The role of CYP11A1 in the production of vitamin D metabolites and their role in the regulation of epidermal functions

Abstract

Research over the last decade has revealed that CYP11A1 can hydroxylate the side chain of vitamin D3 at carbons 17, 20, 22 and 23 to produce at least 10 metabolites, with 20(OH)D3, 20,23(OH)2D3, 20,22(OH)2D3, 17,20(OH)2D3 and 17,20,23(OH)3D3 being the main products. However, CYP11A1 does not act on 25(OH)D3. The placenta, adrenal glands and epidermal keratinocytes have been shown to metabolize vitamin D3 via this CYP11A1-mediated pathway that is modified by the activity of CYP27B1, with 20(OH)D3 (the major metabolite), 20,23(OH)2D3, 1,20(OH)2D3, 1,20,23(OH)3D3 and 17,20,23(OH)3D3 being detected, defining these secosteroids as endogenous regulators/natural products. This is supported by the detection of a mono-hydroxyvitamin D3 with the retention time of 20(OH)D3 in human serum. In new work presented here we demonstrate that the CYP11A1-initiated pathways also occurs in Caco-2 colon cells. Our previous studies show that 20(OH)D3 and 20,23(OH)2D3 are non-calcemic at pharmacological doses, dependent in part on their lack of a C1α hydroxyl group. In epidermal keratinocytes, 20(OH)D3, 20(OH)D2 and 20,23(OH)2D3 inhibited cell proliferation, stimulated differentiation and inhibited NF-κB activity with potencies comparable to 1,25(OH)2D3, acting as partial agonists on the VDR. 22(OH)D3 and 20,22(OH)2D3, as well as secosteroids with a short or no side chain, showed antiproliferative and prodifferentiation effects, however, with lower potency than 20(OH)D3 and 20,23(OH)2D3. The CYP11A1-derived secosteroids also inhibited melanocyte proliferation while having no effect on melanogenesis, and showed anti-melanoma activities in terms of inhibiting proliferation and the ability to grow in soft agar. Furthermore, 20(OH)D3 and 20,23(OH)2D3 showed anti-fibrosing effects in vitro, and also in vivo for the former. New data presented here shows that 20(OH)D3 inhibits LPS-induced production of TNFα in the J774 line, TNFα and IL-6 in peritoneal macrophages and suppresses the production of proinflammatory Th1/Th17-related cytokines, while promoting the production of the anti-inflammatory cytokine IL-10 in vivo. In summary, CYP11A1 initiates new pathways of vitamin D metabolism in a range of tissues and products could have important physiological roles at the local or systemic level. In the skin, CYP11A1-derived secosteroids could serve both as endogenous regulators of skin functions and as excellent candidates for treatment of hyperproliferative and inflammatory skin disorders, and skin cancer. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

Keywords: CYP11A1; Epidermis; Immune system; Keratinocytes; P450scc; Vitamin D.

Fig. 1

Summary of the major pathways for metabolism of vitamin D3 by CYP11A1 and the role of CYP27A1, CYP27B1 and CYP24A1 in the further hydroxylation of products. See the text for published references to the reactions listed.

Fig. 2

Production of vitamin D3 hydroxy-derivatives by human Caco-2 colon cells. Cells were incubated with 50 μM vitamin D3 for 16 h as described before [52]. After extraction of cell suspensions with dichloromethane the products were analyzed by LC–MS using single ion monitoring (SIM) mode [52] for mono-hydroxyvitamin D3 (A) and di-hydroxyvitamin D3 (D) (top panels). Peaks with retention times corresponding to authentic standards were collected and subjected to further analysis by LC–MS for mono-hydroxyvitamin D3 (B1) and di-hydroxyvitamin D3 (E1) (upper panels). Middle panels of B2 and E2 show negative control (no substrate added), while bottom panels show mass spectra for each tested fraction of hydroxyvitamin D3. Arrows identify retention times corresponding to 20(OH)D3, 22(OH)D3, 25(OH)D3, 1,20(OH)₂D3, 20,23(OH)₂D3 and 1,25(OH)₂D3 standards. For A and D, LC was run with a gradient of methanol in water (85–100%) for 20 min and 100% methanol for 10 min at a flow rate of 0.5 ml/min. MS was performed using an ESI source and SIM at m/z = 383.3 [M+1-H₂O] for mono-hydroxyvitamin D3 (A, B1 and B2) and 399.3 [M + 1-H₂O] for dihydroxyvitamin D3 (D, E1 and E2). The fractions with retention times corresponding to 20(OH)D3, 22(OH)D3, 25(OH)D3, 1,20(OH)₂D3, 20,23(OH)₂D3 and 1,25(OH)₂D3 standards were collected and analyzed separately (B1, B2, E1 and E2) by LC–MS isocratically using 96% methanol in water at a flow rate of 0.05 ml/min for 10 min, on a Zorbax Eclipse Plus C18 column.

Fig. 3

UVB-induced transformation of steroidal 5,7-dienes with a short or full-length side chain to novel secosteroids.

Fig. 4

20(OH)D3 suppress TLR4 ligand (LPS)-induced proinflammatory cytokine production in mouse macrophages in a VDR-dependent manner. (A) RT-PCR demonstrates that kidney, J774 cells and WEHI-231 cells express VDR. In contrast VDR mRNA in RAW264.7 cells was only just detectable. The sequences of RT-PCR primers for murine VDR and PCR conditions have been previously described [93]. (B) J774 cells (1 × 10⁶ cell/ml) and RAW264.7 cells (1 × 10⁶ cell/ml) were pre-treated with vehicle (propylene glycol; PG) or the indicated dose of 20(OH)D3. Forty-eight h later, these cells were stimulated with LPS (25 ng/ml) for 24 h and then TNFα in the culture supernatants was measured by ELISA as described previously [94]. Data represent mean (pg/ml) ± S.D. of triplicates. LPS-induced TNFα production was significantly suppressed in J774 cells, but not in RAW264.7 cells (**p < 0.005, *p < 0.05). C. Peritoneal macrophages were isolated from CIA mice (CII-immunized DBA1 mice that fully developed arthritis) as previously described [95]. Peritoneal macrophages (1 × 10⁶ cell/ml) were pre-treated with vehicle (PG) or the indicated dose of 20(OH)D3. Forty-eight h later, cells were stimulated with LPS (5 ng/ml) for 24 h and then levels of TNFα and IL-6 in the culture supernatants were measured by ELISA. Data represent mean (pg/ml) ± S.D. of triplicates. LPS-induced production of TNFα and IL-6 was significantly suppressed by 20(OH)D3 compared to that in vehicle-treated cells (*p <0.05).

Fig. 5

Potential binding poses of novel secosteroids, docking scores, and hydrogen bonding interactions with the VDR based on molecular modeling. (A) Crystal structure of VDR and its native ligand, 1,25(OH)₂D3. The dotted surface within the rectangle box shows the ligand surface. Docking score (–16.99) is shown in the parenthesis. (B)–(F), predicted binding poses for each of the novel secosteroid in VDR and their hydrogen bonding interactions (dotted line between the atoms in the ligand and the VDR) to the six residues that can form hydrogen bonds to 1,25(OH)₂D3. Molecule modeling studies were performed using the Extra Precision (XP) Glide function within the Schrodinger's Small Molecule Drug Discovery Suite 2012 (Schrodinger Inc., New York, NY, USA). Due to the large number of rotatable bonds in these secosteroids, it is challenging for the software to predict the most favorable binding poses in one run since XP requires a good starting conformation. Thus, for each novel secosteroid, we performed docking calculations iteratively, using the best pose generated from the previous run as the new starting pose for the next run until there was no improvement in the docking score. The optimized docking scores obtained from this process are shown in parenthesis for each panel, along with the predicted hydrogen bonding interactions between the secosteroid and the VDR.