Angiotensin II Inhibits Human Trophoblast Invasion through AT1 Receptor Activation

Abstract

Trophoblast implantation depends, in part, on the controlled production of plasmin from plasminogen, a process regulated by plasminogen activators and plasminogen activator inhibitors. We have determined that angiotensin II (Ang II) stimulates plasminogen activator inhibitor-1 (PAI-1) synthesis and secretion in human trophoblasts in a time-and concentration-dependent manner. Our results indicate that Ang II activates PAI-1 gene expression through the AT1 receptor and involves the calcium-dependent activation of calcineurin and the nuclear translocation of NFAT. Increased PAI-1 synthesis and secretion is associated with reduced trophoblast invasion as judged by an in vitro invasion assay. These studies are the first to link the renin-angiotensin system with the fibrinolytic system to regulate trophoblast invasion.The fibrinolytic system is best known for its role in the regulated digestion of fibrin clots. A key component of this system is plasminogen, an inactive zymogen, which is converted into the active protease, plasmin, by the action of a plasminogen activator termed tissue-type plasminogen activator (tPA).1 The ability of tPA to activate plasminogen is substantially enhanced by its association with the substrate, fibrin (1). Plasminogen can also be activated (i.e. converted to plasmin) by the action of another plasminogen activator, urokinase-type plasminogen activator (uPA) (2-6). In contrast to tPA, uPA is not associated with fibrin clots but instead is often associated with a specific cell surface receptor (uPAR) (7), which is thought to play a key role in cell migration and invasion (8). In this role, uPA is synthesized as an inactive single-chain proenzyme that can be stored or secreted. Secreted uPA is cleaved into the active two-chain molecule upon binding to uPAR on the surface of cells. Following activation, receptorbound uPA is capable of converting plasminogen into plasmin, the latter of which is able to degrade several key components of the extracellular matrix (ECM) (9). It is widely accepted that uPA initiates a cascade of proteolysis at the cell surface, which in turn leads to the degradation of the ECM, thereby promoting cellular migration. It is now clear that the uPA-regulated aspect of the fibrinolytic system plays a key role in mediating ECM degradation and cell invasion. Plasminogen activator activity is controlled by plasminogen activator inhibitors (PAIs) of which PAI-1 is the predominant physiological inhibitor (1, 10, 11). PAI-1 is an ECM glycoprotein that is capable of inhibiting both free and receptor-bound uPA through the formation of an irreversible covalent complex (12). In this way PAI-1 is believed to control cell migration and invasion in tumor growth and angiogenesis (13-16).Numerous studies suggest that PAI-1 production by human trophoblasts plays an important role in trophoblast invasion and placental development (17, 18). The human hemochorial placenta is a highly invasive structure in which a subpopulation of placental trophoblast cells invades the uterus, its underlyi...

Differential expression of angiotensin II type 1 and type 2 receptors at the maternal–fetal interface: potential roles in early placental development

“…In vitro studies using a transformed cytotrophoblast cell line (HTR-8/SVneo) demonstrated a potent inhibition of invasive potential, which is abrogated by selective inhibition of type 1 (AGTR1, AT1) receptor signalling (Xia et al 2002). The physiological significance of this result was supported by a recent report demonstrating Ang II stimulates proliferation, but inhibits differentiation, of cytotrophoblast cells from first trimester villous tip outgrowths in culture (Araki-Taguchi et al 2008).…”

Protein Kinase-mediated Regulation of Calcineurin through the Phosphorylation of Modulatory Calcineurin-interacting Protein 1

“…CHO-AT1 cells were cultured as described previously (33) and plated at 1.5 ϫ 10 6 in 10-cm wells in RPMI 1640 medium supplemented with 5% fetal bovine serum. The suspended cells were transiently transfected with various expression plasmids.…”

Mammalian Target of Rapamycin and Protein Kinase A Signaling Mediate the Cardiac Transcriptional Response to Glutamine

“…Cells were plated in six-well dishes (21 cm 2 per well) at a density of 5 ϫ 10 5 cells per well for RNA isolation, total cellular protein isolation, and transient transfection experiments (34). For some experiments cells were plated on coverslips for fluorescent microscopy.…”

“…The cell lysate was centrifuged at 14,000 rpm for 30 min, and the protein concentration in the supernatant was determined using BCA (Pierce) reagent (34).…”