Pannexins in the musculoskeletal system: new targets for development and disease progression

Abstract

During cell differentiation, growth, and development, cells can respond to extracellular stimuli through communication channels. Pannexin (Panx) family and connexin (Cx) family are two important types of channel-forming proteins. Panx family contains three members (Panx1-3) and is expressed widely in bone, cartilage and muscle. Although there is no sequence homology between Panx family and Cx family, they exhibit similar configurations and functions. Similar to Cxs, the key roles of Panxs in the maintenance of physiological functions of the musculoskeletal system and disease progression were gradually revealed later. Here, we seek to elucidate the structure of Panxs and their roles in regulating processes such as osteogenesis, chondrogenesis, and muscle growth. We also focus on the comparison between Cx and Panx. As a new key target, Panxs expression imbalance and dysfunction in muscle and the therapeutic potentials of Panxs in joint diseases are also discussed.

References

1. Allen M. R.& Burr, D. B. in Basic and Applied Bone Biology (eds David B. Burr & Matthew R. Allen) 75-90
(Academic Press, 2014)..
2. Plotkin, L. I. & Bellido, T. Beyond gap junctions: connexin43 and bone cell signaling. Bone 52, 157-166 (2013).
3. Stains, J. P. & Civitelli, R. Gap junctions in skeletal development and function. Biochim. Biophys. Acta1719, 69-81 (2005).
4. Bellido, T., Plotkin, L. I.& Bruzzaniti, A. in Basic and Applied Bone Biology (eds David B. Burr & Matthew R. Allen) 27-45
(Academic Press, 2014)..
5. Sáez J. C., Cisterna B. A., Vargas A.& Cardozo, C. P. Regulation of pannexin and connexin channels and their functional role in skeletal muscles. Cell Mol. Life Sci. 72, 2929-2935 (2015).
6. Tomei E. J.& Wolniak, S. M. Kinesin-2 and kinesin-9 have atypical functions during ciliogenesis in the male gametophyte of Marsilea vestita. BMC Cell Biol. 17, 29(2016).
7. Bhat E. A.& Sajjad, N. Human Pannexin 1 channel: Insight in structure-function mechanism and its potential physiological roles. Mol. Cell Biochem. 476, 1529-1540 (2021).
8. Pelegrin P.& Surprenant, A. Pannexin-1 mediates large pore formation and interleukin-1beta release by the ATP-gated P2X7 receptor. EMBO J. 25, 5071-5082 (2006).
9. Scemes E., Suadicani S. O., Dahl G.& Spray, D. C. Connexin and pannexin mediated cell-cell communication. Neuron Glia Biol. 3, 199-208 (2007).
10. Alhouayek M., Sorti R., Gilthorpe J. D.& Fowler, C. J. Role of pannexin-1 in the cellular uptake, release and hydrolysis of anandamide by T84 colon cancer cells. Sci. Rep. 9, 7622(2019).
11. Chekeni, F. B.et al. Pannexin 1 channels mediate ‘find-me' signal release and membrane permeability during apoptosis. Nature 467, 863-867 (2010).
12. Moorer, M. C.et al.Defective signaling, osteoblastogenesis and bone remodeling in a mouse model of connexin 43 C-terminal truncation. J. Cell Sci. 130, 531-540 (2017).
13. Narahari, A. K.et al.ATP and large signaling metabolites flux through caspaseactivated Pannexin 1 channels. Elife 10, e64787 (2021).
14. Pacheco-Costa, R.et al. Defective cancellous bone structure and abnormal response to PTH in cortical bone of mice lacking Cx43 cytoplasmic C-terminus domain. Bone 81, 632-643 (2015).
15. Dubyak G. R.Both sides now: multiple interactions of ATP with pannexin-1 hemichannels. Focus on “A permeant regulating its permeation pore: inhibition of pannexin 1 channels by ATP”. Am. J. Physiol. Cell Physiol. 296, C235-C241 (2009).
16. Panchin Y. V.Evolution of gap junction proteins-the pannexin alternative. J. Exp. Biol. 208, 1415-1419 (2005).
17. Panchin, Y.et al.A ubiquitous family of putative gap junction molecules. Curr. Biol. 10, R473-R474 (2000).
18. Donahue, H. J., Qu, R. W.& Genetos, D. C. Joint diseases: from connexins to gap junctions. Nat. Rev. Rheumatol. 14, 42-51 (2017).
19. Bond, S. R.et al.Pannexin 3 is a novel target for Runx2, expressed by osteoblasts and mature growth plate chondrocytes. J. Bone Min. Res. 26, 2911-2922 (2011).
20. Penuela, S.et al.Pannexin 1 and pannexin 3 are glycoproteins that exhibit many distinct characteristics from the connexin family of gap junction proteins. J. Cell Sci. 120, 3772-3783 (2007).
21. Xiao, Z.et al.Analysis of the extracellular matrix vesicle proteome in mineralizing osteoblasts. J. Cell Physiol. 210, 325-335 (2007).
22. Baranova, A.et al. The mammalian pannexin family is homologous to the invertebrate innexin gap junction proteins. Genomics 83, 706-716 (2004).
23. Harber P.& McCoy, J. M. Predicate calculus, artificial intelligence, and workers' compensation. J. Occup. Med. 31, 484-489 (1989).
24. Riquelme, M. A.et al. The ATP required for potentiation of skeletal muscle contraction is released via pannexin hemichannels. Neuropharmacology 75, 594-603 (2013).
25. Langlois, S.et al.Pannexin 1 and pannexin 3 channels regulate skeletal muscle myoblast proliferation and differentiation. J. Biol. Chem. 289, 30717-30731 (2014).
26. Buvinic, S.et al.ATP released by electrical stimuli elicits calcium transients and gene expression in skeletal muscle. J. Biol. Chem. 284, 34490-34505 (2009).
27. Cea, L. A.et al. De novo expression of connexin hemichannels in denervated fast skeletal muscles leads to atrophy. Proc. Natl. Acad. Sci. USA 110, 16229-16234 (2013).
28. Kanjanamekanant, K., Luckprom, P.& Pavasant, P. P2X7 receptor-Pannexin1 interaction mediates stress-induced interleukin-1 beta expression in human periodontal ligament cells. J. Periodontal. Res. 49, 595-602 (2014).
29. Vogt, A., Hormuzdi, S. G.& Monyer, H. Pannexin1 and Pannexin2 expression in the developing and mature rat brain. Brain Res. Mol. Brain Res. 141, 113-120 (2005).
30. Ishikawa, M.et al.Pannexin 3 functions as an ER Ca²+ channel, hemichannel, and gap junction to promote osteoblast differentiation. J. Cell Biol. 193, 1257-1274 (2011).
31. Iwamoto, T.et al.Pannexin 3 regulates proliferation and differentiation of odontoblasts via its hemichannel activities. PLoS One 12, e0177557 (2017).
32. Le Vasseur, M., Lelowski, J., Bechberger, J. F., Sin, W. C. & Naus, C. C. Pannexin 2 protein expression is not restricted to the CNS. Front. Cell Neurosci. 8, 392(2014).
33. Pillon, N. J.et al. Nucleotides released from palmitate-challenged muscle cells through pannexin-3 attract monocytes. Diabetes 63, 3815-3826 (2014).
34. Deng, Z.et al.Cryo-EM structures of the ATP release channel pannexin 1. Nat. Struct. Mol. Biol. 27, 373-381 (2020).
35. Jin, Q.et al.Cryo-EM structures of human pannexin 1 channel. Cell Res. 30, 449-451 (2020).
36. Michalski, K.et al.The Cryo-EM structure of pannexin 1 reveals unique motifs for ion selection and inhibition. Elife 9, e54670 (2020).
37. Mou, L.et al.Structural basis for gating mechanism of Pannexin 1 channel. Cell Res. 30, 452-454 (2020).
38. Qu, R.et al.Cryo-EM structure of human heptameric Pannexin 1 channel. Cell Res. 30, 446-448 (2020).
39. Ruan Z., Orozco I. J., Du, J. & Lu, W. Structures of human pannexin 1 reveal ion pathways and mechanism of gating. Nature 584, 646-651 (2020).
40. Ambrosi, C.et al.Pannexin1 and Pannexin2 channels show quaternary similarities to connexons and different oligomerization numbers from each other. J. Biol. Chem. 285, 24420-24431 (2010).
41. Zhang, H.et al.Cryo-EM structure of human heptameric pannexin 2 channel. Nat. Commun. 14, 1118(2023).
42. Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673-681 (2006).
43. Milks L. C., Kumar N. M., Houghten R., Unwin N.& Gilula, N. B. Topology of the 32-kd liver gap junction protein determined by site-directed antibody localizations. EMBO J. 7, 2967-2975 (1988).
44. Yeager M.& Gilula, N. B. Membrane topology and quaternary structure of cardiac gap junction ion channels. J. Mol. Biol. 223, 929-948 (1992).
45. Beckmann A., Grissmer A., Krause E., Tschernig T.& Meier, C. Pannexin-1 channels show distinct morphology and no gap junction characteristics in mammalian cells. Cell Tissue Res. 363, 751-763 (2016).
46. Huang Y., Grinspan J. B., Abrams, C. K. & Scherer, S. S. Pannexin1 is expressed by neurons and glia but does not form functional gap junctions. Glia 55, 46-56 (2007).
47. Wang, N.et al. Connexin targeting peptides as inhibitors of voltage- and intracellular Ca2+ -triggered Cx43 hemichannel opening. Neuropharmacology 75, 506-516 (2013).
48. Wang, N.et al. Paracrine signaling through plasma membrane hemichannels. Biochim. Biophys. Acta1828, 35-50 (2013).
49. Weinger J. M.& Holtrop, M. E. An ultrastructural study of bone cells: the occurrence of microtubules, microfilaments and tight junctions. Calcif. Tissue Res. 14, 15-29 (1974).
50. Bruzzone R., Hormuzdi S. G., Barbe M. T., Herb, A. & Monyer, H. Pannexins, a family of gap junction proteins expressed in brain. Proc. Natl. Acad. Sci. USA 100, 13644-13649 (2003).
51. Ishikawa, M.et al.Pannexin 3 and connexin 43 modulate skeletal development through their distinct functions and expression patterns. J. Cell Sci. 129, 1018-1030 (2016).
52. Cooreman, A.et al. Connexin and pannexin (hemi)channels: emerging targets in the treatment of liver disease. Hepatology 69, 1317-1323 (2019).
53. Vinken, M.et al. Connexins and their channels in cell growth and cell death. Cell Signal 18, 592-600 (2006).
54. Flores, J. A.et al.Connexin-46/50 in a dynamic lipid environment resolved by CryoEM at 1.9 A. Nat. Commun. 11, 4331(2020).
55. Lee, H. J.et al.Cryo-EM structure of human Cx31.3/GJC3 connexin hemichannel. Sci. Adv. 6, eaba4996 (2020).
56. Maeda, S.et al. Structure of the connexin 26 gap junction channel at 3.5 A resolution. Nature 458, 597-602 (2009).
57. Myers, J. B.et al. Structure of native lens connexin 46/50 intercellular channels by cryo-EM. Nature 564, 372-377 (2018).
58. Unger V. M., Kumar N. M., Gilula, N. B. & Yeager, M. Three-dimensional structure of a recombinant gap junction membrane channel. Science 283,1176-1180 (1999).
59. Bhaskaracharya, A.et al.Probenecid blocks human P2X7 receptor-induced dye uptake via a pannexin-1 independent mechanism. PLoS ONE 9, e93058 (2014).
60. Chiu Y. H., Schappe M. S., Desai B. N.& Bayliss, D. A. Revisiting multimodal activation and channel properties of Pannexin 1. J. Gen. Physiol. 150, 19-39 (2018).
61. Iwamoto, T.et al.Pannexin 3 regulates intracellular ATP/cAMP levels and promotes chondrocyte differentiation. J. Biol. Chem. 285, 18948-18958 (2010).
62. Ma W., Hui H., Pelegrin P.& Surprenant, A. Pharmacological characterization of pannexin-1 currents expressed in mammalian cells. J. Pharm. Exp. Ther. 328, 409-418 (2009).
63. Li, S., Bjelobaba, I.& Stojilkovic, S. S. Interactions of Pannexin1 channels with purinergic and NMDA receptor channels. Biochim. Biophys. Acta Biomembr. 1860, 166-173 (2018).
64. Velasquez S.& Eugenin, E. A. Role of Pannexin-1 hemichannels and purinergic receptors in the pathogenesis of human diseases. Front. Physiol. 5, 96(2014).
65. Silverman, W. R.et al.The pannexin 1 channel activates the inflammasome in neurons and astrocytes. J. Biol. Chem. 284, 18143-18151 (2009).
66. Locovei, S., Wang, J.& Dahl, G. Activation of pannexin 1 channels by ATP through P2Y receptors and by cytoplasmic calcium. FEBS Lett. 580, 239-244 (2006).
67. Prochnow, N.et al.Pannexin1 stabilizes synaptic plasticity and is needed for learning. PLoS One 7, e51767 (2012).
68. Burboa P. C., Puebla M., Gaete P. S., Duran W. N.& Lillo, M. A. Connexin and pannexin large-pore channels in microcirculation and neurovascular coupling function. Int. J. Mol. Sci. 23, 7303(2022).
69. Qu, Y.et al.Pannexin-1 is required for ATP release during apoptosis but not for inflammasome activation. J. Immunol. 186, 6553-6561 (2011).
70. Kanneganti, T. D.et al. Pannexin-1-mediated recognition of bacterial molecules activates the cryopyrin inflammasome independent of Toll-like receptor signaling. Immunity 26, 433-443 (2007).
71. Seminario-Vidal, L. et al. Rho signaling regulates pannexin 1-mediated ATP release from airway epithelia. J. Biol. Chem. 286, 26277-26286 (2011).
72. Phelan, P. Innexins: members of an evolutionarily conserved family of gapjunction proteins. Biochim. Biophys. Acta1711, 225-245 (2005).
73. Berendsen, A. D. & Olsen, B. R. Bone development. Bone 80, 14-18 (2015).
74. Yang L., Tsang K. Y., Tang H. C., Chan, D. & Cheah, K. S. Hypertrophic chondrocytes can become osteoblasts and osteocytes in endochondral bone formation. Proc. Natl. Acad. Sci. USA 111, 12097-12102 (2014).
75. Cheung, W. Y.et al.Pannexin-1 and P2X7-receptor are required for apoptotic osteocytes in fatigued bone to trigger RANKL production in neighboring bystander osteocytes. J. Bone Min. Res. 31, 890-899 (2016).
76. Caskenette, D.et al.Global deletion of Panx3 produces multiple phenotypic effects in mouse humeri and femora. J. Anat. 228, 746-756 (2016).
77. Shao, Q.et al.A germline variant in the PANX1 gene has reduced channel function and is associated with multisystem dysfunction. J. Biol. Chem. 291, 12432-12443 (2016).
78. Ishikawa M., Iwamoto T., Fukumoto S.& Yamada, Y. Pannexin 3 inhibits proliferation of osteoprogenitor cells by regulating Wnt and p21 signaling. J. Biol. Chem. 289, 2839-2851 (2014).
79. Delgado-Calle, J. & Bellido, T. Osteocytes and skeletal pathophysiology. Curr. Mol. Biol. Rep. 1, 157-167 (2015).
80. Aguirre, J. I.et al.Osteocyte apoptosis is induced by weightlessness in mice and precedes osteoclast recruitment and bone loss. J. Bone Min. Res. 21, 605-615 (2006).
81. Delgado-Calle, J. & Bellido, T. The osteocyte as a signaling cell. Physiol. Rev. 102, 379-410 (2022).
82. Emerton, K. B.et al. Osteocyte apoptosis and control of bone resorption following ovariectomy in mice. Bone 46, 577-583 (2010).
83. Kennedy, O. D.et al. Activation of resorption in fatigue-loaded bone involves both apoptosis and active pro-osteoclastogenic signaling by distinct osteocyte populations. Bone 50, 1115-1122 (2012).
84. Kennedy O. D., Laudier D. M., Majeska R. J., Sun, H. B. & Schaffler, M. B. Osteocyte apoptosis is required for production of osteoclastogenic signals following bone fatigue in vivo. Bone 64, 132-137 (2014).
85. Bakker A.,Klein-Nulend, J. & Burger, E. Shear stress inhibits while disuse promotes osteocyte apoptosis. Biochem. Biophys. Res. Commun. 320, 1163-1168 (2004).
86. Noble, B. Bone microdamage and cell apoptosis. Eur. Cell Mater. 6, 46-55 (2003). discusssion 55.
87. Tomkinson A., Gevers E. F., Wit J. M., Reeve J.& Noble, B. S. The role of estrogen in the control of rat osteocyte apoptosis. J. Bone Min. Res. 13, 1243-1250 (1998).
88. Verborgt, O., Gibson, G. J.& Schaffler, M. B. Loss of osteocyte integrity in association with microdamage and bone remodeling after fatigue in vivo. J. Bone Min. Res. 15, 60-67 (2000).
89. McCutcheon, S., Majeska, R. J., Spray, D. C., Schaffler, M. B. & Vazquez, M. Apoptotic osteocytes induce RANKL production in bystanders via purinergic signaling and activation of pannexin channels. J. Bone Min. Res. 35, 966-977 (2020).
90. Liu, W.et al.TGF-beta1 facilitates cell-cell communication in osteocytes via connexin43- and pannexin1-dependent gap junctions. Cell Death Discov. 5, 141(2019).
91. Aguilar-Perez, A. et al. Age- and sex-dependent role of osteocytic pannexin1 on bone and muscle mass and strength. Sci. Rep. 9, 13903(2019).
92. Courvoisier A., Sailhan F., Laffenetre O.& Obert, L., French Study Group of, B. M. P. i. O. S. Bone morphogenetic protein and orthopaedic surgery: can we legitimate its off-label use? Int. Orthop. 38, 2601-2605 (2014).
93. Kalitina T. A.[Survival of Coxsackie viruses of group B 3 and 5 serotypes in sausage meat]. Vopr. Pitan. 25, 74-77 (1966).
94. Song, K.et al.Identification of a key residue mediating bone morphogenetic protein (BMP)-6 resistance to noggin inhibition allows for engineered BMPs with superior agonist activity. J. Biol. Chem. 285, 12169-12180 (2010).
95. Wang, L.et al.Bone formation induced by BMP-2 in human osteosarcoma cells. Int. J. Oncol. 43, 1095-1102 (2013).
96. Takeno A., Kanazawa I., Tanaka K. I., Notsu M.& Sugimoto, T. Phloretin suppresses bone morphogenetic protein-2-induced osteoblastogenesis and mineralization via inhibition of phosphatidylinositol 3-kinases/Akt pathway. Int. J. Mol. Sci. 20, 2481(2019).
97. Ghosh-Choudhury, N. et al. c-Abl-dependent molecular circuitry involving Smad5 and phosphatidylinositol 3-kinase regulates bone morphogenetic protein-2-induced osteogenesis. J. Biol. Chem. 288, 24503-24517 (2013).
98. Mukherjee, A., Wilson, E. M.& Rotwein, P. Selective signaling by Akt2 promotes bone morphogenetic protein 2-mediated osteoblast differentiation. Mol. Cell Biol. 30, 1018-1027 (2010).
99. Sun, M.et al.Effects of matrix stiffness on the morphology, adhesion, proliferation and osteogenic differentiation of mesenchymal stem cells. Int. J. Med. Sci. 15, 257-268 (2018).
100. Afzal, F.et al.Smad function and intranuclear targeting share a Runx2 motif required for osteogenic lineage induction and BMP2 responsive transcription. J. Cell Physiol. 204, 63-72 (2005).
101. Zaidi, S. K.et al. Integration of Runx and Smad regulatory signals at transcriptionally active subnuclear sites. Proc. Natl. Acad. Sci. USA 99, 8048-8053 (2002).
102. Lohman, A. W.et al.Expression of pannexin isoforms in the systemic murine arterial network. J. Vasc. Res. 49, 405-416 (2012).
103. Plotkin L. I.& Stains, J. P. Connexins and pannexins in the skeleton: gap junctions, hemichannels and more. Cell Mol. Life Sci. 72, 2853-2867 (2015).
104. Rauch, A.et al.Glucocorticoids suppress bone formation by attenuating osteoblast differentiation via the monomeric glucocorticoid receptor. Cell Metab. 11, 517-531 (2010).
105. Song F., Sun H., Huang L., Fu D.& Huang, C. The role of Pannexin3-modified human dental pulp-derived mesenchymal stromal cells in repairing rat cranial critical-sized bone defects. Cell Physiol. Biochem. 44, 2174-2188 (2017).
106. Tomita M., Reinhold M. I., Molkentin J. D.& Naski, M. C. Calcineurin and NFAT4 induce chondrogenesis. J. Biol. Chem. 277, 42214-42218 (2002).
107. Beals C. R., Clipstone N. A., Ho S. N.& Crabtree, G. R. Nuclear localization of NFATc by a calcineurin-dependent, cyclosporin-sensitive intramolecular interaction. Genes Dev. 11, 824-834 (1997).
108. Esseltine J. L.& Laird, D. W. Next-generation connexin and pannexin cell biology. Trends Cell Biol. 26, 944-955 (2016).
109. Koga, T.et al.NFAT and osterix cooperatively regulate bone formation. Nat. Med. 11, 880-885 (2005).
110. Nakashima, K.et al. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108, 17-29 (2002).
111. Ishikawa, M.et al.Pannexin 3 ER Ca²+ channel gating is regulated by phosphorylation at the Serine 68 residue in osteoblast differentiation. Sci. Rep. 9, 18759(2019).
112. Boyden, L. M.et al.High bone density due to a mutation in LDL-receptor-related protein 5. N. Engl. J. Med. 346, 1513-1521 (2002).
113. Guntur A. R.& Rosen, C. J. Bone as an endocrine organ. Endocr. Pr. 18, 758-762 (2012).
114. Cheung, W. Y.et al.in Journal Of Bone And Mineral Research. S288-S288 (WILEYBLACKWELL 111 RIVER ST, HOBOKEN 07030-5774, NJ USA).
115. Deosthale, P.et al.Sex-specific differences in direct osteoclastic versus indirect osteoblastic effects underlay the low bone mass of Pannexin1 deletion in TRAPexpressing cells in mice. Bone Rep. 16, 101164(2022).
116. Oh, S. K.et al.Pannexin 3 is required for normal progression of skeletal development in vertebrates. FASEB J. 29, 4473-4484 (2015).
117. Bond S. R., Abramyan J., Fu K., Naus C. C.& Richman, J. M. Pannexin 3 is required for late stage bone growth but not for initiation of ossification in avian embryos. Dev. Dyn. 245, 913-924 (2016).
118. Charge S. B.& Rudnicki, M. A. Cellular and molecular regulation of muscle regeneration. Physiol. Rev. 84, 209-238 (2004).
119. Riquelme, M. A.et al.Pannexin channels mediate the acquisition of myogenic commitment in C2C12 reserve cells promoted by P2 receptor activation. Front. Cell Dev. Biol. 3, 25(2015).
120. Jorquera, G.et al.Cav1.1 controls frequency-dependent events regulating adult skeletal muscle plasticity. J. Cell Sci. 126, 1189-1198 (2013).
121. Rose A. J., Alsted T. J., Kobbero J. B.& Richter, E. A. Regulation and function of Ca2+ -calmodulin-dependent protein kinase II of fast-twitch rat skeletal muscle. J. Physiol. 580, 993-1005 (2007).
122. Suarez-Berumen, K. et al. Pannexin 1 regulates skeletal muscle regeneration by promoting bleb-based myoblast migration and fusion through a novel lipid based signaling mechanism. Front. Cell Dev. Biol. 9, 736813(2021).
123. Freeman, E.et al.Sex-dependent role of Pannexin 1 in regulating skeletal muscle and satellite cell function. J. Cell Physiol. 237, 3944-3959 (2022).
124. Martel-Pelletier, J. et al. Osteoarthritis. Nat. Rev. Dis. Prim. 2, 16072(2016).
125. Glyn-Jones, S.et al. Osteoarthritis. Lancet 386, 376-387 (2015).
126. Jiang Y.Osteoarthritis year in review 2021: biology. Osteoarthr. Cartil. 30, 207-215 (2022).
127. Appleton C. T., Pitelka V., Henry J.& Beier, F. Global analyses of gene expression in early experimental osteoarthritis. Arthritis Rheum. 56, 1854-1868 (2007).
128. O'Donnell, B. L. & Penuela, S. Pannexin 3 channels in health and disease. Purinergic Signal 17, 577-589 (2021).
129. Moon, P. M.et al.Deletion of Panx3 prevents the development of surgically induced osteoarthritis. J. Mol. Med. 93, 845-856 (2015).
130. Moon, P. M.et al.Global deletion of Pannexin 3 resulting in accelerated development of aging-induced osteoarthritis in mice. Arthritis Rheumatol. 73, 1178-1188 (2021).
131. Xiao J., Li Y., Zhang J., Xu G.& Zhang, J. Pannexin 3 activates P2X7 receptor to mediate inflammation and cartilage matrix degradation in temporomandibular joint osteoarthritis. Cell Biol. Int. 47, 1183-1197 (2023).
132. Francisco, V.et al.A new immunometabolic perspective of intervertebral disc degeneration. Nat. Rev. Rheumatol. 18, 47-60 (2022).
133. Serjeant, M.et al.The role of Panx3 in age-associated and injury-induced intervertebral disc degeneration. Int. J. Mol. Sci. 22, 1080(2021).
134. Liu, N.et al.microRNA-206 promotes skeletal muscle regeneration and delays progression of Duchenne muscular dystrophy in mice. J. Clin. Invest. 122, 2054-2065 (2012).
135. McNally, E. M. & Pytel, P. Muscle diseases: the muscular dystrophies. Annu. Rev. Pathol. 2, 87-109 (2007).
136. Arias-Calderon, M. et al. Characterization of a multiprotein complex involved in excitation-transcription coupling of skeletal muscle. Skelet. Muscle 6, 15 (2016).
137. Cea, L. A.et al.Fast skeletal myofibers of MDX mouse, model of Duchenne muscular dystrophy, express connexin hemichannels that lead to apoptosis. Cell Mol. Life Sci. 73, 2583-2599 (2016).
138. Valladares, D.et al.Electrical stimuli are anti-apoptotic in skeletal muscle via extracellular ATP. Alteration of this signal in Mdx mice is a likely cause of dystrophy. PLoS One 8, e75340 (2013).
139. Pham, T. L.et al.Expression of Pannexin 1 and Pannexin 3 during skeletal muscle development, regeneration, and Duchenne muscular dystrophy. J. Cell Physiol. 233, 7057-7070 (2018).
140. An, S.et al.Connexin43 in musculoskeletal system: new targets for development and disease progression. Aging Dis. 13, 1715-1732 (2022).
141. Lohman A. W.& Isakson, B. E. Differentiating connexin hemichannels and pannexin channels in cellular ATP release. FEBS Lett. 588, 1379-1388 (2014).
142. Brigid Hogan, R. B. Manipulating the mouse embryo. a laboratory manual, 2nd Edition. Genet. Res. 66, 296-300 (1994).
143. Lecanda, F.et al.Connexin43 deficiency causes delayed ossification, craniofacial abnormalities, and osteoblast dysfunction. J. Cell Biol. 151, 931-944 (2000).
144. Ishikawa M.& Yamada, Y. The role of Pannexin 3 in bone biology. J. Dent. Res. 96, 372-379 (2017).
145. Lengner, C. J.et al.Osteoblast differentiation and skeletal development are regulated by Mdm2-p53 signaling. J. Cell Biol. 172, 909-921 (2006).
146. Uribe, P.et al.Soluble silica stimulates osteogenic differentiation and gap junction communication in human dental follicle cells. Sci. Rep. 10, 9923(2020).
147. van der Heyden, M. A.et al. Identification of connexin43 as a functional target for Wnt signalling. J. Cell Sci. 111, 1741-1749 (1998).
148. Lohman, A. W.et al.Pannexin 1 channels regulate leukocyte emigration through the venous endothelium during acute inflammation. Nat. Commun. 6, 7965(2015).
149. Wang J., Ma M., Locovei S., Keane R. W.& Dahl, G. Modulation of membrane channel currents by gap junction protein mimetic peptides: size matters. Am. J. Physiol. Cell Physiol. 293, C1112-C1119 (2007).
150. Weilinger, N. L., Tang, P. L.& Thompson, R. J. Anoxia-induced NMDA receptor activation opens pannexin channels via Src family kinases. J. Neurosci. 32, 12579-12588 (2012).
151. Plotkin, L. I.et al.Connexin 43 is required for the anti-apoptotic effect of bisphosphonates on osteocytes and osteoblasts in vivo. J. Bone Min. Res. 23, 1712-1721 (2008).
152. Poornima V., Vallabhaneni S., Mukhopadhyay, M. & Bera, A. K. Nitric oxide inhibits the pannexin 1 channel through a cGMP-PKG dependent pathway. Nitric Oxide 47, 77-84 (2015).
153. Dobrowolski, R., Sommershof, A.& Willecke, K. Some oculodentodigital dysplasia-associated Cx43 mutations cause increased hemichannel activity in addition to deficient gap junction channels. J. Membr. Biol. 219, 9-17 (2007).
154. Esseltine, J. L.et al.Connexin43 mutant patient-derived induced pluripotent stem cells exhibit altered differentiation potential. J. Bone Min. Res. 32, 1368-1385 (2017).
155. Katz, J. N., Arant, K. R. & Loeser, R. F. Diagnosis and treatment of hip and knee osteoarthritis: a review. JAMA 325, 568-578 (2021).
156. Hochberg M. C.Serious joint-related adverse events in randomized controlled trials of anti-nerve growth factor monoclonal antibodies. Osteoarthr. Cartil. 23, S18-S21 (2015).
157. van der Kraan, P. M. & van den Berg, W. B. Chondrocyte hypertrophy and osteoarthritis: role in initiation and progression of cartilage degeneration? Osteoarthr. Cartil. 20, 223-232 (2012).
158. Billaud, M.et al.Pannexin1 regulates alpha1-adrenergic receptor- mediated vasoconstriction. Circ. Res. 109, 80-85 (2011).
159. Masuda, T.et al.Dorsal horn neurons release extracellular ATP in a VNUT-dependent manner that underlies neuropathic pain. Nat. Commun. 7, 12529(2016).
160. Conesa-Buendia, F. M. et al. Tenofovir causes bone loss via decreased bone formation and increased bone resorption, which can be counteracted by dipyridamole in mice. J. Bone Min. Res. 34, 923-938 (2019).
161. Hirata, H.et al.Connexin 39.9 protein is necessary for coordinated activation of slow-twitch muscle and normal behavior in zebrafish. J. Biol. Chem. 287, 1080-1089 (2012).
162. Naus C. C.& Giaume, C. Bridging the gap to therapeutic strategies based on connexin/pannexin biology. J. Transl. Med. 14, 330(2016).
163. Dahl, G., Qiu, F. & Wang, J. The bizarre pharmacology of the ATP release channel pannexin1. Neuropharmacology 75, 583-593 (2013).
164. Bruzzone R., Barbe M. T., Jakob N. J.& Monyer, H. Pharmacological properties of homomeric and heteromeric pannexin hemichannels expressed in Xenopus oocytes. J. Neurochem. 92, 1033-1043 (2005).
165. Molica, F.et al.Selective inhibition of Panx1 channels decreases hemostasis and thrombosis in vivo. Thromb. Res. 183, 56-62 (2019).
166. Michalski K.& Kawate, T. Carbenoxolone inhibits Pannexin1 channels through interactions in the first extracellular loop. J. Gen. Physiol. 147, 165-174 (2016).
167. Freeman, T. J.et al.Inhibition of Pannexin 1 reduces the tumorigenic properties of human melanoma cells. Cancers 11, 102 (2019).
168. Chen, K. W., Demarco, B.& Broz, P. Pannexin-1 promotes NLRP3 activation during apoptosis but is dispensable for canonical or noncanonical inflammasome activation. Eur. J. Immunol. 50, 170-177 (2020).
169. Jankowski, J.et al.Epithelial and endothelial pannexin1 channels mediate AKI. J. Am. Soc. Nephrol. 29, 1887-1899 (2018).
170. Sharma, A. K.et al.Pannexin-1 channels on endothelial cells mediate vascular inflammation during lung ischemia-reperfusion injury. Am. J. Physiol. Lung Cell Mol. Physiol. 315, L301-L312 (2018).
171. Furukawa, M., Matsueda, M.& Takagai, Y. Ultrasonic mist generation assist argon-nitrogen mix gas effect on radioactive strontium quantification by online solid-phase extraction with inductively coupled plasma mass spectrometry. Anal. Sci. 34, 471-476 (2018).
172. Feig, J. L.et al.The antiviral drug tenofovir, an inhibitor of Pannexin-1-mediated ATP release, prevents liver and skin fibrosis by downregulating adenosine levels in the liver and skin. PLoS One 12, e0188135 (2017).
173. Davidson J. S.& Baumgarten, I. M. Glycyrrhetinic acid derivatives: a novel class of inhibitors of gap-junctional intercellular communication. Structure-activity relationships. J. Pharm. Exp. Ther. 246, 1104-1107 (1988).
174. Davidson, J. S., Baumgarten, I. M.& Harley, E. H. Reversible inhibition of intercellular junctional communication by glycyrrhetinic acid. Biochem. Biophys. Res. Commun. 134, 29-36 (1986).
175. Goldberg, G. S.et al.Evidence that disruption of connexon particle arrangements in gap junction plaques is associated with inhibition of gap junctional communication by a glycyrrhetinic acid derivative. Exp. Cell Res. 222, 48-53 (1996).
176. Guan X., Wilson S., Schlender K. K.& Ruch, R. J. Gap-junction disassembly and connexin 43 dephosphorylation induced by 18 beta-glycyrrhetinic acid. Mol. Carcinog. 16, 157-164 (1996).
177. Armanini D., Karbowiak I., Krozowski Z., Funder, J. W. & Adam, W. R. The mechanism of mineralocorticoid action of carbenoxolone. Endocrinology 111,1683-1686 (1982).
178. Kuzuya, M.et al.Structures of human pannexin-1 in nanodiscs reveal gating mediated by dynamic movement of the N terminus and phospholipids. Sci. Signal 15, eabg6941 (2022).
179. Willebrords J., Maes M., Crespo Yanguas, S. & Vinken, M. Inhibitors of connexin and pannexin channels as potential therapeutics. Pharm. Ther. 180, 144-160 (2017).
180. Chen X., Yuan S., Mi L., Long Y.& He, H. Pannexin1: insight into inflammatory conditions and its potential involvement in multiple organ dysfunction syndrome. Front. Immunol. 14, 1217366(2023).
181. Grek, C. L.et al.Targeting connexin 43 with alpha-connexin carboxyl-terminal (ACT1) peptide enhances the activity of the targeted inhibitors, tamoxifen and lapatinib, in breast cancer: clinical implication for ACT1. BMC Cancer 15, 296 (2015).
182. Caufriez, A.et al.Determination of structural features that underpin the pannexin1 channel inhibitory activity of the peptide ¹⁰Panx1. Bioorg. Chem. 138, 106612(2023).
183. Zhang Y., Sun Y., Wang Z.& Huang, L. Fluorescein-5-thiosemicarbazide as a probe for directly imaging of mucin-type O-linked glycoprotein within living cells. Glycoconj. J. 29, 445-452 (2012).
184. Boassa D., Nguyen P., Hu J., Ellisman M. H.& Sosinsky, G. E. Pannexin2 oligomers localize in the membranes of endosomal vesicles in mammalian cells while Pannexin1 channels traffic to the plasma membrane. Front. Cell Neurosci. 8, 468(2014).
185. Hedskog, L.et al. Modulation of the endoplasmic reticulum-mitochondria interface in Alzheimer's disease and related models. Proc. Natl. Acad. Sci. USA 110, 7916-7921 (2013).
186. Yang Y., Wang L., Chen L.& Li, L. Pannexin-2, a novel mitochondrialassociated membrane protein, may become the new strategy to treat and prevent neurological disorders. Acta Biochim. Biophys. Sin. 52, 1178-1180 (2020).
187. Grimston S. K., Watkins M. P., Brodt M. D., Silva M. J.& Civitelli, R. Enhanced per

Funding

Hang Zhang ([e-mail]) or Yusheng Li ([e-mail])