Overexpression of Protein Kinase C α and β1 Has Distinct Effects on Bovine Aortic Endothelial Cell Growth

Abstract

Protein kinase C (PKC) plays an important role in the mitogenic response of endothelial cells to growth factors. PKC α and β1 are the predominant classical isoforms expressed by bovine aortic endothelial cells (BAECs). The present studies were undertaken to elucidate the effect of PKC α and β1 overexpression in BAEC growth. A series of BAEC lines that stably overexpress the full-length PKC α and β1 cDNA were generated by using a replication-defective recombinant retrovirus. The level of PKC α and β1 cDNA expression was determined by assaying for PKC α and β1 mRNA transcripts. PKC α and β1 protein levels were analysed by Western blotting. Functional analysis of these overexpressing lines was performed by measuring PKC activity and phorbol ester-binding assays. PKC α and β1 overexpression had distinctive effects on BAEC growth and cell-cycle progression. Relative to untransfected BAECs and BAECs transfected with the viral vector alone, BAECs that overproduced PKC α exhibited reduced proliferation in vitro and increased accumulation of cells in the G 2/M phase of the cell cycle. Growth inhibition was greater in cell lines overexpressing higher levels of PKC α. Conversely, a 5-fold greater increase in PKC β1 activity promoted BAEC growth and shortened BAEC doubling time, whereas cells with a 2- to 4-fold increase in enzyme activity had growth profiles similar to those of both control groups. These results suggest that PKC α and β1 overexpression has reciprocal effects on BAEC growth.