Abstract
It is well established that bone forming cells (osteoblasts) secrete proteins with autocrine, paracrine, and endocrine function. However, the identity and functional role for the majority of these secreted and differentially expressed proteins during the osteoblast (OB) differentiation process, is not fully established. To address these questions, we quantified the temporal dynamics of the human stromal (mesenchymal, skeletal) stem cell (hMSC) secretome during ex vivo OB differentiation using stable isotope labeling by amino acids in cell culture (SILAC). In addition, we employed pulsed SILAC labeling to distinguish genuine secreted proteins from intracellular contaminants. We identified 466 potentially secreted proteins that were quantified at 5 time-points during 14-days ex vivo OB differentiation including 41 proteins known to be involved in OB functions. Among these, 315 proteins exhibited more than 2-fold up or down-regulation. The pulsed SILAC method revealed a strong correlation between the fraction of isotope labeling and the subset of proteins known to be secreted and involved in OB differentiation. We verified SILAC data using qRT-PCR analysis of 9 identified potential novel regulators of OB differentiation. Furthermore, we studied the biological effects of one of these proteins, the hormone stanniocalcin 2 (STC2) and demonstrated its autocrine effects in enhancing osteoblastic differentiation of hMSC. In conclusion, combining complete and pulsed SILAC labeling facilitated the identification of novel factors produced by hMSC with potential role in OB differentiation. Our study demonstrates that the secretome of osteoblastic cells is more complex than previously reported and supports the emerging evidence that osteoblastic cells secrete proteins with endocrine functions and regulate cellular processes beyond bone formation.
Highlights
From the ‡Center for Experimental Bioinformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense; §Molecular Endocrinology Unit (KMEB), Department of Endocrinology, University Hospital of Odense, Denmark; ¶Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud University, KSA
Among the identified proteins, we demonstrated that stanniocalcin 2 (STC2) stimulates osteoblast differentiation of hMSC in an autocrine fashion
HMSC Cells Maintain Their Ex Vivo OB Differentiation Capacity in SILAC Medium—To identify and quantify changes of proteins secreted by hMSC during OB differentiation, we employed hMSC-TERT as a model system [23]. hMSC-TERT fulfill the standard criteria of the hMSC phenotype: they differentiate into osteoblasts in ex vivo cultures and form heterotopic bone in vivo [24]
Summary
Control unstimulated cells were referred to as day 0 At these time points, the cells were washed with DMEM and incubated for 18 h with serum free OB induction medium with either Lys0/Arg0, Lys4/Arg, or Lys8/Arg amino acids (Fig. 2A). The raw data files from the LTQ-Orbitrap Velos (the second biological replicate of the complete SILAC labeled samples) were processed using the MaxQuant software v. All protein identifications were required to have at least one (unique ϩ razor) peptide quantified in each of the five time-points for clustering based on the pulsed labeling analysis. Because of the high correlation of the quantified values (supplemental Fig. S3) the pulsed SILAC data from the experiments using in-gel and in-solution digestion the two data sets were combined to improve the number of proteins quantified at all time-points. Statistical Analysis—Comparison between groups was performed using 2-tailed unpaired Student’s t test. p Ͻ 0.05 was considered significant
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