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Characterization of a Bcl-XL-Interacting Protein FKBP8 and Its Splice Variant in Human RPE Cells

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Characterization of a Bcl-XL-Interacting Protein FKBP8 and Its Splice Variant in Human RPE Cells Characterization of a Bcl-XL-Interacting Protein FKBP8 and Its Splice Variant in Human RPE Cells Yan Chen, Paul Sternberg, and Jiyang Cai PURPOSE. The immunophilin protein FKBP8 interacts with ling mitochondria- and death receptor-mediated apoptosis have 5–7 Bcl2/Bcl-XL and is essential for mouse eye development. The been studied extensively and it has been clearly demon- purpose of this study was to define the expression of the strated that the Bcl2 family of proteins are central regulators of FKBP8 gene in cultured human RPE cells and explore its apoptosis.8–10 Results from recent studies indicate that an involvement in the control of apoptosis. immunophilin protein, FKBP8, interacts with Bcl2/ Bcl-XL and 11–14 METHODS. Rapid amplification of cDNA ends (RACE) was per- regulates the downstream signaling events. formed on RNA isolated from human RPE cells. The existence FK506-binding proteins (FKBPs) belong to a major family of of FKBP8 and a splice variant was confirmed by RT-PCR. The immunophilins that bind to immunosuppressive drugs, FK506 interaction between Bcl-X and FKBP8 was measured by coim- and rapamycin, and convey their effects on immune responses L and apoptosis.15 At least 15 FKBPs have been identified in munoprecipitation. ARPE-19 cells stably overexpressing FKBP8 16,17 and its splice variant were established. Their responses to humans. Despite the distinct subcellular localization, all thapsigargin and t-butyl hydroperoxide–induced cell death FKBP proteins have a conserved motif, the FK506-binding domain, that mediates peptidyl-prolyl cis-trans isomerase were measured by flow cytometry. Apoptosis was determined 16,17 by terminal deoxyribonucleotidyl transferase-mediated fluores- (PPIase) activity and FK506 binding. FKBPs often function cein-dUTP nick-end labeling (TUNEL) assay. The activities of as chaperones or co-chaperones by directly interacting with the nuclear factor of activated T cells (NFAT) were measured other proteins. For instance, the prototypical FKBP family by reporter assay after transient transfection. member FKBP12 binds to ryanodine receptor (RyR) and inosi- tol 1,4,5-trisphosphate (IP3) receptor, both of which are major RESULTS. RACE and RT-PCR identified a splice variant of FKBP8 intracellular calcium channels.18,19 Accordingly, it plays a key lacking exons 3, 4, and 5 in human RPE cells. Both the full- regulatory role in intracellular Ca2ϩ signaling, and most length and the short form of FKBP8 proteins showed mito- FKBP12-knockout mice die during embryonic development chondrial distribution and directly interacted with Bcl-XL. because of severe cardiomyopathy.20 Overexpression of FKBP8 caused increased sensitivity to apo- FKBP8 (also known as FKBP38) was originally isolated from ptosis induced by thapsigargin. The transcriptional activity of a T-cell cDNA library by a degenerative PCR approach.21 It is NFAT was not affected by FKBP8. expressed in a variety of tissues with the highest abundance in 21 CONCLUSIONS. FKBP8 and its novel splice variant are Bcl-XL- the brain. The gene is located on chromosome 19, area p12, interacting proteins and regulate the apoptotic signaling path- and consists of 9 exons.22 Compared with other FKBP family ways in the RPE. (Invest Ophthalmol Vis Sci. 2008;49: proteins, FKBP8 displays unique features in FK506 binding 1721–1727) DOI:10.1167/iovs.07-1121 ability and PPIase activity. Instead of being constitutively active or inactive, its PPIase activity requires prior binding of calcium and calmodulin (CaM) via a CaM binding domain.13 FKBP8 he human retinal pigment epithelial (RPE) cells have low binds to FK506 only in the presence of Ca/CaM.13,23 FKBP8- Tturnover rate and often survive for an individual’s life- knockout mice died at embryonic day (E)13.5 and showed 1 span. However, when there is loss of RPE cells via apoptosis, defects in eye development.24 Another unique function of which may occur in chronic eye diseases such as age-related 11–13 FKBP8 is that it interacts with Bcl2 and Bcl-XL. Although macular degeneration (AMD), retinal function can be compro- controversial, such interaction has been demonstrated in dif- mised. Patchy loss of RPE is one of the classic features of AMD2 3 ferent types of cells and is believed to be important for the and it has been shown that this process involves apoptosis. In localization and protein stability of Bcl2/Bcl-X .11–14 eyes with wet AMD, apoptotic cells are present in choroidal L 4 The high abundance of FKBP8 in the retina and the require- neovascular membranes. The molecular mechanisms control- ment of FKBP8 for normal eye development24 suggest that the protein performs important functions in this structure. In the present study, we showed that the FKBP8 gene is expressed in From the Vanderbilt Eye Institute, Vanderbilt University Medical cultured human RPE cells, along with a splice variant lacking Center, Nashville, Tennessee. the FK506-binding domain. Overexpression of both the full- Supported by the American Health Association Foundation, the length and the short form of FKBP8 sensitized RPE cells to Alston Callahan Postdoctoral Scholar Award (YC) from International thapsigargin-induced apoptosis, but did not affect the tran- Retinal Research Foundation, National Eye Institute Grants EY07892 scriptional function of the nuclear factor of activated T cells and EY08126, National Institute of Environmental Health Sciences (NFAT). These data indicate that, by interacting with Bcl2 Grant ES014668, and Research to Prevent Blindness, Inc. family proteins, FKBP8 may be a functional component of the Submitted for publication August 28, 2007; revised December 17, apoptotic signaling network in the RPE cells and that such an 2007; accepted February 22, 2008. Disclosure: Y. Chen, None; P. Sternberg, None; J. Cai, None effect could be independent of FK506. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- MATERIALS AND METHODS ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. Corresponding author: Jiyang Cai, Vanderbilt Eye Institute, Cell Culture Vanderbilt University Medical Center, 1115B Medical Research Build- ing IV, 2215B Garland Avenue, Nashville, TN 37232; Cultures of human retinal pigment epithelial (hRPE) cells were estab- [email protected]. lished as described previously.25 ARPE-19 cells were obtained from the Investigative Ophthalmology & Visual Science, April 2008, Vol. 49, No. 4 Copyright © Association for Research in Vision and Ophthalmology 1721 Downloaded from iovs.arvojournals.org on 10/01/2021 1722 Chen et al. IOVS, April 2008, Vol. 49, No. 4 TABLE 1. Primers Used for PCR Amplifications Product Size Gene Primer Sequence (bp) NFAT-c1 F: 5Ј-AGC CGA ATT CTC TGG TGG TTG AGA-3Ј 247 R: 5Ј-AGC TGC TGG CTG TAG TAT GGT CTT-3Ј NFAT-c2 F: 5Ј-AAC TGT CAC CAC CAC CAG CTA TGA-3Ј 321 R: 5Ј-AGG CAG CTG TCT GTG TCT TGT CTT-3Ј NFAT-c3 F: 5Ј-TCA AGT CAC ACC AAC ACC TCC TGT-3Ј 264 R: 5Ј-TGA GGC AGG GTA TGC ACA GAA TGA-3Ј NFAT-c4 F: 5Ј-AGA CCC TGC TTG CGA AAC TCC TTA-3Ј 316 R: 5Ј-ATG CAC ATC ACT CTG GGA AGG GAA-3Ј NFAT5 F: 5Ј-TGC TGT GTT ACA GGG CTC TTC AGT-3Ј 239 R: 5Ј-GTT GTG CCT GTT CTT GGG AAA GCA-3Ј FKBP8 F: 5Ј-ATG GCA TCG TGT GCT GAA CCC TC-3Ј Full length: 1239; Primer pair 1 R: 5Ј-GTT CCT GGC AGC GAT GAC CAC ACA-3Ј Variant: 759 FKBP8 F: 5Ј-CAT GGG ACA ACC CCC GGC GGA G-3Ј 133 Primer pair 2 R: 5Ј-CGT CAT GTC CAC CAG AAT GTC C-3Ј FKBP8 F: 5Ј-GGA CAT TCT GGT GGA CAT GAC G-3Ј 478 Primer pair 3 R: 5Ј-GTT CCT GGC AGC GAT GAC CAC ACA-3Ј American Type Culture Collection (ATCC, Manassas, VA). The cells permeabilization with 100% methanol. The recombinant FLAG-FKBP8 were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal and FLAG-FKBP8s were stained with anti-FLAG antibody (Sigma-Al- bovine serum in a humidified CO2 incubator at 37°C. drich, St. Louis, MO). Images were acquired with a confocal micro- scope (LSM 510; Carl Zeiss Meditec, Inc., Dublin, CA)28 provided by RNA Isolation, cDNA Synthesis, and Rapid the Vanderbilt Cell Imaging Shared Resource. Amplification of cDNA Ends Coimmunoprecipitation Total RNA was prepared from human RPE cells (RNeasy Mini Kit; Qiagen, Valencia, CA) and was reverse transcribed into cDNA by using The cells were grown on a 100-mm dish for 48 hours before they were random hexamer primers (Applied Biosystems, Inc. [ABI], Foster City, harvested. FLAG-tagged FKBP8 and FKBP8s were immunoprecipitated CA). To perform 5Ј rapid amplification of cDNA ends (RACE), we used (FLAG Tagged protein immunoprecipitation kit; Sigma-Aldrich), ac- a primer (5Ј-GCA GCA AGG AAC TCT CGG GCC AGG G-3Ј) corre- cording to the manufacturer’s protocol. Briefly, the cells were lysed sponding to the region of 301-325 (GenBank accession number with 1 mL of lysis buffer provided with the kit and incubated with the NM_012181; http://www.ncbi.nlm.nih.gov/Genbank; provided in the anti-FLAG M2 affinity gel overnight. After stringent washing, immuno- public domain by the National Center for Biotechnology Information, precipitates were eluted by 3ϫ FLAG peptide. To immunoprecipitate Ј Bethesda, MD) to amplify the 5 end of FKBP8 cDNA (Smart RACE Bcl-XL, the cell lysates were incubated with antibodies to Bcl-XL (Santa cDNA amplification kit; BD-Clontech, Mountain View, CA). PCR prod- Cruz Biotechnology, Santa Cruz, CA) and protein G-sepharose beads. ucts were ligated into the TOPO TA cloning vector (Invitrogen, Carls- The immunoprecipitates were boiled in 2ϫ SDS loading buffer and bad, CA), and multiple clones were chosen for sequencing.
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