Characterization of a new pathway that activates lumisterol in vivo to biologically active hydroxylumisterols

Abstract

Using LC/qTOF-MS we detected lumisterol, 20-hydroxylumisterol, 22-hydroxylumisterol, 24-hydroxylumisterol, 20,22-dihydroxylumisterol, pregnalumisterol, 17-hydroxypregnalumisterol and 17,20-dihydroxypregnalumisterol in human serum and epidermis, and the porcine adrenal gland. The hydroxylumisterols inhibited proliferation of human skin cells in a cell type-dependent fashion with predominant effects on epidermal keratinocytes. They also inhibited melanoma proliferation in both monolayer and soft agar. 20-Hydroxylumisterol stimulated the expression of several genes, including those associated with keratinocyte differentiation and antioxidative responses, while inhibiting the expression of others including RORA and RORC. Molecular modeling and studies on VDRE-transcriptional activity excludes action through the genomic site of the VDR. However, their favorable interactions with the A-pocket in conjunction with VDR translocation studies suggest they may act on this non-genomic VDR site. Inhibition of RORα and RORγ transactivation activities in a Tet-on CHO cell reporter system, RORα co-activator assays and inhibition of (RORE)-LUC reporter activity in skin cells, in conjunction with molecular modeling, identified RORα and RORγ as excellent receptor candidates for the hydroxylumisterols. Thus, we have discovered a new biologically relevant, lumisterogenic pathway, the metabolites of which display biological activity. This opens a new area of endocrine research on the effects of the hydroxylumisterols on different pathways in different cells and the mechanisms involved.

Figure 1

Detection of vitamin D3 (D3), lumisterol3 (L3) and 7-dehydrocholesterol (7DHC) in human epidermis (A), human serum (B) and pig adrenal grand (C). Extracted ion chromatograms (EIC) on qTOF LC-MS using m/z = 367.3 [M + H-H₂O]⁺ are shown. The extraction and LC-MS conditions are described in Materials and Methods.

Figure 2

Detection of hydroxyl L3 derivatives in human epidermis (A) and human serum (B). Extracted ion chromatograms (EIC) on qTOF LC-MS are shown using m/z = 383.3 [M + H-H₂O]⁺ for 20(OH)L3 (epidermis and serum), 22(OH)L3 (serum) and 24(OH)L3 (epidermis); 401.3 [M + H]⁺ for 22(OH)L3 (serum) and 24(OH)L3 (epidermis); 439.3 [M + Na]⁺ for 20,22(OH)₂L3 (epidermis); 417.3 [M + H]⁺ for 20,22(OH)₂L3 (serum). Inserts are mass spectra recorded on each indicated peak. The extraction and LC-MS conditions are described in Materials and Methods.

Figure 3

Detection of hydroxyl pL derivatives in human epidermis (A) and human serum (B). Extracted ion chromatograms (EIC) on qTOF LC-MS are shown using m/z = 297.2 [M + H-H₂O]⁺ for pL (epidermis and serum); 313.2 [M + H-H₂O]⁺ for 17(OH)pL (epidermis and serum); 333.2 [M + H]⁺ for 17,20(OH)₂pL (epidermis); 315.2 [M + H-H₂O]⁺ for 17,20(OH)₂pL (serum). Mass spectra detected in each samples are shown below EICs. The extraction and LC-MS conditions are described in Materials and Methods.

Figure 4

Inhibition of keratinocytes proliferation by 20(OH)L3, 22(OH)L3, 24(OH)L3 and 20,22(OH)₂L3. (A), MTS assay with HaCaT keratinocytes. The cells were synchronized by precincubation with serum-free media for 24 h, which was then replaced with DMEM plus 5% charcoal-treated FBS, and graded concentrations of hydroxylumisterols. After 48 h, the plates were used for the MTS assay performed at 490 nm. (B), SRB assay with primary normal human epidermal keratinocytes. After 24 h of culture, fresh keratinocyte media containing graded concentrations of 20(OH)L3 were added. After 24 or 48 h, the plates were processed for SRB assays performed at 570 nm. Data represent means ± SE (n ≥ 3) where *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 at student t-test; and #p < 0.05, ##p < 0.01 and ####p < 0.0001 at one-way ANOVA test and general ANOVA tests are shown.

Figure 5

Inhibition of SKMEL-188 human melanoma growth by 20(OH)L3, 22(OH)L3, 24(OH)L3 and 20,22(OH)₂L3. (A), Inhibition of proliferation in monolayer assed by MTS assay. After 24 h of culture, the cells were exposed to graded concentrations of hydroxylumisterols suspended in Ham’s F10 plus 10% charcoal-treated FBS. After 48 h, the plates were used for MTS assay performed at 490 nm. (B), Inhibition of growth in soft agar (anchorage independent growth). Melanoma cells were suspended in medium containing 0.4% agarose (American Bioanalytical, Natick, MA) and 5% charcoal-treated FBS, and seeded at 1,000 cells/well in a 0.8% agar layer in 24-well plates and treated with the graded concentrations of the listed compounds which were freshly added every 72 h over 13 days. The colonies stained with MTT reagent (Promega, Madison, WI) were analyzed using the Cytation 5 Cell Imaging Multi-Mode Reader in three different z-planes and scored using Gen5 software. Data represent means ± SE (n ≥ 3) where *p < 0.05, **p < 0.01 and ***p < 0.001 by the student t-test, and general ANOVA tests are shown.

Figure 6

Modulation of RORα and RORγ activities by 20(OH)L3, 22(OH)L3, 24(OH)L3 and 20,22(OH)₂L3. (A), RORγ transactivation assay in Tet-on CHO cells. To induce expression of RORγ protein expression, CHO cells were treated with 1 μM doxycycline for 24 h. To measure the transactivation the cells were treated with graded concentrations of the hydroxylumisterols listed, and the RORE-mediated activation of the luciferase reporter activity was assayed with a Luciferase Assay Substrate kit (Promega) as described previously. Assays were performed in triplicate. (B), RORα coactivator assay using LanthaScreen TR-FRET RORα Coactivator kit assay. RORα-LBD was added to graded concentrations of hydroxylumisterols followed by the addition of a mixture of peptide (TRAP220/DRIP2) and antibody (Tb-anti-GST). The reaction mixture was incubated at room temperature for 2 h and the TR-FRET ratio was calculated by dividing the fluorescein emission at 520 nm by the Terbium emission at 495 nm using Synergy neo2 (BioTek Instruments, Inc., Winooski, VT). Data represent means ± SE (n ≥ 3) where ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001 student t-test; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 and ^####p < 0.0001 by one-way ANOVA and general ANOVA tests are shown. (C), RORE luciferase assay in HaCaT keratinocytes. The cells were cotransfected with the reporter plasmids pGL4.27-(RORE)₅ and phRL-TK (Promega) using Lipofectamine (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. After transfection, the cells were treated with hydroxylumisterols for 48 h. Luciferase reporter activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). Firefly and Renilla signals were read using Cytation 5 (BioTek Instruments, Inc., Winooski, VT), and the ratios were calculated.

Figure 7

Docking results using the crystal structure of RORα. (a). The ligand binding site of RORα with the docked pose of lumisterol. Residues mapping the active site are shown; protein carbons are colored dark green, ligand carbons pink, all other atoms by atom type (O red, N blue, S yellow, H white). The dashed line indicates hydrogen bonding between lumisterol and Tyr380 (2.1 Å distance between interacting atoms). (b-c). The overlap of docked poses is illustrated in the RORα binding site in comparison with the co-crystallized 20-hydroxycholesterol. (b) Cholesterol analogs (purple carbons) and (c) Hydroxylumisterol analogs (pink carbons). The co-crystallized cholesterol is shown with light green carbons. Only residues that may contribute to polar interactions are shown; dashed lines indicate hydrogen bonds. (d) The ‘flipped’ pose of hydroxylated pregnalumisterol (pL) analogs binding to RORα. Carbon atoms of ligands are color-coded as shown; protein carbons are dark green (all other atoms are colored by atom type). Hydrogen bonding interactions are shown with dashed lines. Interactions contributed by the 20-OH group of 17, 20(OH)₂pL enantiomers: (S)20-OH forms a water-bridged hydrogen bond with R370; (R)20-OH participates in hydrogen bonding with the backbone carbonyl of Val364.

Figure 8

Docked poses in the RORγ binding site and comparison with inverse agonist bound crystal structures. (a) Docked lumisterol in the RORγ active site is shown in comparison with the co-crystallized 20-hydroxycholesterol (light green colored carbons). Similar view angle and the same color coding is used as in case of RORα (Fig. 7a). Hydrogen bonding interactions are indicated with dashed lines. (b) The overlap of docked poses is illustrated for hydroxylumisterols (20(OH)L3, 22(OH)L3, 24(OH)L3, 20,22(OH)₂L3) with pink color carbons while cholesterol is shown with thick bonds and carbons colored maroon. Key residues involved in inverse agonism of co-crystallized ligands are shown only. Residues in the RORγ crystal structure used in our docking study are displayed with dark green carbons (PDB code 3KYT); residues in RORγ structures co-crystallized with inverse agonist ligands are shown with brown colored carbons (PDB codes 3B0W, 4NB6, 4WQP). Using the same view angle and orientation the inset illustrates 20-hydroxylumisterol in comparison with an inverse agonist ligand co-crystallized in RORγ (PDB code 4WQP), shown with brown colored carbons. The analog of this ligand lacking the one-carbon linker marked with a red arrow is a RORγ agonist. (c) RORγ crystal structures with inverse agonists are aligned onto the structure with PDB code 3KYT. The three key residues (as in Fig. 8b) are also shown, along with co-crystallized ligands from two structures: PDB codes 3KYT and 4WQP. Font colors of PDB codes listed correspond to the coloring of secondary structures and carbon atoms of co-crystallized ligands shown.