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As the bile acid receptor sphingosine 1-phosphate receptor 2 (S1PR2) plays a critical role in recruitment of bone marrow-derived monocytes\/macrophages to sites of cholestatic liver injury, S1PR2 expression was examined using cultured macrophages and patient tissues. Bile canaliculi destruction precedes intrahepatic ductopenia; therefore, we focused on hepatocyte S1PR2 and the downstream RhoA\/Rho kinase 1 (ROCK1) signaling pathway and bile canaliculi alterations using three-dimensional hepatocyte culture models that form obvious bile canaliculus-like networks. Multiplex immunohistochemistry revealed increased numbers of S1PR2<jats:sup>+<\/jats:sup>CD45<jats:sup>+<\/jats:sup>CD68<jats:sup>+<\/jats:sup>FCN1<jats:sup>+<\/jats:sup> inflammatory macrophages and S1PR2<jats:sup>+<\/jats:sup>CD45<jats:sup>+<\/jats:sup>CD68<jats:sup>+<\/jats:sup>MARCO<jats:sup>+<\/jats:sup> Kupffer cells in liver tissues showing ductopenia due to graft-versus-host disease and rejection post-liver transplant compared with normal liver. Macrophage expression of proinflammatory cytokines, including MCP1, was reduced following S1PR2 inhibition. Taurocholic acid and S1P2 agonist induced hepatocyte S1PR2 and reduced RhoA\/ROCK1 expression, resulting in bile canaliculi dilatation. S1PR2 inhibition reversed the effect on RhoA\/ROCK1 expression, resulting in maintenance of bile canaliculi through myosin light chain 2 (MLC2) phosphorylation. Activation of S1PR2 on macrophages and S1PR2 on hepatocytes may disrupt bile canaliculi dynamics in VBDS under regulation by RhoA\/ROCK1 through MLC2 phosphorylation.<\/jats:p>","DOI":"10.1371\/journal.pone.0317568","type":"journal-article","created":{"date-parts":[[2025,1,24]],"date-time":"2025-01-24T18:31:13Z","timestamp":1737743473000},"page":"e0317568","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":9,"title":["Activation of S1PR2 on macrophages and the hepatocyte S1PR2\/RhoA\/ROCK1\/MLC2 pathway in vanishing bile duct syndrome"],"prefix":"10.1371","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-8679-4800","authenticated-orcid":true,"given":"Aya","family":"Miyagawa-Hayashino","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tetsuya","family":"Imura","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Toshiaki","family":"Takezawa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maki","family":"Hirai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Saya","family":"Shibata","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroshi","family":"Ogi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1647-342X","authenticated-orcid":true,"given":"Takahiro","family":"Tsujikawa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eiichi","family":"Konishi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2025,1,24]]},"reference":[{"key":"pone.0317568.ref001","first-page":"556","volume-title":"MacSween\u2019s 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