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Mouse lanosterol-14a-demethylase (CYP51) mRNA expression and the CYP51 promoter-CAT reporter activity

Regulation of lanosterol 14alpha-demethylase (CYP51) gene expression was so far studied primarily in humans. However, in future in vivo mouse studies will be necessary to fully understand the regulation of CYP51 in physiological conditions. The purpose of this study was to investigate the sterol-dependent and cAMP-dependent regulation of mouse CYP51 gene ant to evaluate potential interactions between the two signalling pathways by measuriing CYP51 mRNA levels as well as CYP51 promoter-CAT reporter activity. A portion of the mouse CYP51 promoter was amplified with polymerase chain reaction and cloned into the pCAT3 basic vector to produce CYP51m-CAT. This plasmid was used for transfections of the human cell line JEG-3. Different factors of sterol-dependent and cAMP-dependent signalling pathways were used to manipulate the activity of the promoter. CYP51 mRNA levels have been deduced from experiments applying DNA microaaray technology to study expression of genes involved in cholesterol homeostasis in the mouse pre-adipocyte cell line 3T3-L1. CAT reporter studies lead to the following conclusions: mouse CYP51 promoter is activated by the sterol-dependent and by cAMP-dependent pathway, with predominant regulation by the sterol signalling pathway ; promoter activity decreases with increased lipid concentration in the serum and is activated with increased SREBP concentration in the cell ; promoter can be activated by the cAMP-dependent pathway in the sterol-independent manner. Preliminary studies on CYP51-mRNA level confirmed most of the CAT reporter results. High-lipid conditions (repressed sterol-dependent signalling pathway) resulted in lower expression of CYP51-mRNA while addition of forskolin, a mediator of cAMP-dependent signalling pathway, resulted in elevated CYP mRNA levels. By measuring CYP51mRNA level we were so far unable to confirm that cAMP-dependent signalling pathway is able to overcome the lipid repression. Further studies are necessary to get a complete picture of interactions between the two signalling pathways.

lanosterol 14alpha-demethylase; mRNA expression; cAMP-dependent and sterol-dependent signalling pathways; CYP51 promoter-CAT reporter activity

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