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Molecular, cellular and in vivo analyses of the effects of genetic polymorphisms in the 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) on peripheral cortisol metabolism in bone

  • In Germany, around 5.7 million people suffer from osteoporosis. Osteoporosis is characterised by a reduced bone mineral density that leads to an increased risk of fractures. The 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) is an important regulator of local cortisol metabolism. It converts biologically inactive cortisone to biologically active cortisol, but can also catalyse the reverse reaction. 11β-HSD1 is strongly expressed in liver, but 11β-HSD1 expression and activity were also reported in bone. Moreover, polymorphisms in intron 5 of HSD11B1 (the gene encoding for 11β-HSD1) are associated with bone mineral density (BMD) and risk of fractures. This work aimed to confirm and refine the associations between polymorphisms in intron 5 of HSD11B1 and BMD, and to identify the underlying molecular and cellular mechanisms. To this end, analyses were performed on three different levels: i) studies in humans, to confirm and refine the association of polymorphisms in intron 5 of HSD11B1 with BMD, suppressed cortisol levels (PDC) and stiffness index, ii) cellular analyses, to identify the role of 11β-HSD1 in differentiation of the immortalised human mesenchymal stem cell line SCP-1, iii) molecular genetic analyses, to reveal the effect of intron 5 polymorphisms on transcriptional regulation. Fine-mapping analyses of already existing clinical data from 452 osteoporosis patients (HSD study) did not point to another intron 5 SNP as being causative for the observed clinical association. A second prospective clinical study (OsteoGene) was performed to confirm the association of rs11811440 and rs932335 with PDC levels and BMD. A trend to decreased PDC levels and increased BMD was observed in homozygous carriers of the minor A-allele of rs11811440 in patients above the age of 65 years. Pooled analyses of the HSD and the OsteoGene studies revealed a significant association of the minor A-allele with increased Z-scores of the left femoral neck. No associations of rs11811440 and rs932335 with stiffness index, BMI and fat depots were detected the general population using data from the SHIP study. To analyse the effect of 11β-HSD1 on differentiation of mesenchymal stem cells, HSD11B1 overexpressing and HSD11B1 knockout SCP-1 cells were generated. HSD11B1 was stably overexpressed in SCP-1 cells using targeted chromosomal integration. The successful overexpression was shown by 243-fold increased HSD11B1 mRNA expression levels and a 9 fold increased 11β-HSD1 activity, compared to the wildtype cells. Knockout cells were generated by CRISPR-Cas9 mediated gene editing targeting exon 2 and exon 5 of HSD11B1. Using next generation sequencing, the clones 1C4 and 2D10 were confirmed to carry two inactive HSD11B1 alleles and were chosen for further analyses. mRNA expression was unchanged in both knockout clones. However, a clear enzyme activity was detected in the 2D10 clone, whereas no cortisol production was detected in the 1C4 clone. SNaPshot analyses revealed the presence of wildtype cells in the 2D10 clone that became predominant with increased passages. Therefore, further analyses were focused on the 1C4 clone only. The protein expression in the 1C4 clone decreased to 30% of the expression of the wildtype cells. HSD11B1 expression and cortisol production were compared between wildtype, knockout and overexpressing SCP-1 cells under three differentiation conditions: adipogenic, osteogenic with 1α,25-dihydroxyvitamin D3 and osteogenic with dexamethasone. HSD11B1 expression increased upon adipogenic differentiation and in the presence of cortisone in the wildtype and the overexpressing, but not in the knockout cells. Also, the cortisol production from cortisone increased over time in the overexpressing and the wildtype cells, but not in the knockout cells. The increase was dependent on the differentiation used between 3-fold and 9-fold higher in the overexpressing than in the wildtype cells. The generated and validated overexpressing and knockout cell lines were used to analyse the influence of 11β-HSD1 on adipogenic and osteogenic differentiation. Upon adipogenic differentiation, the overexpressing cells accumulated significantly more lipid droplets than the wildtype cells. The accumulation of lipid droplets was not abolished in the knockout. However, when dexamethasone was substituted by cortisone, the knockout cells accumulated less lipid droplets than in the presence of dexamethasone, supporting the involvement of 11β-HSD1 in adipogenic differentiation. Expression of the adipogenic markers FABP4 and LPL increased upon adipogenic differentiation, but a distinct influence of the presence or absence of HSD11B1 on the FABP4 and LPL expression was not detected. Upon osteogenic differentiation with 1α,25-dihydroxyvitamin D3, ALP activity increased only in the knockout cells (more than 5-fold). Accordingly, the strongest increase in ALPL expression was detected also in the knockout cells. Both, ALP activity and gene expression were independent of cortisone. Addtionally, BGLAP expression was increased upon osteogenic differentiation. Unexpectedly, in the presence of cortisone, BGLAP expression increased in the overexpressing cells. Expression of the Wnt inhibitor DKK1 also increased in the overexpressing cells in the presence of cortisone indicating a decreased osteogenic differentiation. Moreover, expression of the adipogenic markers FABP4 and LPL increased in the overexpressing cells in the presence of cortisone indicating a switch from osteogenic to adipogenic differentiation. Upon osteogenic differentiation with dexamethasone, ALP activity and matrix mineralisation was lowest in the overexpressing cells. Finally, the effects of the SNPs rs11811440, rs11119328, rs1000283 and rs932335 in intron 5 of HSD11B1 on transcriptional regulation were analysed by reporter gene assays and electrophoretic mobility shift assays. All four SNPs are genetically linked and are localized within evolutionary conserved regions. The minor C-allele of rs932335 significantly increased luciferase activity. In contrast, the major G-allele of rs932335 showed strong protein binding. However, no transcription factor binding sites were identified at the SNP sites. Additionally, bioinformatics analyses of publicly available RNA-Seq data of adipose tissue and liver confirmed the absence of alternative splicing. Alignment of HSD11B1 intron 5 to the Rfam database predicted the presence of non-coding RNAs (ncRNAs) in intron 5. However, none of the ncRNAs overlapped with the SNP sites. In conclusion, 11β-HSD1 was shown to be involved in adipogenic differentiation and peripheral cortisol production by 11β-HSD1 promotes a switch from osteogenic to adipogenic differentiation. Moreover, among osteoporosis patients, homozygous carriers of the minor A-allele of rs11811440 have increased Z-scores of the femoral neck. Furthermore, HSD11B1 knockout and overexpressing cell lines were successfully generated and validated. These cell lines could be a useful tool in future analyses of the role of peripheral cortisol activation by 11β-HSD1 in differentiation of mesenchymal stem cells.

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Metadaten
Author: Angelique Kragl
URN:urn:nbn:de:gbv:9-opus-60897
Title Additional (German):Molekulare, zelluläre und in vivo Analysen der Effekte genetischer Polymorphismen in der 11β-Hydroxysteroid-Dehydrogenase 1 (11β-HSD1) auf den peripheren Cortisolmetabolismus im Knochen
Referee:Prof. Dr. Werner Weitschies, Prof. Dr. Mladen V. Tzvetkov, Prof. Dr. Uwe Kornak
Document Type:Doctoral Thesis
Language:English
Year of Completion:2022
Granting Institution:Universität Greifswald, Mathematisch-Naturwissenschaftliche Fakultät
Date of final exam:2022/03/04
Release Date:2022/03/28
GND Keyword:Hydrocortison, Hydroxysteroid-Dehydrogenasen, Osteoporose
Page Number:298
Faculties:Universitätsmedizin / Institut für Pharmakologie
DDC class:500 Naturwissenschaften und Mathematik / 570 Biowissenschaften; Biologie
600 Technik, Medizin, angewandte Wissenschaften / 610 Medizin und Gesundheit