Proteomic and transcriptomic analyses of retinal pigment epithelial cells exposed to REF-1/TFPI-2.
OBJECTIVE
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.
Shibuya
Masahiko
M
Laboratory of Cellular and Molecular Biology, National Institute of Sensory Organs, National Hospital Organization Tokyo Medical Center, Tokyo, Japan.
Okamoto
Haru
H
Nozawa
Takehiro
T
Utsumi
Jun
J
Reddy
Venkat N
VN
Echizen
Hirotoshi
H
Tanaka
Yasuhiko
Y
Iwata
Takeshi
T
D049109:Cell Proliferation; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D050699:E2F3 Transcription Factor / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling; D006023:Glycoproteins / Q000494:pharmacology*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D020543:Proteome; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014158:Transcription, Genetic; D015854:Up-Regulation
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050699:E2F3 Transcription Factor; C098319:E2F3 protein, human; D005136:Eye Proteins; D006023:Glycoproteins; C489427:MYC protein, human; D020543:Proteome; D016271:Proto-Oncogene Proteins c-myc; D016160:Retinoblastoma Protein; C021240:tissue-factor-pathway inhibitor 2