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

Andrzej T Slominski,Zorica Janjetovic,Judith V Hobrath,Robert C Tuckey,Tae-Kang Kim,Arnold Postlethwaite,Anton M Jetten,Wei Li,Zongtao Lin,Allen S W Oak,Yukimasa Takeda

doi:10.1038/s41598-017-10202-7

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.

Highlights

The current study shows that this traditional view must be revised, since lumisterol enters the systemic circulation but it can be hydroxylated in vivo by CYP11A1
The CYP11A1-derived hydroxylumisterols inhibit skin cell proliferation in a cell-type dependent fashion with pronounced effects on keratinocytes, and show anti-melanoma activity as well. 20(OH)L3, as a representative hydroxylumisterol, stimulates expression of genes associated with keratinocyte differentiation and anti-oxidative programs
This study reveals that a CYP11A1–mediated pathway of lumisterol metabolism occurs in vivo, the products of which have phenotypic/biological activities determined by their structure and cellular target

Summary

Introduction

The in silico predictions provide additional support for the functional studies on RORs and, as reported in Fig. 6 and supplemental Figure 10, that 20(OH)L3, 22(OH)L3, 24(OH)L3, and 20, 22(OH)2L3 can interact and modify the activities of RORα and RORγ. Functional testing of binding to the VDR has shown that the hydroxylumisterols lack any effect on VDRE- transcriptional activity in HaCaT cells (Supplemental Figure 11) and do not bind to the genomic LBD of the VDR using the LanthaScreen TR-FRET competition kit (not shown). We conclude that CYP11A1-derived hydroxylumisterols are not involved in the regulation of genomic VDR activity.

Results

Conclusion