Adrenomedullin stimulates human trophoblast invasion through the upregulation of nitric oxide and urokinase plasminogen activator activity

Abstract

Extravillous cytotrophoblasts (EVCTs) invasion into the uterine endometrium is important to placentation and successful pregnancy. Dysregulation of the process is associated with a wide range of pregnancy complications, like intrauterine growth restriction, choriocarcinoma and preeclampsia. EVCTs produce urokinase plasminogen activator (uPA) that activates plasmin to degrade the extracellular matrix of the endometrium for invasion. Adrenomedullin is a 52-amino acid polypeptide belonging to the calcitonin gene-related peptide family which has diverse physiological functions including vasodilation, differentiation, hormone secretion and cell invasion. Nitric oxide (NO) signaling has been implicated to play a role in the biological functions of adrenomedullin in different cell types. In pregnant women, adrenomedullin is most abundantly expressed in the first-trimester placenta when the EVCTs exhibit the maximal invasive behavior, suggesting a possible role of the peptide in EVCT invasion during early pregnancy. The objective of this study is to investigate the effect of adrenomedullin on human EVCT invasion. JEG3, a choriocarcinoma cell line derived from first trimester human EVCT, was used as the study model. Adrenomedullin treatment significantly enhanced the invasiveness and uPA expression and activity of the JEG3 cells as demonstrated by transwell invasion assay, RT-PCR and uPA activity assay respectively. Immunostaining, western blotting and PCR demonstrated the presence of adrenomedullin receptor components in JEG3 cells. We further demonstrated that the stimulatory effect of adrenomedullin on trophoblast invasion is mediated by NO signaling pathway. Adrenomedullin treatment was shown to increase the NO production and nitric oxide synthase (NOS) expression in JEG3 cells. NOS inhibitors significantly suppressed the stimulatory effect of adrenomedullin on JEG3 invasion, uPA expression and activity. In contrast, NO donors were able to mimic the biological activities of adrenomedullin. The present results suggest that adrenomedullin enhances EVCT invasion by upregulating the uPA expression and activity through a NO-dependent manner.