Urokinase-type plasminogen activator (uPA) system and invasion of early human extravillous trophoblast is regulated by hypoxia induced factor (HIF) pathway

Abstract

Objective:Urokinase-type plasminogen activator (uPA) system is a critical protease pathway in human pregnancy. We have been reported the importance of uPA system on decidua and extravillous trophoblast (EVT) in early pregnancy, but the controlling molecule of the system remains unclear. In this study, we investigated the connection of hypoxia-induced factor (HIF) and uPA on primary EVTs to define the intracellular and extracellular protease pathway on early pregnancy. Methods: Human placental villi of 5–10 weeks gestation (obtainedafter informedconsent onartificial abortion) were minced, digested and cytotrophoblast fraction was separated with Percoll-based method. Cells were cultured on Matrigel® for 24h under 20%, 5% and 0.1% (three times of one hour hypoxic stimulation) oxygen condition with siRNA designed for knock-down of HIF. The invasive capacity of EVT (Matrigel-coated invasion assay), localization of HIF (immunofluorescence staining), and productions of HIF, uPA and receptor uPAR protein on EVT (Western Blot and ELISA) were studied. Results: HIF protein was increased under 5% oxygen condition than 20%. Invasive capacity was significantly decreased in the presence of siRNA for HIF. Productions of uPA and uPAR were also decreased in the presence of the siRNA. Conclusions: Hypoxic condition increased HIF expression and invasiveness of human primary EVT like cells, but upon addition of siRNA and knock-down of HIF in the cells, invasive capacity of the cells and expression of uPAR were reduced. It was thus suggested that uPA system in human trophoblast was induced by HIF and enhanced invasive capacity under hypoxic conditions.