In vivo production of novel vitamin D2 hydroxy-derivatives by human placentas, epidermal keratinocytes, Caco-2 colon cells and the adrenal gland

The Journal of Steroid Biochemistry and Molecular Biology

Tuckey

2014

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20-Hydroxyvitamin D3 [20(OH)D3], the major product of CYP11A1 action on vitamin D3, is biologically active and like 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] can inhibit proliferation and promote differentiation of a range of cells, and has anti-inflammatory properties. However, unlike 1,25(OH)2D3, it does not cause toxic hypercalcemia at high doses and is therefore a good candidate for therapeutic use to treat hyperproliferative and autoimmune disorders. In this study we analyzed the ability of mouse liver microsomes to metabolize 20(OH)D3. The two major products were identified from authentic standards as 20,24-dihydroxyvitamin D3 [20,24(OH)2D3] and 20,25-dihydroxyvitamin D3 [20,25(OH)2D3]. The reactions for synthesis of these two products from 20(OH)D3 displayed similar Km values suggesting that they were catalyzed by the same cytochrome P450. Some minor metabolites were produced by reactions with higher Km values for 20(OH)D3. Some metabolites gave mass spectra suggesting that they were the result of hydroxylation followed by dehydrogenation. One product had an increase in the wavelength for maximum absorbance from 263 nm seen for 20(OH)D3, to 290 nm, suggesting a new double bond was interacting with the vitamin D-triene chromophore. The two major products, 20,24(OH)2D3 and 20,25(OH)2D3 have both previously been shown to have higher potency for inhibition of colony formation by melanoma cells than 20(OH)D3, thus it appears that metabolism of 20(OH)D3 by mouse liver microsomes can generate products with enhanced activity.

Section: Methodsmentioning

confidence: 96%

Metabolism of 20-hydroxyvitamin D3 by mouse liver microsomes

Cheng

The Journal of Steroid Biochemistry and Molecular Biology

Tuckey

2014

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“…We also demonstrated the ability of CYP11A1 to hydroxylate vitamin D3 at these positions and further showed that C23 is a major site of hydroxylation with 20,23-dihydroxyvitamin D3 being the next most abundant product after 20-hydroxyvitamin D3 (Slominski et al, 2005a; Tuckey et al, 2008a, 2008b, 2011). Importantly, we have provided initial evidence that CYP11A1-initiated metabolism of 7-DHC, vitamin D3 and vitamin D2 can occur under in vivo conditions (Slominski et al, 2009, 2012a, 2012b, 2014a, 2014b). The novel CYP11A1-derived hydroxy-derivatives of vitamin D3 are active in inhibiting proliferation and stimulating differentiation in a range of cell types cultured in vitro (reviewed in (Slominski et al, 2014a)), and in reducing inflammation and skin fibrosis in mice in vivo (Slominski et al, 2013, 2014a), but lack the toxic and calcemic effects seen with high doses of 1,25(OH) 2 D3 (Slominski et al, 2010; Wang et al, 2012).…”

Section: Introductionmentioning

confidence: 88%

“…The organs were collected using standard procedures (Slominski et al, 2014c). The incubations of adrenal fragments followed modifications of the protocols described previously (Slominski et al, 2009, 2014b). Briefly, after dissection from the adipose, the adrenals were cut with scissors into small fragments and washed with PBS.…”

Section: Methodsmentioning

confidence: 99%

“…We and others have also reported the presence of CYP11A1 in human skin (Slominski et al, 1996, 2004, 2014a; Thiboutot et al, 2003;) and have detected CYP11A1-dependent production of 20(OH)D3 by cultured keratinocytes in the absence of exogenous vitamin D3 substrate (Slominski et al, 2012a). These findings, together with the ability of CYP11A1 to act on 7-DHC (Slominski et al, 2004, 2009, 2012b), ergosterol (Slominski et al, 2005b; Tuckey et al, 2012) and vitamin D2 (Nguyen et al, 2009; Slominski et al, 2006, 2014b) has prompted us to test the ability of this enzyme to act on L3.…”

Section: Introductionmentioning

confidence: 99%

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Lumisterol is metabolized by CYP11A1: Discovery of a new pathway

Tuckey

The International Journal of Biochemistry & Cell Biology

Cheng

et al. 2014

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Lumisterol3 (L3) is produced by photochemical transformation of 7-dehydrocholesterol (7-DHC) during exposure to high doses of ultraviolet B radiation. It has been assumed that L3 is biologically inactive and is not metabolized in the body. However, some synthetic derivatives of L3 display biological activity. The aim of this study was to test the ability of CYP11A1 to metabolize L3. Incubation of L3 with bovine or human CYP11A1 resulted in the formation of three major and a number of minor products. The catalytic efficiency of bovine CYP11A1 for metabolism of L3 dissolved in 2-hydroxypropyl-β-cyclodextrin was approximately 20% of that reported for vitamin D3 and cholesterol. The structures of the three major products were identified as 24-hydroxy-L3, 22-hydroxy-L3 and 20,22-dihydroxy-L3 by NMR. 22-hydroxy-L3 was further metabolized by bovine CYP11A1 to 20,22-dihydroxy-L3. Both 22-hydroxy-L3 and 20,22-dihydroxy-L3 gave rise to a minor metabolite identified from authentic standard and mass spectrometry as pregnalumisterol (pL) (product of C20-C22 side chain cleavage of L3) and two trihydroxy-L3 products. The capability of tissues expressing CYP11A1 to metabolize L3 was demonstrated using pig adrenal fragments where 20,22-dihydroxy-L3, 22-hydroxy-L3, 24-hydroxy-L3 and pL were detected by LC/MS. Thus, we have established that L3 is metabolized by CYP11A1 to 22- and 24-hydroxy-L3 and 20,22-dihydroxy-L3 as major products, as well as to pL and other minor products. The previously reported biological activity of pL and the presence of CYP11A1 in skin suggest that this pathway may serve to produce biologically active products from L3, emphasizing a novel role of CYP11A1 in sterol metabolism.

“…Production of 20(OH)D3, 22(OH)D3, 20,23(OH) 2 D3, 20,22(OH) 2 D3, 1,20(OH) 2 D3 also occurs in human dermal fibroblasts incubated with D3 substrate [71]. Similarly, production of 20(OH)D2, 17,20(OH) 2 D2 and 1,20(OH) 2 D2 was observed in human placentas ex-utero , adrenal glands ex-vivo , and isolated human epidermal keratinocytes and colorectal Caco-2 cells incubated with D2 [59]. …”

Section: New Pathways Of Vitamin D Activationmentioning

confidence: 99%

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

The Journal of Steroid Biochemistry and Molecular Biology

Kim²,

Hobrath

et al. 2017

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126

143

The classical pathway of vitamin D activation follows the sequence D3→25(OH)D3→1,25(OH)2D3 with the final product acting on the receptor for vitamin D (VDR). An alternative pathway can be started by the action of CYP11A1 on the side chain of D3, primarily producing 20(OH)D3, 22(OH)D3, 20,23(OH)2D3, 20,22(OH)2D3 and 17,20,23(OH)3D3. Some of these metabolites are hydroxylated by CYP27B1 at C1α, by CYP24A1 at C24 and C25, and by CYP27A1 at C25 and C26. The products of these pathways are biologically active. In the epidermis and/or serum or adrenals we detected 20(OH)D3, 22(OH)D3, 20,22(OH)2D3, 20,23(OH)2D3, 17,20,23(OH)3D3, 1,20(OH)2D3, 1,20,23(OH)3D3, 1,20,22(OH)3D3, 20,24(OH)2D3, 1,20,24(OH)3D3, 20,25(OH)2D3, 1,20,25(OH)3D3, 20,26(OH)2D3 and 1,20,26(OH)3D3. 20(OH)D3 and 20,23(OH)2D3 are non-calcemic, while the addition of an OH at C1α confers some calcemic activity. Molecular modeling and functional assays show that the major products of the pathway can act as “biased” agonists for the VDR with high docking scores to the ligand binding domain (LBD), but lower than that of 1,25(OH)2D3. Importantly, cell based functional receptor studies and molecular modeling have identified the novel secosteroids as inverse agonists of both RORα and RORγ receptors. Specifically, they have high docking scores using crystal structures of RORα and RORγ LBDs. Furthermore, 20(OH)D3 and 20,23(OH)2D3 have been tested in cell model that expresses a Tet-on RORα or RORγ vector and a RORE-LUC reporter (ROR-responsive element), and in a mammalian 2-hybrid model that test interactions between an LBD-interacting LXXLL-peptide and the LBD of RORα/γ. These assays demonstrated that the novel secosteroids have ROR-antagonist activities that were further confirmed by the inhibition of IL17 promoter activity in cells overexpressing RORα/γ. In conclusion, endogenously produced novel D3 hydroxy-derivatives can act both as “biased” agonists of the VDR and/or inverse agonists of RORα/γ. We suggest that the identification of large number of endogenously produced alternative hydroxy-metabolites of D3 that are biologically active, and of possible alternative receptors, may offer an explanation for the pleiotropic and diverse activities of vitamin D, previously assigned solely to 1,25(OH)2D3 and VDR.