HepG2細胞由来分泌タンパク質のプロテオーム解析

抄録

It is known that adipocytes secrete proteins called adipocytokines, which take part in the regulation of the lipid and glucose metabolism. Similar to adipocytes tissue, liver is recently speculated to secrete proteins, which have some physiological functions. The hepatic lipid homeostasis is regulated by a cross talk between peroxisome proliferator-activated receptor (PPAR) alpha and liver X receptor (LXR), both of which are nuclear receptors. We attempted to identify the secreted proteins, which are regulated by PPAR-alpha or LXR in hepatocytes using proteomic analysis. Human hepatoma HepG2 cells were treated with TO-901317 (LXR agonist) or Wy-14, 643 (PPAR-alpha agonist). After 36-48 h, media were collected and concentrated by centrifugal filter devices. Paired samples from cells treated with vehicle or ligands were labeled with fluorescent Cy3 or Cy5 dyes and electrophoresed on the same two-dimensional gels. Some spots from the ligand-treated sample were found to be upregulated or downregulated as compared with those from vehicle sample. For identification of these secreted proteins by mass spectrometry, the 2-DE gel, loaded with 0.5-1 mg of total protein, was stained with Sypro Ruby. After in-gel digestion, the peptides from the protein spots were analyzed using RPLC-ESI MS/MS. We found that one of the proteins with significant differences was identified as apolipoproteinE, secretion of which was previously shown to be increased by treatment with LXR agonist. Now, we are trying to identify other secreted proteins from hepatocytes, which may have some roles in regulating lipid metabolism.