RAGE and phospho‐ATM correlation during DNA Double Strand Breaks in trophoblast cells

Abstract

Trophoblasts are critical for successful pregnancies and they mediate important steps including implantation, immune protection of the fetus, maternal blood flow to the placenta, and delivery. Increased DNA double strand breaks (DNA‐DSBs) in trophoblasts has been implicated in complicated pregnancies such as intrauterine growth restriction. Previous studies in our laboratory showed increased expression of the receptor for advanced glycation end‐products (RAGE) following cigarette smoke extract (CSE) treatment of trophoblast cells. More recently, a role for nuclear RAGE (nRAGE) has been implicated in DNA‐DSB repair in these cells. ATM is a regulator known to be involved in the repair of DNA‐DSBs through interaction with molecules associated with DNA repair. We tested the hypothesis that RAGE associates with ATM in facilitating DNA‐DSB repair in trophoblast cells. DSBs were induced in human trophoblasts by exposure to cigarette smoke extract (CSE) or bleomycin (BLM). Assessment of nRAGE and g‐H2AX (involved in DNA‐DSB repair) was done by immunoblotting. Immunoprecipitation was used to quantify RAGE and p‐ATM complexing in cells. Invasion was measured after CSE and BLM treatment in the presence or absence of a RAGE neutralizing antibody (nAb).Increased expression of RAGE and g‐H2AX was detected (1.4‐fold and 1.6‐fold; p<0.05) in treated trophoblast cells compared to controls. Trophoblast invasion was decreased by 65% with BLM treatment compared to controls (p<0.0005) and diminished invasion was further enhanced (73%, p<0.0004) when RAGE nAb was added. Similarly, trophoblast invasion was decreased by 58% (p<0.002) when treated with 1% CSE and further decreased (79%, p<0.0006) in the presence of RAGE nAb. There was also a significant increase (56%, p<0.002) in RAGE expression when p‐ATM was immunoprecipitated from trophoblasts treated with CSE.We conclude that CSE and BLM cause DNA‐DSBs in trophoblast cells and that DNA‐DSB repair may plausibly involve nRAGE association with ATM. We further conclude that DNA‐DSBs could be a factor that inhibits trophoblast invasion. Nuclear RAGE may be a potential target that maintains trophoblastic invasion by assisting in the repair of DNA‐DSBs. These studies provide a critical insight into dissecting tobacco‐mediated placental deficiency.Support or Funding InformationThis work was supported by a grant from the Flight Attendant’s Medical Research Institute (FAMRI, PRR and JAA) and a BYU Mentoring Environment Grant (JAA and PRR).