Decreased FABP5 and DSG1 protein expression following PAX6 knockdown of differentiated human limbal epithelial cells

Highlights

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PAX6, DSG1 and RA signaling component FABP5 expression is significantly upregulated in presence of Ca²⁺ in LECs.

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DSG1 protein expression is only visible under Ca²⁺ growth conditions, proving enhanced differentiation in LECs through Ca²⁺.

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Ca²⁺ supplementation can be combined with PAX6 siRNA treatment to mimic aniridia cellular behavior observed in patients.

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DSG1 and FABP5 seem to be PAX6 dependent or driven by PAX6 in LECs.

Abstract

PAX6 haploinsufficiency related aniridia is characterized by disorder of limbal epithelial cells (LECs) and aniridia related keratopathy. In the limbal epithelial cells of aniridia patients, deregulated retinoic acid (RA) signaling components were identified. We aimed to visualize differentiation marker and RA signaling component expression in LECs, combining a differentiation triggering growth condition with a small interfering RNA (siRNA) based aniridia cell model (PAX6 knock down).

Primary LECs were isolated from corneoscleral rims of healthy donors and cultured in serum free low Ca²⁺ medium (KSFM) and in KSFM supplemented with 0.9 mmol/L Ca²⁺. In addition, LECs were treated with siRNA against PAX6. DSG1, PAX6, KRT12, KRT 3, ADH7, RDH10, ALDH1A1, ALDH3A1, STRA6, CYP1B1, RBP1, CRABP2, FABP5, PPARG, VEGFA and ELOVL7 expression was determined using qPCR and western blot.

DSG1, FABP5, ADH7, ALDH1A1, RBP1, CRABP2 and PAX6 mRNA and FABP5 protein expression increased (p ≤ 0.03), PPARG, CYP1B1 mRNA expression decreased (p ≤ 0.0003) and DSG1 protein expression was only visible after Ca²⁺ supplementation. After PAX6 knock down and Ca²⁺ supplementation, ADH7 and ALDH1A1 mRNA and DSG1 and FABP5 protein expression decreased (p ≤ 0.04), compared to Ca²⁺ supplementation alone.

Using our cell model, with Ca²⁺ supplementation and PAX6 knockdown with siRNA treatment against PAX6, we provide evidence that haploinsufficiency of the master regulatory gene PAX6 contributes to differentiation defect in the corneal epithelium through alterations of RA signalling. Upon PAX6 knockdown, DSG1 differentiation marker and FABP5 RA signaling component mRNA expression decreases. A similar effect becomes apparent at protein level though differentiation triggering Ca²⁺ supplementation in the siRNA-based aniridia cell model. Expression data from this cell model and from our siRNA aniridia cell model strongly indicate that FABP5 expression is PAX6 dependent. These new findings may lead to a better understanding of differentiation processes in LECs and are able to explain the insufficient cell function in AAK.