Cholesterol is the main sterol in all mammalian cells and is one of the key components that make up cell membranes, determining the structural and functional parameters of the membrane, which is important for cell survival. Cholesterol is a precursor for steroid hormones, bile acids, and oxidized forms of cholesterol. Its roles include interactions with proteins and involvement in various signalling pathways, such as the regulation of the cell cycle. Cholesterol synthesis is a process precisely regulated at multiple stages, and disruptions in this process are consequently linked to numerous pathological conditions.
Cholesterol synthesis is active in all tissues, especially in the liver, which is also responsible for maintaining cholesterol balance in the body. The synthesis of cholesterol is a complex process involving multiple reactions and numerous enzymes. In the post-squalene part, synthesis begins with lanosterol, which is then converted into cholesterol through at least 11 enzymatic reactions. After the conversion of lanosterol, cholesterol synthesis is divided into two proposed pathways: the Bloch pathway and the Kandutsch-Russell pathway, which together involve at least 19 different sterol molecules. It is known that several sterols have other functions unrelated to cholesterol synthesis. One of the more important functions is that cholesterol intermediates can act as ligands for the RORC nuclear receptor, which regulates the transcription of target genes by binding to RORE in DNA promoters. It is not yet clear which sterols serve as physiological ligands for RORC in different cell types and how RORC-activated signalling via sterols affects the circadian clock in the liver and consequently, how it affects metabolic processes associated with the circadian clock. The circadian clock is a transcriptional-translational mechanism that regulates the 24-hour rhythm in organisms. At the genetic level, clock is regulated by genes and proteins of the circadian clock, such as CLOCK, BMAL1, PER and CRY gene families. It is known that the circadian expression of genes at the secondary level is also regulated by transcription factors ROR and RevErb, which activate or deactivate the transcription of circadian genes by binding to RORE.
In doctoral thesis, we focused on sterols that are found in the cholesterol synthesis pathway between lanosterol and cholesterol. The aim was to identify their role as potential ligands for the nuclear receptor RORC in liver cells. To achieve this, we prepared HepG2 cell lines with KO genes involved in cholesterol synthesis, resulting in the enrichment of specific sterols. Additional goal was to establish a methodology for the detection and quantification of sterols.
Using the CRISPR-Cas9 method, we modified the DNA sequence of the CYP51A1, DHCR24, SC5D and HSD17B7 target genes, thereby disrupting cholesterol synthesis at various stages. With the newly developed LC-MS method, which allows for the identification and quantification of 11 nonpolar sterol intermediates from cholesterol synthesis, we demonstrated that none of the target enzymes were functional. Sterols, usually produced after the enzymatic step, were no longer present in the cells, while precursor sterols accumulated in high concentrations. The accumulation of sterols affected the differential expression of genes, with most pathways being altered in a single KO, indicating specific mechanisms involving individual sterols.
Analysis of enriched metabolic pathways, genes, and transcription factors revealed numerous statistically significant altered pathways, but not RORC signalling or pathways connected to RORC signalling. Measurement of genes under RORC control using RT-qPCR and microarrays, did not show the expected differences in the expression of target genes. Successful overexpression of RORC in cell lines and immunoprecipitation of the RORC fraction were achieved, but we were unable to identify sterols specifically bound to RORC from the immunoprecipitate. However, we demonstrated the role of sterols in NFKB/WNT signalling via the LEF1 protein and we have shown that early sterols such as 24,25-dihydrolanosterol promote cell proliferation and changes in the cell cycle. Studies on the circadian expression of genes in HepG2 cells showed that these cells had a low basal oscillation of gene expression. We identified changes in the expression of certain central circadian clock genes in cells with KO genes involved in cholesterol synthesis, compared to control cells. The results indicated altered oscillation of BMAL1 and CRY1 genes and a phase shift in the PER2 gene, but we cannot attribute this to altered RORC signalling. It is most likely influenced by other signalling pathways, such as WNT and NF-KB, as well as changes in the cell cycle, which have already been shown to affect the expression of circadian genes. Our findings suggest that sterols from cholesterol synthesis control various signalling pathways, and only early sterols stimulate cell proliferation
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