Purpose: Human corneal endothelial cells (HCECs) are considered to be nonreplicative in vivo; however, isolated HCECs can be cultured and grown successfully, indicating that they retain proliferative capacity. This capacity to replicate tends to decrease with donor age. Cyclin-dependent kinase inhibitors (CKIs) are important negative regulators of the cell cycle. Of those CKIs, p16INK4a, p21WAF1/Cip1, and p27Kip1 are expressed in corneal endothelium. To help reveal the mechanism of this age-related difference, the relative expression of those CKIs and the kinetics of hyperphosphorylation of the retinoblastoma protein, Rb, were analyzed in HCECs from various aged donors.
Methods: Fresh-frozen sections of corneas from an 18-year-old and a 74-year-old donor were immunostained to reveal the expression and localization of the three CKIs in corneal endothelium in situ. HCECs from eight donors of various ages were isolated and cultured until they reached passage 4. After the cells reached confluence, total protein was extracted, and the relative expression of p16(INK4a), p21WAF1/Cip1, and p27Kip1 was determined by Western blot analysis. A parallel analysis was performed with primary cultures of HCECs obtained from eight different donors. Subconfluent passage 2 HCECs from eight donors were serum starved and, at different times after growth factor stimulation, protein was extracted, and Western blot analysis was used to compare the overall expression of Rb protein and the kinetics of Rb hyperphosphorylation.
Results: Immunocytochemistry confirmed the expression and nuclear localization of p16(INK4a), p21WAF1/Cip1, and p27Kip1 in HCECs in situ. Western blot studies revealed an age-related increase in p16INK4a and p21WAF1/Cip1 protein expression in cultured HCECs. Expression of p27Kip1 tended to decrease with the donor's age in passage-4 cells; however, there was no significant difference in p27Kip1 expression level between young and older donors in primary cultured HCECs. No age-related difference in total Rb protein was observed in the Western blots; however, the rate of Rb hyperphosphorylation was significantly slower in HCECs from older donors.
Conclusions: p16(INK4a), p21WAF1/Cip1, p27Kip1, and Rb were all expressed in HCECs, regardless of donor age. Age-related differences in the relative expression of p16INK4a and p21WAF1/Cip1 and in the kinetics of Rb hyperphosphorylation led to the conclusion that, in addition to the normal inhibitory activity of p27Kip1, there is an age-dependent increase in negative regulation of the cell cycle by p16INK4a and p21WAF1/Cip1. This additional molecular mechanism may be responsible, at least in part, for the reduced proliferative response observed in HCECs from older donors.