Temporal Profiling and Pulsed SILAC Labeling Identify Novel Secreted Proteins During Ex Vivo Osteoblast Differentiation of Human Stromal Stem Cells

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. Molecular & Cellular

Osteoblastic cell secretome: A novel role for progranulin during risedronate treatment

“…As expected, the best protein recovery was found in gel bands of those samples containing proteins ranging from 60 to 100 kDa. In agreement with a previous publication [24], the Hobit cells secrete proteins also found in the secretome of human stromal bone cells such as osteonectin, lumican, fibrillin-1 and nidogen-1 which, together with the collagen type I, play a direct role in bone formation.…”

“…The majority of all previously reported protein constituents of human stromal stem cells were found [24], confirming our cellular model is suitable to study the osteoblasts biological processes. Among the identified proteins, we show here for the first time that progranulin exerts proliferative and pro-survival effects on osteocyte-like cells, suggesting that this growth factor merits further investigation in the context of osteoblast/osteocyte biology.…”

Cyclic-RGD Peptides Increase the Adenoviral Transduction of Human Mesenchymal Stem Cells

“…Multiple secreted molecules have been implicated in directing stem cell osteogenic differentiation, including BMP family members [41], glucocorticoids [42], parathyroid hormone [43], and oxysterols [44]. Furthermore, recent studies have identified hundreds of proteins in hMSCs' secretome during medium-induced osteogenic differentiation [45,46]. Therefore, in future studies, it will be interesting to harness these techniques to measure the change in proteins secreted by hMSCs when they interact with cyclic-RGD peptides presented in different contexts.…”

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) for Quantitative Proteomics

“…A similar approach has been applied to the bacterium Escherichia coli [ 96 ], human HeLa cells [ 97 -99 ], human A549 lung carcinoma cells [ 92 , 100 ], human stromal stem cells [ 101 ], mouse NIH3T3 and C2C12 cells [ 98 , 102 ] and chicken [ 91 ]. Selbach and coworkers have compared protein translation rates between two samples by pulsed SILAC labeling with two different sets of stable isotopes, medium and heavy [ 102 , 103 ].…”