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. 2018 Jan 11;9(6):6707-6717.
doi: 10.18632/oncotarget.24158. eCollection 2018 Jan 23.

Endoplasmic reticulum stress stimulates the release of extracellular vesicles carrying danger-associated molecular pattern (DAMP) molecules

Gavin P Collett  1 , Christopher W Redman  1 , Ian L Sargent  1 , Manu Vatish  1
Affiliations

Endoplasmic reticulum stress stimulates the release of extracellular vesicles carrying danger-associated molecular pattern (DAMP) molecules

Gavin P Collett et al. Oncotarget. .

Abstract

Disturbances in endoplasmic reticulum (ER) function lead to ER stress which, when severe or prolonged, may result in apoptosis. Severe ER stress has been implicated in several pathological conditions including pre-eclampsia, a multisystem disorder of pregnancy associated with the release of pro-inflammatory factors from the placenta into the maternal circulation. Here, we show that severe ER stress induced by two distinct mechanisms in BeWo choriocarcinoma cells leads to the release of extracellular vesicles (EVs) carrying pro-inflammatory damage-associated molecular pattern (DAMP) molecules. Co-treatment with the antioxidant pyrrolidine dithiocarbamate results in a reduction in ER stress-induced EV-associated DAMP release. We further demonstrate that severe ER stress is associated with changes in the expression of several stress-related proteins, notably Cited-2 and phosphorylated JNK. Together, these data indicate that severe ER stress-mediated release of EV-associated DAMPs may contribute to the heightened systemic maternal inflammatory response characteristic of pre-eclampsia and may also be relevant to other chronic inflammatory diseases which display elevated ER stress.

Keywords: ER stress; endoplasmic reticulum stress; extracellular vesicles; pathology.

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Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Induction of mild and severe ER stress by tunicamycin in BeWo cells
BeWo cells were treated with growth medium alone (G), vehicle (DMSO) or the indicated concentrations of tunicamycin for 48h (a) or 24h (b). Cell lysates were analysed for the expression of GRP78, phospho-eIF2α, CHOP, cleaved PARP and β-actin by immunoblotting. Representative blots from three separate experiments are shown. (c) Blots were analysed by densitometric image analysis (Image J), normalising to β-actin, for GRP78, phospho-eIF2α, CHOP & cleaved PARP. Results are represented as mean ± SEM for three separate experiments. *p<0.05, **p<0.01, ***p<0.001 compared with control (ANOVA). (d) Cells were treated with 1μg/ml or 10μg/ml tunicamycin for 48h, representing mild and severe ER stress respectively. Cell death was analysed by release of lactate dehydrogenase (LDH) into the culture medium. Results are represented as mean ± SEM for four separate experiments. ***p<0.001 compared with control (ANOVA).
Figure 2
Figure 2. Severe ER stress stimulates the release of extracellular vesicles carrying danger-associated molecular pattern (DAMP) molecules
BeWo cells were treated with 1μg/ml or 10μg/ml tunicamycin, or DMSO control, for 48h. Extracellular vesicles were isolated from the culture medium by ultracentrifugation and analysed by NTA for quantity (a) and size (b). Equal volumes of EV pellet suspensions were analysed for expression of DAMPs HMGB1, hsp70 and histone H3, and exosome markers syntenin and alix by immunoblotting (c). (d) EV pellets from severely ER-stressed cells were resuspended in PBS (500μl) and overlaid onto 5-40% discontinuous iodixanol gradients. Ultracentrifugation was carried out at 150,000xg (maximum) for 18 h at 4°C. Fractions (12 × 1ml) were collected manually and EV concentrations were analysed by NTA. Results are represented as mean ± SEM for six (a-c) or three (d) separate experiments. ***p<0.001 compared with control (ANOVA).
Figure 3
Figure 3. Antioxidant treatment attenuates ER stress-induced release of extracellular vesicles
BeWo cells were treated with 1μg/ml or 10μg/ml tunicamycin, or DMSO control, for 48h in the presence or absence of the ROS scavenging antioxidant pyrrolidine dithiocarbamate (PDTC). Extracellular vesicles were isolated from the culture medium by ultracentrifugation and analysed by NTA for quantity (a) and size (b). Equal volumes of EV pellet suspensions were analysed for expression of HMGB1 and hsp70 by immunoblotting (c). Results are represented as mean ± SEM for three separate experiments. ***p<0.001 compared with control (ANOVA).
Figure 4
Figure 4. Thapsigargin-induced severe ER stress stimulates release of EV-associated DAMPs
BeWo cells were treated with 10μM thapsigargin or DMSO control for 48h. Cell lysates were analysed for the expression of CHOP, cleaved PARP and β-actin by immunoblotting (a). Extracellular vesicles were isolated from the culture medium by ultracentrifugation and analysed by NTA for quantity (b) and size (c). Equal volumes of EV pellet suspensions were analysed for expression of HMGB1, hsp70 and histone H3 by immunoblotting (d). Results are represented as mean ± SEM for three separate experiments. ***p<0.001 compared with control (Students t test).
Figure 5
Figure 5. Human cell stress arrays
BeWo cells were treated with 1μg/ml or 10μg/ml tunicamycin, or DMSO control, for 48h. (a) Cell lysate samples containing equal amounts of protein were analysed on human cell stress arrays. (b) The blots shown in (A) were analysed by densitometric image analysis (Image J). The graph displays the proteins in the order on the dot blots, from top left to bottom right excluding the three positive control pairs of dots on the top left, top right and bottom left.
Figure 6
Figure 6. Cited-2 and phospho-JNK expression is altered in severe ER stress
BeWo cells were treated with 1μg/ml or 10μg/ml tunicamycin, or DMSO control, for 24h and 48h. At each time point cell lysates were made and analysed for the expression of Cited-2 (a) and phospho-JNK (c). Blots were analysed by densitometric image analysis (Image J), normalising to β-actin for Cited-2 (b) and total JNK for phospho-JNK (d). Results are represented as mean ± SEM for three separate experiments. *p<0.05, ***p<0.001 compared with control (ANOVA).
Figure 7
Figure 7. Proposed mechanism of action of ER stress-induced EV-associated DAMPs
Increased placental ER stress, via the production of reactive oxygen species, leads to the release of extracellular vesicles carrying DAMPs into the maternal circulation. These EV-associated DAMPs may interact with maternal endothelial and immune cells, resulting in exacerbated endothelial cell permeability and pro-inflammatory cytokine production.
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