Adrenomedullin as a modulator of human trophoblast invasion

During placental development, human trophoblasts differentiate along two pathways leading to the formation of the extravillous cytotrophoblasts (EVCT) and the villous cytotrophoblasts. EVCTis responsible for trophoblast invasion, which is an important process for successfulplacentation in early pregnancy.Dysregulation of the process is associated with a wide range of pregnancy complications, including intrauterine growth restriction, preeclampsia and choriocarcinoma. EVCTsproducematrix metalloproteinase and urokinase plasminogen activator(uPA)that degrade the extracellular matrix of the endometrium for the invasion process. Adrenomedullin is a 52-amino acid polypeptide belonging to the calcitonin/calcitoningene-related peptide/amylin peptide family. The expression of adrenomedullin in trophoblasts is most abundant in the first-trimester human placentas, consistent with the involvement of the molecule in early placentation. Most of the studies on adrenomedullin concentrate on its vasodilatory activity. Studies on the action of adrenomedullin in human trophoblast functions are limited. Therefore the role of adrenomedullin in early placentation is not known. The hypothesis of this project is that adrenomedullin regulates the invasion of human EVCT. Two human EVCT cell lines, JEG-3 and TEV-1 were used as study models in this project. The functions of adrenomedullin aremediated through a receptor system composedof two transmembrane components, the calcitonin receptor-likereceptor and receptor activity-modifying protein(RAMP). Adrenomedullin binds to calcitoninreceptor-likereceptorwhen coupled with RAMP2 or RAMP3. Immunostaining, western blotting and RT-PCR demonstrated the presence of these components on EVCT cell lines. Adrenomedullin treatment significantly enhanced invasiveness, migration, but not proliferation in EVCT as demonstrated by transwell invasion assay, transwell migration assay and cell proliferation assayrespectively. The adrenomedullin antagonist, adrenomedullin22-52, blocked the effects of adrenomedullin on EVCT cell lines, confirming adrenomedullin exerted its biological effects through its classical receptors. RT-PCR results further demonstrated that while adrenomedullin treatment had no effect on matrix metalloproteinase-2 expression, it up-regulated uPA expression and activity. Silencingof uPA by siRNA transfection abolished the stimulatory effect of adrenomedullin, suggesting uPA is the key mediator for adrenomedullin-induced invasion. Adrenomedullin increased nitric oxide synthase expression and nitric oxide (NO) production in JEG-3 cells. The involvement of NO in adrenomedullin-induced EVCT invasion and biosynthesis of uPAwas confirmed bypharmacologicalstudy using nitric oxide synthase inhibitorsor NO donors.Protein kinase G was shown to be one of the downstream regulator of NO that mediated the adrenomedullin-induced EVCT invasion. By using a biotin-switch based method to purify theS-nitrosylated proteins, adrenomedullin was further demonstrate to up-regulate the NO-dependent S-nitrosylation in EVCTs.Interestingly, S-nitrosylation of uPA in vitro induced a higher proteinaseactivitywhich may be responsiblefor the stimulatory action of adrenomedullin. In conclusion, thisstudyhas provided evidence that adrenomedullin modulates EVCT invasion by regulating the uPA expression andactivity through NO signaling pathway. Abnormal trophoblast invasion can lead to a range of major pregnancy complications. The outcome of this project enhanced our understanding of the mechanisms that regulate trophoblast invasion. These data could constitute the basis for new therapeutic strategies of the diseases in the future.published_or_final_versionObstetrics and GynaecologyDoctoralDoctor of Philosoph