Targeted inactivation of vitamin D hydroxylases in mice

Abstract

Vitamin D undergoes a first hydroxylation in the liver to generate 25-hydroxyvitamin D, then this metabolite is further hydroxylated in the kidney to yield either 1alpha,25-dihydroxyvitamin D [1alpha,25(OH)2D], or 24R,25-dihydroxyvitamin D[24,25(OH)2D]. The production of 1alpha,25(OH)2D is catalyzed by the enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-OHase), while the synthesis of 24,25(OH)2D is catalyzed by the enzyme 25-hydroxyvitamin D-24-hydroxylase (24-OHase). To determine the role of each of these enzymes in vivo and their putative role during development, we have inactivated each gene by homologous recombination in embryonic stem cells. The targeting vector for the 1alpha-OHase gene was constructed to allow tissue-specific gene inactivation in order to study the hypothesized paracrine/autocrine roles of the 1alpha-OHase enzyme in particular target tissues such as skin, brain, or macrophages. The targeting vector for the 24-OHase gene utilized standard methodology, and analysis of the phenotype of 24-OHase-deficient mice confirmed the role of the 24-OHase enzyme in the catabolism of 1alpha,25(OH)2D. The phenotype of the second generation 24-OHase-null mice also suggests a key role for 24,25(OH)2D in intramembranous bone formation during development.