Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Liu, L.
Molecular regulation of adrenal function by bile acids
PhD-Studium (Doctor of Philosophy); Humanmedizin; [ Dissertation ] Medical University of Graz; 2019. pp. 105
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Fickert Peter
- Heinemann Akos
- Höfler Gerald
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- Abstract:
- Background and Aims: Bile acids, the main constituents of bile exclusively secreted by the liver, are considered as signaling molecules except for their function of facilitating digestion and absorption. They are involved in the regulation of multiple physiological activities including glucose, amino acid and lipid metabolism. Some known bile acid binding receptors such as the farnesoid X receptor (FXR), the G protein-coupled bile acid receptor (TGR5), and the sphingosine-1-phosphate receptor 2 (S1PR2) are also found to be expressed in adrenals. However, whether bile acids directly act on adrenocortical cells and regulate steroidogenesis is still unclear. In this thesis, we aimed to study the effects of cholestasis and more specifically of the bile acids on the glucocorticoid synthesis of adrenal glands, and to unravel the underlying molecular mechanisms of regulation of bile acids on steroidogenesis in adrenocortical cells. Methods: FXR and TGR5 knock out C57BL/6 mice, together with their corresponding wild type littermates were subjected to common bile duct ligation or 1% chenodeoxycholic acid supplemented diet to model cholestasis. Human adrenocortical carcinoma cell line H295R were cultured with various bile acids agonists or antagonists of engaged pathways were applied. siRNA interference was conducted for genetic inhibition when necessary. Systematic influences of bile acids on steroidogenesis were evaluated by serological tests, real time PCR and Western Blotting assays. Transactivation activity of transcription factors affected by bile acids was studied by luciferase activity assay. Results: We found that both CBDL and CDCA-fed mice had significantly elevated levels of corticosterone, the main glucocorticoid in rodent, which is equivalent to human cortisol, in an FXR and TGR5 independent manner. mRNA and protein levels of most steroidogenesis-related enzymes were also increased by CBDL and CDCA feeding independent of FXR and TGR5. In addition, genes related to transport and de novo synthesis of cholesterol in adrenal gland were found to be increased by CBDL and CDCA feeding as well, while cholesteryl ester concentrations in adrenal glands of CBDL and CDCA-fed mice were strikingly decreased. In cell culture assays, cortisol secretion of H295R cells was substantially enhanced by conjugated CDCA, especially taurine-conjugated CDCA (TCDCA). mRNA and protein expressions of steroidogenesis-related enzymes were also increased upon TCDCA treatment. In contrast, FXR and TGR5 agonists failed to activate cortisol level secreted by H295R cells. ERK phosphorylation was dramatically elevated by TCDCA treatment and was indispensable for TCDCA-induced steroidogenesis in H295R cells. Application of JTE-013, a specific pharmacological inhibitor of S1PR2 successfully attenuated phosphorylation of ERK and abrogated cortisol secretion caused by TCDCA in H295R cells. As a result, enhanced expressions of steroidogenesis-related genes by TCDCA were also abolished by JTE-013. Likewise, siRNA interference significantly inhibited the mRNA and protein levels of S1PR2, thus leading to lower phosphorylation level of ERK, decreased cortisol secretion and reduced mRNA levels of steroidogenesis-related genes in H295R cells. Luciferase activity assays demonstrated that steroidogenic factor-1 (SF-1) transactivation activity was increased upon TCDCA treatment in H295R cells. Accordingly, addition of a SF-1 inverse agonist also decreased steroidogenesis caused by TCDCA. Protein level of SF-1 was found to be decreased by pharmacological inhibition of S1PR2 and ERK phosphorylation in H295R cells. Conclusions: Our combined in vivo and in vitro experimental results indicate that supraphysiological bile acids directly stimulate steroidogenesis in adrenocortical cells via an S1PR2-ERK-SF-1 signaling pathway. These findings help to understand the mechanisms of some diseases such as hepato-adrenal syndrome and osteoporosis encountered in cholestasis.