Dysfunction of cholesterol sensor SCAP promotes inflammasome activation by up-regulating P2X7R in THP-1 macrophages

Shasha WU; Nan OUYANG; Quan HE; Chao ZHOU

doi:10.16016/j.1000-5404.201610063

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Dysfunction of cholesterol sensor SCAP promotes inflammasome activation by up-regulating P2X7R in THP-1 macrophages

Basic Medicine | 更新时间：2026-01-09

- Dysfunction of cholesterol sensor SCAP promotes inflammasome activation by up-regulating P2X7R in THP-1 macrophages
- Journal of Third Military Medical University Vol. 39, Issue 13, Pages: 1327-1332(2017)
- 作者机构：
  
  1.400016 重庆，重庆医科大学附属第一医院, 心血管内科
  2.400016 重庆，重庆医科大学附属第一医院, 肾脏内科
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(81500341)
- DOI：10.16016/j.1000-5404.201610063
  CLC： R392.12;R392.32;R394.3
- Received：06 October 2016，
  
  Revised：2017-01-19，
  
  Published：15 July 2017
- 稿件说明：
移动端阅览
Shasha WU, Nan OUYANG, Quan HE, et al. Dysfunction of cholesterol sensor SCAP promotes inflammasome activation by up-regulating P2X7R in THP-1 macrophages[J]. Journal of Third Military Medical University, 2017, 39(13): 1327-1332.
DOI：

Shasha WU, Nan OUYANG, Quan HE, et al. Dysfunction of cholesterol sensor SCAP promotes inflammasome activation by up-regulating P2X7R in THP-1 macrophages[J]. Journal of Third Military Medical University, 2017, 39(13): 1327-1332. DOI： 10.16016/j.1000-5404.201610063.

摘要

目的

探讨胆固醇调节原件结合蛋白裂解激活蛋白(sterol regulatory element binding proteins cleavage-activating protein，SCAP)功能失调促进THP-1源性巨噬细胞炎症小体活化及IL-1β成熟的分子机制。

方法

采用基因转染方法在THP-1源性巨噬细胞中过表达SCAP，结合使用P2X7受体(P2X7 receptor，P2X7R)激动剂ATP(5 mmol/L)或抑制剂A438079 (100 μmol/L)处理THP-1源性巨噬细胞，将细胞分为：① 对照组、② ATP处理组、③ A438079处理组、④ ATP+A438079组、⑤ 过表达SCAP组、⑥ 过表达SCAP+ATP组、⑦ 过表达SCAP+A438079组、⑧ 过表达SCAP+ATP+A438079组。RT-PCR检测过表达SCAP以及激动或抑制P2X7R对P2X7R、pro-IL-1β、NLRP3、pro-caspase-1基因表达水平的影响。流式细胞术检测细胞表面P2X7R的表达。Western blot检测SCAP、pro-IL-1β、NLRP3、caspase-1(p20) 以及培养上清中IL-1β的蛋白水平。同一实验在细胞水平重复4次。

结果

① 过表达SCAP组与对照组比较，其pro-IL-1β、P2X7R基因表达水平显著升高(

0.01)，细胞表面P2X7R表达明显上调。② P2X7R激动剂ATP与抑制剂A438079对THP-1源性巨噬细胞SCAP及P2X7R mRNA表达无影响(

0.05)。P2X7R激动剂ATP能够显著促进THP-1源性巨噬细胞pro-IL-1β mRNA表达(

0.05)，在过表达SCAP基础上激动P2X7R能够进一步激活pro-IL-1β基因转录(

0.01)，同时显著促进细胞内pro-IL-1β剪切成熟并向细胞外分泌(

0.01)，抑制P2X7R活性并不影响过表达SCAP致pro-IL-1β表达上调的作用，但将减少上清中成熟IL-1β的含量。过表达SCAP并不影响NLRP3及caspase-1表达(

0.05)，但在过表达SCAP基础上充分激动P2X7R将显著上调N

LRP3及caspase-1基因及蛋白水平(

0.01)，上述变化能够被P2X7R抑制剂A438079抵消。

结论

胆固醇敏感器SCAP功能失调能够促进THP-1源性巨噬细胞内pro-IL-1β表达，同时通过上调细胞表面P2X7R表达激活NLRP3炎症小体，促进pro-IL-1β成熟及其向细胞外分泌。

Abstract

Objective

To investigate the molecular mechanism of inflammasome activation and IL-1β maturation promoted by sterol regulatory element binding proteins cleavage-activating protein (SCAP) dysfunction in THP-1 macrophages.

Methods

Full length SCAP over-expression plasmid pCMV-scap was transfected into THP-1 macrophages. THP-1 macrophages were treated with P2X7 receptor (P2X7R) agonist ATP (5 mmol/L) or antagonist A438079 (100μmol/L). Thus

the THP-1 macrophages were divided into control group

ATP group

A438079 group

SCAP over-expression group

SCAP over-expression plus ATP group

SCAP over-expression plus A438079 group

and SCAP over-expression plus both ATP and A438079 group. The mRNA expression levels of P2X7R

pro-IL-1β

NLRP3 and pro-caspase-1 were investigated by real time-PCR. The expression of P2X7R on the cell surface was detected by flow cytometry. The protein expression and the mature IL-1β level in the supernatants were estimated by Western blotting. The experiments were repeated for 4 times at the cellular level.

Results

① Over-expression of SCAP significantly increased the expression of pro-IL-1β and P2X7R at mRNA level (

0.01)

and enhanced the P2X7R protein expression on the cell surface. ② The agonist ATP or the antagonist A438079 of P2X7R did not affect the mRNA expression levels of SCAP and P2X7R in THP-1 macrophages (

0.05). The agonist ATP could promote the mRNA level of pro-IL-1β (

0.05)

which would be further enhanced when combined with over-expression of SCAP (

0.05)

and the combination also improved the cleavage

maturation a

nd secretion of intracellular pro-IL-1β (

0.01)

while suppressing the activity of P2X7R by A438079 had no effect on the up-regulation of pro-IL-1β induced by SCAP over-expression

but decreased the production of mature IL-1β in the supernatant. ③ Though over-expression of SCAP showed no effect on the expression of NLRP3 and caspase-1 at mRNA and protein levels (

0.05)

it combined with P2X7R agonist ATP up-regulated the expression of the 2 molecules dramatically (

0.01). The up-regulating effects of SCAP over-expression on NLRP3 and caspase-1 above were reconciled by the treatment with P2X7R antagonist A438079.

Conclusion

The dysfunction of cholesterol sensor SCAP increases pro-IL-1β production

promotes NLRP3 inflammasome activation

via

up-regulating P2X7R

and then facilitates the maturation and secretion of pro-IL-1β in THP-1 macrophages.

关键词

Keywords

references

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