# FOXC1 Regulates Cytokine Signaling, Inflammatory Pathways, and Retinoid Metabolism to Maintain Limbal Epithelial Cell Homeostasis In Vitro

## TL;DR

This study shows that FOXC1 helps maintain eye surface cell health by regulating inflammation, cell differentiation, and retinoid metabolism.

## Contribution

The study reveals FOXC1's role in regulating inflammatory and retinoid pathways in limbal epithelial cells.

## Key findings

- FOXC1 silencing reduced epithelial markers KRT12 and KRT13 at mRNA and protein levels.
- FOXC1 knockdown altered inflammatory signaling, reducing IL-6 and IL-8 mRNA but increasing IL-1α.
- Genes involved in retinoid metabolism were significantly downregulated with reduced protein levels.

## Abstract

This study aimed to evaluate FOXC1-mediated regulatory mechanisms on gene and protein expression profiles in primary human limbal epithelial cells (pLECs) using siRNA-mediated FOXC1 knockdown under basal conditions and following lipopolysaccharide (LPS) and interleukin-1β (IL-1β)-induced inflammatory conditions. The gene expression related to inflammation, epithelial differentiation, cell proliferation and remodeling, and retinoic acid metabolism was analyzed using qPCR. Corresponding protein levels were assessed through Western blotting and ELISA. FOXC1 silencing significantly downregulated epithelial differentiation markers KRT12 and KRT13 at the mRNA and protein levels (p ≤ 0.045), whereas KRT3 and KRT19 were unaffected. Inflammatory signaling was markedly altered, with a reduced IL-6 and IL-8 mRNA expression (p ≤ 0.029), increased IL-1α expression (p ≤ 0.015), and condition-dependent changes in IL-6 and IL-8 protein secretion. CCL2 was increased at the mRNA level only (p = 0.007). VEGFA mRNA was consistently reduced (p ≤ 0.022) without corresponding protein changes, while TGF-β protein was increased under non-inflammatory and LPS conditions (p ≤ 0.011). Genes involved in retinoid metabolism, including CYP1B1, FABP5, CRABP2, RDH10, STRA6, and ALDH3A1, were significantly downregulated (p ≤ 0.037), with reduced CRABP2 and RDH10 protein levels (p ≤ 0.017) and a decreased FABP5/CRABP2 ratio under IL-1β stimulation (p = 0.006). FOXC1 knockdown affected proliferation-related genes, with decreased FOSL2 (p = 0.048) and increased MKi67 (p = 0.006). FOXC1 silencing disrupts epithelial differentiation, inflammatory signaling, retinoid metabolism, and selected proliferation-related pathways at the transcriptional level, with more selective effects on protein levels. Such changes may potentially predispose the ocular surface to lineage instability, fibrosis, and impaired regenerative capacity.