Cloning and characterization of mink plasminogen activator inhibitor type 1 (PAI-1) cDNA and the regulation of mink PAI-1 expression at mRNA level
Mink plasminogen activator inhibitor type 1 (PAI-1) is a glycoprotein with molecular weight about 50 kD and is highly regulated in cultured cells. In mink lung CCL64 epithelial cells, epidermal growth factor (EGF), transforming growth factor-[beta] (TGF-[beta]), and 12-O-tetradecanoyl phorbol-13-acetate (TPA) increase the synthesis of PAI-1. Epidermal growth factor and TGF-[beta] synergistically stimulate the synthesis of PAI-1. The synergistic effect and factors which govern the PAI-1 expression in mink lung CCL64 cells were studied by using a mink PAI-1 cDNA as a probe to detect PAI-1 mRNA;The mink PAI-1 cDNA was cloned from a cDNA library constructed from mink lung CCL64 cells. The deduced amino acid sequence of mink PAI-1 contains 400 amino acids. Identities between the mink PAI-1 protein sequence and the sequence of the human, bovine, mouse, and rat PAI-1 proteins are 86%, 87%, 79% and 77%, respectively;The synergistic effect of EGF and TGF-[beta] on PAI-1 induction is due to a combination of transcriptional activation of PAI-1 gene by TGF-[beta] and stabilization of mRNA by EGF. The production of PAI-1 protein increased in parallel with the accumulation of PAI-1 mRNA. However, the synergism was not observed on the transcriptional activity of the PAI-1 gene. This suggested that EGF inhibits the turnover of PAI-1 mRNA. More direct evidence for the effect of EGF on PAI-1 mRNA turnover is that the half-life of PAI-1 mRNA in cells treated with TGF-[beta] is 30 min whereas the half-life of PAI-1 mRNA in cells treated with EGF plus TGF-[beta] is 46 min;In this study, turnover rate of PAI-1 mRNA was found to be an important factor regulating the time course of PAI-1 expression in mink lung CCL64 cells. The time course of accumulation of PAI-1 mRNA showed a parallel profile with the change in the production of PAI-1 protein. The fast decay rate of PAI-1 mRNA is responsible for the rapid increase and decrease in PAI-1 mRNA accumulation which is seen as a brief spurt of production of PAI-1 protein after stimulation by TGF-[beta] or TPA. It is concluded that the regulation of mRNA turnover is important for the regulation PAI-1 expression.