Endogenously produced nonclassical vitamin D hydroxy-metabolites act as “biased” agonists on VDR and inverse agonists on RORα and RORγ
Graphical abstract
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).
References (109)
The cutaneous photosynthesis of previtamin D3: a unique photoendocrine system
J. Invest. Dermatol.
(1981)Cytochromes P450 are essential players in the vitamin D signaling system
Biochim. Biophys. Acta
(2011)Vitamin D: newly discovered actions require reconsideration of physiologic requirements
Trends Endocrinol. Metab.
(2010)- et al.
Cytochrome P450-mediated metabolism of vitamin d
J. Lipid Res.
(2014) Vitamin D metabolism, mechanism of action, and clinical applications
Chem. Biol.
(2014)- et al.
Novel mechanisms for the vitamin D receptor (VDR) in the skin and in skin cancer
J. Steroid Biochem. Mol. Biol.
(2015) - et al.
CYP11A1 in skin: an alternative route to photoprotection by vitamin D compounds
J. Steroid Biochem. Mol. Biol.
(2015) - et al.
Novel non-calcemic secosteroids that are produced by human epidermal keratinocytes protect against solar radiation
J. Steroid Biochem. Mol. Biol.
(2015) - et al.
The role of vitamin D in cancer prevention
Chin. J. Nat. Med.
(2015) Intervention in autoimmunity: the potential of vitamin D receptor agonists
Cell. Immunol.
(2005)
1 alpha, 25-(OH)2-vitamin D3 analogs with minimal in vivo calcemic activity can stimulate significant transepithelial calcium transport and mRNA expression in vitro
Arch. Biochem. Biophys.
Metabolism of 1alpha,25-dihydroxyvitamin D(3) and its C-3 epimer 1alpha,25-dihydroxy-3-epi-vitamin D(3) in neonatal human keratinocytes
Steroids
C-3 epimerization of vitamin D3 metabolites and further metabolism of C-3 epimers: 25-hydroxyvitamin D3 is metabolized to 3-epi-25-hydroxyvitamin D3 and subsequently metabolized through C-1alpha or C-24 hydroxylation
J. Biol. Chem.
1alpha, 25-dihydroxy-3-epi-vitamin D3: in vivo metabolite of 1alpha, 25-dihydroxyvitamin D3 in rats
FEBS Lett.
Differential activities of 1alpha,25-dihydroxy-16-ene-vitamin D(3) analogs and their 3-epimers on human promyelocytic leukemia (HL-60) cell differentiation and apoptosis
Steroids
Progesterone synthesis by the human placenta
Placenta
On the role of skin in the regulation of local and systemic steroidogenic activities
Steroids
Enzymatic metabolism of ergosterol by cytochrome p450scc to biologically active 17alpha,24-dihydroxyergosterol
Chem. Biol.
Lumisterol is metabolized by CYP11A1: discovery of a new pathway
Int. J. Biochem. Cell Biol.
Novel activities of CYP11A1 and their potential physiological significance
J. Steroid Biochem. Mol. Biol.
Metabolism of 20-hydroxyvitamin D3 and 20,23-dihydroxyvitamin D3 by rat and human CYP24A1
J. Steroid Biochem. Mol. Biol.
Metabolism of cholesterol, vitamin D3 and 20-hydroxyvitamin D3 incorporated into phospholipid vesicles by human CYP27A1
J. Steroid Biochem. Mol. Biol.
In vivo production of novel vitamin D2 hydroxy-derivatives by human placentas, epidermal keratinocytes Caco-2 colon cells and the adrenal gland
Mol. Cell. Endocrinol.
Kinetics of vitamin D3 metabolism by cytochrome P450scc (CYP11A1) in phospholipid vesicles and cyclodextrin
Int. J. Biochem. Cell Biol.
Metabolism of 1alpha-hydroxyvitamin D3 by cytochrome P450scc to biologically active 1alpha,20-dihydroxyvitamin D3
J. Steroid Biochem. Mol. Biol.
Metabolism of substrates incorporated into phospholipid vesicles by mouse 25-hydroxyvitamin D3 1alpha-hydroxylase (CYP27B1)
J. Steroid Biochem. Mol. Biol.
Hydroxylation of 20-hydroxyvitamin D3 by human CYP3A4
J. Steroid Biochem. Mol. Biol.
Chemical synthesis of 20S-hydroxyvitamin D3, which shows antiproliferative activity
Steroids
Total synthesis of biologically active 20S-hydroxyvitamin D3
Steroids
The role of CYP11A1 in the production of vitamin D metabolites and their role in the regulation of epidermal functions
J. Steroid Biochem. Mol. Biol.
Cloning of a cDNA encoding the murine orphan receptor RZR/ROR gamma and characterization of its response element
Gene
X-ray structure of the hRORalpha LBD at 1.63 A: structural and functional data that cholesterol or a cholesterol derivative is the natural ligand of RORalpha
Structure
Ursolic acid suppresses interleukin-17 (IL-17) production by selectively antagonizing the function of RORgamma t protein
J. Biol. Chem.
20-Hydroxyvitamin D3 a product of vitamin D3 hydroxylation by cytochrome P450scc, stimulates keratinocyte differentiation
J. Invest. Dermatol.
Vitamin D analogs 17,20S(OH)2pD and 17,20R(OH)2pD are noncalcemic and exhibit antifibrotic activity
J. Invest. Dermatol.
Vitamin D derivatives enhance cytotoxic effects of H2O2 or cisplatin on human keratinocytes
Steroids
Correlation between secosteroid-induced vitamin D receptor activity in melanoma cells and computer-modeled receptor binding strength
Mol. Cell. Endocrinol.
What is pharmacological ‘affinity'? Relevance to biased agonism and antagonism
Trends Pharmacol. Sci.
Regulation of cutaneous previtamin D3 photosynthesis in man: skin pigment is not an essential regulator
Science
Biological effects of sunlight, ultraviolet radiation visible light, infrared radiation and vitamin D for health
Anticancer Res.
A millenium perspective
J. Cell. Biochem.
Vitamin D: an ancient hormone
Exp. Dermatol.
Genetic disorders of Vitamin D biosynthesis and degradation
J Steroid Biochem Mol Biol.
Vitamin D deficiency
N. Engl. J. Med.
Dual metabolic pathway of 25-hydroxyvitamin D3 catalyzed by human CYP24
Eur. J. Biochem.
Human 25-hydroxyvitamin D3-24-hydroxylase, a multicatalytic enzyme
Biochemistry
Kinetic analysis of human CYP24A1 metabolism of vitamin D via the C24-oxidation pathway
FEBS J.
Vitamin D, disease and therapeutic opportunities
Nat. Rev. Drug Discov.
Vitamin D and cancer: the promise not yet fulfilled
Endocrine
Vitamin D: metabolism, molecular mechanism of action, and pleiotropic effects
Physiol. Rev.
Cited by (118)
1,25(OH)<inf>2</inf>D<inf>3</inf> improves diabetic wound healing by modulating inflammation and promoting angiogenesis
2024, Journal of Steroid Biochemistry and Molecular BiologyDissection of an impact of VDR and RXRA on the genomic activity of 1,25(OH)<inf>2</inf>D<inf>3</inf> in A431 squamous cell carcinoma
2024, Molecular and Cellular EndocrinologyA review of the critical role of vitamin D axis on the immune system
2023, Experimental and Molecular PathologyEnzymatic activation in vitamin D signaling – Past, present and future
2023, Archives of Biochemistry and BiophysicsVitamin D attenuates elevated oxidative DNA damage in scleroderma patients with organ involvement: A prospective study
2023, Journal of Steroid Biochemistry and Molecular BiologyPhotobiology of vitamin D
2023, Feldman and Pike's Vitamin D: Volume One: Biochemistry, Physiology and Diagnostics