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

https://doi.org/10.1016/j.jsbmb.2016.09.024Get rights and content

Highlights

  • CYP11A1- derived hydroxyvitamin D derivatives are present in human skin and serum.

  • CYP11A1- derived hydroxyvitamin D derivatives are biologically active.

  • CYP11A1- derived hydroxyvitamin D derivatives act as partial/biased agonists on VDR.

  • CYP11A1- derived hydroxyvitamin D derivatives as “inverse” agonists on RORα and RORγ.

  • 20(OH)D3, 20(OH)D2 and 20,23(OH)2D3 are noncalcemic at pharmacological doses.

Abstract

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 a 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.

Introduction

Vitamin D is generated from the photochemical transformation of 7-dehydrocholesterol (7DHC) that requires UVB energy (λ = 280–320 nm) and represents the most fundamental reaction in photobiology, not requiring any enzyme [1], [2], [3]. After exposure to UVB, the B ring of 7DHC absorbs the electromagnetic energy leading to the breakage of the C9-C10 bond, opening the B-ring and thereby producing previtamin D3. The latter subsequently undergoes thermal isomerization to form D3, or with high doses of UVB produces lumisterol (L3) and tachysterol (T3) [1], [2], [3], [4]. These reactions are dependent on the temperature and the UVB dose and are reversible.

It is well established that D3 can be activated by two sequential hydroxylations, the first at C25 (catalyzed by CYP2R1 and CYP27A1) and the second at C1α (catalyzed by CYP27B1) to generate biologically active 1,25(OH)2D3 as a final product [4], [5], [6], [7]. In addition, circulating 25(OH)D3 can be activated in target tissues by ubiquitously expressed CYP27B1 (reviewed in [6], [8], [9]). 1,25(OH)2D3 is inactivated by CYP24A1 which initially hydroxylates it at C24 then catalyzes subsequent oxidations leading to shortening of side chain and the production calcitroic acid [10], [11], [12], [13].

In addition to regulating calcium homeostasis, 1,25(OH)2D3 displays a variety of pleiotropic activities, which include inhibition of proliferation and stimulation of the differentiation program in cells of different lineage, anticancerogenic effects, and enhancement of innate, and attenuation of adaptive immune activities and inflammation [4], [8], [14], [15], [16], [17]. Its effects are mediated via the vitamin D receptor (VDR), which after agonist activation and dimer formation with RXR binds to the VDR responsive element (VDRE) to influence expression of responsive genes [14], [17], [18], [19].

In the skin, 1,25(OH)2D3 plays an important role in the regulation of skin barrier functions and in the regulation of hair follicle growth and cycling, and has anti-cancerogenic, anti-proliferative and anti-inflammatory effects [3], [17], [20]. Most recently, it was reported that it can inhibit skin cell death and DNA damage induced by exposure to UVR [20], [21], [22], [23]. Because of the toxic effect secondary to calcemia, the pharmacological use of 1,25(OH)2D3 is limited.

Many analogs of 1,25(OH)2D3 have been chemically synthesized with the aim of reducing calcemic activity, without the loss of therapeutically useful anticancer activities (reviewed in [14], [24], [25], [26], [27]) or immunoregulatory properties (reviewed in [28], [29]). Modification of the A-ring, CD ring and side chain have all produced analogs with reduced calcemic activity. Key changes include replacement of the C1α-hydroxyl group with a 1β-CH2OH, C3-epimerization, removal of C19, epimerization at C20, addition of a second side chain at C20 (Gemini analogs), insertion of a double bond at C16 and a triple bond at C23, and insertion of an oxygen in place of C22. Many side chain modifications between C22 and C26 have also been aimed at reducing metabolism by CYP24A1 rather than reducing calcemic activity. The effects of isomerization of the two hydroxyl groups in the A-ring of 1,25(OH)2D3 was reported by Fleet et al. [30], and is of particular interest since one of the resulting diastereomers, 3-epi-25-dihydroxyvitamin D3, is a natural metabolite of 1,25(OH)2D3. 1,25(OH)2D3 (where hydroxyl groups are 1α and 3β) was compared to 1β,3β; 1α,3α and 1β,3α diastereomers. The 1α,3α isomer (3-epi-1,25(OH)2D3) is produced in vivo by the action of vitamin D 3-epimerase on 1,25(OH)2D3 [31], [32], [33]. All three of these diastereomers showed reduced binding to the VDR with binding strength only partially correlating with their ability to stimulate calcium transport. The 3-epi-1,25(OH)2D3 diastereomer stimulated calcium transport in excess of its relative ability to bind to the VDR. Other studies on 3-epi-1,25(OH)2D3 indicate that its reduced binding to the VDR does generally correlate with its reduced biological activity [30], [32], [34], [35], but there are notable exceptions such as the maintenance of the ability to suppress parathyroid hormone secretion by cultured parathyroid cells and enhancement of the ability to stimulate HL-60 cell apoptosis, relative to 1,25(OH)2D3 [36], [37].

While several of the low-calcemic synthetic analogs discussed above show some promise for the treatment of hyperproliferative and immunological disorders, hypercalcemia resulting from long-term high therapeutic doses remains a significant problem [24], [26], [29]. The studies with synthetic analogs also illustrate the possibility of designing specific analogs for specific therapeutic applications. It is well established that 1α-hydroxylation of the A-ring of 25(OH) D3 dramatically enhances its binding to the VDR and its calcemic activity. However, until our studies on CYP11A1-derived secosteroids that lack the 1α-hydroxyl group (described below) little was done to explore the possibility that active metabolites might be synthesized without the 1α-hydroxyl group (or equivalent), as it was generally thought to be indispensable for tight binding to the VDR.

The consensus conveyed by the majority of the literature is that all biologically relevant phenotypic effects of D3 can been assigned to one molecule, 1,25(OH)2D3, and one receptor, VDR [3], [15], [17], [38]. This makes both 1,25(OH)2D3 and VDR a bioregulatory couple, which would regulate vastly unrelated or sometime contradictory effects, which is highly unusual for endogenous ligands and their respective receptor. The existence of an alternative membrane bound receptor for 1,25(OH)2D3, e.g., 1,25D3-membrane-associated, rapid response steroid-binding protein (1,25D3-MARRS), has been proposed by some authors [39], [40]. This review, supplemented by new data and molecular modeling, will offer an additional explanation for the pleiotropic phenotypic effects of D3 by identifying both a family of novel bioactive D3 hydroxy-derivatives and the retinoid acid-related orphan receptors (RORs) α and γ, which function as alternative nuclear receptors for these compounds in addition to the VDR.acid

Section snippets

New pathways of vitamin D activation

Until recently, the traditional role of CYP11A1 was believed to be to initiate steroid synthesis, solely in steroidogenic organs using cholesterol as the substrate. This involved hydroxylations at C22 and C20 followed by oxidative cleavage of the bond between C20 and C22 to produce pregnenolone, a precursor to all steroids [41], [42]. However, the expression of CYP11A1 in peripheral tissues, albeit at low levels, has now been documented [43] and alternative substrates to cholesterol have been

RORs (retinoid-related orphan receptors), an overview

There are three members of the ROR subfamily of nuclear receptors, RORα-γ (NR1F1-3) [73], [74]. The ROR transcription factors exhibit a domain structure containing an N-terminal domain, a highly conserved DNA-binding domain (DBD) with two C2-C2 zinc finger motifs, a ligand-binding domain (LBD), and a hinge domain between the DBD and LBD. Transcriptional regulation by RORs is mediated through monomeric interaction with ROREs (ROR response elements) in the regulatory regions of target genes [73],

An overview of biological activity

The biological activity of 20S(OH)D3 in the skin was the subject of a recent review [70], therefore the description below is brief. 20(OH)D3 and its hydroxymetabolites exert prodifferentiation, antiproliferative, and antiinflammatory activities on skin cells, comparable or better than that of 1,25(OH)2D3 [53], [64], [65], [67], [70], [83], [84], [85], [86], [87], [88], [89], [90], [91], [92]. 20(OH)D3 shows antifibrotic properties both in vitro [87], [88], [89] and in an in vivo mouse model of

CYP11A1-derived D3 hydroxymetabolites act as “partial/biased” VDR agonists

Our previous studies have documented that 20S(OH)D3 and 20,23(OH)2D3 can act as “partial agonists on the VDR (discussed in [70]). They may also be termed biased agonists, a term now commonly applied to some ligands for G-protein coupled receptors which are functionally selective (biased) for certain response pathways from a particular receptor [98], [99]. The involvement of VDR in the regulation of differentiation, proliferation and immune functions of keratinocytes was demonstrated by

Concluding remarks

Over 12 years we have documented the existence of new pathways of vitamin D3 metabolism started by the action of CYP11A1 and further modified by the actions of CYP27B1, CYP27A1, CYP24A1 and CYP3A4, generating at least 21 hydroxymetabolites with additional ones still to be experimentally defined (Table 1) [43], [50]. At least 13 of them are endogenously produced [72]. These metabolites display biological activity by acting both as “biased” agonists of the VDR and/or inverse agonists of RORα and

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

We acknowledge the support by NIH grants R21AR066505, 1R01AR056666 and 2R01AR052190 to AS. 1R21AR063242, 1S10OD010678, and RR-026377 to WL, and the University of Western Australia to RCT; and the Intramural Research Program of the NIEHS, NIH (Z01-ES-101586 to AMJ).

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