Characterization of a Bcl-XL-interacting protein FKBP8 and its splice variant in human RPE cells.
OBJECTIVE
The immunophilin protein FKBP8 interacts with Bcl2/Bcl-XL and is essential for mouse eye development. The purpose of this study was to define the expression of the FKBP8 gene in cultured human RPE cells and explore its involvement in the control of apoptosis.


METHODS
Rapid amplification of cDNA ends (RACE) was performed on RNA isolated from human RPE cells. The existence of FKBP8 and a splice variant was confirmed by RT-PCR. The interaction between Bcl-XL and FKBP8 was measured by coimmunoprecipitation. ARPE-19 cells stably overexpressing FKBP8 and its splice variant were established. Their responses to thapsigargin and t-butyl hydroperoxide-induced cell death were measured by flow cytometry. Apoptosis was determined by terminal deoxyribonucleotidyl transferase-mediated fluorescein-dUTP nick-end labeling (TUNEL) assay. The activities of the nuclear factor of activated T cells (NFAT) were measured by reporter assay after transient transfection.


RESULTS
RACE and RT-PCR identified a splice variant of FKBP8 lacking exons 3, 4, and 5 in human RPE cells. Both the full-length and the short form of FKBP8 proteins showed mitochondrial distribution and directly interacted with Bcl-XL. Overexpression of FKBP8 caused increased sensitivity to apoptosis induced by thapsigargin. The transcriptional activity of NFAT was not affected by FKBP8.


CONCLUSIONS
FKBP8 and its novel splice variant are Bcl-XL-interacting proteins and regulate the apoptotic signaling pathways in the RPE.
Chen
Yan
Y
Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
Sternberg
Paul
P
Cai
Jiyang
J
D017209:Apoptosis / Q000187:drug effects; D002478:Cells, Cultured; D005091:Exons / Q000235:genetics; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D020287:In Situ Nick-End Labeling; D018613:Microscopy, Confocal; D050778:NFATC Transcription Factors / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000378:metabolism* / Q000473:pathology; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D022021:Tacrolimus Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection; D051020:bcl-X Protein / Q000378:metabolism*; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
C494811:BCL2L1 protein, human; C471205:FKBP8 protein, human; D050778:NFATC Transcription Factors; D020033:Protein Isoforms; D012333:RNA, Messenger; D051020:bcl-X Protein; D019284:Thapsigargin; D020122:tert-Butylhydroperoxide; D022021:Tacrolimus Binding Proteins