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-Cblmediated 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.

Cbl-b and c-Cbl negatively regulate osteoblast differentiation by enhancing ubiquitination and degradation of Osterix

“…Another mechanism by which Cbl-b/c-Cbl controls osteoblast differentiation is through the regulation of osteogenic transcription factors. Recently, c-Cbl-mediated ubiquitination and degradation of the transcription factor STAT5 have been shown to decrease the interaction between STAT5 and Runx2 and to reduce osteoblast differentiation of MSCs [25]. Cbl-b also increased the protein stability and transcriptional activity of Runx2 in osteoblast cell lines [35].…”

“…Cbl-b and c-Cbl proteins, members of mammalian Cbl (Casitas B-lineage lymphoma) family, act as cytoplasmic adaptors and E3 ubiquitin ligases, and they regulate bone formation both positively and negatively [21][22][23][24][25][26][27][28]. Other members of Cbl family have not been shown to possess ubiquitination-related function.…”

“…N-terminal proline-rich region (PRR) mediates the protein-protein interaction. Osterix acts downstream of Runx2 and it, in turn, regulates the expression of many osteoblast differentiation markers including Alkaline phosphatase, Osteocalcin, Osteonectin, Osteopontin and Runx2 [14,16,20].Cbl-b and c-Cbl proteins, members of mammalian Cbl (Casitas B-lineage lymphoma) family, act as cytoplasmic adaptors and E3 ubiquitin ligases, and they regulate bone formation both positively and negatively [21][22][23][24][25][26][27][28]. Other members of Cbl family have not been shown to possess ubiquitination-related function.…”

“…E3 ubiquitin ligases control the ubiquitination and proteasome-mediated degradation of target proteins. Ubiquitin ligase activity of Cbl proteins has been shown to regulate bone formation both positively and negatively [25,26,34]. In addition, down-regulation of Cbl-b or c-Cbl has been shown to affect osteoblast proliferation through the regulation of PI3K-Akt signaling [23,24].…”

STAT5 in Cancer and Immunity

“…Recently, a functional role for the E3 ubiquitin ligase c-Cbl was demonstrated in differentiation of osteoblasts and that blocking c-Cbl activity, downregulates STAT5 ubiquitination and promotes bone regeneration [74].…”