Hydroxylation of CYP11A1-Derived Products of Vitamin D3 Metabolism by Human and Mouse CYP27B1

Abstract

CYP11A1 can hydroxylate vitamin D3 at carbons 17, 20, 22, and 23, producing a range of secosteroids which are biologically active with respect to their ability to inhibit proliferation and stimulate differentiation of various cell types, including cancer cells. As 1a-hydroxylation of the primary metabolite of CYP11A1 action, 20S-hydroxyvitamin D3 [20(OH)D3], greatly influences its properties, we examined the ability of both human and mouse CYP27B1 to 1a-hydroxylate six secosteroids generated by CYP11A1. Based on their k cat /K m values, all CYP11A1-derived metabolites are poor substrates for CYP27B1 from both species compared with 25-hydroxyvitamin D3. No hydroxylation of metabolites with a 17a-hydroxyl group was observed. 17a,20-Dihydroxyvitamin D3 acted as an inhibitor on human CYP27B1 but not the mouse enzyme. We also tested CYP27B1 activity on 20,24-, 20,25-, and 20,26-dihydroxyvitamin D3, which are products of CYP24A1 or CYP27A1 activity on 20(OH)D3. All three compounds were metabolized with higher catalytic efficiency (k cat /K m ) by both mouse and human CYP27B1 than 25-hydroxyvitamin D3. CYP27B1 action on these new dihydroxy derivatives was confirmed to be 1a-hydroxylation by mass spectrometry and nuclear magnetic resonance analyses. Both 1,20,25-and 1,20,26-trihydroxyvitamin D3 were tested for their ability to inhibit melanoma (SKMEL-188) colony formation, and were significantly more active than 20(OH)D3. This study shows that CYP11A1-derived secosteroids are 1a-hydroxylated by both human and mouse CYP27B1 with low catalytic efficiency, and that the presence of a 17a-hydroxyl group completely blocks 1a-hydroxylation. In contrast, the secondary metabolites produced by subsequent hydroxylation of 20(OH)D3 at C24, C25, or C26 are very good substrates for CYP27B1.

Endogenously produced nonclassical vitamin D hydroxy-metabolites act as “biased” agonists on VDR and inverse agonists on RORα and RORγ

“…The mechanism may be similar to those proposed for classical vitamin D ligands [16–20, 38, 104]. It must also be noted that 20(OH)D is a relatively poor substrate for CYP27B1, however, its hydroxylation by CYP27A1 or CYP24A1 at either the 24, 25 or 26 position make the resulting dihydroderivative an excellent substrate for 1α hydroxylation [53, 54]. Thus, the challenge posed by the novel hydroxyderivatives on the nature of their activation of the VDR can be properly answered using CYP27B1 −/− mice.…”

“…20(OH)D3, 22(OH)D3, 20,22(OH) 2 D3 and 20,23(OH) 2 D3 can be hydroxylated by CYP27B1 at C1α to the corresponding di- or trihydroxyderivatives (Table 1) [54, 56, 58, 63, 64]. The main product of D3 metabolism initiated by CYP11A1, 20(OH)D3, can be hydroxylated on the side chain by CYP27A1, CYP24A1 and CYP3A4 to 20,24(OH) 2 D3, 20,25(OH) 2 D3 and/or 20,26(OH) 2 D3, which are further 1α hydroxylated by CYP27B1 to the corresponding trihydroxyderivatives (Table 1)[55–57, 64–66].…”

“…Most recently it was shown that both 20(OH)D3 and 20,23(OH) 2 D3 enhance defense mechanisms against UVB-induced oxidative stress and DNA damage in cultured human keratinocytes [23] and murine skin in vivo [22]. 20(OH)D3 and its hydroxymetabolites also show anti-cancer properties that are cell-lineage dependent [53, 54, 64, 68, 69, 88, 90, 92–97]. …”

Metabolism of 20-hydroxyvitamin D3 by mouse liver microsomes

“…These major metabolites did not undergo further transformation by the microsomal fraction. However, we have reported previously that both of these metabolites can be converted to their 1α-hydroxy derivatives by the mitochondrial enzyme, CYP27B1, of which highest activity is expressed in kidney (17). Both 20,24(OH) 2 D3 and 20,25(OH) 2 D3, as well as 1,24,25-trihydroxyvitamin D3, have previously been shown to display higher biological activity than 20(OH)D3 towards melanoma cells (18).…”

“…CYP27B1 produces 1α,20-dihydroxyvitamin D3 (16,17), and the addition of the 1α-hydroxyl group confers some calcemic activity to the derivative (13). We detected production of 1α-hydroxy derivatives of 20(OH)D3 and 20,23(OH) 2 D3 in vivo in human placenta and epidermal keratinocytes (8).…”