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Mitchell Watsky, Rodolfo Elizondo, Zhaohong Yin; Vitamin D Receptor Knockout Affects Mouse Corneal Tight Junctions. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2583.
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Vitamin D is typically known to be produced in the skin and activated through successive hydroxylation steps in the liver and kidneys. Our lab has recently determined that vitamin D can also be produced in the cornea and can influence the barrier function of the corneal epithelium. The purpose of this study was to determine if proteins associated with corneal tight junctions are affected by vitamin D receptor (VDR) knockout and if the junctions themselves are altered as observed using transmission electron microscopy (TEM).
Wild type 2.5 month-old C57BL/6 mice along with heterozygous (+/-) and homozygous (-/-) VDR null mice (Jackson Labs) were used in this study. Total RNA was obtained from freshly isolated mouse cornea epithelium. Real-time PCR was used to quantify mRNA levels of the tight junction-associated proteins occludin and ZO-1. 2ug total RNA was used for the subsequent synthesis of cDNA using the ThermoScript RT-PCR system for first-strand synthesis at 25°C 10min, 50°C 50min, and was inactivated by heating to 85°C for 5 min. Equal amounts of cDNA were applied for PCR amplification in quadruplicate at a final volume of 25 μl of 2X RT2 Real-Time SYBR Green master mix. Amplification was performed for 95°C 10min, followed by 40 cycles at 94°C 15 s and 60°C 60 s. Quantitative values were obtained from the threshold cycle value (Ct), which is the point where a significant increase of fluorescence is first detected. GAPDH was used as an internal RNA control, and each sample was normalized on the basis of its GAPDH gene content (ΔCt). Transmission electron microscopy (TEM) was used to examine tight junction structures in the cornea epithelium and endothelium.
Occludin levels in -/- mice were significantly lower than wild type and +/- mice. ZO-1 was significantly lower in -/- versus wild type mice. TEM demonstrated significant reductions in epithelial and endothelial tight junction structures in -/- corneas versus those from wild type mice.
VDR ablation results in significant loss of tight junction-associated proteins and tight junction structures in mouse corneas.
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