IOVS Circulation
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(Investigative Ophthalmology and Visual Science. 2007;48:516-521.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.06-0434
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Proteomic and Transcriptomic Analyses of Retinal Pigment Epithelial Cells Exposed to REF-1/TFPI-2

Masahiko Shibuya,1,2 Haru Okamoto,1 Takehiro Nozawa,3 Jun Utsumi,4 Venkat N. Reddy,5 Hirotoshi Echizen,2 Yasuhiko Tanaka,6 and Takeshi Iwata1

1From the Laboratory of Cellular and Molecular Biology, National Institute of Sensory Organs, National Hospital Organization Tokyo Medical Center, Tokyo, Japan; 2Department of Pharmacotherapy, Meiji Pharmaceutical University, Tokyo, Japan; 3Analytical Instrument Division, AMR Inc., Tokyo, Japan; 4R&D Division, Toray Industries, Inc., Tokyo, Japan; 5Department of Ophthalmology, Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan; and 6International University of Health and Welfare, Mita Hospital, Tokyo, Japan.

PURPOSE. The authors previously reported a growth-promoting factor, REF-1/TFPI-2, that is specific to retinal pigment epithelial (RPE) cells. The purpose of this study was to determine the genes and proteins of human RPE cells that are altered by exposure to TFPI-2.

METHODS. Human primary RPE cells were cultured with or without TFPI-2. Cell extracts and isolated RNA were subjected to proteomic and transcriptomic analyses, respectively. Proteins were separated by two-dimensional gel electrophoresis followed by gel staining and ion spray tandem mass spectrometry analyses. Transcriptomic analysis was performed using a DNA microarray to detect 27,868 gene expressions.

RESULTS. Proteomic analysis revealed c-Myc binding proteins and ribosomal proteins L11 preferentially induced by TFPI-2 in human RPE cells. Transcriptomic analysis detected 10,773 of 33,096 probes in the TFPI-2 treated samples, whereas only 2186 probes were detected in the nontreated samples. Among the genes up-regulated by TFPI-2 at the protein level were c-myc, Mdm2, transcription factor E2F3, retinoblastoma binding protein, and the p21 gene, which is associated with the c-myc binding protein and ribosomal protein L11.

CONCLUSIONS. The mechanisms by which TFPI-2 promotes the proliferation of RPE cells may be associated with augmented c-myc synthesis and the activation of E2F in the retinoblastoma protein (Rb)/E2F pathway at the G1 phase of the RPE cells. Activation of ribosomal protein L11 and the Mdm2 complex of the p53 pathway may be counterbalanced by the hyperproliferative conditions.






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