PAD4 silencing inhibits inflammation whilst promoting trophoblast cell invasion and migration by inactivating the NEMO/NF‑κB pathway

Abstract

Preeclampsia (PE), presenting with onset hypertension and proteinuria, is a pregnancy-specific disorder that can result in maternal and fetal morbidity and mortality. Insufficient trophoblast invasion and migration has been considered to be an important cause of this disease. The present study aimed to investigate the role of peptidyl arginine deiminase 4 (PAD4), whose knockdown has been previously indicated to reduce inflammation and susceptibility to pregnancy loss in mice, in the development of PE in vitro. Lipopolysaccharide (LPS) was used to treat a human trophoblast cell line (HTR8/SVneo). After PAD4 silencing via transfection with short hairpin RNA against PAD4, the concentrations of inflammatory factors IL-6, IL-12 and monocyte chemoattractant protein (MCP)-1 were measured using ELISA. Cell viability was also measured using Cell Counting Kit-8 assay. HTR8/SVneo cell invasion and migration were detected using Transwell and wound healing assays, respectively. Western blotting was used to measure the expression of citrullinated NF-κB essential modulator (NEMO) and nuclear NF-κB p65 protein levels. TNF-α was applied for evaluating the potential regulatory effects of PAD4 on NF-κB in LPS-stimulated HTR8/SVneo cells. LPS increased the levels of IL-6, IL-12 and MCP-1 and reduced the migration and invasion of HTR8/SVneo cells. PAD4-knockdown was found to markedly reduce the levels of IL-6, IL-12 and MCP-1 secretion. HTR8/SVneo cell invasion and migration was also significantly elevated after PAD4 silencing following LPS exposure. In addition, LPS stimulation notably upregulated the protein levels of citrullinated NEMO and nuclear NF-κB p65, which was restored by PAD4 knockdown. Furthermore, TNF-α treatment partially counteracted the effects of PAD4 knockdown on the secretion of IL-6, MCP-1 and IL-12, which are markers of inflammation, and invasion and migration in LPS-induced HTR8/SVneo cells. To conclude, these results suggest that PAD4 silencing can suppress inflammation whilst promoting invasion and migration by trophoblast cells through inhibiting the NEMO/NF-κB pathway. These findings furthered the understanding in the complex molecular mechanism that can trigger PE and provide a promising target for the treatment of this disease.

Regulation of IkappaB Protein Expression by Early Gestation in the Thymus of Ewes

“…The upregulation of the NEMO gene in the maternal blood of women with preeclampsia suggests that NEMO can be associated with preeclampsia development [ 47 ]. It is through suppressing the NEMO/NF-κB pathway that peptidyl arginine deiminase 4 knockdown inhibits inflammation in trophoblast cells to reduce preeclampsia development in vitro [ 48 ]. The NF-κB-essential-modulator (NEMO) is involved in the regulation of thymocyte selection by the cylidromatosis tumor suppressor [ 49 ].…”