{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T21:36:39Z","timestamp":1792791399820,"version":"4.5.0"},"reference-count":48,"publisher":"Wiley","issue":"8","license":[{"start":{"date-parts":[[2015,3,26]],"date-time":"2015-03-26T00:00:00Z","timestamp":1427328000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["J Cellular Molecular Medi"],"published-print":{"date-parts":[[2015,8]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>In patients with inflammatory arthritis, tumour necrosis factor (<jats:styled-content style=\"fixed-case\">TNF<\/jats:styled-content>)\u2010\u03b1 are overproduced in inflamed joints. This leads to local erosion of cartilage and bone, periarticular osteopenia, as well as osteoporosis. But less is known regarding the molecular mechanisms that mediate the effect of <jats:styled-content style=\"fixed-case\">TNF<\/jats:styled-content>\u2010\u03b1 on osteoblast function. The purpose of this study was to test that C terminus of Hsc70\u2010interacting protein (<jats:styled-content style=\"fixed-case\">CHIP<\/jats:styled-content>) has a specific role in suppressing the osteogenic activity of osteoblasts under inflammatory conditions. C2C12, <jats:styled-content style=\"fixed-case\">MC<\/jats:styled-content>3T3\u2010E1 and <jats:styled-content style=\"fixed-case\">HEK<\/jats:styled-content>293T cell lines were cultured and cotransfected with related plasmids. After transfection, the cells were cultured further in the presence or absence of murine <jats:styled-content style=\"fixed-case\">TNF<\/jats:styled-content>\u2010\u03b1 and subjected to real time <jats:styled-content style=\"fixed-case\">RT<\/jats:styled-content>\u2010<jats:styled-content style=\"fixed-case\">PCR<\/jats:styled-content>, Western blot, Ubiquitination assay, Co\u2010immunoprecipitation, Luciferase reporter assay, Small interfering <jats:styled-content style=\"fixed-case\">RNA<\/jats:styled-content>s and Mineralization assay. The expression levels of <jats:styled-content style=\"fixed-case\">TNF<\/jats:styled-content>\u2010\u03b1\u2010induced <jats:styled-content style=\"fixed-case\">CHIP<\/jats:styled-content> and Osx were examined by <jats:styled-content style=\"fixed-case\">RT<\/jats:styled-content>\u2010<jats:styled-content style=\"fixed-case\">PCR<\/jats:styled-content> and Western blot analysis. Co\u2010immunoprecipitation and ubiquitination assays revealed ubiquitinated Osx, confirmed that <jats:styled-content style=\"fixed-case\">CHIP<\/jats:styled-content> indeed interacted with Osx and identified K55 and K386 residues as the ubiquitination sites in Osx, Luciferase reporter assay and Small interfering <jats:styled-content style=\"fixed-case\">RNA<\/jats:styled-content>s examined whether <jats:styled-content style=\"fixed-case\">TNF<\/jats:styled-content>\u2010\u03b1 target the bone morphogenetic protein signalling through <jats:styled-content style=\"fixed-case\">CHIP<\/jats:styled-content>. We established stable cell lines with the overexpression of <jats:styled-content style=\"fixed-case\">HA<\/jats:styled-content>\u2010<jats:styled-content style=\"fixed-case\">CHIP<\/jats:styled-content>, Mineralization assay and <jats:styled-content style=\"fixed-case\">CHIP<\/jats:styled-content> si<jats:styled-content style=\"fixed-case\">RNA<\/jats:styled-content> demonstrated the important roles of <jats:styled-content style=\"fixed-case\">CHIP<\/jats:styled-content> on osteoblast function in conditions in which <jats:styled-content style=\"fixed-case\">TNF<\/jats:styled-content>\u2010\u03b1 is overexpressed. We found that the K55 and K386 residues are ubiquitination site(s) in Osx, and that <jats:styled-content style=\"fixed-case\">TNF<\/jats:styled-content>\u2010\u03b1 inhibits osteoblast differentiation by promoting Osx degradation through up\u2010regulation of E3 ubiquitin ligase <jats:styled-content style=\"fixed-case\">CHIP<\/jats:styled-content> in osteoblast. Thus, <jats:styled-content style=\"fixed-case\">CHIP<\/jats:styled-content> targets Osx for ubiquitination and degradation in osteoblasts after chronic exposure to <jats:styled-content style=\"fixed-case\">TNF<\/jats:styled-content>\u2010\u03b1, and inhibition of <jats:styled-content style=\"fixed-case\">CHIP<\/jats:styled-content> expression in osteoblasts may be a new mechanism to limit inflammation\u2010mediated osteoporosis by promoting their differentiation into osteoblasts.<\/jats:p>","DOI":"10.1111\/jcmm.12553","type":"journal-article","created":{"date-parts":[[2015,3,27]],"date-time":"2015-03-27T08:39:49Z","timestamp":1427445589000},"page":"1814-1824","source":"Crossref","is-referenced-by-count":15,"title":["Identification of C\u2010terminal Hsp70\u2010interacting protein as a mediator of tumour necrosis factor action in osteoblast\u00a0differentiation by targeting osterix for 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