Silica nanoparticles induce endoplasmic reticulum stress response, oxidative stress and activate the mitogen-activated protein kinase (MAPK) signaling pathway

Abstract

Application of silica nanoparticles (SiO₂-NPs) may result in human exposure. Here we investigate unexplored modes of action by which SiO₂-NPs with average size of 225 nm act on human hepatoma cells (Huh7). We focused on the endoplasmic (ER) stress response and on mitogen-activated protein kinase (MAPK) signaling pathways. Both pathways were induced. ER stress and the associated three unfolded protein response (UPR) pathways were activated as demonstrated by significant inductions of BiP and XBP-1s and a moderate but significant induction of ATF-4 at 0.05 and 0.5 mg/ml. In addition to activation of NFкB interferon stimulated genes IP-10, IRF-9, and ISG-15 were up-regulated. As a consequence of ER stress, the pro-inflammatory cytokine TNFα and PP2Ac were induced following exposure to 0.05 mg/ml SiO₂-NPs. Additionally, this occurred at 0.005 mg/ml SiO₂-NPs for TNFα at 24 h. This in turn led to a strong transcriptional induction of MAP-kinases and its target genes cJun, cMyc and CREB. A strong transcriptional down-regulation of the proapoptotic gene p53 occurred at 0.05 and 0.5 mg/ml SiO₂-NP. Exposure of Huh7 cells to the anti-oxidant N-acetyl cysteine reduced transcriptional induction of ER stress markers demonstrating a link between the induction of oxidative stress and ER stress. Our study demonstrates that SiO₂-NPs lead to strong ER stress and UPR induction, oxidative stress, activation of MAPK signaling and down-regulation of p53. All of these activated pathways, which are analyzed here for the first time in detail, inhibit apoptosis and induce cell proliferation, which may contribute to a hepatotoxic, inflammatory and tumorigenic action of SiO₂-NPs.

Keywords: ATF-4, Activating transcription factor 4; ATF-6, activating transcription factor 6; BiP, binding immunoglobulin protein; CHOP, CCAAT/enhancer binding protein-homologous protein; CREB, cAMP response element-binding protein; Huh7, human hepatoma cells; Human hepatoma cells; IFN α, interferon α; IFN β, interferon β; IP-10, interferon gamma-induced protein 10; IRE-1, inositol-requiring protein 1; IRF-9, interferon regulatory factor 9; ISG-15, interferon-induced 17 kDa protein; ISGs, interferon stiulated genes; MAPK, mitogen-activated protein kinase signaling pathway; NFκB, nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells; Noxa, phorbol-12-myristate-13-acetate-induced protein 1; PERK, protein kinase like ER kinase; PP2A, protein phosphatase 2a; Proinflammatory response ;Iinterferon-stimulated genes; STAT1, signal transducer and activator of transcription 1; SiO2-NPs, silica nanoparticles; TNFα, tumor necrosis factor α; Tumor necrosis factor alpha; UPR, unfolded protein response; XBP-1, X-box binding protein 1; eIF2α, eukaryotic initiation factor 2α; p53, TP53-tumorsuppressor-gene.

Fig. 1

Induction of ER stress in Huh7 cells after SiO₂-NP exposure. (A) Huh7 cells were exposed to 0.005 (blank bars), 0.05 (vertical strips) and 0.5 mg/ml (horizontal strips) SiO₂-NPs for 24 h followed by the investigation of mRNA level of ATF-4, BiP and XBP-1s. (B) Huh7 cells were exposed to 0.05 mg/ml SiO₂-NPs for 24 h followed by Western blot analysis for BiP (upper panel). As loading control the Western blot membrane was stained with Ponceau S solution (lower panel). (C) Huh7 cells were exposed to 0.05 (blank bar) and 0.5 mg/ml (vertical strips) SiO₂-NPs for 24 h followed by the detection of mRNA levels of Noxa. Shown are the results of three independent experiments. Significant differences with p-value of ≤0.05 are marked with asterisks.

Fig. 2

Induction of TNF-α and PP2Ac in Huh7 cells after exposure to SiO₂-NPs. (A) Huh7 cells were exposed to 0.0005 (blank bars), 0.005 (diagonal strips), 0.05 (horizontal strips) and 0.5 mg/ml (vertical strips) SiO₂-NPs for 24 h followed by determination of mRNA levels of TNF-α. (B) Huh7 cells were exposed to 0.005 and 0.05 mg/ml SiO₂-NPs for 24 h followed by the investigation of TNF-α protein level in the cell culture supernatant. (C) Huh7 cells were exposed to 0.005 (blank bars), 0.05 (diagonal strips) and 0.5 mg/ml (horizontal strips) SiO₂-NPs for 24 h followed by detection of mRNA level of PP2Ac. (D) Huh7 cells were exposed to 0.05 mg/ml SiO₂-NPs for 24 h followed by Western blot analysis for PP2Ac (upper panel). As loading control the Western blot membrane was stained with Ponceau S solution (lower panel). Shown are the results of three independent experiments. Significant differences with p-value of ≤0.05 are marked with asterisks.

Fig. 3

Activation of NFκB and induction of interferon stimulated genes in Huh7 cells after exposure to SiO₂-NPs. (A) Huh7 cells were exposed to 0.05 mg/ml SiO₂-NPs for 24 h followed by NFκB activation assay. (B) Huh7 cells were exposed to 0.005 (blank bars), 0.05 (vertical strips) and 0.5 mg/ml (horizontal strips) SiO₂-NPs for 24 h followed by the investigation of mRNA level of IRF-9, ISG-15 and IP-10. Shown are the results of three independent experiments. Significant differences with p-value of ≤0.05 are marked with asterisks.

Fig. 4

Activation of MAPK signaling pathway and inhibition of p53 in Huh7 cells after exposure to SiO₂-NPs. (A) Huh7 cells were exposed to 0.005 (blank bars), 0.05 (vertical strips) and 0.5 mg/ml (diagonal strips) SiO₂-NPs for 24 h followed by the investigation of mRNA level of STAT1, CREB, c-Jun and c-Myc. (B) Huh7 cells were exposed to 0.005 (blank bars), 0.05 (horizontal strips) and 0.5 mg/ml (diagonal strips) SiO₂-NPs for 24 h followed by the investigation of mRNA level of p53. Shown are the results of three independent experiments. Significant differences with p-value of ≤0.05 are marked with asterisks.

Fig. 5

Induction of oxidative stress in Huh7 cells upon exposure to SiO₂-NPs. (A) Huh7 cells were exposed to 0.005, 0.05 and 0.5 mg/ml SiO₂-NPs alone (blank bars), or pretreated for 30 min with NAC followed by 24 h exposure to SiO₂-NP (horizontal bars) or co-exposed to NAC and SiO₂-NPs for 24 h (diagonal bars) followed by the investigation of oxidative stress. (B) Huh7 cells were exposed to 0.05 mg/ml SiO₂-NPs alone (blank bars), or pretreated for 30 min with NAC followed by 24 h exposure to SiO₂-NP (horizontal bars) or co-exposed to NAC and SiO₂-NPs for 24 h (diagonal bars) followed by the investigation of the mRNA of BiP, XBP-1s and TNF-α. Shown are the results of three independent experiments. Significant differences with p-value of ≤0.05 are marked with asterisks.