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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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Oxidized low-density lipoprotein (oxLDL) promotes THP-1 macrophage production and the release of interleukin-1β (IL-1β) by activating the purinergic 2X7 receptor (P2X7R)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) pathway. (A) IL-1β concentration in medium after THP-1 cells were stimulated with different concentrations of oxLDL. (B) IL-1β mRNA expression after THP-1 cells were stimulated with different concentrations of oxLDL. (C) IL-1β expression after THP-1 cells were stimulated with different concentrations of oxLDL. (D) IL-1β concentration in medium at different time points after THP-1 cells were stimulated with 100 μg/ml oxLDL. (E) IL-1β mRNA expression at different time points after THP-1 cells were stimulated with 100 μg/ml oxLDL. (F) IL-1β expression at different time points after THP-1 cells were stimulated with 100 μg/ml oxLDL. (G and H) Effect of oxLDL on NLRP3 expression in THP-1 macrophages. (I and J) Effect of oxLDL on P2X7R expression in THP-1 macrophages.
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f5-ijmm-35-05-1179: Oxidized low-density lipoprotein (oxLDL) promotes THP-1 macrophage production and the release of interleukin-1β (IL-1β) by activating the purinergic 2X7 receptor (P2X7R)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) pathway. (A) IL-1β concentration in medium after THP-1 cells were stimulated with different concentrations of oxLDL. (B) IL-1β mRNA expression after THP-1 cells were stimulated with different concentrations of oxLDL. (C) IL-1β expression after THP-1 cells were stimulated with different concentrations of oxLDL. (D) IL-1β concentration in medium at different time points after THP-1 cells were stimulated with 100 μg/ml oxLDL. (E) IL-1β mRNA expression at different time points after THP-1 cells were stimulated with 100 μg/ml oxLDL. (F) IL-1β expression at different time points after THP-1 cells were stimulated with 100 μg/ml oxLDL. (G and H) Effect of oxLDL on NLRP3 expression in THP-1 macrophages. (I and J) Effect of oxLDL on P2X7R expression in THP-1 macrophages.

Mentions: To demonstrate that oxLDL upregulates P2X7R and NLRP3 in THP-1 macrophages and promotes the secretion of IL-1β, the THP-1 macrophages were stimulated with 25, 50, 100 and 200 μg/ml oxLDL for 24 h. The IL-1β concentrations were significantly higher in the oxLDL-treated groups compared to the control group (Fig. 5A). The cytoplasmic proIL-1β mRNA and protein levels, as well as the mature IL-1β protein levels, were increased in the THP-1 macrophages treated with oxLDL (Fig. 5B and C). A concentration of 100 μg/ml of oxLDL was selected to treat the THP-1 macrophages for 6, 12, 24 and 48 h to examine the IL-1β expression and secretion. Following treatment with 100 μg/ml oxLDL for 24 h, the expression of mature IL-1β and proIL-1β increased compared to the control group (Fig. 5E and F). IL-1β medium concentration was also significantly increased (Fig. 5D). These results indicated that oxLDL upregulated IL-1β expression in THP-1 macrophages and promoted proIL-1β hydrolysis to activate IL-1β. oxLDL also upregulated the the mRNA and protein expression levels of P2X7R and NLRP3 in the THP-1 macrophages (Fig. 5G–J).


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)

Oxidized low-density lipoprotein (oxLDL) promotes THP-1 macrophage production and the release of interleukin-1β (IL-1β) by activating the purinergic 2X7 receptor (P2X7R)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) pathway. (A) IL-1β concentration in medium after THP-1 cells were stimulated with different concentrations of oxLDL. (B) IL-1β mRNA expression after THP-1 cells were stimulated with different concentrations of oxLDL. (C) IL-1β expression after THP-1 cells were stimulated with different concentrations of oxLDL. (D) IL-1β concentration in medium at different time points after THP-1 cells were stimulated with 100 μg/ml oxLDL. (E) IL-1β mRNA expression at different time points after THP-1 cells were stimulated with 100 μg/ml oxLDL. (F) IL-1β expression at different time points after THP-1 cells were stimulated with 100 μg/ml oxLDL. (G and H) Effect of oxLDL on NLRP3 expression in THP-1 macrophages. (I and J) Effect of oxLDL on P2X7R expression in THP-1 macrophages.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5-ijmm-35-05-1179: Oxidized low-density lipoprotein (oxLDL) promotes THP-1 macrophage production and the release of interleukin-1β (IL-1β) by activating the purinergic 2X7 receptor (P2X7R)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) pathway. (A) IL-1β concentration in medium after THP-1 cells were stimulated with different concentrations of oxLDL. (B) IL-1β mRNA expression after THP-1 cells were stimulated with different concentrations of oxLDL. (C) IL-1β expression after THP-1 cells were stimulated with different concentrations of oxLDL. (D) IL-1β concentration in medium at different time points after THP-1 cells were stimulated with 100 μg/ml oxLDL. (E) IL-1β mRNA expression at different time points after THP-1 cells were stimulated with 100 μg/ml oxLDL. (F) IL-1β expression at different time points after THP-1 cells were stimulated with 100 μg/ml oxLDL. (G and H) Effect of oxLDL on NLRP3 expression in THP-1 macrophages. (I and J) Effect of oxLDL on P2X7R expression in THP-1 macrophages.
Mentions: To demonstrate that oxLDL upregulates P2X7R and NLRP3 in THP-1 macrophages and promotes the secretion of IL-1β, the THP-1 macrophages were stimulated with 25, 50, 100 and 200 μg/ml oxLDL for 24 h. The IL-1β concentrations were significantly higher in the oxLDL-treated groups compared to the control group (Fig. 5A). The cytoplasmic proIL-1β mRNA and protein levels, as well as the mature IL-1β protein levels, were increased in the THP-1 macrophages treated with oxLDL (Fig. 5B and C). A concentration of 100 μg/ml of oxLDL was selected to treat the THP-1 macrophages for 6, 12, 24 and 48 h to examine the IL-1β expression and secretion. Following treatment with 100 μg/ml oxLDL for 24 h, the expression of mature IL-1β and proIL-1β increased compared to the control group (Fig. 5E and F). IL-1β medium concentration was also significantly increased (Fig. 5D). These results indicated that oxLDL upregulated IL-1β expression in THP-1 macrophages and promoted proIL-1β hydrolysis to activate IL-1β. oxLDL also upregulated the the mRNA and protein expression levels of P2X7R and NLRP3 in the THP-1 macrophages (Fig. 5G–J).

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