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K. M. Crawford, L. R. Bollu; The Effect of Endothelin-1 on the Expression of the Cyclin Dependent Kinase Inhibitors p21 and p27 in Bovine Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1803.
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Mammalian corneal endothelial cells are considered to be non-proliferative due to the arrest of the cell cycle at the G1 phase. The purpose of this study is to determine whether the down regulation of cyclin dependant kinase inhibitors (p21cip1 and p27kip1) levels by Endothelin-1, will overcome the G1 phase arrest and promote cell cycle progression and wound healing in cultured bovine corneal endothelial cells (BCEC). The relative expression of cyclin dependant kinase inhibitors (CDKIs) in response to Endothelin-1, a purported mitogen, was determined by western blot.
BCECs were isolated from bovine corneas and cultured in DMEM with 10% serum and antibiotics for two passages. The confluent second passage cells were serum starved for 24 hours and then treated with 1nM, 20nM, 50nM or 100nM Endothelin-1 in serum free medium for 24 hours. The control cells were left untreated in serum free medium. Total cellular protein was isolated using RIPA buffer and quantified according to Peterson modified Lowry method. An equal amount of each protein sample was used to determine the expression of p21cip1 and p27kip1 proteins relative to actin by western blotting analysis.
We observed that cell division in sub-confluent cultures increases with Endothelin-1 treatment. This is supported by a dose dependent increase in tritiated thymidine incorporation in response to Endothelin-1. Densitometry of immunoblots revealed a 123% increase in the expression of p27 in confluent cultures when compared to sub-confluent, dividing cells. P21 was undetectable in sub-confluent, actively dividing cultures. Endothelin-1 treatment for 24 hr led to a dose-dependent decrease in p27 expression in confluent cultures as well as in sub-confluent cultures. The decline in p27 in response to ET-1 was greatest at 48 hrs.
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