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Physiol."],"abstract":"<jats:p>Increased pre-osteoblast cell density within bone resorption areas is a key factor in promoting osteoblast differentiation; however, the molecular mechanisms underlying this process remain poorly understood. In this study, we investigated the role of ATP release and purinergic receptor P2X4 signaling in osteoblast differentiation using the murine pre-osteoblastic cell line MC3T3-E1. Under high cell density conditions, which were essential for the induction of osteoblast differentiation, we observed a significant accumulation of extracellular ATP released via the hemichannel Pannexin 3 (Panx3). This ATP accumulation subsequently activated the P2X4. Pharmacological inhibition and siRNA-mediated knockdown Panx3 markedly reduced extracellular ATP levels and decreased expression and activity of alkaline phosphatase (ALP), an osteoblast differentiation marker. Similarly, pharmacological inhibition and siRNA-mediated knockdown of P2X4 suppressed ALP expression and activity. Functional analyses using calcium imaging and whole-cell patch-clamp recording revealed that P2X4 activity was enhanced during osteoblast differentiation. Notably, this functional upregulation correlated with enhanced localization of P2X4 to the plasma membrane. Moreover, pharmacological inhibition of Panx3 or P2X4 impaired the endochondral ossification of isolated mouse metatarsal bone. These findings suggest that Panx3-mediated ATP release and the subsequent activation of P2X4 signaling constitute a crucial pathway linking cell density to osteoblast differentiation. Our study provides new insights into the purinergic signaling mechanisms underlying bone formation and identifies potential molecular targets for promoting osteogenesis.<\/jats:p>","DOI":"10.3389\/fphys.2026.1880201","type":"journal-article","created":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T04:28:39Z","timestamp":1789964919000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Pannexin 3-mediated adenosine triphosphate release from pre-osteoblasts promotes osteoblast differentiation through P2X4 receptor activation"],"prefix":"10.3389","volume":"17","author":[{"given":"Hiroaki","family":"Kito","sequence":"first","affiliation":[{"name":"Department of Pharmacology, Graduate School of Medical Sciences, Nagoya City University","place":["Nagoya, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ayano","family":"Niwa","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Graduate School of Medical Sciences, Nagoya City University","place":["Nagoya, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takusei","family":"Ryu","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Graduate School of Medical Sciences, Nagoya City University","place":["Nagoya, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yohei","family":"Yamaguchi","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Graduate School of Medical Sciences, Nagoya City University","place":["Nagoya, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junko","family":"Kajikuri","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Graduate School of Medical Sciences, Nagoya City University","place":["Nagoya, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Susumu","family":"Ohya","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Graduate School of Medical Sciences, Nagoya City University","place":["Nagoya, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,9,21]]},"reference":[{"key":"B57","doi-asserted-by":"publisher","first-page":"32770","DOI":"10.1074\/jbc.M110.148940","article-title":"Lipid raft association and cholesterol sensitivity of P2X1-4 receptors for ATP: chimeras and point mutants identify intracellular amino-terminal residues involved in lipid regulation of P2X1 receptors","volume":"285","author":"Allsopp","year":"2010","journal-title":"J. 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