Interleukin-11 up-regulates endoplasmic reticulum stress induced target, PDIA4 in human first trimester placenta and in vivo in mice

doi:10.1016/j.placenta.2017.04.007

Placenta

Volume 53, May 2017, Pages 92-100

https://doi.org/10.1016/j.placenta.2017.04.007 Get rights and content

Highlights

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Interleukin-(IL)11 is regulated by IL1β, but not TNFα, or low oxygen tension placental villous.
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HPLC MS/MS identified protein disulfide isomerase 4 (PDIA4) in IL11-treated first trimester villous.
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PDIA4 is an ER stress induced protein.
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IL11 upregulates PDIA4 protein in human villous, HTR8-SVneo trophoblasts and in vivo in IL11-treated mouse placenta.

Abstract

Interleukin (IL)11 is a crucial factor for human trophoblast function and placentation. Elevated levels are associated with pregnancy complications including preeclampsia, intrauterine growth restriction (IUGR) and preterm birth. However, the regulation of IL11 in the placenta has not been investigated. We examined the effect of pro-inflammatory cytokines IL1β and TNFα, as well as low oxygen tension (2%) on IL11 levels in first trimester placental villous explants. IL1β upregulated IL11 mRNA and protein, while TNFα and low oxygen had no effect. Using mass spectrometry, we identified protein disulfide isomerase 4 (PDIA4) in IL11-treated first trimester human placental explants (100 ng/ml, 24 h, n = 3), but not PBS control tissues. PDIA4 is a member of the PDI family, also known as endoplasmic reticulum (ER) stress protein (ERP)72. We previously identified GRP78 (a master regulator for ER stress) in human placenta for the first time and demonstrated that IL11 up-regulates GRP78 in the placenta. In this report, we demonstrated that IL11 upregulates PDIA4 protein in human placental villous tissue, HTR8-SVneo trophoblasts (cell line) and in vivo in IL11-treated mouse placenta. We aimed to determine whether IL11 upregulates other ER stress proteins in human first trimester placental villous. IL11 stimulated ERP44, but not GRP94, or PDI. Placental endoplasmic reticulum stress has been postulated in the pathophysiology of preeclampsia and IUGR, but its activation remains elusive. Together, these data suggest that IL11 could trigger an ER stress response in the placenta, which may contribute to obstetric complications such as preeclampsia.

Introduction

Interleukin (IL)11 is a critical regulator of placental development. IL11 impedes primary human first trimester extravillous trophoblast (EVT) cell invasion [1] and villous outgrowth [2] in vitro, required for normal placentation. In vivo, elevated levels of IL11 during early placentation impair trophoblast invasion and spiral artery remodeling in mice, leading to onset of preeclamptic features, including hypertension, proteinuria and intrauterine growth restriction (IUGR) [3]. In women with preeclampsia, placental villous IL11 mRNA and protein levels [3] and decidual IL11 protein levels [4] are up-regulated at term. Furthermore, circulating IL11 is elevated as early as the first trimester in pregnant women who subsequently develop preeclampsia [3].

Preeclampsia affects approximately 8% of all pregnancies [5]. It is widely accepted that inadequate trophoblast invasion and spiral artery remodeling are key initiating factors [6], [7], leading to reduced utero-placental arterial flow [8] and pro-inflammatory cytokine production, such as tumour necrosis factor-α (TNFα) and interleukin-1β (IL1β) [9], [10]. We previously established that IL11 partially inhibits first trimester placental villous outgrowth via the protease pregnancy-associated plasma protein A2 (PAPPA2). However other placental targets of IL11.

In this study we investigated the regulation of IL11 by pro-inflammatory cytokines and low oxygen tension in first trimester placental villous explants. Using high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS), we identified protein disulfide isomerase 4 (PDIA4) in IL11-treated first trimester placental samples versus control tissues. PDIA4 is a member of the PDI family, also known as endoplasmic reticulum (ER) stress protein (ERP)72. This is the first study to identify PDIA4 in the placenta.

We hypothesized that IL11 regulates PDIA4 protein in first trimester human placental villous explants and trophoblasts cells in vitro and mouse placenta in vivo. We aimed to determine whether IL11 alters other ER stress proteins in human first trimester placental villous.

Section snippets

Participants and patient placental samples

First trimester human placenta was obtained by suction curettage from women undergoing elective termination of pregnancy for psycho-social reasons at 6–12 gestation weeks. The Human Research and Ethics Committee of Monash Medical Centre approved the study and informed written consent was obtained from all patients prior to surgery.

First-trimester villous explant culture and treatments

Tissues were processed immediately upon arrival at the laboratory. Tissues were rinsed in DMEM/Ham's, F12 (Invitrogen, Victoria, Australia) and explants were

IL11 is regulated by IL1β, but not TNFα, or low oxygen tension in human first trimester placental villous

First trimester placental villous explants were cultured in 20% O₂ or 2% O₂, or with pro-inflammatory cytokines IL1β and TNFα. Low oxygen tension (2% O₂) did not alter IL11 mRNA expression versus 20% O₂ (Fig. 1A). Low oxygen tension did not change the localisation pattern or abundance of IL11 receptor-α (IL11Rα) protein in villous tissue compared to control (Fig. 1B). LDH secretions were unchanged between villous tissue explants cultured under 20% O₂ or, 2% O₂, confirming the viability of all

Discussion

IL11 is a crucial regulator of human trophoblast function in vitro [1], [2], [15] and placental formation in vivo [3]. We recently demonstrated that elevated IL11 causes placental syncytial damage, reduced trophoblast invasion and impaired spiral artery remodeling in mice [3]. Abnormal placentation during the first trimester is associated with decreased utero-placental arterial flow, placental oxidative stress, the generation of reactive oxygen species and pro-inflammatory cytokines, all

Funding

This work was supported by NHMRC Australia Project Grant (1078674) and the Victorian Government's Operational Infrastructure Support Program. AW was supported by a Cancer Council Victoria Postdoctoral Fellowship. ED was supported by a NHMRC Australia Senior Research Fellowship (550905).

Authors' roles

AW - performed experiments (tissue and cell culture, immunohistochemistry, Western blot), data analysis, wrote manuscript. KS - performed experiments (immunohistochemistry, Western blot). JC - performed experiments (tissue culture, and preparation for protein identification). AR - performed experiments (protein identification). JY - performed experiments (tissue culture, ELISA, immunohistochemistry). ED – designed study, critical analysis of manuscript.

Conflict of interest

The authors have nothing to disclose.

Acknowledgements

The authors acknowledge all the women that donated tissue and Judi Hocking for collection of donated tissues.

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Cited by (13)

The regulatory network of lncRNA DLX6-AS1/miR-149–5p/ERP44 is possibly related to the progression of preeclampsia
2020, Placenta
Citation Excerpt :
Down-regulation of ERP44 induced apoptosis of HeLa cells [32]. ERP44 was reported to be stimulated by IL11, a key factor in trophoblast function and placentation, which triggered a stress response leading to obstetric complications such as PE [33,34]. In the present study, ERP44 turned out to be a target gene of miR-149–5p and was inversely modulated by miR-149–5p.
Long noncoding RNA DLX6 antisense RNA 1 (DLX6-AS1) has been reported to be involved in various human diseases, however, its potential role in the pathogenesis of preeclampsia (PE) has not been fully explored.
The levels of DLX6-AS1, microRNA-149–5p (miR-149–5p) and endoplasmic reticulum protein 44 (ERP44) were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Some clinicopathological parameters of PE were statistically analyzed. The cell proliferation, invasion and angiogenesis were assessed by methylthiazolyldiphenyl-tetrazolium bromide (MTT), transwell and tube formation assays, respectively. Levels of all protein were detected by western blot. The target relationship was predicted by StarBase v2.0 and confirmed by dual-luciferase reporter assay.
Higher levels of DLX6-AS1 and ERP44, lower level of miR-149–5p were observed in PE placenta tissues. Compared with PE group with low DLX6-AS1 expression, the systolic blood pressure, diastolic blood pressure and proteinuria levels in the group with high DLX6-AS1 expression were higher, and the infant body weight level was lower. The role of miR-149–5p on these clinicopathological parameters of PE patients was opposite to that of DLX6-AS1, while ERP44 had the same effect as DLX6-AS1. Besides, DLX6-AS1 directly targeted miR-149–5p and miR-149–5p targeted ERP44. The inhibitory impact of DLX6-AS1 overexpression or ERP44 overexpression on proliferation and invasion of trophoblast cells as well as angiogenesis of HUVEC cells was reversed by up-regulating miR-149–5p. We also found that DLX6-AS1 could enhance ERP44 expression by sponging miR-149–5p.
DLX6-AS1 inhibited proliferation and invasion of trophoblast cells, and suppressed angiogenesis of HUVEC cells by miR-149–5p/ERP44 pathway.
PDIA4: The basic characteristics, functions and its potential connection with cancer
2020, Biomedicine and Pharmacotherapy
Citation Excerpt :
PDIA4, therefore, acts as a bridge between activated platelet and tumor growth. Abnormal PDIA4 expression and endoplasmic reticulum stress (ERS) response have been proved as a self-protection response to various diseases [20–23]. There are also a bunch of evidences demonstrated the connection between ERS response and tumor.
Disulfide bond formation is catalyzed by the protein disulfide Isomerases (PDI) family. This is a critical step in protein folding which occurs within the endoplasmic reticulum. PDIA4, as a member of the PDI family, can cause the adjustment of αIIβ 3 affinities which activate platelet and promote thrombosis formation. Endoplasmic reticulum response is triggered by accumulation of abnormal folding proteins concomitant with increasing PDIA4 expression. Besides, current researches indicate that activated platelets and ERS response affect tumor progression. And PDIA4, as previous reported, also participates in tumor progression by affecting cell apoptosis and DNA repair machinery without specific mechanisms revealed.Therefore, PDI inhibitor might possess great potential value in against tumor progression. In this review, we summarize information on PDIA4 including its the basic characteristics and its implication on tumor.
Small extracellular vesicles-transported lncRNA TDRKH-AS1 derived from AOPPs-treated trophoblasts initiates endothelial cells pyroptosis through PDIA4/DDIT4 axis in preeclampsia
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2023, Heart Surgery Forum
IL11 activates the placental inflammasome to drive preeclampsia
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High expression of PDIA4 promotes malignant cell behavior and predicts reduced survival in cervical cancer
2022, Oncology Reports

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View full text

Interleukin-11 up-regulates endoplasmic reticulum stress induced target, PDIA4 in human first trimester placenta and in vivo in mice

Highlights

Abstract

Introduction

Section snippets

Participants and patient placental samples

First-trimester villous explant culture and treatments

IL11 is regulated by IL1β, but not TNFα, or low oxygen tension in human first trimester placental villous

Discussion

Funding

Authors' roles

Conflict of interest

Acknowledgements

Lancet

Am. J. Obstetrics Gynecol.

J. Reproductive Immunol.

Placenta

J. Biol. Chem.

Methods

Placenta

Interleukin-11 promotes migration, but not proliferation, of human trophoblast cells, implying a role in placentation

Endocrinology

Interleukin (IL)11 mediates protein secretion and modification in human extravillous trophoblasts

Hum. Reprod.

Interleukin-11 alters placentation and causes preeclampsia features in mice

Proc. Natl. Acad. Sci. U. S. A.

Preeclampsia-related increase of interleukin-11 expression in human decidual cells

Reproduction

Trohpoblast invasion

Reprod. Med. Rev.

The role of the spiral arteries in the pathogenesis of preeclampsia

Obstet. Gynecol. Annu.