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Investigation of proteome changes in osteoclastogenesis in low serum culture system using quantitative proteomics.

ABSTRACT: BACKGROUND:RAW 264.7 cells can differentiate into osteoclasts when cultured in medium supplemented with 1 % FBS. However, the proteomic changes in the development of RAW 264.7 cells into osteoclasts in low serum culture system have not been elucidated. Therefore, we conducted quantitative proteomics analysis to investigate proteomic changes during osteoclastogenesis in low serum culture system. RESULTS:Our study confirmed that mature multinucleated osteoclasts were generated in a low serum culture system, validated by upregulated expression of 15 characteristic marker proteins, including TRAP, CTSK, MMP9, V-ATPase and ITGAV. Proteomics results demonstrated that 549 proteins expressed differentially in osteoclastogenesis in low serum culture system. In-depth bioinformatics analysis suggested that the differentially expressed proteins were mainly involved in mitochondrial activities and energy metabolism, including the electron transport chain pathway, TCA cycle pathway, mitochondrial LC-fatty acid beta-oxidation pathway and fatty acid biosynthesis pathway. The data have been deposited to the ProteomeXchange with identifier PXD001935. CONCLUSION:Osteoclast formation is an ATP consuming procedure, whether occurring in a low serum culture system or a conventional culture system. In contrast to osteoclasts formed in conventional culture system, the fatty acid biosynthesis pathway was upregulated in osteoclasts cultured in low serum condition.

SUBMITTER: Xiong Q

PROVIDER: S-EPMC4815185 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Investigation of proteome changes in osteoclastogenesis in low serum culture system using quantitative proteomics.

Xiong Qi Q Zhang Lihai L Zhan Shaohua S Ge Wei W Tang Peifu P

Proteome science 20160331

<h4>Background</h4>RAW 264.7 cells can differentiate into osteoclasts when cultured in medium supplemented with 1 % FBS. However, the proteomic changes in the development of RAW 264.7 cells into osteoclasts in low serum culture system have not been elucidated. Therefore, we conducted quantitative proteomics analysis to investigate proteomic changes during osteoclastogenesis in low serum culture system.<h4>Results</h4>Our study confirmed that mature multinucleated osteoclasts were generated in a ...[more]

PMID: 27034619

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Project description:Because of their antagonistic catalytic functions, protein-tyrosine phosphatases (PTPs) and protein-tyrosine kinases act together to control phosphotyrosine-mediated signaling processes in mammalian cells. However, unlike for protein-tyrosine kinases, little is known about the cellular substrate specificity of many PTPs because of the lack of appropriate methods for the systematic and detailed analysis of cellular PTP function. Even for the most intensely studied, prototypic family member PTP1B many of its physiological functions cannot be explained by its known substrates. To gain better insights into cellular PTP1B function, we used quantitative MS to monitor alterations in the global tyrosine phosphorylation of PTP1B-deficient mouse embryonic fibroblasts in comparison with their wild-type counterparts. In total, we quantified 124 proteins containing 301 phosphotyrosine sites under basal, epidermal growth factor-, or platelet-derived growth factor-stimulated conditions. A subset of 18 proteins was found to harbor hyperphosphorylated phosphotyrosine sites in knock-out cells and was functionally linked to PTP1B. Among these proteins, regulators of cell motility and adhesion are overrepresented, such as cortactin, lipoma-preferred partner, ZO-1, or p120ctn. In addition, regulators of proliferation like p62DOK or p120RasGAP also showed increased cellular tyrosine phosphorylation. Physical interactions of these proteins with PTP1B were further demonstrated by using phosphatase-inactive substrate-trapping mutants in a parallel MS-based analysis. Our results correlate well with the described phenotype of PTP1B-deficient fibroblasts that is characterized by an increase in motility and reduced cell proliferation. The presented study provides a broad overview about phosphotyrosine signaling processes in mouse fibroblasts and, supported by the identification of various new potential substrate proteins, indicates a central role of PTP1B within cellular signaling networks. Importantly the MS-based strategies described here are entirely generic and can be used to address the poorly understood aspects of cellular PTP function.

Dataset Information

Investigation of proteome changes in osteoclastogenesis in low serum culture system using quantitative proteomics.

Publications

Investigation of proteome changes in osteoclastogenesis in low serum culture system using quantitative proteomics.

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