Interactions of Xenobiotics and Oxysterols with 11β-Hydroxysteroid Dehydrogenase 2 and Retinoic Acid-Related Orphan Receptor Gamma

Steroid hormones are regulators of a wide variety of biochemical mechanisms, mainly initiated by the activation of nuclear receptors that regulate the transcription of various target genes. The activation of steroid hormone receptors can be controlled via enzymes serving as pre-receptor control mechanisms. 11β-hydroxysteroid dehydrogenase type 1 and 2 (11β-HSD1/2) represent such enzymes that interconvert the well-investigated glucocorticoid substrates cortisol and cortisone, thereby controlling nuclear receptor activity. Additional 11β-HSD-substrates from the oxysterol family were described, and recent research revealed oxysterols as bioactive ligands at nuclear receptors, including the retinoic acid-related orphan receptor gamma (RORγ) and its isoform RORγt that are involved in multiple (patho-)physiological situations.
This thesis aimed to elucidate the effect of xenobiotics on 11β-HSD2 expression, investigated species-specific susceptibility to xenobiotic-dependent 11β-HSD2 inhibition, and studied interference of xenobiotics with RORγ(t) activity. Furthermore, the goal was to explore the role of 11β-HSDs in pre-receptor control of oxysterol-mediated RORγ(t) activation.
The first project of this thesis included the investigation of the effect of the environmental chemicals tributyltin (TBT) and triphenyltin (TPT) on the regulation of 11β-HSD2 expression. 11β-HSD2 has an essential role in the placenta by inactivating cortisol, thereby controlling access of maternal glucocorticoids reaching the fetus. We revealed that nanomolar concentrations of TBT and TPT increase 11β-HSD2 expression in a placental cell line and confirmed the effect in primary human trophoblast cells. Inhibition or knockdown of the retinoid X receptor alpha (RXRα) significantly diminished this effect, indicating the involvement of this nuclear receptor in the mechanism. After RNA and protein synthesis inhibition, the influence of TBT and TPT on 11β-HSD2 expression was revealed as a direct effect initiated by enhanced gene transcription. Increased 11β-HSD2 expression by TBT, TPT, and further RXRα agonists may disturb glucocorticoid balance at the placental barrier, resulting in reduced exposure of the fetus to active glucocorticoids, which are essential for fetal development.
Clinical case studies described patients suffering from pseudohyperaldosteronism during treatment with the azole antifungal drugs itraconazole and posaconazole. This adverse drug effect is partially caused by 11β-HSD2 inhibition and disturbed pre-receptor control of the mineralocorticoid receptor and has been missed during preclinical investigations. In the second project, we revealed significant species-specific differences in the inhibition of human, mouse, rat, and zebrafish 11β-HSD2 by these azole fungicides. While the tested compounds potently inhibited the human enzyme, the rat enzyme was moderately inhibited, and the moue and the zebrafish 11β-HSD2 were very weakly inhibited. Investigation of chimeric 11β-HSD2 proteins and novel homology models of the human and mouse enzyme increased the structural understanding of the species-specific variability in inhibition. Residues at position 170/172 and the C-terminus were revealed as essential structural elements, mediating potent inhibition of human 11β-HSD2 by the assessed azole antifungals. This structure-activity relationship information will be helpful in future investigations of 11β-HSD2 inhibitors and will aid in selecting suitable animal models for drug safety and efficacy studies.
In the third project, the focus was a toxicological assessment of RORγ and RORγt. RORγt controls the expression of pro-inflammatory cytokines, such as interleukin-17 in T helper 17 (Th17) cells that are linked to inflammation in autoimmune skin diseases, such as psoriasis. We tested parabens and UV filters that are frequently used compounds in cosmetics with attributed endocrine disrupting properties for their potential to interfere with RORγ(t) activity. Hexylparaben, benzylparaben, and benzophenone-10 were the most potent agonists of RORγ(t) with EC50 values of 144 ± 97 nM, 3.39 ± 1.74 µM, and 1.67 ± 1.04 µM, respectively. These substances enhanced pro-inflammatory cytokine expression in EL4 mouse T lymphocyte cells, a cell model for human Th17 cells. By virtual screening of a cosmetics database for structurally similar chemicals, benzylbenzoate, benzylsalicylate, and 4-methylphenylbenzoate were identified as additional substances that can activate these receptors. Dermal application of RORγ(t) agonists may cause adverse outcomes in sensitive human populations with autoimmune skin diseases or interfere with other situations of the skin where Th17 cells are of importance, such as acne.
The fourth project aimed to further explore the role of the steroid metabolizing enzymes 11β-HSD1 and 2 in oxysterol metabolism. We identified 7-keto,27-hydroxycholesterol and 7β,27-dihydroxycholesterol as substrates of 11β-HSD1 and 2, respectively. The detected apparent enzyme affinities were equal or higher than those reported for glucocorticoids. Potent inhibition of 11β-HSD-mediated glucocorticoid metabolism by the investigated oxysterols supported this finding. Additionally, molecular docking calculations were applied to explain the stereospecificity of the enzymes. Importantly, we described a 11β-HSD-dependent pre-receptor control mechanism of RORγ(t), which may have a function in inflammatory bowel disease.
In conclusion, this thesis presents new insight into the transcriptional regulation of 11β-HSD2 and novel structural comprehension of azole fungicide-mediated 11β-HSD2 inhibition. Furthermore, cosmetic additives with the potential to interfere with RORγ(t) were identified. Additionally, this work presents further understanding of oxysterol metabolism by 11β-HSDs and suggests their function in pre-receptor regulation of RORγ(t) activity. Overall, these findings expand the knowledge about the effects of xenobiotics and oxysterols on 11β-HSDs and RORγ(t), as well as their interplay.