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Xiaowen Lu, Mitchell Watsky; Megalin and Cubilin Levels are Altered in Tear-Producing Glands of Vitamin D Receptor Null Mice. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4311. doi: https://doi.org/.
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Vitamin D is known to be produced in the skin and activated in the liver and kidneys. Megalin and cubilin are endocytosis -associated proteins believed to act as Vit D transporters. Our lab has recently determined that Vit D can also be produced in the cornea and is found in tear, aqueous humor, and vitreous humor of rabbits. Dietary Vit D supplementation results in increased Vit D concentrations in the plasma and in the tear and aqueous humor. The purpose of this study was to determine if megalin or cubilin levels are altered in Vit D receptor (VDR) null mice and if feeding them a special diet rich in calcium, P, and lactose reverses VDR-induced changes.
Real-time PCR was used to explore the level of mRNA of megalin and cubilin in mouse Harderian glands, which secrete a highly lipid-based product into tear. Glands were isolated from wild type C57BL/6 mice (+/+) and from heterozygous (+/-) and homozygous (-/-) VDR null mice (Jackson Labs) that were 1 and 2.5 months old. A subset of 2.5 mo (-/-) mice were fed a replenishment diet consisting of high Ca, P, and lactose. mRNA was isolated and cDNA was synthesized at 55 °C for 60 min, and reverse transcriptase was inactivated by heating to 85 °C for 5 min. Equal amounts of cDNA were applied for PCR amplification in triplicate in the thermocycler using the LightCycler® 480 System and UPL probes. The amplification was normalized by reference to that of the ribosomal protein S19 gene. PCR conditions were set to 95°C for 10 min initialization, followed by 40 cycles at 95 °C for 10 sec, 60 °C for 30 sec, 72 °C for 10 sec, followed by cool down to 40 °C for 30 sec.
Megalin mRNA levels were decreased in VDR -/- mice at 1 mo and were further decreased in 2.5 mo mice. VDR +/- mice had lower levels at 1 mo but no change at 2.5 mo. The special diet resulted in an increase in megalin levels as compared to the 2.5 mo mice that was lower than control levels. Cubilin mRNA levels were lower in +/- and -/- mice at both time points with no effect of the special diet. 2.5 mo -/- mice had lower levels than 1 mo mice.
VDR knockout lowers both megalin and cubilin mRNA levels in mouse Harderian gland. The special diet resulted in an increase in megalin mRNA levels but not cubilin, indicating that cubilin is more dependent on VDR receptor activity.
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