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New insights in osteogenic differentiation revealed by mass spectrometric assessment of phosphorylated substrates in murine skin mesenchymal cells

Erik Halcsik1, Maria Fernanda Forni1, Andre Fujita2, Thiago Verano-Braga3, Ole Nørregaard Jensen3 and Mari Cleide Sogayar1*

Author Affiliations

1 Chemistry Institute, Department of Biochemistry, Cell and Molecular Therapy Center (NUCEL/NETCEM), School of Medicine, University of São Paulo, São Paulo 05508-000, SP, Brazil

2 Department of Computer Science, Institute of Mathematics and Statistics, University of São Paulo, Rua do Matão 1010, São Paulo, SP 05508-090, Brazil

3 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark

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BMC Cell Biology 2013, 14:47  doi:10.1186/1471-2121-14-47

Published: 22 October 2013



Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNFα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated murine skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS.


From 150 μg of starting material, 2,264 proteins were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied.


Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during differentiation to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells.