'Two-stage double-technique hybrid (TSDTH)' identification strategy for the analysis of BMP2-induced transdifferentiation of premyoblast C2C12 cells to osteoblast.
Transdifferentiation offers new opportunities in the area of cell replacement therapy; however, the molecular mechanism by which transdifferentiation occurs is not fully understood. Our understanding about the sophisticated regulations of transdifferentiation is limited yet since their comprehensive proteome regulations have not been fully elucidated. Studies on bone morphogenic protein-2 (BMP2)-induced transdifferentiation of murine C2C12 cells, a myogenic lineage committed premyoblast, to osteogenic cells can provide a full picture of the dynamic events that occur at the level of protein activity and/or expression. Here, we investigated the overall dynamic regulatory proteome associated with BMP2-induced osteoblast transdifferentiation in premyoblast C2C12 cells using a novel Two-Stage Double-Technique Hybrid (TSDTH) strategy for proteomic analysis. Here, we took the approach of a TSDTH involving phosphoproteomic analysis after a short-term treatment (stage one, 30 min) and a long-term treatment (stage two, 3 days); SILAC (Stable isotope labeling with amino acids in cell culture)-proteomics was used to map the proteins. In these experiments, a total of 1321 potential phosphoproteins were identified in stage one analysis and 433 proteins were quantified in stage two analysis. Among them, 374 BMP2-specific phosphoproteins and 54 up- or down-regulated proteins were selected. In first stage analysis, several deubiquitination enzymes including Uch-l3 as well as ubiquitination related proteins were newly identified, and its inhibitor reduced the stability of phosphorylated Smad1, and the BMP2-induced ALP levels of C2C12 cells were detected. In second stage analysis, Thrombospondin1 was identified as the highest up-regulated protein by BMP2-long time stimulation and this was confirmed with immunoblot analysis. Furthermore, pathway enrichment and network analyses revealed that insulin-like growth factor (IGF) and calcium signaling pathways as well as TGFbeta/BMP signaling proteins are found to be potentially involved in the early and long-term actions of BMP2. Collectively, our TSDTH is a useful simple strategy to obtain comprehensive molecular mechanism of cellular processes such as transdifferentiation.
Kim
Byung-Gyu
BG
Department of Biochemistry, School of Dentistry, Kyungpook National University, Daegu, Korea.
Lee
Ji-Hyun
JH
Ahn
Jung-Mo
JM
Park
Sung Kyu
SK
Cho
Ji-Hoon
JH
Hwang
Daehee
D
Yoo
Jong-Shin
JS
Yates
John R
JR
Ryoo
Hyun-Mo
HM
Cho
Je-Yoel
JY
D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000378:metabolism*; D002460:Cell Line; D054338:Cell Transdifferentiation / Q000502:physiology*; D051379:Mice; D032446:Myoblasts / Q000378:metabolism*; D010006:Osteoblasts / Q000378:metabolism*; D010748:Phosphopeptides / Q000032:analysis; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D019699:Thrombospondins / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism; D054875:Ubiquitination; D015854:Up-Regulation
C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D010748:Phosphopeptides; D010750:Phosphoproteins; D019699:Thrombospondins; D016212:Transforming Growth Factor beta; D011494:Protein Kinases