Abstract
Purpose :
Previously we demonstrated that the Secreted Ly-6/uPAR Related Protein-1 (SLURP1), abundantly expressed in the cornea and secreted to the tear film, is an anti-proliferative and pro-differentiative protein. Here we have studied the mechanistic basis for its anti-proliferative effect on the Human Corneal Limbal Epithelial (HCLE) cells.
Methods :
HCLE cells were stably transfected with pCMV-SLURP1 and two different clones overexpressing SLURP1 were selected for the present studies. The effect of SLURP1 overexpression on HCLE cell cycle progression was quantified by flow cytometry. The expression, quantity and subcellular localization of cyclins, cyclin-dependent kinases (CDKs) and cyclin inhibitors were determined by QPCR, immunoblots, and immunofluorescent staining, respectively.
Results :
Flow cytometry revealed that the percent of cells in G0/G1 phase is significantly higher in SLURP1-expressing HCLE clones (73.8% and 81.6%) compared with the WT HCLE (63%). QPCR revealed an upregulation of p21 (1.1- and 2-fold), and downregulation of Cyclin-B1 (0.66- and 0.68-fold), and Cyclin D2 (0.48- and 0.64-fold) in HCLE-SLURP1 clones relative to the WT control. Immunoblots revealed elevated expression of cell cycle regulators p15 (1.5- and 1.6-fold), lower CDK-4 (0.9- and 0.57-fold) and cyclin-E (0.93- and 0.77-fold) in HCLE-SLURP1 relative to WT HCLE cells. HCLE-SLURP1 clone with a higher level of SLURP1 expression displayed decreased CDK6 (69% of the WT) and Cyclin-D1/D2 (60% of the WT) expression. Immunofluorescent staining revealed an increased cytoplasmic and decreased nuclear localization of CDK-2, -4 and -6 in HCLE-SLURP1 cells compared with the WT.
Conclusions :
SLURP1 serves as an anti-proliferative protein by promoting the expression of cell cycle inhibitors p15 and p21 and suppressing the expression of cyclins-D1/D2 and -E, and CDK-4 and -6. Further cell cycle regulation is facilitated by altered sub-cellular localization of CDKs in the cytoplasm of SLURP1-overexpressing cells.
This is a 2021 ARVO Annual Meeting abstract.