HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Induction of plasminogen activator inhibitor I gene expression by intracellular calcium via hypoxia-inducible factor-1

The plasminogen activator inhibitor-1 (PAI-1) expression can be enhanced by hypoxia and other stimuli leading to the mobilization of intracellular calcium. Thus, it was the aim of the present study to investigate the role of calcium in the hypoxia-dependent PAI-1 expression. It was shown that the Ca2 -ionophore A23187 and the cell permeable Ca2 chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N ,N -tetraacetic acidacetoxymethyl ester) induced PAI-1 mRNA and protein expression under normoxia and hypoxia in HepG2 cells. Transfection experiments with wild-type and hypoxia response element (HRE)– mutated PAI promoter constructs revealed that the HRE binding hypoxiainducible factor-1 (HIF-1) mediated the response to A23187 and BAPTA-AM. Although A23187 induced a striking and stable induction of HIF-1 , BAPTA-AM only mediated a fast and transient increase. By using actinomycin D and cycloheximide we showed that A23187 induced HIF-1 mRNA expression, whereas BAPTA-AM acted after transcription. Although A23187 activated extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK), as well as protein kinase B, it appeared that the enhancement of HIF-1 by A23187 was only mediated via the ERK pathway. By contrast, BAPTA-AM exerted its effects via inhibition of HIFprolyl hydroxylase activity and von Hippel-Lindau tumor repressor protein (VHL) interaction. Thus, calcium appeared to have a critical role in the regulation of the HIF system and subsequent activation of the PAI-1 gene expression. (Blood. 2004;104:3993-4001)