Characterization of a Bcl-XL-interacting protein FKBP8 and its splice variant in human RPE cells.

The human retinal pigment epithelial (RPE) cells have low turnover rate and often survive for an individual’s lifespan.1 However, when there is loss of RPE cells via apoptosis, which may occur in chronic eye diseases such as age-related macular degeneration (AMD), retinal function can be compromised. Patchy loss of RPE is one of the classic features of AMD2 and it has been shown that this process involves apoptosis.3 In eyes with wet AMD, apoptotic cells are present in choroidal neovascular membranes.4 The molecular mechanisms controlling mitochondria- and death receptor-mediated apoptosis have been studied extensively5-7 and it has been clearly demonstrated that the Bcl2 family of proteins are central regulators of apoptosis.8-10 Results from recent studies indicate that an immunophilin protein, FKBP8, interacts with Bcl2/ Bcl-XL and regulates the downstream signaling events.11-14 FK506-binding proteins (FKBPs) belong to a major family of immunophilins that bind to immunosuppressive drugs, FK506 and rapamycin, and convey their effects on immune responses and apoptosis.15 At least 15 FKBPs have been identified in humans.16,17 Despite the distinct subcellular localization, all FKBP proteins have a conserved motif, the FK506-binding domain, that mediates peptidyl-prolyl cis-trans isomerase (PPIase) activity and FK506 binding.16,17 FKBPs often function as chaperones or co-chaperones by directly interacting with other proteins. For instance, the prototypical FKBP family member FKBP12 binds to ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate (IP3) receptor, both of which are major intracellular calcium channels.18,19 Accordingly, it plays a key regulatory role in intracellular Ca2+ signaling, and most FKBP12-knockout mice die during embryonic development because of severe cardiomyopathy.20 FKBP8 (also known as FKBP38) was originally isolated from a T-cell cDNA library by a degenerative PCR approach.21 It is expressed in a variety of tissues with the highest abundance in the brain.21 The gene is located on chromosome 19, area p12, and consists of 9 exons.22 Compared with other FKBP family proteins, FKBP8 displays unique features in FK506 binding 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 binds to FK506 only in the presence of Ca/CaM.13,23 FKBP8-knockout mice died at embryonic day (E)13.5 and showed defects in eye development.24 Another unique function of FKBP8 is that it interacts with Bcl2 and Bcl-XL.11-13 Although controversial, such interaction has been demonstrated in different types of cells and is believed to be important for the localization and protein stability of Bcl2/Bcl-XL.11-14 The high abundance of FKBP8 in the retina and the requirement 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 cultured human RPE cells, along with a splice variant lacking the FK506-binding domain. Overexpression of both the full-length and the short form of FKBP8 sensitized RPE cells to thapsigargin-induced apoptosis, but did not affect the transcriptional function of the nuclear factor of activated T cells (NFAT). These data indicate that, by interacting with Bcl2 family proteins, FKBP8 may be a functional component of the apoptotic signaling network in the RPE cells and that such an effect could be independent of FK506.