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

Abstract

BackgroundBone 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.ResultsFrom 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.ConclusionsTaken 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.

HMGB1 Osteo‐Modulatory Action on Osteosarcoma SaOS‐2 Cell Line: An Integrated Study From Biochemical and ‐Omics Approaches

“…The observations from SWATH-MS analysis are strictly in accordance with previous observations concerning the upregulation of transcription factors and DNA-binding motifs in osteoblast differentiation process [Halcsik et al, 2013] as well as the important modifications of cell metabolism of cells undergoing osteogenic pathway [Hakelien et al, 2014].…”

p38 MAPK Signaling in Osteoblast Differentiation

“…The involvement of these MAPKs has been observed in different osteogenic in vitro models (Gallea et al, 2001 ; Viñals et al, 2002 ; Guicheux et al, 2003 ). A recent report showed that p38 was predicted to account for 20% of the phospho-residues identified after treatment of MSCs with BMP (Halcsik et al, 2013 ). TGF-β provokes the phosphorylation of p38, ERK and JNK in a very rapid manner in MC3T3 and primary osteoblasts (Arnott et al, 2008 ).…”

Simultaneous Isolation of Three Different Stem Cell Populations from Murine Skin

“…Osteogenesis was also addressed and achieved, being corroborated by Alizarin Red positive staining and increased levels of alkaline phosphatase mRNA, two standard methods for this type of differentiation. In addition, these cells also show a typical osteogenecic-associated phosphorylation pattern when inquired through phosphoproteomics [ 19 ]. Chondrogenesis was screened by Toluidine blue staining and collagen type II expression, with both parameters being positive in our model, suggesting that chondrocyte-like cells were obtained.…”

“…Chondrogenesis: DMEM-F12 (Gibco, Rockville, MD) 3:1, 0.5ug/ml Insulin (catalog number # I1882-100MG, Invitrogen, USA), 50uM ascorbic acid (catalog number # A4544-25G, Sigma, USA), 10ng/mL TGFβ-1 (catalog number # 7666-MB-005, R&D Systems, USA) and 1mM sodium piruvate (catalog number # 11360–070, Sigma, USA). Osteogenesis: αMEM (Gibco, Rockville, MD), 10% FBS, 0.1uM dexamethasone (catalog number # D4902-25MG, Sigma, USA), 2mM ascorbic acid (catalog number # A4544-25G, Sigma, USA), and 10mM Glycerol 2-Phosphate (catalog number # G9422-10G, Sigma, USA) as described in [ 19 ].…”