P27 Expression in Endothelin-1 Stimulated Mitogenesis

Bovine corneal endothelial cells (BCECs) have been shown in our lab to synthesize and secrete endothelin-1 (ET-1). The endothelin-1 binds ETA receptors, this elevates intracellular calcium, stimulate DNA replication and cell division. This investigation explores ET-l's role as a mitogenic factor in corneal endothelial cells and addresses whether mitogenic activity is mediated through the cyclin dependent kinase inhibitor p27. BCEC were isolated from bovine eyes and grown in DMEM-10% serum in a 5% C02, 37° C incubator. Confluent cultures of BCEC were incubated for 24 hours with lOOnM Endothelin-1, luM BQ123 and BQ123 +100nM Endothelin-1 and some left untreated. Total cellular protein was isolated and the concentration quantified by the Peterson modification of the Lowry protein assay. Protein samples and prestained molecular weight standards were separated by 4-15% gradient SDS-PAGE and transferred overnight to PVDF Hyperbond membrane by western blot. The relative abundance of p27 was detected using a polyclonal antibody for p27 (1:100) and HRP conjugated secondary antibody (1:500) and alkaline phosphatase conjugated secondary antibody (goat-anti rabbit). The membrane was then washed thoroughly with PBST and treated with enhanced chemiluminescence (ECL) or enhanced chemifluorescence (ECF). The data show a marked decrease in cellular p27 in cells treated with 1 OOnM endothelin-1. ET-1 appears to have mitogenic properties in BCEC by downregulating the expression of p27. Stimulation of cell proliferation may be mediated by suppressing the expression of p27, a cyclin dependent kinase inhibitor.

Name thesis:degree_name

Master of Science in Biology

Discipline thesis:degree_discipline

Department of Biology

Year

2007

Author dc:creator

• Kiriu, Joseph

• Medical Sciences

Repository record dc:identifier

https://digitalcommons.wku.edu/theses/428

OAI identifier oai:identifier

oai:digitalcommons.wku.edu:theses-1431