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SREBF2 triggers endoplasmic reticulum stress and Bax dysregulation to promote lipopolysaccharide-induced endothelial cell injury

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Abstract

An increased lipopolysaccharide (LPS) level in patients with cirrhosis induced the dysregulation of sterol regulatory element–binding transcription factor 2 (SREBF2), which participated in the modulation of tumor inflammatory microenvironment. However, the role of SREBF2 in the LPS-induced injury of portal vein endothelium was scarcely reported. This study aimed to investigate the effects of SREBF2 on the LPS-induced injury to endothelial cells (ECs) in vitro and in vivo and explore the underlying mechanism. In this study, we found that LPS increased SREBF2 expression through activating the TLR4/JNK/c-Jun pathway and suppressed UBE2I-mediated SREBF2 sumoylation to enhance its transcriptional activity. The dysregulation of SREBF2 induced ER stress by increasing the intracellular cholesterol level and facilitated Bax expression to cause additional damage to LPS-induced ECs. As a potential intervention, miR590-3p negatively regulated SREBF2 expression and upregulated UBE2I expression by targeting TLR4, thus alleviating LPS-induced injury. These results suggest that LPS-induced SREBF2 triggered ER stress and promoted Bax expression to injure ECs, which was reversed by miR590-3p. The mechanisms of SREBF2 mediated LPS-induced endothelial injury of portal vein, which might be the therapeutic target for PVT development in cirrhosis patients.

Graphical abstract

1. LPS promoted SREBF2 expression by activating the TLR4/JNK/c-Jun pathway and suppressed UBE2I-mediated SREBF2 sumoylation to upregulate SREBF2 transcriptional activity

2. SREBF2-mediated ER stress and Bax expression involved in LPS-induced EC injury

3. miR590-3p decreased SREBF2 expression by targeting TLR4 and mitigated LPS-induced EC injury

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Funding

This work was supported by the Shanghai Sailing Program (No. 19YF1406500) and partly supported by the National Natural Science Foundation of China (No. 81900511) and Innovation Fund of Science and Technology Commission of Shanghai Municipality (No. 19411970200).

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Author notes

  1. Gang Dong and Xiaoquan Huang contributed equally to this work.

Authors and Affiliations

  1. Department of Gastroenterology and Hepatology, Zhongshan Hospital, Fudan University, Shanghai, 200032, China

    Gang Dong, Xiaoquan Huang, Ling Wu, Siyu Jiang, Qintian Tan & Shiyao Chen

  2. Center of Evidence-Based Medicine, Fudan University, Shanghai, 200032, People’s Republic of China

    Xiaoquan Huang & Shiyao Chen

  3. Endoscopy Center and Endoscopy Research Institute, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, People’s Republic of China

    Shiyao Chen

Authors
  1. Gang Dong
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  2. Xiaoquan Huang
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  3. Ling Wu
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  4. Siyu Jiang
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Contributions

G.D., X.-Q.H., and S.-Y.C. developed and designed the study concept. G.D. and X.-Q.H. analyzed in vitro experimental data and drafted this article. L.W., S.-Y.J., and Q.-T.T. performed animal experiments and analyzed the data. S.-Y.C. supervised the research and provided critical review and revised version of this manuscript.

Corresponding author

Correspondence to Shiyao Chen.

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The ethics committee of Zhongshan Hospital of Fudan University (Shanghai, China) approved all animal experiments.

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Supplementary Information

Figure S1

SREBF2 interacts with UBE2I or SUMO-1. (A-B) Co-IP assays were used to verify these interactions before and after LPS treatment. (C) Dual-luciferase reporter assays performed in LPS-treated ECs transfected with WT plasmid containing SREBF2-binding sites in the HMGCR promoter using Lipofectamine 2000 after the overexpression of Sumo-1. *P < 0.05, **P < 0.01. (TIF 816 kb)

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Figure S2

Effects of LPS on SCAP intracellular translocation. (A) Confocal microscopy was used to detect SCAP intracellular translocation by staining with the antibody SCAP (red) and GM-130 antibody (Golgi, green). (TIF 19030 kb)

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Figure S3

Effects of LPS on SREBF1expression. (A) SREBF1 expression determined by RT-PCR or (B-C) Western blot. *P < 0.05. (TIF 749 kb)

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Figure S4

The activation of SREBF2 pathway in cells cultured with FCS-free medium treated by LPS. (A-B) SREBF2, HMGCR and LDLR expression determined by Western blot. *P < 0.05, **P < 0.01. (TIF 19248 kb)

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Figure S5

mRNA and protein levels of SREBF2 target genes such as LDLR and HMGCR after c-Jun overexpression. (A-B) Protein levels of LDLR and HMGCR after c-Jun overexpression were detected by Western blot. (C) mRNA levels of LDLR and HMGCR after c-Jun overexpression were detected by qRT-RCR. *P < 0.05, **P < 0.01. (TIF 18999 kb)

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Figure S6

mRNA levels of UBE2I after LPS treatment detected by qRT-RCR. *P < 0.05. (TIFF 128 kb)

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Figure S7

TLR4 and miR590-3p expression in control and LPS-treated cells. RT-qPCR analysis of TLR4 and miR590-3p in control and LPS-treated cells. *P < 0.05, **P < 0.01. (TIF 19049 kb)

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Figure S8

Quantitative analysis of western blot bands from Figure 1, 2, 3, 4, 5, 6, 7 and 8. (A) Quantitative analysis of western blot bands Figure 1. (B-E) Quantitative analysis of western blot bands Figure 2. (F-J) Quantitative analysis of western blot bands Figure 3. (K-P) Quantitative analysis of western blot bands Figure 4. (Q-T) Quantitative analysis of western blot bands Figure 5. (U-V) Quantitative analysis of western blot bands Figure 6. (W-X) Quantitative analysis of western blot bands Figure 7. (Y-b) Quantitative analysis of western blot bands Figure 8. *P < 0.05, **P < 0.01. (TIF 19291 kb)

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Figure S9

Quantitative analysis of ECs apoptosis induced by various treatment measured by TUNEL assay (from Figure 4-Figure 8). (A-E) Quantitative analysis of ECs apoptosis in Figure 4. (F) Quantitative analysis of ECs apoptosis in Figure 5. (G-H) Quantitative analysis of ECs apoptosis in Figure 6. (I) Quantitative analysis of ECs apoptosis in Figure 7. (J-K) Quantitative analysis of ECs apoptosis in Figure 8. *P < 0.05, **P < 0.01. (TIF 19123 kb)

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Figure S10

Effects of SREBF2 on LPS-induced HUVECs injury. (A-B) SREBF2 and SCAP expression in LPS-treated (100ng/ml) HUVECs for 24 hour detected by Western blot. (C-D) Apoptosis of HUVECs (green) was measured by TUNEL assay. Nuclei were counterstained into blue. *P < 0.05, **P < 0.01. (TIF 19145 kb)

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Dong, G., Huang, X., Wu, L. et al. SREBF2 triggers endoplasmic reticulum stress and Bax dysregulation to promote lipopolysaccharide-induced endothelial cell injury. Cell Biol Toxicol 38, 185–201 (2022). https://doi.org/10.1007/s10565-021-09593-1

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