Faulty oxygen sensing disrupts angiomotin function in trophoblast cell migration and predisposes to preeclampsia
Abby Farrell, Sruthi Alahari, Leonardo Ermini, Andrea Tagliaferro, Michael Litvack, Martin Post, Isabella Caniggia
Abby Farrell, Sruthi Alahari, Leonardo Ermini, Andrea Tagliaferro, Michael Litvack, Martin Post, Isabella Caniggia
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Research Article Cell biology Reproductive biology

Faulty oxygen sensing disrupts angiomotin function in trophoblast cell migration and predisposes to preeclampsia

Abstract

Human placenta development and a successful pregnancy is incumbent upon precise oxygen-dependent control of trophoblast migration/invasion. Persistent low oxygen leading to failed trophoblast invasion promotes inadequate spiral artery remodeling, a characteristic of preeclampsia. Angiomotin (AMOT) is a multifaceted scaffolding protein involved in cell polarity and migration, yet its upstream regulation and significance in the human placenta remain unknown. Herein, we show that AMOT is primarily expressed in migratory extravillous trophoblast cells (EVTs) of the intermediate and distal anchoring column. Its expression increases after 10 weeks of gestation when oxygen tension rises and EVT migration/invasion peaks. Time-lapse imaging confirmed that the AMOT 80-kDa isoform promotes migration of trophoblastic JEG3 and HTR-8/SVneo cells. In preeclampsia, however, AMOT expression is decreased and its localization to migratory fetomaternal interface EVTs is disrupted. We demonstrate that Jumonji C domain–containing protein 6 (JMJD6), an oxygen sensor, positively regulates AMOT via oxygen-dependent lysyl hydroxylation. Furthermore, in vitro and ex vivo studies show that transforming growth factor-β (TGF-β) regulates AMOT expression, its interaction with polarity protein PAR6, and its subcellular redistribution from tight junctions to cytoskeleton. Our data reveal an oxygen- and TGF-β–driven migratory function for AMOT in the human placenta, and implicate its deficiency in impaired trophoblast migration that plagues preeclampsia.

Authors

Abby Farrell, Sruthi Alahari, Leonardo Ermini, Andrea Tagliaferro, Michael Litvack, Martin Post, Isabella Caniggia

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Figure 3

AMOT interacts with PAR6 under the regulation of TGF-β.

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AMOT interacts with PAR6 under the regulation of TGF-β. (A) Representati...
(A) Representative images depicting in situ proximity ligation assay (PLA) of AMOT with PAR6 in JEG3 cells following treatment with TGF-β1, TGF-β3, or vehicle control. Negative control was PLA reaction missing the minus (–) PLA probe and nuclei were stained with DAPI (blue). Original magnification, ×63. Data were quantified as number of PLA signals per nucleus (cell) and expressed as fold change relative to vehicle control. *P < 0.05 by nonparametric Mann-Whitney U test (n = 4). (B) IP of PAR6 followed by Western blot (WB) of AMOT in JEG3 cells after co-overexpression of PARD6 with AMOT 130, AMOT 80, AMOT 130 delta PDZ, or AMOT 80 delta PDZ (n = 3). (C) Representative images depicting in situ PLA of AMOT with PAR6 in JEG3 cells following overexpression of empty vector (EV), AMOT 80, or AMOT 80delta PDZ. Negative control was PLA reaction missing the minus (–) PLA probe. Nuclei were stained with DAPI (blue). Original magnification, ×40 and ×20 (for EV). Data were quantified as number of PLA signals per nucleus (cell) and expressed as fold change relative to EV control. **P < 0.01 by 2-tailed unpaired Student’s t test (n = 3). (D) Representative WB of RhoA and AMOT 80 protein levels following overexpression of EV or AMOT 80 in JEG3 cells (lanes were run on the same gel but were noncontiguous). Densitometric analysis of AMOT protein levels, which were normalized to β-actin (ACTB) and expressed as fold change relative to EV control. *P < 0.05 by unpaired Mann-Whitney U test (n = 4 separate experiments).