A disintegrin and metalloproteinase 12 (ADAM12) localizes to invasive trophoblast, promotes cell invasion and directs column outgrowth in early placental development

Abstract

During pregnancy, stromal- and vascular-remodeling trophoblasts serve critical roles in directing placental development acquiring pro-invasive characteristics. The A Disintegrin and Metalloproteinase (ADAM) family of multifunctional proteins direct cellular processes across multiple organ systems via their intrinsic catalytic, cell adhesive and intracellular signaling properties. ADAM12, existing as two distinct splice variants (ADAM12L and ADAM12S), is highly expressed in the human placenta and promotes cell migration and invasion in several tumor cell lines; however, its role in trophoblast biology is unknown. In this study, ADAM12 was localized to anchoring trophoblast columns in first trimester placentas and to highly invasive extracellular matrix-degrading trophoblasts in placental villous explants. The importance of ADAM12 in directing trophoblast invasion was tested using loss-of and gain-of-function strategies, where siRNA-directed knockdown of ADAM12 inhibited trophoblast cell invasion while over-expression promoted migration and invasion in two trophoblastic cell models. In placental villous explant cultures, siRNA-directed loss of ADAM12 significantly dampened trophoblast column outgrowth. Additionally, we provide functional evidence for the ADAM12S variant in promoting trophoblast invasion and column outgrowth through a mechanism requiring its catalytic activity. This is the first study to assign a function for ADAM12 in trophoblast biology, where ADAM12 may play a central role regulating the behavior of invasive trophoblast subsets in early pregnancy. This study also underlines the importance of ADAM12L and ADAM12S in directing cell motility in normal developmental processes outside of cancer, specifically highlighting a potentially important function of ADAM12S in directing early placental development.

ADAM12-directed ectodomain shedding of E-cadherin potentiates trophoblast fusion

“…22 Further, we 20 and others 21 have localized ADAM12 to EVTs; together these data show that ADAM12 is expressed by multiple trophoblast subtypes and imply that ADAM12 has diverse functions in placentation. In this study, FACS purification followed by gene expression analysis of ADAM12L and ADAM12S in vCTs and column EVTs clearly shows that ADAM12S is restricted to EVTs, while ADAM12L levels are less defined between these cell types.…”

“…Previous work localized ADAM12 to EVT subsets within distal anchoring columns 20,21 and to the synCT. 22 To confirm these findings, we re-examined ADAM12 localization in early (5-7 weeks gestation) and late (10-12 weeks gestation) first trimester placentae by immunofluorescence microscopy; the trophoblast markers hepatocyte growth factor activator inhibitor-1 (HAI-1), HLA-G and KRT7, as well as the proliferation marker Ki67 helped identify distinct trophoblast populations.…”

“…Discrepancies exist related to ADAM12 expression within the placenta, where previous studies have localized ADAM12 to the apical membrane 20,22,39 and cytoplasm 22 of the synCT, as well as stochastically within vCTs. 22 Further, we 20 and others 21 have localized ADAM12 to EVTs; together these data show that ADAM12 is expressed by multiple trophoblast subtypes and imply that ADAM12 has diverse functions in placentation.…”

“…Immunofluorescence was performed as described elsewhere. 20 Briefly, cells or placental tissues underwent antigen retrieval by heating slides in a microwave for 5 × 2 min intervals in a citrate buffer (pH 6.0). Sections were incubated with sodium borohydride for 5 min, RT, followed by Triton X-100 permeabilization for 5 min, RT.…”

“…19 Recently, our work has focused on the importance of ADAM12 in controlling trophoblast invasion and placental column outgrowth. 20 ADAM12, expressed as two distinct catalytically active splice variants, a full-length membranebound isoform (ADAM12L) and a truncated secreted variant (ADAM12S), is highly expressed in the placenta and localizes to invasive extravillous cytotrophoblast (EVT) subsets 20,21 and to the synCT layer. 22 Though ADAM12's localization to the syncytium suggests that it may play a role in directing trophoblast differentiation into multinucleated structures, the importance of ADAM12 in synCT formation has not yet been tested.…”