN2 expression in aged epithelial cells.

## Abstract

Senescent cells are characterized by the up‐regulation of senescence markers and exhibit key features, such as irreversible growth arrest and the senescence‐associated secretory phenotype (SASP), which is mainly regulated by transcription factors of nuclear factor κB (NF‐κB). Lipocalin‐2 (LCN2), a glycoprotein secreted by immune cells, astrocytes, and epithelial cells, is present in saliva and gingival crevicular fluid and possesses antimicrobial and immunomodulatory properties. Although LCN2 expression is mainly regulated by NF‐κB, the effects of aging and cellular senescence on salivary LCN2 protein concentrations remain unknown. We herein demonstrated that LCN2 protein levels in the serous acinar cells of salivary glands, oral epithelial cells, and saliva were higher in aged mice than in young mice. However, in primary oral keratinocytes and salivary gland epithelial cells, replicative senescence and DNA damage‐induced senescence did not increase LCN2 expression, with similar results being obtained for the SASP factors tumor necrosis factor‐alpha and interleukin‐1β (IL‐1β). Although the cyclic GMP‐AMP synthase‐mediated induction of LCN2 expression has been reported in astrocytes, its expression decreased with cellular senescence, and its ligand did not induce LCN2 expression in these oral‐related epithelial cells. Conversely, an IL‐1β treatment significantly induced LCN2 expression and secretion, even in senescent epithelial cells. The source of IL‐1β was not senescent fibroblasts, but M1 macrophages that accumulate with inflammaging. Collectively, these results suggest that aging up‐regulates LCN2 expression in oral‐related epithelial cells mainly via IL‐1β secreted from M1 macrophages, rather than through the induction of their senescence.

The age‐related increase in salivary LCN2 levels is unlikely to be directly attributable to cellular senescence of oral mucosal or salivary gland epithelial cells, nor to SASP factors derived from surrounding senescent fibroblasts. Instead, it is more plausibly driven by IL‐1β secreted from M1 macrophages that accumulate with inflammaging.