Abrogation of Cbl–PI3K Interaction Increases Bone Formation and Osteoblast Proliferation

Cbl is an adaptor protein and E3 ligase that plays both positive and negative roles in several signaling pathways that affect various cellular functions. Tyrosine 737 is unique to Cbl and phosphorylated by Src family kinases. Phosphorylated CblY737 creates a binding site for the p85 regulatory subunit of phosphatidylinositol 3 kinase (PI3K) that also plays an important role in the regulation of bone homeostasis. To investigate the role of Cbl–PI3K interaction in bone homeostasis, we examined knock-in mice in which the PI3K binding site on Cbl was ablated due to the substitution of tyrosine 737 to phenylalanine (CblYF/YF, YF mice). We previously reported that bone volume in these mice is increased due to decreased osteoclast function (Adapala et al., J Biol Chem 285:36745–36758, 19). Here, we report that YF mice also have increased bone formation and osteoblast numbers. In ex vivo cultures bone marrow-derived YF osteoblasts showed increased Col1A expression and their proliferation was also significantly augmented. Moreover, proliferation of MC3T3-E1 cells was increased after treatment with conditioned medium generated by culturing YF bone marrow stromal cells. Expression of stromal derived factor-1 (SDF-1) was increased in YF bone marrow stromal cells compared to wild type. Increased immunostaining of SDF-1 and CXCR4 was observed in YF bone marrow stromal cells compared to wild type. Treatment of YF condition medium with neutralizing anti-SDF-1 and anti-CXCR4 antibodies attenuated MC3T3-E1 cell proliferation. Cumulatively, these results show that abrogation of Cbl–PI3K interaction perturbs bone homeostasis, affecting both osteoclast function and osteoblast proliferation

The Ubiquitin/Proteasome System Mediates Entry and Endosomal Trafficking of Kaposi's Sarcoma-Associated Herpesvirus in Endothelial Cells

Ubiquitination, a post-translational modification, mediates diverse cellular functions including endocytic transport of molecules. Kaposi's sarcoma-associated herpesvirus (KSHV), an enveloped herpesvirus, enters endothelial cells primarily through clathrin-mediated endocytosis. Whether ubiquitination and proteasome activity regulates KSHV entry and endocytosis remains unknown. We showed that inhibition of proteasome activity reduced KSHV entry into endothelial cells and intracellular trafficking to nuclei, thus preventing KSHV infection of the cells. Three-dimensional (3-D) analyses revealed accumulation of KSHV particles in a cytoplasmic compartment identified as EEA1+ endosomal vesicles upon proteasome inhibition. KSHV particles are colocalized with ubiquitin-binding proteins epsin and eps15. Furthermore, ubiquitination mediates internalization of both KSHV and one of its receptors integrin β1. KSHV particles are colocalized with activated forms of the E3 ligase c-Cbl. Knock-down of c-Cbl or inhibition of its phosphorylation reduced viral entry and intracellular trafficking, resulting in decreased KSHV infectivity. These results demonstrate that ubiquitination mediates internalization of both KSHV and one of its cognate receptors integrin β1, and identify c-Cbl as a potential E3 ligase that facilitates this process

iTRAQ-Based Proteomic Analysis reveals possible target-related proteins and signal networks in human osteoblasts overexpressing FGFR2

Abstract Background Fibroblast growth factor receptor 2 (FGFR2) play a vital role in skeletogenesis. However, the molecular mechanisms triggered by FGFR2 in osteoblasts are still not fully understood. In this study, proteomics and bioinformatics analysis were performed to investigate changes in the protein profiles regulated by FGFR2, with the goal of characterizing the molecular mechanisms of FGFR2 function in osteoblasts. Methods In this study, FGFR2-overexpression cell line was established using the lentivirus-packaging vector in human osteoblasts (hFOB1.19). Next, the isobaric tags for relative and absolute quantitation (iTRAQ) in combination with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to compare the proteomic changes between control and FGFR2-overexpression cells. Thresholds (fold-change of ≥ 1.5 and a P-value of < 0.05) were selected to determine differentially expressed proteins (DEPs). The bioinformatics analysis including GO and pathway analysis were done to identify the key pathways underlying the molecular mechanism. Results A Total of 149 DEPs was identified. The DEPs mainly located within organelles and involved in protein binding and extracellular regulation of signal transduction. ColI, TNC, FN1 and CDKN1A were strikingly downregulated while UBE2E3, ADNP2 and HSP70 were significantly upregulated in FGFR2-overexpression cells. KEEG analysis suggested the key pathways included cell death, PI3K-Akt signaling, focal adhesion and cell cycle. Conclusions To our knowledge, this is the first protomic research to investigate alterations in protein levels and affected pathways in FGFR2-overexpression osteoblasts. Thus, this study not only provides a comprehensive dataset on overall protein changes regulated by FGFR2, but also shed light on its potential molecular mechanism in human osteoblasts