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Shimin Li, Tracy T. Nguyen, Joseph A. Bonanno; CD147 Expression Required for Lactate Transporters MCT1 and MCT4 in Rabbit Corneal Endothelium. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6009.
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CD147 is a broadly expressed plasma membrane glycoprotein with particularly high levels in corneal endothelium and retinal pigment epithelium. It has been reported that CD147 is associated with lactate transporters MCT1 and MCT4 and acts to facilitate their cell surface expression. To demonstrate that CD147 plays a pivotal role in corneal endothelial lactate transport, we knocked down CD147 expression via siRNA transfection in ex vivo cultured rabbit corneas, and examined the MCT expression as well as the effect on lactate-H+ transport.
A corneo-scleral skirt was excised and placed into a 6 well plate with adaptations that allow the tissue to sit upright and retain normal curvature. The endothelial surface was transfected with 100 nM of CD147 specific or negative control siRNA. Three days post-transfection, the endothelium was peeled to extract protein and RNA. The expression of CD147 and MCTs (1, 2 and 4) were examined by real-time RT-PCR and immunoblotting. Physiological effects were determined by measuring: (1) intracellular pH (pHi) and lactate induced acidification (LIA) from peeled tissue mounted in a double-sided perfusion chamber, (2) endogenous lactate efflux from corneas in [HCO3-]-rich Ringer and (3) total corneal lactate concentration in CD147 knockdown and control corneas.
The results showed that CD147 expression was reduced up to 85% by the siRNA transfection at days 3-4. Correspondingly, expression of monocarboxylate transporters MCT1 and MCT4 were reduced 73% and 80% in CD147 knockdown corneas, while MCT2 was unaffected. As a consequence, steady state endothelial pHi was decreased from 7.15 to 6.80 in CD147 knockdown corneas. The rate and amount of LIA was reduced 53% and 44% respectively, when lactate was perfused on both apical and basolateral (BL) surfaces. When lactate was perfused only on the BL surface, there was little or no LIA. Moreover, endogenous lactate efflux was decreased by 60% across the endothelial surface and there was a 3-fold greater accumulation of lactate in CD147 siRNA transfected corneas compared to control corneas.
(1) CD147 is required for expression of monocarboxylate transporters-MCT1 and MCT4 on the basolateral membrane of corneal endothelium. (2) A significant proportion of corneal lactate efflux is transcellular.
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