Novel vitamin D photoproducts and their precursors in the skin

Abstract

Novel metabolic pathways initiated by the enzymatic action of CYP11A1 on 7DHC (7-dehydrocholesterol), ergosterol, vitamins D3 and D2 were characterized with help of chemical synthesis, UV and mass spectrometry and NMR analyses. The first pathway follows the sequence 7DHC→22(OH)7DHC → 20,22(OH)27DHC → 7DHP (7-dehydropregnenolone), which can further be metabolized by steroidogenic enzymes. The resulting 5,7-dienes can be transformed by UVB to corresponding, biologically active, secosteroids. Action of CYP11A1 on vitamin D3 and D2 produces novel hydroxyderivatives with OH added at positions C17, C20, C22, C23 and C24, some of which can be hydroxylated by CYP27B1 and/or by CYP27A1 and/ or by CYP24A1.The main products of these pathways are biologically active with a potency related to their chemical structure and the target cell type. Main products of CYP11A1-mediated metabolism on vitamin D are non-calcemic and non-toxic at relatively high doses and serve as partial agonists on the vitamin D receptor. New secosteroids are excellent candidates for therapy of fibrosing, inflammatory or hyperproliferative disorders including cancers and psoriasis.

Keywords: 5,7-dienes; dermal fibroblasts; keratinocytes; melanocytes; melanoma cells; skin; vitamin D.

Figure 1. ∆7 Steroidogenic pathways with the enzymes involved and major products.

Figure 2. The synthesis of the 5,7-diene precursors of secosteroid analogs.

Figure 3. The synthesis of 20R(OH)7DHC.

Figure 4. The plausible synthetic route of 20(OH)ergosterol

Figure 5. UVB-induced transformation of steroidal 5,7-dienes to secosteroids

Figure 6. pL (3β-hydroxy-9β,10α-pregna-5,7-dien-20-one) and 20(OH)pL (9β,10α-pregna-5,7-diene-3β,20-diol) inhibit the anchorage independent growth (ability to form colonies in soft agar) of human melanoma cells in a dose-dependent manner. SKMel-188 human melanoma cells were grown in soft agar in the presence of graded concentrations of 20(OH)pL (A and B) or pL (C) as described previously. After three weeks colonies with a diameter larger than 0.2 mm (A) or 0.5 mm (B and C) were counted. Data are shown as mean ± SD (n = 4); statistical significance was estimated using the t-test and is presented as *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 7. Comparison of the effects of vitamin D derivatives having a full-length site chain to those of 7DHP and pD, on the proliferation of the immortalized line, PIG1, of human melanocytes. The immortalized melanocytes were cultured as described previously. To study DNA synthesis, immortalized melanocytes were plated in 96-well plates at 10,000 cells/well. After overnight incubation, 20(OH)D₃, 1,25(OH)₂D₃, 20,23(OH)₂D₃, 25(OH)D₃, pD, 7DHP or ethanol as a control were added to the cells which were processed as described previously. Briefly, after 12 h of incubation [³H]-thymidine was added to a final concentration of 1 μCi/mL medium. After an additional 12 h, media were discarded, cells washed with cold PBS, lysed and processed for final measurement of the incorporated of ³H-radioactivity into DNA with a β counter. Significant differences between treated and non-treated (ethanol control) cells were measured using the t-test (*p < 0.05, **p < 0.01 and ***p < 0.001). Data are expressed as % of control.

Figure 8. 7DHP, pD, 17,20R(OH)₂7DHP and 17,20S(OH)₂7DHP inhibit TGF-β-induced collagen synthesis. Human dermal fibroblasts grown from explant skin cultures at less than 10 subpassages were used as described previously. After a 2 h preincubation with the compounds being tested at concentrations of 10⁻⁹ or 10⁻¹⁰ M, or the vehicle control (ethanol), TGF-β1 (R and D systems) was added to each well (except control) at a final concentration of 5 ng/ml. After 48 h of culture, plate wells were pulsed with 1 μCi ³[H]-proline. After 24 h, culture supernatants were harvested and collagenase sensitive protein was determined.^, Data are shown as mean ± SD (n = 4); statistical significance was estimated using the t-test and showed that TGF-β1 stimulated collagen synthesis, while the 5,7-dienes and pD inhibited it (p < 0.001). pD was more potent than its precursor, 7DHP, at both concentrations: 1 nM (p < 0.01) and 0.1 nM (p < 0.05).

Figure 9. 20S(OH)7DHC (precursor to 20S(OH)D₃) inhibits proliferation of neonatal human epidermal keratinocytes (HEKn). HEKn in their third passage were treated with graded concentrations of 20S(OH)7DHC for 24 h (A) or 48 h (B), as described previously. Incorporation of radioactive thymidine (³H) into DNA was determined 4 h after incubation. Data are presented as mean ± SD (n = 4). DNA incorporation was calculated as a percentage of the control (ethanol treated cells). Statistical significance was measured using one-way ANOVA presented as *p < 0.05, **p < 0.01 and p < 0.001.

Figure 10. 7DHC and 7DHP attenuate oxidative protein damage in isolated rat liver mitochondria treated with iron/ascorbate. (A) 7DHC and 7DHP are more effective than cholesterol, vitamin D₃ and vitamin D₂ in attenuating oxidative protein modification. (B) Relative antioxidant abilities of 7-DHC and 7-DHP in comparison to GSH, melatonin and its metabolites (6-hydroxymelatonin, N1-acetyl-N2-formyl-5-methoxykynuramine(AFMK) and its precursor, N-acetylserotonin (NAS). The carbonyl content of mitochondrial proteins following treatment with iron/ascorbate was measured as described in the supplemental file. Data are presented as means ± SEM (n = 3), statistical significance was estimated using the t-test. The basal level of protein-bound carbonyls in intact mitochondria was 2.20 ± 0.13 nmol/mg protein. The content of protein carbonyls increased 3.6 times over the basal level after incubation of mitochondria in the iron/ascorbate system for 45 min.