Exploring the impact of inducible-whole body and tissue-specific removal of CYP24A1 on vitamin D metabolism in mice

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2023-05

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Abstract

CYP24A1 is an enzyme that initiates the catabolism of 1,25 Hydroxyvitamin D3 (1,25(OH)₂D₃ ;1,25D), the active hormonal form of Vitamin D that regulates bone and calcium homeostasis. In the following two experiments, we aim to elucidate the specific roles of whole-body versus intestinal CYP24A1 in the metabolism of 1,25(OH)₂D₃. We generated inducible-Cyp24a1 KO and HT (UBC [superscript Cre+/-] xCyp24a1 [superscript flox/flox]; UBC [superscript Cre+/-] xCyp24a1 [superscript flox/wt]) mice to understand the impact of CYP24A1 dose variation in healthy, adult mice. The Cyp24a1 KO, HT, and Control (Cyp24a1 [superscript flox/flox]) littermates were fed a standard Chow diet from weaning until 11-weeks of age. Following a series of intraperitoneal injections of 2.5ul/g(body weight) of Tamoxifen daily for 7 days to induce genetic recombination the mice were randomized to AIN93G diets (0.4% P, 200 IU/kg Vitamin D3) with either adequate (0.5%) or deficient (0.2%) calcium (Ca) levels (n=6/sex/group). At 12-weeks old, all mice were euthanized, and tissues collected and stored for RNA isolation. Duodenal (Dd) and renal (Kd) mRNA levels were quantified using qPCR. As expected, we observed a trend towards an allele-dependent suppression of Cyp27b1 regardless of diet compared to controls supporting alterations in 1,25D metabolism. Interestingly, there was no change to Trpv6 and s100g mRNA by diet or genotype, possibly due to balancing circulating 1,25D production through Cyp27b1 suppression. In the second study, control (Cyp24a1 [superscript flox/flox]) and intestinal-epithelial-cell-specific knock-out (Villin [superscript Cre+/-] x Cyp24a1 [superscript flox/flox], IEC KO) mice were fed a standard Chow diet from weaning until 11 weeks old, they were then randomized to AIN93G diets (0.4% P, 200 IU/kg Vitamin D3) with 0.5% or 0.2% Ca (n=5/sex/group). One week later, mice were euthanized, and tissues collected and stored for RNA isolation. Dd and Kd mRNA levels were quantified using qPCR. Surprisingly, while we saw trends of Cyp27b1 mRNA suppression and intestinal Trpv6 mRNA elevation, there were no significant changes to mRNA expression.

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