Robert C. Tuckey

Differential expression of HPA axis homolog in the skin

Human skin expresses elements of the hypothalamo-pituitary-adrenal (HPA) axis including pro-opiomelanocortin (POMC), corticotropin releasing hormone (CRH), the CRH receptor-1 (CRH-R1), key enzymes of corticosteroid synthesis and synthesizes glucocorticoids. Expression of these elements is organized in functional, cell type-specific regulatory loops, which imitate the signaling hierarchy of the HPA axis. In melanocytes and fibroblasts CRH-induced CRH-R1 stimulation upregulates POMC expression and production of ACTH through activation of cAMP dependent pathway(s). Melanocytes respond with enhanced production of cortisol and corticosterone, which is dependent on POMC activity. Fibroblasts respond to CRH and ACTH with enhanced production of corticosterone, but not cortisol, which is produced constitutively. Organ-cultured human scalp hair follicles also show a fully functional HPA axis equivalent, including cortisol synthesis and secretion and negative feedback regulation by cortisol on CRH expression. Thus, differential, CRH-driven responses of defined cutaneous cell populations reproduce key features of the central HPA axis at the tissue/single cell levels.

In vivo evidence for a novel pathway of vitamin D3 metabolism initiated by P450scc and modified by CYP27B1

We define previously unrecognized in vivo pathways of vitamin D3 (D3) metabolism generating novel D3‐hydroxyderivatives different from 25‐hydroxyvitamin D3 [25(OH)D3] and 1,25(OH)2D3. Their novel products include 20‐hydroxyvitamin D3 [20(OH)D3], 22(OH)D3, 20,23(OH)2D3, 20,22(OH)2D3, 1,20(OH)2D3,1,20,23(OH)3D3, and 17,20,23(OH)3D3 and were produced by placenta, adrenal glands, and epidermal keratinocytes. We detected the predominant metabolite [20(OH)D3] in human serum with a relative concentration ~20 times lower than 25(OH)D3. Use of inhibitors and studies performed with isolated mitochondria and purified enzymes demonstrated involvement of the steroidogenic enzyme cytochrome P450scc (CYP11A1) as well as CYP27B1 (1α‐hydroxylase). In placenta and adrenal glands with high CYP11A1 expression, the predominant pathway was D3 → 20(OH)D3 → 20,23(OH)2D3 → 17,20,23(OH)3D3 with further 1α‐hydroxylation, and minor pathways were D3 → 25(OH)D3 → 1,25(OH)2D3 and D3 → 22(OH)D3 → 20,22(OH)2D3. In epidermal keratinocytes, we observed higher proportions of 22(OH)D3 and 20,22(OH)2D3. We also detected endogenous production of 20(OH)D3, 22(OH) D3, 20,23(OH)2D3, 20,22(OH)2D3, and 17,20,23(OH)3D3 by immortalized human keratinocytes. Thus, we provide in vivo evidence for novel pathways of D3 metabolism initiated by CYP11A1, with the product profile showing organ/cell type specificity and being modified by CYP27B1 activity. These findings define the pathway intermediates as natural products/endogenous bioregulators and break the current dogma that vitamin D is solely activated through the sequence D3 → 25(OH)D3 → 1,25(OH)2D3.—Slominski, A. T., Km, T.‐K., Shehabi, H. Z., Semak, I., Tang, E. K. Y., Nguyen, M. N., Benson, H. A. E., Korik, E., Janjetovic, Z., Chen, J., Yates, C. R., Postlethwaite, A., Li, W., Tuckey, R. C. In vivo evidence for a novel pathway of vitamin D3 metabolism initiated by P450scc and modified by CYP27B1. FASEB J. 26, 3901–3915 (2012). www.fasebj.org

The cytochrome P450scc system opens an alternate pathway of vitamin D3 metabolism

We show that cytochrome P450scc (CYP11A1) in either a reconstituted system or in isolated adrenal mitochondria can metabolize vitamin D3. The major products of the reaction with reconstituted enzyme were 20‐hydroxycholecalciferol and 20,22‐dihydroxycholecalciferol, with yields of 16 and 4%, respectively, of the original vitamin D3 substrate. Trihydroxycholecalciferol was a minor product, likely arising from further metabolism of dihydroxycholecalciferol. Based on NMR analysis and known properties of P450scc we propose that hydroxylation of vitamin D3 by P450scc occurs sequentially and stereospecifically with initial formation of 20(S)‐hydroxyvitamin D3. P450scc did not metabolize 25‐hydroxyvitamin D3, indicating that modification of C25 protected it against P450scc action. Adrenal mitochondria also metabolized vitamin D3 yielding 10 hydroxyderivatives, with UV spectra typical of vitamin D triene chromophores. Aminogluthimide inhibition showed that the three major metabolites, but not the others, resulted from P450scc action. It therefore appears that non‐P450scc enzymes present in the adrenal cortex to some extent contribute to metabolism of vitamin D3. We conclude that purified P450scc in a reconstituted system or P450scc in adrenal mitochondria can add one hydroxyl group to vitamin D3 with subsequent hydroxylation being observed for reconstituted enzyme but not for adrenal mitochondria. Additional vitamin D3 metabolites arise from the action of other enzymes in adrenal mitochondria. These findings appear to define novel metabolic pathways involving vitamin D3 that remain to be characterized.

20-Hydroxyvitamin D3, a Product of Vitamin D3 Hydroxylation by Cytochrome P450scc, Stimulates Keratinocyte Differentiation

It has been shown that mammalian cytochrome P450scc can metabolize vitamin D3 to 20-hydroxyvitamin D3 (20(OH)D3) and 20,22(OH)2D3. To define the biological significance of this pathway, we tested the effects of 20(OH)D3 on the differentiation program of keratinocytes and on the expression of enzymes engaged in vitamin D3 metabolism. Immortalized HaCaT and adult human epidermal keratinocytes were used as a model and the effects of 20(OH)D3 were compared with those of 25(OH)D3 and 1,25(OH)2D3. 20(OH)D3 inhibited proliferation and caused G2/M arrest. 20(OH)D3 stimulated involucrin and inhibited cytokeratin 14 expression. The potency of 20(OH)D3 was comparable to that of 1,25(OH)2D3. 20(OH)D3 decreased the expression of cytochrome P450 enzyme (CYP)27A1 and CYP27B1, however, having only slight effect on CYP24. The effect of 20(OH)D3 was dependent on the vitamin D receptor (VDR). As shown by electrophoretic mobility shift assay, 20(OH)D3 stimulated the binding of nuclear proteins to the VDRE. Transfection of cells with VDR-specific siRNA decreased 20(OH)D3-stimulated transcriptional activity of the VDRE promoter and the expression of involucrin and CYP24 mRNA. Therefore, the above studies identify 20(OH)D3 as a biologically active secosteroid that induces keratinocyte differentiation. These data imply that the previously unreported pathway of vitamin D3 metabolism by P450scc may have wider biological implications depending, for example, on the extent of adrenal gland or cutaneous metabolism.

20-Hydroxycholecalciferol, Product of Vitamin D3 Hydroxylation by P450scc, Decreases NF-κB Activity by Increasing IκBα Levels in Human Keratinocytes

The side chain of vitamin D3 is hydroxylated in a sequential manner by cytochrome P450scc (CYP11A1) to form 20-hydroxycholecalciferol, which can induce growth arrest and differentiation of both primary and immortalized epidermal keratinocytes. Since nuclear factor-κB (NF-κB) plays a pivotal role in the regulation of cell proliferation, differentiation and apoptosis, we examined the capability of 20-hydroxycholecalciferol to modulate the activity of NF-κB, using 1,25-dihydroxycholecalciferol (calcitriol) as a positive control. 20-hydroxycholecalciferol inhibits the activation of NFκB DNA binding activity as well as NF-κB-driven reporter gene activity in keratinocytes. Also, 20-hydroxycholecalciferol induced significant increases in the mRNA and protein levels of the NF-κB inhibitor protein, IκBα, in a time dependent manner, while no changes in total NF-κB-p65 mRNA or protein levels were observed. Another measure of NF-κB activity, p65 translocation from the cytoplasm into the nucleus was also inhibited in extracts of 20-hydroxycholecalciferol treated keratinocytes. Increased IκBα was concomitantly observed in cytosolic extracts of 20-hydroxycholecalciferol treated keratinocytes, as determined by immunoblotting and immunofluorescent staining. In keratinocytes lacking vitamin D receptor (VDR), 20-hydroxycholecalciferol did not affect IκBα mRNA levels, indicating that it requires VDR for its action on NF-κB activity. Comparison of the effects of calcitrol, hormonally active form of vitamin D3, with 20-hydrocholecalciferol show that both agents have a similar potency in inhibiting NF-κB. Since NF-κB is a major transcription factor for the induction of inflammatory mediators, our findings indicate that 20-hydroxycholecalciferol may be an effective therapeutic agent for inflammatory and hyperproliferative skin diseases.

Products of vitamin D3 or 7-dehydrocholesterol metabolism by cytochrome P450scc show anti-leukemia effects, having low or absent calcemic activity.

Background Cytochrome P450scc metabolizes vitamin D3 to 20-hydroxyvitamin D3 (20(OH)D3) and 20,23(OH)2D3, as well as 1-hydroxyvitamin D3 to 1α,20-dihydroxyvitamin D3 (1,20(OH)2D3). It also cleaves the side chain of 7-dehydrocholesterol producing 7-dehydropregnenolone (7DHP), which can be transformed to 20(OH)7DHP. UVB induces transformation of the steroidal 5,7-dienes to pregnacalciferol (pD) and a lumisterol-like compounds (pL). Methods and Findings To define the biological significance of these P450scc-initiated pathways, we tested the effects of their 5,7-diene precursors and secosteroidal products on leukemia cell differentiation and proliferation in comparison to 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). These secosteroids inhibited proliferation and induced erythroid differentiation of K562 human chronic myeloid and MEL mouse leukemia cells with 20(OH)D3 and 20,23(OH)2D3 being either equipotent or slightly less potent than 1,25(OH)2D3, while 1,20(OH)2D3, pD and pL compounds were slightly or moderately less potent. The compounds also inhibited proliferation and induced monocytic differentiation of HL-60 promyelocytic and U937 promonocytic human leukemia cells. Among them 1,25(OH)2D3 was the most potent, 20(OH)D3, 20,23(OH)2D3 and 1,20(OH)2D3 were less active, and pD and pL compounds were the least potent. Since it had been previously proven that secosteroids without the side chain (pD) have no effect on systemic calcium levels we performed additional testing in rats and found that 20(OH)D3 had no calcemic activity at concentration as high as 1 µg/kg, whereas, 1,20(OH)2D3 was slightly to moderately calcemic and 1,25(OH)2D3 had strong calcemic activity. Conclusions We identified novel secosteroids that are excellent candidates for anti-leukemia therapy with 20(OH)D3 deserving special attention because of its relatively high potency and lack of calcemic activity.

20-Hydroxyvitamin D2 is a noncalcemic analog of vitamin D with potent antiproliferative and prodifferentiation activities in normal and malignant cells

20-hydroxyvitamin D(2) [20(OH)D(2)] inhibits DNA synthesis in epidermal keratinocytes, melanocytes, and melanoma cells in a dose- and time-dependent manner. This inhibition is dependent on cell type, with keratinocytes and melanoma cells being more sensitive than normal melanocytes. The antiproliferative activity of 20(OH)D(2) is similar to that of 1,25(OH)(2)D(3) and of newly synthesized 1,20(OH)(2)D(2) but significantly higher than that of 25(OH)D(3). 20(OH)D(2) also displays tumorostatic effects. In keratinocytes 20(OH)D(2) inhibits expression of cyclins and stimulates involucrin expression. It also stimulates CYP24 expression, however, to a significantly lower degree than that by 1,25(OH)(2)D(3) or 25(OH)D(3). 20(OH)D(2) is a poor substrate for CYP27B1 with overall catalytic efficiency being 24- and 41-fold lower than for 25(OH)D(3) with the mouse and human enzymes, respectively. No conversion of 20(OH)D(2) to 1,20(OH)(2)D(2) was detected in intact HaCaT keratinocytes. 20(OH)D(2) also demonstrates anti-leukemic activity but with lower potency than 1,25(OH)(2)D(3). The phenotypic effects of 20(OH)D(2) are mediated through interaction with the vitamin D receptor (VDR) as documented by attenuation of cell proliferation after silencing of VDR, by enhancement of the inhibitory effect through stable overexpression of VDR and by the demonstration that 20(OH)D(2) induces time-dependent translocation of VDR from the cytoplasm to the nucleus at a comparable rate to that for 1,25(OH)(2)D(3). In vivo tests show that while 1,25(OH)(2)D(3) at doses as low as 0.8 μg/kg induces calcium deposits in the kidney and heart, 20(OH)D(2) is devoid of such activity even at doses as high as 4 μg/kg. Silencing of CY27B1 in human keratinocytes showed that 20(OH)D(2) does not require its transformation to 1,20(OH)(2)D(2) for its biological activity. Thus 20(OH)D(2) shows cell-type dependent antiproliferative and prodifferentiation activities through activation of VDR, while having no detectable toxic calcemic activity, and is a poor substrate for CYP27B1.

RORα and ROR γ are expressed in human skin and serve as receptors for endogenously produced noncalcemic 20-hydroxy- and 20,23-dihydroxyvitamin D

RORα and RORγ are expressed in human skin cells that produce the noncalcemic 20‐hydroxyvitamin D3 [20(OH)D3] and 20,23‐dihydroxyvitamin D3 [20,23(OH)2D3]. Chinese hamster ovary (CHO) cells stably expressing a Tet‐on RORα or RORγ expression vector and a ROR‐responsive element (RORE)‐LUC reporter, and a mammalian 2‐hybrid model examining the interaction between the ligand binding domain (LBD) of RORα or RORγ with an LBD‐interacting LXXLL‐peptide, were used to study ROR‐antagonist activities. These assays revealed that 20(OH)D3 and 20,23(OH)2D3 function as antagonists of RORα and RORγ. Moreover, 20(OH)D3 inhibited the activation of the promoter of the Bmal1 and G6pase genes, targets of RORα, and 20(OH)D3 and 20,23(OH)2D3 inhibited Il17 promoter activity in Jurkat cells overexpressing RORα or RORγ. Molecular modeling using crystal structures of the LBDs of RORα and RORγ revealed docking scores for 20(OH)D3, 20, 23(OH)2D3 and 1,25(OH)2D3 similar to those of the natural ligands, predicting good binding to the receptor. Notably, 20(OH)D3, 20,23(OH)2D3, and 1,25(OH)2D3 inhibited RORE‐mediated activation of a reporter in keratinocytes and melanoma cells and inhibited IL‐17 production by immune cells. Our study identifies a novel signaling pathway, in which 20(OH)D3 and 20,23(OH)2D3 act as antagonists or inverse agonists of RORα and RORγ, that opens new possibilities for local (skin) or systemic regulation.—Slominski, A. T., Kim, T.‐K., Takeda, Y., Janjetovic, Z., Brożyna, A. A., Skobowiat, C., Wang, J., Postlethwaite, A., Li, W., Tuckey, R. C., Jetten, A. M. RORα and ROR γ are expressed in human skin and serve as receptors for endogenously produced noncalcemic 20‐hydroxy‐ and 20,23‐dihydroxyvitamin D. FASEB J. 28, 2775–2789 (2014). www.fasebj.org

Pathways and products for the metabolism of vitamin D3 by cytochrome P450scc

Cytochrome P450scc (CYP11A1) can hydroxylate vitamin D3 to produce 20‐hydroxyvitamin D3 and other poorly characterized hydroxylated products. The present study aimed to identify all the products of vitamin D3 metabolism by P450scc, as well as the pathways leading to their formation. Besides 20‐hydroxyvitamin D3, other major metabolites of vitamin D3 were a dihydroxyvitamin D3 and a trihydroxyvitamin D3 product. The dihydroxyvitamin D3 was clearly identified as 20,23‐dihydroxyvitamin D3 by NMR, in contrast to previous reports that postulated hydroxyl groups in positions 20 and 22. NMR of the trihydroxy product identified it as 17α,20,23‐trihydroxyvitamin D3. This product could be directly produced by P450scc acting on 20,23‐dihydroxyvitamin D3, confirming that hydroxyl groups are present at positions 20 and 23. Three minor products of D3 metabolism by P450scc were identified by MS and by examining their subsequent metabolism by P450scc. These products were 23‐hydroxyvitamin D3, 17α‐hydroxyvitamin D3 and 17α,20‐dihydroxyvitamin D3 and arise from the three P450scc‐catalysed hydroxylations occurring in a different order. We conclude that the major pathway of vitamin D3 metabolism by P450scc is: vitamin D3 → 20‐hydroxyvitamin D3 → 20,23‐dihydroxyvitamin D3 → 17α,20,23‐trihydroxyvitamin D3. The major products dissociate from the P450scc active site and accumulate at a concentration well above the P450scc concentration. Our new identification of the major dihydroxyvitamin D3 product as 20,23‐dihydroxyvitamin D3, rather than 20,22‐dihydroxyvitamin D3, explains why there is no cleavage of the vitamin D3 side chain, unlike the metabolism of cholesterol by P450scc.

An alternative pathway of vitamin D2 metabolism Cytochrome P450scc (CYP11A1)-mediated conversion to 20-hydroxyvitamin D2 and 17,20-dihydroxyvitamin D2

We report an alternative, hydroxylating pathway for the metabolism of vitamin D2 in a cytochrome P450 side chain cleavage (P450scc; CYP11A1) reconstituted system. NMR analyses identified solely 20‐hydroxyvitamin D2 and 17,20‐dihydroxyvitamin D2 derivatives. 20‐Hydroxyvitamin D2 was produced at a rate of 0.34 mol·min−1·mol−1 P450scc, and 17,20‐dihydroxyvitamin D2 was produced at a rate of 0.13 mol·min−1·mol−1. In adrenal mitochondria, vitamin D2 was metabolized to six monohydroxy products. Nevertheless, aminoglutethimide (a P450scc inhibitor) inhibited this adrenal metabolite formation. Initial testing of metabolites for biological activity showed that, similar to vitamin D2, 20‐hydroxyvitamin D2 and 17,20‐dihydroxyvitamin D2 inhibited DNA synthesis in human epidermal HaCaT keratinocytes, although to a greater degree. 17,20‐Dihydroxyvitamin D2 stimulated transcriptional activity of the involucrin promoter, again to a significantly greater extent than vitamin D2, while the effect of 20‐hydroxyvitamin D2 was statistically insignificant. Thus, P450scc can metabolize vitamin D2 to generate novel products, with intrinsic biological activity (at least in keratinocytes).