Retinol fails to compensate for disrupted retinoic acid signaling in PAX6-deficient limbal epithelial cells, in vitro

PURPOSE: Congenital aniridia is a rare ocular disease marked by limbal stem cell deficiency and aniridia-associated keratopathy (AAK). This study investigated gene and protein expression of key regulators in the retinoic acid (RA) signaling pathway using a Paired box gene 6 (PAX6) knockdown in vitro model of limbal epithelial cells (LECs) following retinol treatment. MATERIALS AND METHODS: Primary human LECs from healthy donors were cultured in keratinocyte medium, treated with small interfering RNA targeting PAX6 (siPAX6) for 24 h, then with 0, 1, or 5 μM retinol for 48 h. PAX6, retinol dehydrogenase 10 (RDH10), Alcohol dehydrogenase 7 (ADH7), Aldehyde dehydrogenase 1 family member A1 (ALDH1A1), Cytochrome P450 family 26 subfamily A member 1 (CYP26A1), Cellular retinoic acid-binding protein 2 (CRABP2) , fatty acid-binding protein 5 ( FABP5 ), Retinol binding protein 1 (RBP1), Peroxisome proliferator-activated receptor gamma (PPARG), Retinoid X receptor alpha (RXRA), Retinoid X receptor beta (RXRB), Retinoic acid receptor alpha (RARA), Retinoic acid receptor beta (RARB) , and vascular endothelial growth factor A ( VEGFA ) messenger ribonucleic acid (mRNA) levels were analyzed using real-time–quantitative polymerase chain reaction. PAX6, RDH10, ADH7, ALDH1A1, CYP26A1, Cellular retinoic acid-binding protein 2 (CRABP2), FABP5, Peroxisome proliferator-activated receptor gamma 2 (PPARG2), and VEGFA protein levels were assessed using enzyme-linked immunosorbent assay or Western blotting and cell proliferation by the bromodeoxyuridine (BrdU) assay. RESULTS: PAX6 knockdown significantly reduced PAX6, ADH7, ALDH1A1, CRABP2 , and FABP5 mRNA levels and PAX6, ALDH1A1, and PPARG2 protein levels ( P < 0.05). RDH10 and VEGFA mRNA were significantly upregulated ( P < 0.05). CYP26A1 mRNA and the FABP5-to-CRABP2 protein ratio were significantly increased after 5 μM retinol in controls only ( P = 0.021, P = 0.038). BrdU assay revealed a significant reduction in cell proliferation in the PAX6 knockdown group ( P < 0.001). CONCLUSIONS: Consistent with previous reports, PAX6 haploinsufficiency is associated with dysregulation of RA pathway gene and protein expression and reduced LEC proliferation, recapitulating key features of AAK. Retinol does not appear to exert a compensatory effect on PAX6-associated alterations in RA metabolism; however, its potential impact on LEC differentiation, stemness, and junctional markers warrants further investigation.