Abstract
Purpose :
Caveolin (CAV) 1 and 2 are integral membrane proteins that constitute the major components of small membrane pouches termed caveolae. Despite the recognized pleiotropic functions in diverse tissues, their roles in the ocular surface compartments are not fully understood. In the cornea, CAV1 expression was previously found to increase with aging and was negatively associated with wound healing. However, the exact molecular roles of CAV1 and CAV2 in corneal epithelium remain to be elucidated. In the current study, we investigated the contribution of CAV1 and CAV2 to the maintenance of the corneal epithelial phenotype.
Methods :
Human donor corneas were obtained from the Saving Sight eye bank, Kansas City, MO and CorneaGen, Seattle, WA. Immunostaining was performed to determine the distribution of CAV1 and CAV2 expression in the cornea. Limbal epithelial cell cultures were transfected with CAV1- and CAV2-targeting siRNAs designated as CAV1 KD #1, CAV1 KD #2, CAV2 KD #1 and CAV2 KD #2. Western blotting, colony-forming assays and RNA-seq were performed using CAV1 and CAV2 knockdown cells and their controls.
Results :
Immunohistochemical analyses revealed high CAV1 and CAV2 expression by the transit-amplifying cells (TACs) located in the basal epithelial layer of the limbus and to a lesser degree of the central cornea. siRNA-induced CAV1 and CAV2 KD in cultured limbal TACs resulted in significant reduction of their self-renewal potential as evidenced by attenuated
colony-forming efficiency (66.6 +/- 16.8% decrease by CAV1 KD #1 and 36.8 +/- 21.4% decrease by CAV1 KD #2; 72.4 +/- 13.2% decrease by CAV2 KD #1 and 34.8 +/-14.7% decrease by CAV2 KD #2; all compared to controls, Ps = 0.0002, 0.0118, <0.0001 and 0.0024, respectively). RNA-seq analyses showed that loss of CAV1 and CAV2 led to significant downregulation of genes involved in corneal epithelial formation (e.g., IVL, MUC21, and KRT78) and induction of genes involved in stromal development (e.g., FBN1 and SMPD1).
Conclusions :
Our study reveals novel functional roles of CAV1 and CAV2 expressed by TACs in the human cornea. Specifically, CAV1 and CAV2 are essential for TAC self-renewal and the maintenance of the corneal epithelial phenotype.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.