Overexpression of protein kinase C alpha and beta1 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 alpha and beta1 are the predominant classical isoforms expressed by bovine aortic endothelial cells (BAECs). The present studies were undertaken to elucidate the effect of PKC alpha and beta1 overexpression in BAEC growth. A series of BAEC lines that stably overexpress the full-length PKC alpha and beta1 cDNA were generated by using a replication-defective recombinant retrovirus. The level of PKC alpha and beta1 cDNA expression was determined by assaying for PKC alpha and beta1 mRNA transcripts. PKC alpha and beta1 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 alpha and beta1 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 alpha exhibited reduced proliferation in vitro and increased accumulation of cells in the G2/M phase of the cell cycle. Growth inhibition was greater in cell lines overexpressing higher levels of PKC alpha. Conversely, a 5-fold greater increase in PKC beta1 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 alpha and beta1 overexpression has reciprocal effects on BAEC growth.