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P2X7R is involved in the progression of atherosclerosis by promoting NLRP3 inflammasome activation.

Peng K, Liu L, Wei D, Lv Y, Wang G, Xiong W, Wang X, Altaf A, Wang L, He D, Wang H, Qu P - Int. J. Mol. Med. (2015)

Bottom Line: P2X7R knockdown by siRNA suppressed NLRP3 inflammasome activation by inhibiting the PKR phosphorylation mediated by oxLDL.In the atherosclerotic lesions in the aortic sinuses of apoE(-/-) mice, P2X7R expression was found at high levels.In conclusion, our results demonstrate that P2X7R plays a significant role in the development of atherosclerosis and regulates NLRP3 inflammasome activation by promoting PKR phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, The Second Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116023, P.R. China.

ABSTRACT
Purinergic 2X7 receptor (P2X7R) and nucleotide‑binding oligomerization domain-like receptor protein 3 (NLRP3) are expressed in macrophages in atherosclerotic lesions. However, the mechanisms through which P2X7R participates in the inflammatory response in atherosclerosis remain largely unknown. The aim of the present study was to investigate the role of P2X7R in atherosclerosis and the mechanisms of action of the NLRP3 inflammasome following stimulation with oxidized low-density lipoprotein (oxLDL). We observed the expression and distribution of P2X7R in the atherosclerotic plaque in the coronary arteries from an autopsy specimen and in that of the aortic sinuses of apoE(-/-) mice by immunohistochemistry and immunofluorescence staining. The specificity of short interfering RNA (siRNA) was used to suppress P2X7R and NLRP3 mRNA expression. RT-qPCR and western blot analysis were used to analyze mRNA and protein expression, respectively. Co-immunoprecipitation was used to examine the interaction between protein kinase R (PKR) phosphorylation and NLRP3. P2X7R and NLRP3 were expressed at high levels in the atherosclerotic plaque in the coronary arteries. Stimulation with oxLDL upregulated P2X7R, NLRP3 and interleukin (IL)-1β expression. P2X7R knockdown by siRNA suppressed NLRP3 inflammasome activation by inhibiting the PKR phosphorylation mediated by oxLDL. In the atherosclerotic lesions in the aortic sinuses of apoE(-/-) mice, P2X7R expression was found at high levels. Moreover, P2X7R siRNA attenuated the development of atherosclerosis in the apoE(-/-) mice. In conclusion, our results demonstrate that P2X7R plays a significant role in the development of atherosclerosis and regulates NLRP3 inflammasome activation by promoting PKR phosphorylation.

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Effect of purinergic 2X7 receptor (P2X7R) on THP-1 macrophage production and secretion of interleukin-1β (IL-1β). (A) Effect of P2X7R on IL-1β concentration in medium secreted by THP-1 macrophages. P2X7R knockdown by short interfering RNA (siRNA) significantly reduced the IL-1β concentration in medium compared with the control group (P<0.01). (B) Effect of P2X7R on IL-1β mRNA expression in THP-1 macrophages. (C) Effect of P2X7R on THP-1 macrophage nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) mRNA expression in THP-1 macrophages. (D) oxLDL promoted production of IL-1β by THP-1 macrophages, and this was related to the NLRP3 inflammasome activation. (E) Effect of P2X7R on proIL-1β expression in and mature IL-1β production by THP-1 macrophages. (F) Effect of P2X7R on NLRP3 expression in THP-1 macrophages. NC, negative control.
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f6-ijmm-35-05-1179: Effect of purinergic 2X7 receptor (P2X7R) on THP-1 macrophage production and secretion of interleukin-1β (IL-1β). (A) Effect of P2X7R on IL-1β concentration in medium secreted by THP-1 macrophages. P2X7R knockdown by short interfering RNA (siRNA) significantly reduced the IL-1β concentration in medium compared with the control group (P<0.01). (B) Effect of P2X7R on IL-1β mRNA expression in THP-1 macrophages. (C) Effect of P2X7R on THP-1 macrophage nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) mRNA expression in THP-1 macrophages. (D) oxLDL promoted production of IL-1β by THP-1 macrophages, and this was related to the NLRP3 inflammasome activation. (E) Effect of P2X7R on proIL-1β expression in and mature IL-1β production by THP-1 macrophages. (F) Effect of P2X7R on NLRP3 expression in THP-1 macrophages. NC, negative control.

Mentions: The IL-1β mRNA and proIL-1β protein expression levels in the oxLDL-stimulated macrophages did not differ between the negative control group and the P2X7R siRNA-treated group (Fig. 6B and E). However, proIL-1β activity was significantly suppressed (Fig. 6E). The concentration of IL-1β in the medium was also reduced due to the suppression of P2X7R (Fig. 6A), since proIL-1β hydrolysis to mature IL-1β is controlled by NLRP3 inflammasome activation. After NLRP3 expression was suppressed using siRNA, mature IL-1β expression in the oxLDL-stimulated macrophages was significantly lower than that in the negative controls, while proIL-1β expression was unaltered (Fig. 6D). However, NLRP3 expression in the macrophages in which P2X7R was knocked down following oxLDL stimulation did not differ from that observed in the negative control group (Fig. 6C and F). These results indicate that, upon oxLDL stimulation, the expression of P2X7R in macrophages regulates NLRP3 inflammasome function, but not NLRP3 expression.


P2X7R is involved in the progression of atherosclerosis by promoting NLRP3 inflammasome activation.

Peng K, Liu L, Wei D, Lv Y, Wang G, Xiong W, Wang X, Altaf A, Wang L, He D, Wang H, Qu P - Int. J. Mol. Med. (2015)

Effect of purinergic 2X7 receptor (P2X7R) on THP-1 macrophage production and secretion of interleukin-1β (IL-1β). (A) Effect of P2X7R on IL-1β concentration in medium secreted by THP-1 macrophages. P2X7R knockdown by short interfering RNA (siRNA) significantly reduced the IL-1β concentration in medium compared with the control group (P<0.01). (B) Effect of P2X7R on IL-1β mRNA expression in THP-1 macrophages. (C) Effect of P2X7R on THP-1 macrophage nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) mRNA expression in THP-1 macrophages. (D) oxLDL promoted production of IL-1β by THP-1 macrophages, and this was related to the NLRP3 inflammasome activation. (E) Effect of P2X7R on proIL-1β expression in and mature IL-1β production by THP-1 macrophages. (F) Effect of P2X7R on NLRP3 expression in THP-1 macrophages. NC, negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380202&req=5

f6-ijmm-35-05-1179: Effect of purinergic 2X7 receptor (P2X7R) on THP-1 macrophage production and secretion of interleukin-1β (IL-1β). (A) Effect of P2X7R on IL-1β concentration in medium secreted by THP-1 macrophages. P2X7R knockdown by short interfering RNA (siRNA) significantly reduced the IL-1β concentration in medium compared with the control group (P<0.01). (B) Effect of P2X7R on IL-1β mRNA expression in THP-1 macrophages. (C) Effect of P2X7R on THP-1 macrophage nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) mRNA expression in THP-1 macrophages. (D) oxLDL promoted production of IL-1β by THP-1 macrophages, and this was related to the NLRP3 inflammasome activation. (E) Effect of P2X7R on proIL-1β expression in and mature IL-1β production by THP-1 macrophages. (F) Effect of P2X7R on NLRP3 expression in THP-1 macrophages. NC, negative control.
Mentions: The IL-1β mRNA and proIL-1β protein expression levels in the oxLDL-stimulated macrophages did not differ between the negative control group and the P2X7R siRNA-treated group (Fig. 6B and E). However, proIL-1β activity was significantly suppressed (Fig. 6E). The concentration of IL-1β in the medium was also reduced due to the suppression of P2X7R (Fig. 6A), since proIL-1β hydrolysis to mature IL-1β is controlled by NLRP3 inflammasome activation. After NLRP3 expression was suppressed using siRNA, mature IL-1β expression in the oxLDL-stimulated macrophages was significantly lower than that in the negative controls, while proIL-1β expression was unaltered (Fig. 6D). However, NLRP3 expression in the macrophages in which P2X7R was knocked down following oxLDL stimulation did not differ from that observed in the negative control group (Fig. 6C and F). These results indicate that, upon oxLDL stimulation, the expression of P2X7R in macrophages regulates NLRP3 inflammasome function, but not NLRP3 expression.

Bottom Line: P2X7R knockdown by siRNA suppressed NLRP3 inflammasome activation by inhibiting the PKR phosphorylation mediated by oxLDL.In the atherosclerotic lesions in the aortic sinuses of apoE(-/-) mice, P2X7R expression was found at high levels.In conclusion, our results demonstrate that P2X7R plays a significant role in the development of atherosclerosis and regulates NLRP3 inflammasome activation by promoting PKR phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, The Second Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116023, P.R. China.

ABSTRACT
Purinergic 2X7 receptor (P2X7R) and nucleotide‑binding oligomerization domain-like receptor protein 3 (NLRP3) are expressed in macrophages in atherosclerotic lesions. However, the mechanisms through which P2X7R participates in the inflammatory response in atherosclerosis remain largely unknown. The aim of the present study was to investigate the role of P2X7R in atherosclerosis and the mechanisms of action of the NLRP3 inflammasome following stimulation with oxidized low-density lipoprotein (oxLDL). We observed the expression and distribution of P2X7R in the atherosclerotic plaque in the coronary arteries from an autopsy specimen and in that of the aortic sinuses of apoE(-/-) mice by immunohistochemistry and immunofluorescence staining. The specificity of short interfering RNA (siRNA) was used to suppress P2X7R and NLRP3 mRNA expression. RT-qPCR and western blot analysis were used to analyze mRNA and protein expression, respectively. Co-immunoprecipitation was used to examine the interaction between protein kinase R (PKR) phosphorylation and NLRP3. P2X7R and NLRP3 were expressed at high levels in the atherosclerotic plaque in the coronary arteries. Stimulation with oxLDL upregulated P2X7R, NLRP3 and interleukin (IL)-1β expression. P2X7R knockdown by siRNA suppressed NLRP3 inflammasome activation by inhibiting the PKR phosphorylation mediated by oxLDL. In the atherosclerotic lesions in the aortic sinuses of apoE(-/-) mice, P2X7R expression was found at high levels. Moreover, P2X7R siRNA attenuated the development of atherosclerosis in the apoE(-/-) mice. In conclusion, our results demonstrate that P2X7R plays a significant role in the development of atherosclerosis and regulates NLRP3 inflammasome activation by promoting PKR phosphorylation.

Show MeSH
Related in: MedlinePlus