Conditionally Immortal Slc4a11$^{-/-}$ Mouse Corneal Endothelial Cell Line Recapitulates Disrupted Glutaminolysis Seen in Slc4a11$^{-/-}$ Mouse Model

Purpose: To 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. Methods: We 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). Results: The 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. Conclusions: This is the first report of the immortalization of MCECs. Ion transport of the immortalized endothelial cells remains active, except for NH$_3$: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.


Publication Date:
Jul 01 2017
Date Submitted:
Aug 10 2018
Pagination:
3723-3731
ISSN:
1552-5783
Citation:
Investigative ophthalmology & visual science
58
9
Note:
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 Record created 2018-08-10, last modified 2019-04-03


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