Conditionally Immortal Slc4a11-/- Mouse Corneal Endothelial Cell Line Recapitulates Disrupted Glutaminolysis Seen in Slc4a11-/- Mouse Model.

Wenlin Zhang,Jason M Tenessen,Hongde Li,Diego G Ogando,Joseph A Bonanno,Edward T Kim,Moon-Jung Choi

doi:10.1167/iovs.17-21781

Abstract

PurposeTo establish conditionally immortal mouse corneal endothelial cell lines with genetically matched Slc4a11+/+ and Slc4a11−/− mice as a model for investigating pathology and therapies for SLC4A11 associated congenital hereditary endothelial dystrophy (CHED) and Fuchs' endothelial corneal dystrophy.MethodsWe intercrossed H-2Kb-tsA58 mice (Immortomouse) expressing an IFN-γ dependent and temperature-sensitive mutant of the SV40 large T antigen (tsTAg) with Slc4a11+/+ and Slc4a11−/− C57BL/6 mice. The growth characteristics of the cell lines was assessed by doubling time. Ion transport activities (Na+/H+ exchange, bicarbonate, lactate, and Slc4a11 ammonia transport) were analyzed by intracellular pH measurement. The metabolic status of the cell lines was assessed by analyzing TCA cycle intermediates via gas chromatography mass spectrometry (GC-MS).ResultsThe immortalized Slc4a11+/+ and Slc4a11−/− mouse corneal endothelial cells (MCECs) remained proliferative through passage 49 and maintained similar active ion transport activity. As expected, proliferation was temperature sensitive and IFN-γ dependent. Slc4a11−/− MCECs exhibited decreased proliferative capacity, reduced NH3:H+ transport, altered expression of glutaminolysis enzymes similar to the Slc4a11−/− mouse, and reduced proportion of TCA cycle intermediates derived from glutamine with compensatory increases in glucose flux compared with Slc4a11+/+ MCECs.ConclusionsThis is the first report of the immortalization of MCECs. Ion transport of the immortalized endothelial cells remains active, except for NH3:H+ transporter activity in Slc4a11−/− MCECs. Furthermore, Slc4a11−/− MCECs recapitulate the glutaminolysis defects observed in Slc4a11−/− mouse corneal endothelium, providing an excellent tool to study the pathogenesis of SLC4A11 mutations associated with corneal endothelial dystrophies and to screen potential therapeutic agents.

Highlights

Slc4a11À/À mouse corneal endothelial cells (MCECs) exhibited decreased proliferative capacity, reduced NH3:Hþ transport, altered expression of glutaminolysis enzymes similar to the Slc4a11À/À mouse, and reduced proportion of TCA cycle intermediates derived from glutamine with compensatory increases in glucose flux compared with Slc4a11þ/þ MCECs
Slc4a11À/À MCECs recapitulate the glutaminolysis defects observed in Slc4a11À/À mouse corneal endothelium, providing an excellent tool to study the pathogenesis of SLC4A11 mutations associated with corneal endothelial dystrophies and to screen potential therapeutic agents
S LC4A11 mutations are associated with congenital hereditary endothelial corneal dystrophy (CHED), Fuchs’ endothelial corneal dystrophy, Harboyan syndrome (CHED plus perceptive deafness), and Peters anomaly.[1,2,3,4]

Summary

Methods

We intercrossed H-2Kb-tsA58 mice (Immortomouse) expressing an IFN-c dependent and temperature-sensitive mutant of the SV40 large T antigen (tsTAg) with Slc4a11þ/þ and Slc4a11À/À C57BL/6 mice. The metabolic status of the cell lines was assessed by analyzing TCA cycle intermediates via gas chromatography mass spectrometry (GC-MS). Slc4a11À/À C57BL/6 mice have a targeted deletion of exons 9 to 13 of the murine Slc4a11 gene.[31] The offspring were genotyped by PCR of genomic DNA from ear-punch sections following protocols using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Primers 1 and 3 were used to identify the SV40 transgene, Immorto (tsTAg). The first generations were bred, and the second-generation pups were genotyped by PCR to identify Slc4a11þ/þ and Slc4a11À/À animals that carried at least one copy of the SV40 tsTAg transgene to be used for tissue isolation

Results

Discussion

Conclusion