title: Promotion of osteoblast differentiation in mesenchymal cells through Cbl-mediated control of STAT5 activity.
abstract: The identification of the molecular mechanisms controlling the degradation of regulatory proteins in mesenchymal stromal cells (MSC) may provide clues to promote MSC osteogenic differentiation and bone regeneration. Ubiquitin ligase-dependent degradation of proteins is an important process governing cell fate. In this study, we investigated the role of the E3 ubiquitin ligase c-Cbl in MSC osteoblast differentiation and identified the mechanisms involved in this effect. Using distinct shRNA targeting c-Cbl, we showed that c-Cbl silencing promotes osteoblast differentiation in murine and human MSC, as demonstrated by increased alkaline phosphatase activity, expression of phenotypic osteoblast marker genes (RUNX2, ALP, type 1 collagen), and matrix mineralization in vitro. Coimmunoprecipitation analyses showed that c-Cbl interacts with the transcription factor STAT5, and that STAT5 forms a complex with RUNX2, a master transcription factor controlling osteoblastogenesis. Silencing c-Cbl decreased c-Cbl-mediated STAT5 ubiquitination, increased STAT5 protein level and phosphorylation, and enhanced STAT5 and RUNX2 transcriptional activity. The expression of insulin like growth factor-1 (IGF-1), a target gene of STAT5, was increased by c-Cbl silencing in MSC and in bone marrow stromal cells isolated from c-Cbl deficient mice, suggesting that IGF-1 contributes to osteoblast differentiation induced by c-Cbl silencing in MSC. Consistent with these findings, pharmacological inhibition of STAT5 activity, or neutralization of IGF-1 activity, abrogated the positive effect of c-Cbl knockdown on MSC osteogenic differentiation. Taken together, the data provide a novel functional mechanism by which the ubiquitin ligase c-Cbl regulates the osteoblastic differentiation program in mesenchymal cells by controlling Cbl-mediated STAT5 degradation and activity.
authors.affiliation: Laboratory of Osteoblast Biology and Pathology, Paris, France.
authors.forename: François-Xavier
authors.initials: FX
authors.lastname: Dieudonne
authors.forename: Nicolas
authors.initials: N
authors.lastname: Sévère
authors.forename: Martin
authors.initials: M
authors.lastname: Biosse-Duplan
authors.forename: Jing-Jie
authors.initials: JJ
authors.lastname: Weng
authors.forename: Yeu
authors.initials: Y
authors.lastname: Su
authors.forename: Pierre J
authors.initials: PJ
authors.lastname: Marie
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism*; D051379:Mice; D008809:Mice, Inbred C3H; D018345:Mice, Knockout; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism*; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D050799:STAT5 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D050799:STAT5 Transcription Factor; D007334:Insulin-Like Growth Factor I; D044767:Ubiquitin-Protein Ligases
keywords: Mesenchymal cells; Osteoblast differentiation; RUNX2; STAT5; Ubiquitination; c-Cbl ubiquitin ligase