Proteome profiling of mitotic clonal expansion during 3T3-L1 adipocyte differentiation using iTRAQ-2DLC-MS/MS.
Mitotic clonal expansion (MCE) is one of the important events taking place at the early stage during 3T3-L1 adipocyte differentiation. To investigate the mechanism underlying this process, we carried out a temporal proteomic analysis to profile the dynamic changes in MCE. Using 8-plex-iTRAQ-2DLC-MS/MS analysis, 3152 proteins were quantified during the initial 28 h of 3T3-L1 adipogenesis. Functional analysis was performed on 595 proteins with maximum or minimum quantities at 20 h of adipogenic induction that were potentially involved in MCE, which identified PI3K/AKT/mTOR as the most relevant pathway. Among the 595 proteins, PKM2 (Pyruvate kinase M2), a patterned protein identified as a potential target gene of C/EBPβ in our previous work, was selected for further investigation. Network analysis suggested positive correlations among C/EBPβ, PIN1, and PKM2, which may be related with the PI3K-AKT pathway. Knockdown of PKM2 with siRNA inhibited both MCE and adipocyte differentiation of 3T3-L1 cells. Moreover, PKM2 was down-regulated at both the mRNA level and the protein level upon the knockdown of C/EBPβ. And overexpressed PKM2 can partially restore MCE, although it did not restore terminal adipocyte differentiation, which was inhibited by siC/EBPβ. Thus, PKM2, potentially regulated by C/EBPβ, is involved in MCE during adipocyte differentiation. The dynamic proteome changes quantified here provide a promising basis for revealing molecular mechanism regulating adipogenesis.
Key Laboratory of Molecular Medicine, the Ministry of Education, Department of Biochemistry and Molecular Biology, Fudan University Shanghai Medical College , Shanghai 200032, People's Republic of China.
Yan
Y
Jiang
Liang
L
Guo
Li-Qi
LQ
Xie
You-You
YY
Zhang
Xiao-Hui
XH
Liu
Yang
Y
Zhang
Hao
H
Zhu
Peng-Yuan
PY
Yang
Hao-Jie
HJ
Lu
Qi-Qun
QQ
Tang
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D002853:Chromatography, Liquid / Q000379:methods; D002999:Clone Cells; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D051379:Mice; D008938:Mitosis; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D022782:CCAAT-Enhancer-Binding Protein-beta; D019869:Phosphatidylinositol 3-Kinases; D020543:Proteome; D051057:Proto-Oncogene Proteins c-akt; D011770:Pyruvate Kinase; D034741:RNA, Small Interfering; D058570:TOR Serine-Threonine Kinases; C545013:Cebpb protein, mouse; C546843:mTOR protein, mouse