title: New insights in osteogenic differentiation revealed by mass spectrometric assessment of phosphorylated substrates in murine skin mesenchymal cells.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.forename: Erik
authors.initials: E
authors.lastname: Halcsik
authors.forename: Maria Fernanda
authors.initials: MF
authors.lastname: Forni
authors.forename: Andre
authors.initials: A
authors.lastname: Fujita
authors.forename: Thiago
authors.initials: T
authors.lastname: Verano-Braga
authors.forename: Ole Nørregaard
authors.initials: ON
authors.lastname: Jensen
authors.affiliation: 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. mcsoga@iq.usp.br.
authors.forename: Mari Cleide
authors.initials: MC
authors.lastname: Sogayar
mesh_terms: D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics* / Q000378:metabolism; D047388:Casein Kinases / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D048670:MAP Kinase Kinase 4 / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D015398:Signal Transduction; D012867:Skin / Q000166:cytology / Q000378:metabolism*; D051153:Wnt Proteins / Q000235:genetics / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D010750:Phosphoproteins; D051153:Wnt Proteins; D047388:Casein Kinases; D018844:Cyclin-Dependent Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D018631:ras Proteins