Background & Aims
Store-operated calcium channels (SOCs) play a role in various calcium dependent cellular functions. It is well known that SOCs can be activated by Gq-protein coupled receptors through IP3-mediated calcium release. We have demonstrated that neuronal SOCs play an important role in inflammatory pain and that the SOC family is expressed in spinal astrocytes [1, 2, 3, 4]. However, the contribution of astrocytic SOCs to chronic pain remain elusive. The P2X7 receptor (P2X7R), belonging to the ATP-gated cation channel family, is crucial in chronic pain associated with inflammation and nerve injury. Nevertheless, the mechanisms underlying P2X7R involvement in chronic pain remain unclear.
Methods
Calcium imaging recordings were performed to evaluate the effect of SOCs on P2X7R-mediated calcium response. ELISA was conducted to measure P2X7R-mediated cytokine production. Hargreaves’ and von Frey hair tests were performed to measure complete Freund’s adjuvant (CFA)-induced thermal and mechanical sensitivities. Immunohistochemistry was used to examine co-localization of P2X7R and the endoplasmic reticulum (ER) marker. Western blot analysis was conducted to examine activation of mitogen-activated protein kinases.
Results
We observed that P2X7R activation stimulates the production of IL-6 in spinal astrocytes, which is eliminated in Orai1 knockdown or knockout astrocytes. A combination of knockout STIM2 and knockdown of STIM1 largely reduces IL-6 production. Western blot analysis reveals that activation of P2X7 increases the phosphorylation of ERK, p38 and JNK, which is abolished in Orai1 deficiency astrocytes. Calcium imaging results demonstrate that activation of P2X7 leads to both calcium release from the ER calcium stores and extracellular calcium influx. Our immunostaining data show that P2X7R is co-expressed with the ER marker. Using the complete Freund’s adjuvant (CFA) model, we observed a significant reduction in mechanical and thermal hypersensitivity two weeks of CFA injection in Orai1 conditional KO mice. Western blot results reveal that spinal Orai1 is increased in CFA-injected mice and deletion of astrocytic Orai1 reduces CFA-induced the increase of p-ERK, p-JNK, IL-6 and TNF-?.
Conclusions
Our data demonstrate a functional link between SOCs and P2X7R. This study provides novel insights into P2X7-mediated inflammatory pain, potentially paving the way for new avenue to treat inflammatory diseases.
References
1. Gao X, Gao R, Tian Y, McGonigle P, Barrett JE, Dai Y and Hu H*. A store-operated calcium channel inhibitor attenuates collagen-induced arthritis channels in rheumatoid arthritis. British J Pharmacol. 2015, 172: 2991-3002.
2. Gao X, Xia J, Munoz FM, Manners MT, Pan R, Meucci O, Dai Y, Hu H*. STIMs and Orai1 regulate cytokine production in spinal astrocytes. J Neuroinflammation. 2016, 13(1):126.
3. Dou Y, Xia J, Gao R, Gao X, Munoz FM, Wei D, Tian Y, Barrett JE, Ajit S, Meucci O, Putney JW, Dai Y, and Hu H*. Orai1 plays a crucial role in central sensitization by modulating neuronal excitability. Journal of Neuroscience. 2018, 38(4):887-9002017.
4. Birla H, Xia J, Gao X, Zhao H, Wang F, Patel S, Akwasi A, Bekker A, Tao Y, Hu H*. Toll-like receptor 4 activation enhances Orai1-mediated calcium signal promoting cytokine production in spinal astrocytes. Cell Calcium, 2022 Jul;105:102619.
Presenting Author
Huijuan Hu
Poster Authors
Topics
- Mechanisms: Biological-Molecular and Cell Biology