The calcium-sensing receptor regulates the NLRP3 inflammasome through Ca2+ and cAMP

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Abstract

Mutations in the gene encoding NLRP3 cause a spectrum of autoinflammatory diseases known as cryopyrin-associated periodic syndromes (CAPS)1. NLRP3 is a key component of one of several distinct cytoplasmic multiprotein complexes (inflammasomes) that mediate the maturation of the proinflammatory cytokine interleukin-1β (IL-1β) by activating caspase-1. Although several models for inflammasome activation, such as K+ efflux2, generation of reactive oxygen species3 and lysosomal destabilization4, have been proposed, the precise molecular mechanism of NLRP3 inflammasome activation, as well as the mechanism by which CAPS-associated mutations activate NLRP3, remain to be elucidated. Here we show that the murine calcium-sensing receptor (CASR) activates the NLRP3 inflammasome, mediated by increased intracellular Ca2+ and decreased cellular cyclic AMP (cAMP). Ca2+ or other CASR agonists activate the NLRP3 inflammasome in the absence of exogenous ATP, whereas knockdown of CASR reduces inflammasome activation in response to known NLRP3 activators. CASR activates the NLRP3 inflammasome through phospholipase C, which catalyses inositol-1,4,5-trisphosphate production and thereby induces release of Ca2+ from endoplasmic reticulum stores. The increased cytoplasmic Ca2+ promotes the assembly of inflammasome components, and intracellular Ca2+ is required for spontaneous inflammasome activity in cells from patients with CAPS. CASR stimulation also results in reduced intracellular cAMP, which independently activates the NLRP3 inflammasome. cAMP binds to NLRP3 directly to inhibit inflammasome assembly, and downregulation of cAMP relieves this inhibition. The binding affinity of cAMP for CAPS-associated mutant NLRP3 is substantially lower than for wild-type NLRP3, and the uncontrolled mature IL-1β production from CAPS patients’ peripheral blood mononuclear cells is attenuated by increasing cAMP. Taken together, these findings indicate that Ca2+ and cAMP are two key molecular regulators of the NLRP3 inflammasome that have critical roles in the molecular pathogenesis of CAPS.

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Figure 1: Extracellular calcium and ATP activate the NLRP3 inflammasome through CASR.
Figure 2: PLC-InsP 3 -mediated calcium release from the ER triggers NLRP3 inflammasome activation.
Figure 3: cAMP binds to NLRP3 and suppresses inflammasome activation.
Figure 4: The role of cAMP and calcium in the pathogenesis of CAPS.

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Acknowledgements

This work was supported by the Intramural Research Programs of the NIAMS, NHGRI, and NIAID, NIH. We thank E. Remmers for discussion and a thorough review of this manuscript.

Author information

G.-S.L., D.L.K. and J.J.C. designed the research; G.-S.L., N.S., A.I.K. and J.J.C. performed the experiments; G.-S.L., N.S., I.A., D.B.S., R.N.G., D.L.K. and J.J.C. analysed the results; R.G.-M., I.A. and D.L.K. provided patient samples; G.-S.L., J.J.C. and D.L.K. wrote the paper; N.S., I.A., R.G.-M., D.B.S. and R.N.G. edited and commented on the manuscript.

Correspondence to Daniel L. Kastner or Jae Jin Chae.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-12. (PDF 3657 kb)

ATP induces intracellular Ca2+ increase

This video shows intracellular Ca2+ levels of BMDMs treated with 1 μM ATP. Images of untreated cells were acquired (t=0), then ATP was added and cells were imaged for 30 min with acquisition at 15 sec intervals. After 30 min, ionomycin (5 μM) was added to the medium, and then BAPTA-AM (100 μM) was added. By following cells during the course of the video, it is evident that ATP induces intracellular Ca2+ increases in BMDMs. (MOV 16858 kb)

2. 2-APB blocks ATP-induced intracellular Ca2+ increase

This video shows intracellular Ca2+ levels of BMDMs treated with 50 μM 2-APB followed by treatment with 1 μM ATP. Images of cells treated with 2-APB were acquired (t=0), then ATP was added and cells were imaged for 30 min with acquisition at 15 sec intervals. After 30 min, ionomycin (5 μM) was added to the medium, and then BAPTA-AM (100 μM) was added. By following cells during the course of the video, it is evident that ATP-induced intracellular Ca2+ increases are blocked by 2-APB in BMDMs. (MOV 14264 kb)

BAPTA-AM blocks ATP-induced intracellular Ca2+ increase

This video shows intracellular Ca2+ levels of BMDMs treated with 10 μM BAPTA-AM followed by treatment with 1 μM ATP. Images of cells treated with BAPTA-AM were acquired (t=0), then ATP was added and cells were imaged for 30 min with acquisition at 15 sec intervals. After 30 min, ionomycin (5 μM) was added to the medium, and then BAPTA-AM (100 μM) was added. By following cells during the course of the video, it is evident that ATP-induced intracellular Ca2+ increases are blocked by BAPTA-AM in BMDMs. (MOV 9422 kb)

Extracellular Ca2+ induces intracellular Ca2+ increase

This video shows intracellular Ca2+ levels of BMDMs treated with 1 μM CaCl2. Images of untreated cells were acquired (t=0), then CaCl2 was added and cells were imaged for 30 min with acquisition at 15 sec intervals. After 30 min, ionomycin (5 μM) was added to the medium, and then BAPTA-AM (100 μM) was added. By following cells during the course of the video, it is evident that CaCl2 induces intracellular Ca2+ increases in BMDMs. (MOV 9577 kb)

2-APB blocks extracellular Ca2+-induced intracellular Ca2+ increase

This video shows intracellular Ca2+ levels of BMDMs treated with 50 μM 2-APB followed by treatment with 1 μM CaCl2. Images of cells treated with 2-APB were acquired (t=0), then CaCl2 was added and cells were imaged for 30 min with acquisition at 15 sec intervals. After 30 min, ionomycin (5 μM) was added to the medium, and then BAPTA-AM (100 μM) was added. By following cells during the course of the video, it is evident that CaCl2-induced intracellular Ca2+ increases are blocked by 2-APB in BMDMs. (MOV 11061 kb)

BAPTA-AM blocks extracellular Ca2+-induced intracellular Ca2+ increase

This video shows intracellular Ca2+ levels of BMDMs treated with 10 μM BAPTA-AM followed by treatment with 1 μM CaCl2. Images of cells treated with BAPTA-AM were acquired (t=0), then CaCl2 was added and cells were imaged for 30 min with acquisition at 15 sec intervals. After 30 min, ionomycin (5 μM) was added to the medium, and then BAPTA-AM (100 μM) was added. By following cells during the course of the video, it is evident that CaCl2-induced intracellular Ca2+ increases are blocked by BAPTA-AM in BMDMs. (MOV 9061 kb)

Extracellular Gd3+ induces intracellular Ca2+ increase

This video shows intracellular Ca2+ levels of BMDMs treated with 1 μM GdCl3. Images of untreated cells were acquired (t=0), then GdCl3 was added and cells were imaged for 30 min with acquisition at 15 sec intervals. After 30 min, ionomycin (5 μM) was added to the medium, and then BAPTA-AM (100 μM) was added. By following cells during the course of the video, it is evident that GdCl3 induces intracellular Ca2+ increases in BMDMs. (MOV 12425 kb)

2-APB blocks extracellular Gd3+-induced intracellular Ca2+ increase

This video shows intracellular Ca2+ levels of BMDMs treated with 50 μM 2-APB followed by treatment with 1 μM GdCl3. Images of cells treated with 2-APB were acquired (t=0), then GdCl3 was added and cells were imaged for 30 min with acquisition at 15 sec intervals. After 30 min, ionomycin (5 μM) was added to the medium, and then BAPTA-AM (100 μM) was added. By following cells during the course of the video, it is evident that GdCl3-induced intracellular Ca2+ increases are blocked by 2-APB in BMDMs. (MOV 11898 kb)

BAPTA-AM blocks extracellular Gd3+-induced intracellular Ca2+ increase

This video shows intracellular Ca2+ levels of BMDMs treated with 10 μM BAPTA-AM followed by treatment with 1 μM GdCl3. Images of cells treated with BAPTA-AM were acquired (t=0), then GdCl3 was added and cells were imaged for 30 min with acquisition at 15 sec intervals. After 30 min, ionomycin (5 μM) was added to the medium, and then BAPTA-AM (100 μM) was added. By following cells during the course of the video, it is evident that GdCl3-induced intracellular Ca2+ increases are blocked by BAPTA-AM in BMDMs. (MOV 11229 kb)

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Lee, G., Subramanian, N., Kim, A. et al. The calcium-sensing receptor regulates the NLRP3 inflammasome through Ca2+ and cAMP. Nature 492, 123–127 (2012) doi:10.1038/nature11588

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