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M. Shibuya, H. Okamoto, T. Nozawa, Y. Tanaka, J. Utsumi, V.N. Reddy, H. Echizen, T. Iwata; Proteome Analysis of Retinal Pigment Epithelial Cells Treated With Growth Promotive Factor; REF–1/TFPI–2 . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5539.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously reported growth promotive factor REF–1 specific to RPE cell proliferation (IOVS 45:245–252, 2004). This REF–1 was later found identical to tissue factor pathway inhibitor 2 (TFPI–2). To determine the biological effect of TFPI–2, transcriptome and proteome analysis of TFPI–2 treated RPE cells were performed.
Human primary RPE cells treated with TFPI–2 were cultured in DMEM medium with 15% FCS for 24 hours. The cell extract was separated by 2D–gel electrophoresis followed by SYPRO Ruby (BIO–RAD) gel staining. Ten differentially observed spots were in gel digested for LC–MS/MS analysis (LCQ DECA XP plus, Thermo Electron Corporation). The data analysis software (Bioworks v3.1, Thermo Electron) was used for protein identification. Transcriptional analysis of mRNA extracted from TFPI–2 treated and non–treated cells were performed using DNA microarray containing 30,000 genes (1700 Chemiluminescent Microarray Analyzer, Applied Biosystems). Total RNA was isolated from cultured RPE cells using total RNA isolation kit (RNA–Bee–RNA Isolation Reagent; Tel–Test, Friendswood, TX) after 6h, 12h, and 24h of TFPI–2 treatment.
Proteome analysis revealed 2 proteins c–myc binding protein(MYCBP) and ribosomal protein L11(RPL11) induced by TFPI–2 treatment. By transcriptome analysis out of 33,096 genes analyzed 10,773 genes were detected in the TFPI–2 treated samples whereas only 2,186 genes were detected in non–treated cells. Differential gene expression of c–myc, Mdm2, Transcription factor E2F3, retinoblastoma binding protein(Rb), and the p21 gene which all relates to two induced proteins MYCBP and RPL11 were detected in DNA microarray.
The proliferation of human RPE cell is promoted by stimulation of TFPI–2. TFPI–2 treatment triggered c–myc synthesis and the activation of E2F in the Rb/E2F pathway at the G1 phase of cell cycle leading to proliferation of human RPE cells. It is speculated that RPL11 and the Mdm2 complex of p53 pathway may be down regulating to balance the hyper–proliferative condition.
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