In vivo evidence for a novel pathway of vitamin D3 metabolism initiated by CYP11A1 (#185)
It is well recognized that vitamin D3 is activated by sequential hydroxylation at C25 and C1 to produce 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). We have previously reported that purified CYP11A1 can sequentially metabolize vitamin D3 to a number of hydroxy-metabolites with the major ones being 20-hydroxyvitamin D3 (20(OH)D3) and 20,23-dihydroxyvitamin D3 (20,23(OH)2D3), suggestive of an alternative pathway of vitamin D metabolism. We have also shown that these metabolites are biologically active on a range of cells causing inhibition of proliferation and inflammation, and stimulation of differentiation, but unlike 1,25(OH)2D3, do not raise calcium levels in rats and mice. We now show that this alternative novel pathway for vitamin D3 activation occurs in vivo. 20(OH)D3, 20,23(OH)2D3 and more minor metabolites including 22-hydroxyvitamin D3 (22(OH)D3), 20,22-dihydroxyvitamin D3 (20,22(OH)2D3) and 17,20,23-trihydroxyvitamin D3 (17,20,23(OH)3D3) were produced by human placental and mammalian adrenal fragments incubated ex vivo with vitamin D3. Use of inhibitors and studies performed with isolated mitochondria indicated that these products were from CYP11A1 action. 1α-Hydroxy derivatives of 20(OH)D3 and 20,23(OH)2D3 were also detected in the placenta indicating that the CYP11A1-derived products can be acted on by CYP27B1, as previously demonstrated with the purified enzyme. In epidermal keratinocytes, which express CYP11A1, we observed the new pathway with higher proportions of 22(OH)D3 and 20,22(OH)2D3 being produced than in the adrenal or placenta. Importantly, we detected endogenous production of 20(OH)D3, 22(OH)D3, 20,23(OH)2D3, 20,22(OH)2D3 and 17,20,23(OH)3D3 by immortalized human keratinocytes demonstrating that the pathway occurs in the absence of high concentrations of exogenous vitamin D3. As final support that this new pathway occurs in vivo we detected of the predominant metabolite, 20(OH)D3, in human serum. Thus we provide in vivo evidence for a novel pathway of vitamin D3 activation initiated by CYP11A1 with the product profile showing organ/cell type specificity and being modified by CYP27B1 activity.