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Cited by 196S100 Proteins and AnnexinsNeuroinflammation and Neurodegeneration MechanismsAdvanced Glycation End Products research

The DOI matches its Crossref record; 47 of the 48 references it cites were found in a registry or an index.

Proinflammatory effects of S100A8/A9 via TLR4 and RAGE signaling pathways in BV-2 microglial cells

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Ma, Li, Sun, Peng, Zhang, Jian-Cheng, Zhang, Qing, Yao, Shang-Long·2017·International Journal of Molecular Medicine
DOI 10.3892/ijmm.2017.2987PMC PMC5466387
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Abstract

S100A8/A9, a heterodimer of the two calcium-binding proteins S100A8 and S100A9, has emerged as an important proinflammatory mediator in acute and chronic inflammation. However, whether S100A8/A9 is implicated in microglial‑induced neuroinflammatory response remains unclear. Here, we found that S100A8/A9 significantly increased the secretion of proinflammatory cytokines inclu-ding tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in cultured BV-2 microglial cells. Inhibition of the Toll-like receptor 4 (TLR4) and the receptor for advanced glycation end-products (RAGE) with C225 and a RAGE-blocking antibody, respectively significantly reduced the secretion of TNF-α and IL-6 from S100A8/A9-stimulated BV-2 microglial cells. Furthermore, S100A8/A9 markedly enhanced the nuclear translocation of NF-κB p65 and the DNA-binding activities of NF-κB in BV-2 microglial cells, and suppression of ERK and JNK/MAPK signaling pathways by PD98059 or SP600125 significantly inhibited NF-κB activity and the release of TNF-α and IL-6 in the S100A8/A9-treated BV-2 microglial cells. Our data also showed that inhibition of NF-κB with pyrrolidine dithiocarbamate (PDTC) significantly reduced the secretion of TNF-α and IL-6 from BV-2 microglial cells treated with S100A8/A9. Taken together, our data suggest that S100A8/A9 acts directly on BV-2 microglial cells via binding to TLR4 and RAGE on the membrane and then stimulates the secretion of proinflammatory cytokines through ERK and JNK-mediated NF-κB activity in BV-2 microglial cells. Targeting S100A8/A9 may provide a novel therapeutic strategy in microglial-induced neuroinflammatory diseases.

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Figure 1 Proinflammatory cytokine production by BV-2 microglial cells after S100A8/A9 stimulation. Effect of lipopolysaccharide (LPS) (1 µg/ml) and S100A8/A9 (0.01–1.0 µM) on the production of (A) tumor necrosis factor-α (TNF-α) and (B) interleukin-6 (IL-6) in BV-2 microglial cells. TNF-α and IL-6 levels were determined by ELISA after 12 h of incubation. (C) Polymyxin B (25 µg/ml) had no inhibitory effect on S100A8/A9-induced TNF-α expression in BV-2 cells, whereas the effects of LPS were efficiently blocked. TNF-α levels were determined after 12 h of stimulation by ELISA. Each value represents the mean ± SEM of three independent experiments. **P<0.01 and ***P<0.001 compared with the control; ##P<0.01 and ###P<0.001 compared with 0.1 nM S100A8/A9.
Figure 1 Proinflammatory cytokine production by BV-2 microglial cells after S100A8/A9 stimulation. Effect of lipopolysaccharide (LPS) (1 µg/ml) and S100A8/A9 (0.01–1.0 µM) on the production of (A) tumor necrosis factor-α (TNF-α) and (B) interleukin-6 (IL-6) in BV-2 microglial cells. TNF-α and IL-6 levels were determined by ELISA after 12 h of incubation. (C) Polymyxin B (25 µg/ml) had no inhibitory effect on S100A8/A9-induced TNF-α expression in BV-2 cells, whereas the effects of LPS were efficiently blocked. TNF-α levels were determined after 12 h of stimulation by ELISA. Each value represents the mean ± SEM of three independent experiments. **P<0.01 and ***P<0.001 compared with the control; ##P<0.01 and ###P<0.001 compared with 0.1 nM S100A8/A9.CC BY-NC-ND · PubMed Central
Figure 2 Effects of receptor for advanced glycation endproducts (RAGE) and Toll-like receptor 4 (TLR4) blockade on S100A8/A9 stimulation. (A) Tumor necrosis factor-α (TNF-α) and (B) interleukin-6 (IL-6), as determined by ELISA, in BV-2 cells after preincubation for 1 h with 1 µg/ml TLR4 inhibitor (C225) or 166 µg/ml RAGE-blocking antibody followed by 12 h of stimulation with lipopolysaccharide (LPS) or S100A8/A9 as indicated. Each value represents the mean ± SEM of three independent experiments. ***P<0.001 compared with the control; ##P<0.01 and ###P<0.001 compared with S100A8/A9.
Figure 2 Effects of receptor for advanced glycation endproducts (RAGE) and Toll-like receptor 4 (TLR4) blockade on S100A8/A9 stimulation. (A) Tumor necrosis factor-α (TNF-α) and (B) interleukin-6 (IL-6), as determined by ELISA, in BV-2 cells after preincubation for 1 h with 1 µg/ml TLR4 inhibitor (C225) or 166 µg/ml RAGE-blocking antibody followed by 12 h of stimulation with lipopolysaccharide (LPS) or S100A8/A9 as indicated. Each value represents the mean ± SEM of three independent experiments. ***P<0.001 compared with the control; ##P<0.01 and ###P<0.001 compared with S100A8/A9.CC BY-NC-ND · PubMed Central
Figure 3 Involvement of nuclear factor-κB (NF-κB) activation in S100A8/A9-stimulated secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). (A) BV-2 cells were stimulated with lipopolysaccharide (LPS) (1 µg/ml) or S100A8/A9 (0.1 µM) for 1 h. Nuclear extracts were prepared and used to analyze NF-κB DNA-binding activity by EMSA, as described in Materials and methods. Binding specificity was confirmed by unlabelled probe (100-fold in excess; lane 5) to compete with labelled oligonucleotide. Free-labelled probes are also indicated. (B and C) p65 NF-κB inhibitor PDTC (50 µM) was added 1 h before activation. Results were confirmed by three independent experiments. **P<0.01 compared with the control; ###P<0.001 compared with S100A8/A9.
Figure 3 Involvement of nuclear factor-κB (NF-κB) activation in S100A8/A9-stimulated secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). (A) BV-2 cells were stimulated with lipopolysaccharide (LPS) (1 µg/ml) or S100A8/A9 (0.1 µM) for 1 h. Nuclear extracts were prepared and used to analyze NF-κB DNA-binding activity by EMSA, as described in Materials and methods. Binding specificity was confirmed by unlabelled probe (100-fold in excess; lane 5) to compete with labelled oligonucleotide. Free-labelled probes are also indicated. (B and C) p65 NF-κB inhibitor PDTC (50 µM) was added 1 h before activation. Results were confirmed by three independent experiments. **P<0.01 compared with the control; ###P<0.001 compared with S100A8/A9.CC BY-NC-ND · PubMed Central
Figure 4 Involvement of mitogen-activated protein kinase (MAPK) signaling pathways in S100A8/A9-stimulated secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). TNF-α levels determined by (B) ELISA and (C) PCR, and (A) IL-6 levels as determined by PCR in BV-2 cells after 12 h of stimulation with lipopolysaccharide (LPS) (1 µg/ml) or S100A8/A9 (0.1 µM) in the absence or presence of protein kinase inhibitors: extracellular signal-regulated kinase (ERK) inhibitor PD98059 (20 µM), p38 inhibitor SB203580 (7 µM), c-Jun N-terminal kinase (JNK) inhibitor SP600125 (10 µM). Each value represents the mean ± SEM of three independent experiments. ##P<0.01 and ###P<0.001 compared with S100A8/A9.
Figure 4 Involvement of mitogen-activated protein kinase (MAPK) signaling pathways in S100A8/A9-stimulated secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). TNF-α levels determined by (B) ELISA and (C) PCR, and (A) IL-6 levels as determined by PCR in BV-2 cells after 12 h of stimulation with lipopolysaccharide (LPS) (1 µg/ml) or S100A8/A9 (0.1 µM) in the absence or presence of protein kinase inhibitors: extracellular signal-regulated kinase (ERK) inhibitor PD98059 (20 µM), p38 inhibitor SB203580 (7 µM), c-Jun N-terminal kinase (JNK) inhibitor SP600125 (10 µM). Each value represents the mean ± SEM of three independent experiments. ##P<0.01 and ###P<0.001 compared with S100A8/A9.CC BY-NC-ND · PubMed Central
Figure 5 Mitogen-activated protein kinase (MAPK) signaling pathway acts upstream of nuclear factor-κB (NF-κB) in S100A8/A9 stimulation. (A) BV-2 cells were stimulated with lipopolysaccharide (LPS) (1 µg/ml) or S100A8/A9 (0.1 µM). Subcellular location of the p65 subunit was assessed using immunofluorescence assay 60 min after activation. (B) BV-2 cells were stimulated with S100A8/A9 (0.1 µM) in the presence of PD98059 (20 µM), SB203580 (7 µM), SP600125 (10 µM) that were added 60 min before activation. Subcellular location of the p65 subunit was assessed using immunofluorescence assay 60 min after activation. (C and D) Western blot analysis and quantitative data for cytoplasmic and nuclear p65 in BV-2 cells stimulated with S100A8/A9 (0.1 µM) in the presence of PD98059 (20 µM), SB203580 (7 µM) and SP600125 (10 µM). Data are presented as mean ± SEM of three independent experiments. #P<0.05 and ##P<0.01 compared with S100A8/A9;. *P<0.05 and ***P<0.001 compared with the control.
Figure 5 Mitogen-activated protein kinase (MAPK) signaling pathway acts upstream of nuclear factor-κB (NF-κB) in S100A8/A9 stimulation. (A) BV-2 cells were stimulated with lipopolysaccharide (LPS) (1 µg/ml) or S100A8/A9 (0.1 µM). Subcellular location of the p65 subunit was assessed using immunofluorescence assay 60 min after activation. (B) BV-2 cells were stimulated with S100A8/A9 (0.1 µM) in the presence of PD98059 (20 µM), SB203580 (7 µM), SP600125 (10 µM) that were added 60 min before activation. Subcellular location of the p65 subunit was assessed using immunofluorescence assay 60 min after activation. (C and D) Western blot analysis and quantitative data for cytoplasmic and nuclear p65 in BV-2 cells stimulated with S100A8/A9 (0.1 µM) in the presence of PD98059 (20 µM), SB203580 (7 µM) and SP600125 (10 µM). Data are presented as mean ± SEM of three independent experiments. #P<0.05 and ##P<0.01 compared with S100A8/A9;. *P<0.05 and ***P<0.001 compared with the control.CC BY-NC-ND · PubMed Central
Figure 6 Proinflammatory response of the S100A8/A9 contribution to microglial activation. S100A8/A9 activated microglia via binding the receptor for advanced glycation endproducts (RAGE) and Toll-like receptor 4 (TLR4) on the membrane and promoted the production of proinflammatory cytokines in microglia through the activation of c-Jun N-terminal kinase (JNK)/nuclear factor-κB (NF-κB) and extracellular signal-regulated kinase (ERK)/NF-κB signaling pathways.
Figure 6 Proinflammatory response of the S100A8/A9 contribution to microglial activation. S100A8/A9 activated microglia via binding the receptor for advanced glycation endproducts (RAGE) and Toll-like receptor 4 (TLR4) on the membrane and promoted the production of proinflammatory cytokines in microglia through the activation of c-Jun N-terminal kinase (JNK)/nuclear factor-κB (NF-κB) and extracellular signal-regulated kinase (ERK)/NF-κB signaling pathways.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Ma, L., Sun, P., Zhang, J.-C., Zhang, Q., & Yao, S.-L.
Affiliations
Wuhan Union Hospital (CN)
Corresponding author
Zhang, Qing
ORCID
Venue
International Journal of Molecular Medicine
Publisher
Spandidos Publications
Published
2017-05-12
Volume
40
Issue
1
Pages
31-38
ISSN
  • 1107-3756 (ISSN-L)
  • 1791-244X
DOI
10.3892/ijmm.2017.2987
PMC
PMC5466387
OpenAlex
W2613996502
Cited by
196
References
48
Citations by year
2026: 23 · 2025: 32 · 2024: 30 · 2023: 35 · 2022: 23 · 2021: 11 · 2020: 19 · 2019: 10 · 2018: 10 · 2017: 2
FWCI
4.58
Type
journal article
Language
en
Open access
Licence
cc-by-nc-nd
Locations
  • International Journal of Molecular Medicine (publishedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
National Natural Science Foundation of China (81101401)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › S100 Proteins and Annexins
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Clinical Biochemistry › Advanced Glycation End Products research
Keywords
S100A8/A9, microglia, neuroinflammation, TLR4, RAGE, NF-κB, ERK signaling, JNK signaling, TNF-α, interleukin-6, inflammatory cytokines, pyrrolidine dithiocarbamate, C225, BV-2 cells
MeSH terms
Receptor for Advanced Glycation End Products, Animals, Cell Line, Transformed, Inflammation, Signal Transduction, Cytokines, Microglia, Inflammation Mediators, Calgranulin A, Calgranulin B, Toll-Like Receptor 4, Mice

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