Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

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

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

References

  1. Masters, S. L., Simon, A., Aksentijevich, I. & Kastner, D. L. Horror autoinflammaticus: the molecular pathophysiology of autoinflammatory disease. Annu. Rev. Immunol. 27, 621–668 (2009)

    Article  CAS  Google Scholar 

  2. Kahlenberg, J. M. & Dubyak, G. R. Mechanisms of caspase-1 activation by P2X7 receptor-mediated K+ release. Am. J. Physiol. Cell Physiol. 286, C1100–C1108 (2004)

    Article  CAS  Google Scholar 

  3. Zhou, R., Tardivel, A., Thorens, B., Choi, I. & Tschopp, J. Thioredoxin-interacting protein links oxidative stress to inflammasome activation. Nature Immunol. 11, 136–140 (2010)

    Article  CAS  Google Scholar 

  4. Hornung, V. et al. Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nature Immunol. 9, 847–856 (2008)

    Article  CAS  Google Scholar 

  5. Pétrilli, V. et al. Activation of the NALP3 inflammasome is triggered by low intracellular potassium concentration. Cell Death Differ. 14, 1583–1589 (2007)

    Article  Google Scholar 

  6. Brough, D. et al. Ca2+ stores and Ca2+ entry differentially contribute to the release of IL-1β and IL-1α from murine macrophages. J. Immunol. 170, 3029–3036 (2003)

    Article  CAS  Google Scholar 

  7. Cockcroft, S. & Gomperts, B. D. Activation and inhibition of calcium-dependent histamine secretion by ATP ions applied to rat mast cells. J. Physiol. (Lond.) 296, 229–243 (1979)

    Article  CAS  Google Scholar 

  8. Ross, P. E., Ehring, G. R. & Cahalan, M. D. Dynamics of ATP-induced calcium signaling in single mouse thymocytes. J. Cell Biol. 138, 987–998 (1997)

    Article  CAS  Google Scholar 

  9. Murakami, T. et al. Critical role for calcium mobilization in activation of the NLRP3 inflammasome. Proc. Natl. Acad. Sci. USA 109, 11282–11287 (2012)

    Article  ADS  CAS  Google Scholar 

  10. Hofer, A. M. & Brown, E. M. Extracellular calcium sensing and signalling. Nature Rev. Mol. Cell Biol. 4, 530–538 (2003)

    Article  CAS  Google Scholar 

  11. Xi, Y. H. et al. The functional expression of calcium-sensing receptor in the differentiated THP-1 cells. Mol. Cell. Biochem. 342, 233–240 (2010)

    Article  CAS  Google Scholar 

  12. Osawa, Y., Lee, H. T., Hirshman, C. A., Xu, D. & Emala, C. W. Lipopolysaccharide-induced sensitization of adenylyl cyclase activity in murine macrophages. Am. J. Physiol. Cell Physiol. 290, C143–C151 (2006)

    Article  CAS  Google Scholar 

  13. Duncan, J. A. et al. Cryopyrin/NALP3 binds ATP/dATP, is an ATPase, and requires ATP binding to mediate inflammatory signaling. Proc. Natl Acad. Sci. USA 104, 8041–8046 (2007)

    Article  ADS  CAS  Google Scholar 

  14. Gattorno, M. et al. Pattern of interleukin-1β secretion in response to lipopolysaccharide and ATP before and after interleukin-1 blockade in patients with CIAS1 mutations. Arthritis Rheum. 56, 3138–3148 (2007)

    Article  CAS  Google Scholar 

  15. Chae, J. J. et al. Gain-of-function pyrin mutations induce NLRP3 protein-independent interleukin-1β activation and severe autoinflammation in mice. Immunity 34, 755–768 (2011)

    Article  CAS  Google Scholar 

  16. Menkin, V. Biochemical Mechanisms in Inflammation (Charles Thomas Publisher, 1981)

    Google Scholar 

  17. Peters-Golden, M. Putting on the brakes: cyclic AMP as a multipronged controller of macrophage function. Sci. Signal. 2, pe37 (2009)

    Article  Google Scholar 

  18. Torphy, T. J. Phosphodiesterase isozymes: molecular targets for novel antiasthma agents. Am. J. Respir. Crit. Care Med. 157, 351–370 (1998)

    Article  CAS  Google Scholar 

  19. Kim, C., Cheng, C. Y., Saldanha, S. A. & Taylor, S. S. PKA-I holoenzyme structure reveals a mechanism for cAMP-dependent activation. Cell 130, 1032–1043 (2007)

    Article  CAS  Google Scholar 

  20. Bos, J. L. Epac: a new cAMP target and new avenues in cAMP research. Nature Rev. Mol. Cell Biol. 4, 733–738 (2003)

    Article  CAS  Google Scholar 

  21. Martinon, F., Petrilli, V., Mayor, A., Tardivel, A. & Tschopp, J. Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 440, 237–241 (2006)

    Article  ADS  CAS  Google Scholar 

  22. Vandanmagsar, B. et al. The NLRP3 inflammasome instigates obesity-induced inflammation and insulin resistance. Nature Med. 17, 179–188 (2011)

    Article  CAS  Google Scholar 

  23. Duewell, P. et al. NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature 464, 1357–1361 (2010)

    Article  ADS  CAS  Google Scholar 

  24. Halle, A. et al. The NALP3 inflammasome is involved in the innate immune response to amyloid-β. Nature Immunol. 9, 857–865 (2008)

    Article  CAS  Google Scholar 

Download references

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

Authors and Affiliations

Authors

Contributions

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.

Corresponding authors

Correspondence to Daniel L. Kastner or Jae Jin Chae.

Ethics declarations

Competing interests

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)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature11588

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing