Telomerase Immortalization of Human Corneal Endothelial Cells Yields Functional Hexagonal Monolayers

Human corneal endothelial cells (HCEnCs) form a monolayer of hexagonal cells whose main function is to maintain corneal clarity by regulating corneal hydration. HCEnCs are derived from neural crest and are arrested in the post-mitotic state. Thus cell loss due to aging or corneal endothelial disorders leads to corneal edema and blindness–the leading indication for corneal transplantation. Here we show the existence of morphologically distinct subpopulations of HCEnCs that are interspersed among primary cells and exhibit enhanced self-renewal competence and lack of phenotypic signs of cellular senescence. Colonies of these uniform and hexagonal HCEnCs (HCEnC-21) were selectively isolated and demonstrated high proliferative potential that was dependent on endogenous upregulation of telomerase and cyclin D/CDK4. Further transduction of HCEnC-21 with telomerase yielded a highly proliferative corneal endothelial cell line (HCEnT-21T) that was devoid of oncogenic transformation and retained critical corneal endothelial cell characteristics and functionality. This study will significantly impact the fields of corneal cell biology and regenerative medicine

Resonance assignment and secondary structure of the middle MA-3 domain and complete tandem MA-3 region of the tumour suppressor protein Pdcd4

Pdcd4 (Programmed Cell Death Protein 4) is a novel eukaryotic tumour suppressor protein, which is involved in the regulation of both transcription and translation (reviewed in Lankat-Buttgereit and Göke 2009). The protein contains two interacting MA-3 domains (MA-3M and MA-3C), which are linked by a short semi-flexible linker region (Waters et al. 2007; Suzuki et al. 2008). The MA-3 domains are involved in mediating specific protein–protein interactions with functional partners such as eIF4A (Yang et al. 2003). Here we report essentially complete backbone and side chain 15N, 13C and 1H assignments for a construct composed of the middle MA-3 domain and subsequent linker region (MA-3M) and backbone assignments for the entire tandem MA-3 region of Pdcd4 (Pdcd4 MA-3M-C). Analysis of the backbone chemical shift data obtained indicates that Pdcd4 MA-3M contains eight helical regions corresponding to over 74% of the protein backbone and that Pdcd4 MA-3M-C contains fifteen helical regions (72%). Comparison of the position of these helical regions with those observed in the crystal structures suggests that the solution and crystal structures of both proteins are very similar

Decline in DJ-1 and decreased nuclear translocation of Nrf2 in Fuchs endothelial corneal dystrophy

PURPOSE. This study sought to determine factors involved in nuclear factor erythroid 2-related factor 2 (Nrf2) regulation and their response to oxidative stress in Fuchs endothelial corneal dystrophy (FECD) and normal corneal endothelial cells (CECs). METHODS. FECD corneal buttons were obtained from transplantations and normal human corneas from tissue banks. Oxidative stress was induced by tert-butyl hydroperoxide (tBHP). Protein and mRNA levels of Nrf2, DJ-1, p53, and Kelchlike ECH-associated protein1 (Keap1) were investigated using Western blotting and real-time PCR. Immunoprecipitation was used to detect levels of oxidized DJ-1 protein and Cullin 3- (Cul3)-regulated degradation of DJ-1 in immortalized FECD (FECDi) and normal CEC (HCECi) cell lines. Nrf2 subcellular localization was assessed by immunocytochemistry. RESULTS. Nrf2 protein stabilizer, DJ-1, decreased significantly in FECD CECs compared with normal, whereas Nrf2 protein repressor, Keap1, was unchanged at baseline but increased under oxidative stress. Under oxidative stress, normal CECs upregulated DJ-1 protein synthesis, whereas FECD CECs did not. DJ-1 decline correlated with increased DJ-1 oxidative modification and carbonylation in FECDi as compared with HCECi. Increased labeling of immunoprecipitated DJ-1 protein with anti-Cul3 antibody indicated enhanced DJ-1 degradation in FECDi as compared with HCECi. Following tBHP treatment, Nrf2 translocated from cytoplasm to nuclei in normal CECs, whereas Nrf2 nuclear localization was not observed in FECD. CONCLUSIONS. Decreased levels of DJ-1 in FECD at baseline and under oxidative stress correlate with impaired Nrf2 nuclear translocation and heightened cell susceptibility to apoptosis. Targeting the DJ-1/Nrf2 axis could yield a mechanism to slow CEC degeneration in FECD. © 2012 The Association for Research in Vision and Ophthalmology, Inc

Decline in DJ-1 and Decreased Nuclear Translocation of Nrf2 in Fuchs Endothelial Corneal DystrophyRoles of DJ-1 and Nrf2 in the Development of FECD

Purpose: This study sought to determine factors involved in nuclear factor erythroid 2–related factor 2 (Nrf2) regulation and their response to oxidative stress in Fuchs endothelial corneal dystrophy (FECD) and normal corneal endothelial cells (CECs). Methods: FECD corneal buttons were obtained from transplantations and normal human corneas from tissue banks. Oxidative stress was induced by tert-butyl hydroperoxide (tBHP). Protein and mRNA levels of Nrf2, DJ-1, p53, and Kelch-like ECH-associated protein1 (Keap1) were investigated using Western blotting and real-time PCR. Immunoprecipitation was used to detect levels of oxidized DJ-1 protein and Cullin 3- (Cul3)–regulated degradation of DJ-1 in immortalized FECD (FECDi) and normal CEC (HCECi) cell lines. Nrf2 subcellular localization was assessed by immunocytochemistry. Results: Nrf2 protein stabilizer, DJ-1, decreased significantly in FECD CECs compared with normal, whereas Nrf2 protein repressor, Keap1, was unchanged at baseline but increased under oxidative stress. Under oxidative stress, normal CECs upregulated DJ-1 protein synthesis, whereas FECD CECs did not. DJ-1 decline correlated with increased DJ-1 oxidative modification and carbonylation in FECDi as compared with HCECi. Increased labeling of immunoprecipitated DJ-1 protein with anti-Cul3 antibody indicated enhanced DJ-1 degradation in FECDi as compared with HCECi. Following tBHP treatment, Nrf2 translocated from cytoplasm to nuclei in normal CECs, whereas Nrf2 nuclear localization was not observed in FECD. Conclusions: Decreased levels of DJ-1 in FECD at baseline and under oxidative stress correlate with impaired Nrf2 nuclear translocation and heightened cell susceptibility to apoptosis. Targeting the DJ-1/Nrf2 axis could yield a mechanism to slow CEC degeneration in FECD

HCEnC-21 and HCEnC-21T retain typical corneal endothelial barrier integrity and pump function.

<p>(A) Cells were plated in 12-well transwell inserts (0.4 µm) at a density of 100,000 cells per transwell and transendothelial resistance (TER) was measured every 2 or 4 days over the course of 4.5 wk. Note that earlier (32–39) and later (58–67) passages of both HCEnC-21 and HCEnC-21T established a typical corneal endothelial barrier of 15 Ω*cm² after about 2 wk and maintained this barrier for 2.5 more wk. (B,C) HCEnC-21 (B) and HCEnC-21T (C) were grown to confluence (3,000 cells/cm²) in perfusion chambers and 20 mM lactate was applied to their apical, basolateral or both cell membranes. Facilitated lactate uptake is H⁺ coupled and was measured indirectly by detecting intracellular pH changes using the BCECF-AM fluorescent dye. Temporal acidification of both HCEnC-21 and HCEnC-21T was observed after lactate pulses to the apical and basolateral membrane. Error bars indicate mean ± SEM. *, P<0.05.</p

Synthesis of cyclin D, CDK4, p16^INK4 and p53 in HCEnC-21 and HCEnC-21T.

<p>(A) Analysis of p53 functionality. Oxidative stress was induced in confluent monolayers of HCEnC-21 and HCEnC-21T cells using 50 µM <i>tert</i>-Butyl-hydroperoxide for 1 hr. Cell extracts were then separated by SDS-PAGE and immunoblotted with antibodies against p53, phospho-p53 (serine-15), and β-actin. P53 was detected in extracts of both HCEnC-21 and HCEnC-21T and levels of phospho-p53 (activated p53) were increased upon induction of oxidative stress. E: earlier passages <25. L: later passages >45. (B) Synthesis of G1 phase regulatory proteins. Cell extracts were run on SDS-polyacrylamide gels and immunoblotted with antibodies against cyclin D, CDK4, p16^INK4 and β-actin. No changes in p16^INK4 protein levels were detected. Cyclin D and CDK4 levels were increased in both, HCEnC-21 and HCEnC-21T compared to 21M.</p

Primer Sequences for RT-PCR.

<p>Primer Sequences for RT-PCR.</p

Morphologic study of corneal endothelial primary cells.

<p>(A) In vivo confocal microscopy of human corneal endothelium demonstrating the typical hexagonal cell morphology. (B) Phase-contrast micrograph of non-dividing primary cells from a 21-year-old donor (21M) at passage 9, displaying typical signs of senescence. 200x. (C) Phase-contrast micrograph of primary cells from a 63-year-old donor that underwent endothelial-mesenchymal transformation at passage 1. Note that the cells lost the typical corneal endothelial morphology and appear fibroblast-like. 200x. (D) Phase-contrast micrograph showing 2 morphologically distinct subpopulations of cells in the 21M primary culture. The uniform cells growing in colony-like structures were designated HCEnC-21. 40x. (E-H) Phase-contrast micrographs of HCEnC-21 at passages 24 (E) and 46 (F) as well as telomerase-transduced HCEnC-21 (HCEnC-21T) at passages 25 (G) and 50 (H). Importantly, HCEnC-21 and HCEnC-21T cells grew in contact-inhibited monolayers displaying the typical hexagonal cell morphology seen in vivo. 200x. (I) Phase-contrast micrograph of telomerase-transduced 21M (21M+hTERT) at passage 7 showing a senescent phenotype. 200x.</p

Telomerase expression, telomerase activity and cell doubling time.

<p>(A) Telomerase mRNA levels relative to 21M primary cells were detected by real-time PCR. Telomerase-transduced primary (21M+hTERT) and HCEnC-21 cells (HCEnC-21T) showed strongly elevated telomerase mRNA levels. Importantly, non-transduced HCEnC-21 cells also expressed significantly higher levels of telomerase mRNA than 21M primary cells. (B) Telomerase activity indicated as template copy number was measured in cell extracts using the telomeric repeat amplification protocol (TRAPeze RT). Heat-inactivated extracts served as negative controls. No specific telomerase activity was detected in 21M primary cells, while HCEnC-21T cell lysates contained strongly elevated telomerase activity. Note that HCEnC-21 intrinsically upregulated endogenous telomerase activity. (C) Cell doubling time of earlier (19–24) and later (32–41) passages of HCEnC-21 and HCEnC-21T. Cells were plated in 12-well plates at a density of 50,000 cells/well and counted after 2, 3, and 4 days using a hemocytometer. HCEnC-21T showed a higher proliferation rate compared to HCEnC-21 in long-term cultures. Error bars indicate mean ± SEM. *, P<0.05.</p